Discovery of pyrazinone based compounds that potently inhibit the drug-resistant enzyme variant R155K of the hepatitis C virus NS3 protease

Article abstract of DOI:10.1016/j.bmc.2016.03.066

Herein, we present the design and synthesis of 2(1H)-pyrazinone based HCV NS3 protease inhibitors with variations in the C-terminus. Biochemical evaluation was performed using genotype 1a, both the wild-type and the drug resistant enzyme variant, R155K. S

Full text of DOI:10.1016/j.bmc.2016.03.066

Bioorganic & Medicinal Chemistry 24 (2016) 2603–2620
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of pyrazinone based compounds that potently inhibit the
drug-resistant enzyme variant R155K of the hepatitis C virus NS3
protease
Anna Karin Belfrage a, Eldar Abdurakhmanov b, Eva Åkerblom a, Peter Brandt a, Anna Oshalim a,
Johan Gising a, Anna Skogh a, Johan Neyts c, U. Helena Danielson b, Anja Sandström a,
⇑
a Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry, Uppsala University, Box 574, SE-75123 Uppsala, Sweden
b Department of Chemistry-BMC, Uppsala University, Box 576, SE-75123 Uppsala, Sweden
c Rega Institute, Department of Microbiology and Immunology, University of Leuven, B-3000 Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
Herein, we present the design and synthesis of 2(1H)-pyrazinone based HCV NS3 protease inhibitors with
variations in the C-terminus. Biochemical evaluation was performed using genotype 1a, both the wild-
type and the drug resistant enzyme variant, R155K. Surprisingly, compounds without an acidic sulfon-
amide retained good inhibition, challenging our previous molecular docking model. Moreover, selected
compounds in this series showed nanomolar potency against R155K NS3 protease; which generally con-
fer resistance to all HCV NS3 protease inhibitors approved or in clinical trials. These results further
strengthen the potential of this novel substance class, being very different to the approved drugs and
clinical candidates, in the development of inhibitors less sensitive to drug resistance.
Received 22 October 2015
Revised 17 March 2016
Accepted 21 March 2016
Available online 8 April 2016
Keywords:
Hepatitis C virus
Drug resistance
Pyrazinone
Ó 2016 Elsevier Ltd. All rights reserved.
NS3 protease inhibitors
R155K
1. Introduction
(IncivekTM, 1) were approved for the treatment of HCV Gt 1 infec-
tion in combination with ribavirin and pegIFNa. These were fol-
Hepatitis C virus (HCV) is regarded a global health problem with
around 130–150 million people chronically infected worldwide
and approximately 350,000–500,000 related deaths each year.1,2
75–85% of those who get infected will develop a chronic infection
which could lead to development of cirrhosis, end-stage liver dis-
ease and hepatocellular carcinoma if left untreated.3 The medical
treatment has for many years relied on the broad-spectrum antivi-
lowed by two novel drugs being approved in 2013; the product
based NS3 protease inhibitor simeprevir10 (OlysioTM, 2), and the
NS5B polymerase inhibitor sofosbuvir11 (SovaldiTM, 3), as outlined
in Figure 1, both in combination with pegIFNa and ribavirin. These
drugs showed less side effects and improved pharmacokinetic
properties which enabled attractive dosing regimens.12,13 Of par-
ticular note, the first all oral treatments, without the need for rib-
ral agent ribavirin in combination with pegylated interferon-
a
avirin or pegIFNa injections, were recently approved: the
(pegIFN
a
).4 This therapy is associated with serious adverse effects,
combination pill, HarvoniTM (ledipasvir14, NS5A inhibitor/Sofosbu-
and the sustained virological response (SVR) is highly dependent
on the genotype of the virus.4 The SVR is especially low for geno-
type (Gt) 1 infection (40–50%) and higher for Gt 2 and 3 (80%).4,5
During the past decade several drug discovery programs have
focused on the development of direct-acting antivirals (DAAs)
especially directed towards HCV Gt 1 infection.6 DAAs interfering
with various stages of the HCV life cycle, for instance the NS3 pro-
tease, the NS5A phosphoprotein and the NS5B polymerase have
been developed.7 In 2011, the first DAAs, the electrophilic HCV
NS3 protease inhibitors, boceprevir8 (VictrelisTM) and telaprevir9
vir 3), a combination of simeprevir (2) and sofosbuvir (3) and Vie-
kira PakTM,
a combination of three DAAs and ritonavir, i.e.,
paritaprevir15 (4) (NS3 protease inhibitor), ombitasvir16 (NS5A
inhibitor) and dasabuvir17 (non-nucleoside NS5B polymerase inhi-
bitor). Thus, the treatment options have been improved drastically
for HCV infected patients in recent years. Moreover, there are still
many drugs and combinations in the pipeline, including for exam-
ple several NS3 protease inhibitors18,19 (e.g., Danoprevir (5), Fig. 1).
Despite recent success in drug development within the field, the
majority of people infected with HCV is still undiagnosed, which is
a problem that remains.3,13,20,21 Moreover, the high costs of the
anti-HCV drugs will limit the access to effective HCV therapies
for people living in resource-poor countries.6,7 Another concern
⇑
Corresponding author. Tel.: +46 18 4714285; fax: +46 18 4714474.
E-mail address: anja.sandstrom@orgfarm.uu.se (A. Sandström).
http://dx.doi.org/10.1016/j.bmc.2016.03.066
0968-0896/Ó 2016 Elsevier Ltd. All rights reserved.

2604
A. K. Belfrage et al. / Bioorg. Med. Chem. 24 (2016) 2603–2620
which were presumed to interact with the S2 pocket in the NS3
O
protease. The improved potency gained by changing a tert-butyl
carbamate with a tert-butyl urea as a P3-capping group was postu-
lated to be related to an internal hydrogen bond between the urea
and the 4-N on the pyrazinone, stabilizing the bound conformation
of the inhibitor.26
H
N
H
N
O
N
H
N
O
O
N
N
N
H
O
O
We herein present synthesis and further development of
pyrazinone-based HCV NS3 protease inhibitors as well as biochem-
ical evaluation against Gt 1a, both wild-type and the resistant
enzyme variant R155K. Different variations in the R0 (P1P10) region
(Fig. 3), including various regioisomers, substituents, and reversed
acyl sulfonamides were studied.
1
Telaprevir ( ) (Vertex)
S
O
N
O
N
O
N
O
P
NH
NH
O
O
O
P2
H3C
O
O
O
O
O
N
H
N
S
2. Chemistry
N
O
O
H
O
P1
A diverse set of R0-substituents were synthesized (Fig. 3), as
described in Section 2.1. These building blocks were subsequently
combined with P3-pyrazinones with varied R6 moieties yielding
the final inhibitors, as described in Sections 2.2 and 2.3.
F
OH
Simeprevir (2) (Janssen/Medivir)
Sofosbuvir (3) (Gilead)
F
2.1. Synthesis of R0-building blocks
O
N
O
N
O
The R0-building blocks 12–14 (Scheme 1) encompassing a
bromo-, fluoro- or trifluoromethyl substituent, were prepared
starting from the corresponding commercially available aminoben-
zoic acids (7–9) which were N-Boc protected using di-tert-butyl
dicarbonate in the presence of 1 M NaOH in dioxane. The resulting
benzoic acids (12–14) were coupled with 4-(trifluoromethyl) ben-
zene sulfonamide using carbonyl-diimidazole (CDI) and 1,8-diaz-
abicycloundecene (DBU) in dry THF yielding 20, 23–24 (74–80%).
The same coupling procedure, but with various regioisomers of
the trifluoromethyl- or methylbenzene sulfonamide, was followed
in the preparation of the analogs 15–19 and 27 (Scheme 1) starting
from 10–11 and 26 (19–74% yield). The formation of the spirocyclic
analog 30 (Scheme 1) began with hydrolysis of the ethyl ester
2927,28 by LiOH in THF/H2O at room temperature producing the
carboxylic acid 30 in 93% yield. CDI coupling with 4-(trifluo-
romethyl) benzene sulfonamide gave the Boc protected building
block 31 in excellent yield (89%). Compounds 21 and 22 (Scheme 1)
were prepared from 20 in a microwave (MW) assisted Suzuki cou-
pling, using 2,4,6-trivinylcycloboroxane pyridine complex29 and 5-
pyrimidine boronic acid (66% and 48%, respectively). The methy-
lated acyl sulfonamide 25 (Scheme 1) was prepared by treating
1530 with methyl iodide and cesium carbonate in dry DMF (47%
yield). In order to deprotect the free amine of 15–25 and 31 for
subsequent amide coupling, the N-Boc moieties were removed
using 4 M HCl in dioxane yielding the hydrochloride salts of the
corresponding amines, while the nitro group in compound 27
was reduced by catalytic hydrogenation to give 28 in 92% yield.
The synthesis of reversed acyl sulfonamides 38–40 started with
the corresponding nitro benzene sulfonamides 32–34, which were
coupled with 4-trifluoromethyl benzoyl chloride, giving intermedi-
ates 35–37 in moderate yields (45–58%) as depicted in Scheme 2.
O
H
O
O
N
O
H
NH
N
N
S
N
N
N
O
N
O
S
O
NH
H
O
O
O
O
O
O
N
H
Paritaprevir (4) (Abbvie)
Danoprevir (5) (Roche)
Figure 1. Examples of approved drugs for HCV treatment: the HCV NS3/4a protease
inhibitors 1, 2, 4, 5 and the NS5B polymerase inhibitor 3.
with the highly potent antivirals and an error prone viral poly-
merase, such as the one in HCV, is the apparent risk for develop-
ment of drug-resistant enzyme variants.22 Mutations in the
genome corresponding to amino acid positions R155, A156 and
D168 in the NS3 protease are common in Gt 1 infected patients
and, e.g., R155K/T/Q confer resistance to all NS3 protease inhibitors
approved and in clinical trials.23
Most of the advanced NS3 protease inhibitors share structural
similarities, such as a P2-proline/cyclopentyl in combination with
a P1-vinylcyclopropyl/ethylcyclopropyl. In addition they all con-
tain an acyl sulfonamide as a carboxylic acid bioisostere in the C-
terminal (P1P10) and a bulky P2-substituent (2, 4 and 5, Fig. 1).
Most of these structural fragments are associated with the cross-
resistance observed for the compounds.24,25 We have previously
reported on novel achiral compounds based on a 2(1H)-pyrazi-
none, which inhibit the wild-type NS3 protease as well as drug-
resistant enzyme variants (e.g., 6, Fig. 2).26 These inhibitors were
based on the 2(1H)-pyrazinone scaffold encompassing an aromatic
P1P10 substituent in combination with various R6-substituents (P2)
CF3
H
N
P4
P3
O
HBA HBA HBD
O
HBA
O
S
P1´
H
N
H
N
H
N
O
O
3
N
6
P2
O
H
N
H
N
H
N
O
N
4
P1
R´
N
Cl
O
N
O
R6
Cl
6
Lead compound (
)
HBD
HBD
Figure 2. The lead compound (6).26 2(1H)-Pyrazinone based HCV NS3 protease
inhibitor, comprising a P3-pyrazinone scaffold, an P1P10 aromatic acyl sulfonamide
and tert-butyl urea as a hypothetical P3-capping group.
Figure 3. A schematic figure of 2(1H)-pyrazinone based HCV NS3 protease
inhibitors. Possible hydrogen bond interactions (b-strand) between the peptide
backbone and the protease (HBA and HBD) are illustrated.

A. K. Belfrage et al. / Bioorg. Med. Chem. 24 (2016) 2603–2620
2605
BocHN
O
N
H2N
O
BocHN
O
a
O
S
b
O
OH
OH
R3
R2
R1
R1
R1
7 R1= p-Br
10 R1= p-H
8 R1= p-F
15 R1= p-H, R2= o-CF3, R3= H (47%)
16 R1= m-H, R2= p-CF3, R3= H (66%)
17
18
19
11 R1= m-H
1
1
9
R = m-CF3
12
13
14
R = p-Br (50%)
R = p-F (66%)
R = m-CF3 (62%)
1
2
3
1
R = p-H, R = o-CF3, R = H (19%)
R = p-H, R = m-CF3, R = H (47%)
R = p-H, R = p-CH3, R = H (74%)
1
2
3
1
1
2
3
20 R1= p-Br, R2= p-CF3, R3= H (78%)
21 R1= p-ethenyl, R2= p-CF3, R3= H (66%)
22 R1= p-5-pyrimidyl, R2= p-CF3, R3= H (48%)
23 R1= p-F, R2= p-CF3, R3= H (74%)
c
c
1
2
3
24
25
R = m-CF32, R = p-CF3,3 R = H (80%)
d
1
R = p-H, R = p-CF3, R = CH3 (47%)
CF3
e
CF3
H
H
OH
N
N
b
S
S
O
O
O O
O
O
O
O2N
O2N
H2N
26
27
28
(92%)
(26%)
O
O
O
O
O
S
BocHN
BocHN
X
OEt
f
OH
b
N
H
CF3
OBn
OBn
OBn
31
29
30
X= NH-Boc (90%)
Scheme 1. Reagents and conditions: (a) Boc2O, NaOH, dioxane; (b) CDI, THF, 2-CF3-benzene sulfonamide, 3-CF3-benzene sulfonamide, 4-CF3-benzene sulfonamide or
4-CH3-benzene sulfonamide, DBU, rt; (c) 2,4,6-trivinylcycloboroxane pyridine complex or 5-pyrimidine boronic acid, Pd(OAc)2, HP(tBu)3BF4, K2CO3, H2O, DME, MW 100 °C,
15 min; (d) Cs2CO3, MeI, DMF, 65 °C; (e) Pd/C (10%), H2, EtOAc, 1 atm, rt, (f) LiOH, H2O, THF, rt, quant.
The nitro group was next reduced by catalytic hydrogenation,
producing the building blocks 38–40 in 65–87% yields.
the NS4A as a cofactor (Tables 1 and 2).32 Moreover, a selected
set of inhibitors (6, 47, 53–54, 57–61, 64–65 and 68) were evalu-
ated on the R155K mutant form of the protease (NS3 R155K).
The arginine (R) to lysine (K) mutation was introduced by PCR at
position 155, and cloned in a similar way as has been described
previously.26,33 The protease activity of the full-length HCV NS3
protein (protease-helicase/NTPase) was measured using a FRET-
assay as previously described.32,34 In short, 1 nM enzyme was incu-
bated for 10 min at 30 °C in 50 mM HEPES, pH 7.5, 10 mM DTT, 40%
2.2. Amide coupling of R0- substituents to 2(1H)-pyrazinone:
final inhibitors 6, 46–67
The preparation of final inhibitors 6, 46–67 (Scheme 3, Tables 1
and 2) started with hydrolysis30 of the benzylester (41–44)
followed by amide coupling using the hydrochloride salts of the
R0-building blocks 15–25 and 31, as well as the amines 28, 38–40,
4531 (the corresponding R0 in the final inhibitor 47) and methyl
2-aminobenzoate, either by using POCl3 (4-89% over 2 steps) or
N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridine-1-yl-methy-
lene]-N-methylmethanaminium hexafluorophosphate N-oxide
(HATU) (11–68% over 2 steps) for activation of the carboxylic acid,
as described in Scheme 3.
glycerol, 0.1% n-octyl-b-
peptide cofactor 2K-NS4A (KKGSVVIVGRIVLSGK) and inhibitor. The
reaction was started by the addition of 0.5 M substrate (Ac-DED
(Edans)EEAbu -[COO]ASK(Dabcyl)-NH2) obtained from AnaSpec
D-glucoside, 3.3% DMSO with 25 lM of the
l
w
Inc. (San Jose, USA). The non-linear regression analysis was made
using Grafit 5.0.8 (Erithacus Software Limited).
The inhibition measurements were corrected for the com-
pounds’ concentration-dependent auto-fluorescent intensities,
which were determined for each compound by measuring fluores-
cence intensities at each tested concentrations. The percentage of
correction was calculated by dividing the fluorescence intensity
in presence of compound, to fluorescence intensity in the absence
of compound and applied to the enzyme’s measured initial veloci-
2.3. Removal of protecting groups: final inhibitors 68–70
The final inhibitors 68–70 (Tables 1 and 2) were accomplished
as described in Scheme 4. The methylester in 57 as well as the ben-
zyl ester in 44 were hydrolyzed by K2CO3 in CH3CN/H2O and
heated in the microwave reactor at 100 °C for 15 min30 to give
the carboxylic acid derivatives 68 and 69 in 75% and 98% yield,
respectively. Compound 66 was dissolved in MeOH and treated
with Pd/C under hydrogen gas, at 1 atm and room temperature
which yielded compound 70 in 59%. A shorter reaction time was
explored to avoid removal of the chlorine, but these attempts were
not successful.
ties. The presence of 15 lM DABCYL did not affect the auto-fluores-
cence of the compounds meaning that no auto-fluorescence
quenching could be observed. Inhibition of viral replication
(EC50) was determined in a subgenomic HCV replicon assay35 for
compounds 54, 57 and 68 (Table 3).
4. Modeling
3. Biochemical evaluation
Five structurally different but equipotent inhibitors (6, 46, 47,
57, and 68) were studied in a series of molecular modeling exper-
iments to identify a common binding mode. Initially, the previ-
ously proposed binding mode A26 was evaluated by constrained
Compounds 6, 46–70 were biochemically evaluated in an
in vitro assay using the full-length NS3 1a and the central part of

2606
A. K. Belfrage et al. / Bioorg. Med. Chem. 24 (2016) 2603–2620
O2N
O2N
O
O
NH2
H2N
O
O O
HN
O
a
b
S
S
S
O O
HN
32
35
38
ortho
ortho (62%)
ortho (87%)
33 meta
34 para
36 meta (51%)
37 para (45%)
39 meta (65%)
40 para (66%)
CF3
CF3
Scheme 2. Reagents and conditions: (a) 4-pyrrolidino pyridine, pyridine, 4-(CF3)-benzoyl chloride, toluene, rt, N2-atm; (b) Pd/C (10%), H2, EtOAc, 1 atm, rt.
O
O
5. Discussion
H
N
R3
R3
OMe/OBn
N
R´
N
a, b or c
Our previous SAR studies of this class of NS3 protease inhibi-
tors have focused on the P2 substituent, located at the R6-posi-
O
O
N
N
R6
R6
Cl
Cl
R
R
R
tion of the pyrazinone.26 Additionally, a few commonly used
a-
3 = tert-Bu urea, R6 = (CH2)2(cyclo-hexyl)
3 = tert-Bu urea, R6 = (CH2)(1-naphthyl)
3 = tert-Bu urea, R6 = phenethyl
6
46 67
-
(Table 1-3)
,
(4-89%, 2 steps)
41
42
43
amino acid based C-terminal building blocks in HCV NS3 protease
inhibitors, i.e., an electrophilic -keto amide, a carboxylic acid
a
44 R3 = tert-Bu urea, R6 = benzyl
and an acyl sulfonamide were evaluated and compared to an aryl
acyl sulfonamide substituent (same as in lead compound 6,
Fig. 2), of which the latter was identified as the most attractive
moiety based on favorable potency and a less peptide-like char-
acter.26,30 Thus, we felt prompted to study the different optimiza-
tion possibilities of the new aromatic R0-substituent, having two
aryl moieties which synthetically allow for introduction of vari-
ous substituents and evaluation of different regioisomers.26,30,35
Hence, inhibitors 6, 46–70 in Tables 1 and 2 were synthesized
and evaluated towards the full-length NS3 1a. Moreover, a set
of selected inhibitors in the series were evaluated against the
drug resistant variant R155K.
Scheme 3. Reagents and conditions: (a) K2CO3, CH3CN/H2O, MW 100–120 °C, 15–
20 min. (b) (i) 15–25 and 31 in 4 M HCl in dioxane, rt, quant. (ii) POCl3, pyridine,
ꢀ15 °C/rt or HATU, DIEA, DCM, 45 °C; (c) 28, 38–40, 4531 and methyl 2-aminoben-
zoate, POCl3, pyridine, ꢀ15 °C/rt, or HATU, DIEA, DCM, 45 °C.
molecular mechanics conformational searches. As illustrated in
Figure 4 by compound 6 (orange) with the macrocyclic ligand of
4A92 superimposed (green), the acidic acyl sulfonamide moiety
in 6 interacts with the catalytic site (red) of the protease. In addi-
tion, the interaction is stabilized by a series of hydrogen bond
interactions (Fig. 5) between the ligand and the backbone of bE2
(R155, A157, and C159).36 The aryl of the anilide is placed in the
hydrophobic S1 pocket whereas the cyclohexyl and tert-butyl
groups and the aryl on the sulfone are placed in more water
exposed regions of the protease. The OPLS3 energy of this pose
was 20 kJ/mol higher in energy compared to poses where the ani-
lide has moved out from the S1 pocket and the hydrogen bonds to
the anionic group are lost. Thus, the ligand becomes rather strained
in Pose A. Compounds 46, 47, and 57 could not be modeled into
this binding mode.
Alternative binding modes were generated by induced fit dock-
ing of the five compounds using hydrogen bond constraints to bE2
(A157 and C159). The obtained poses were clustered based on pair-
wise RMSD values based on the maximum common substructure,
and clusters containing the highest number of different ligands
were considered further. This generated three new binding modes
of which only one (B) formed interactions to the backbone of bE2
(A157 and C159, see Fig. 5 and Supporting information). Further-
more, both NH groups of the urea interacts with the carbonyl of
C159, and the cyclohexyl group is placed in S4 extending slightly
to what is known as the allosteric site of the protease usually host-
ing hydrophobic extensions from P2. The tert-butyl group of the
To our surprise, compared to the lead compound
6
(Ki = 140 nM), altering the R0-regioisomer into the meta 46
(Ki = 170 nM) and para 47 (Ki = 260 nM) derivatives did not affect
the inhibitory potencies, despite the significant geometrical
changes (Table 1). In analogy, it was allowed to move the electron
withdrawing trifluoromethyl group to the ortho (48) and meta (49)
positions with preserved or slightly higher Ki-values (130 and
230 nM, respectively), as well as replacing the CF3 moiety with
an electron donating methyl group in 50 (Ki = 120 nM).
These results indicate that no specific interactions exist between
the aromatic substituent on the anilide and the enzyme, and that
the CF3-groups possibility to increase acidity of the acyl sulfon-
amide hydrogen do not improve the potency, which has shown
to be the case for other types of NS3 protease inhibitors.35
In order to investigate steric and electronic effects by various
substituents on the anilide, compounds 51 (Ki = 150 nM), 52
(Ki = 120 nM), 53 (Ki = 120 nM), 54 (Ki = 110 nM) and 55
(Ki = 100 nM) were evaluated (Table 1). Again, it was found that
large differences in size and electronic properties had minor
influence on the inhibitory potencies. For example, the bulky
pyrimidine moiety in 53 (120 nM) was tolerated. The vinyl moiety
(52, Ki = 120 nM) as well as the electron withdrawing groups in 51
(Ki = 150 nM), 54 (Ki = 110 nM) and 55 (Ki = 100 nM) did not
increase the potency, partly questioning the importance of an
acidic acyl sulfonamide. Considering the steric bulk tolerable in
the position para to the carboxylic acid in 53, the previously
proposed binding mode A can again be questioned. To investigate
the importance of the acid further, the methylated acyl sulfon-
amide 56 (Ki = 260 nM) was evaluated. Thus, removal of the acidic
hydrogen resulted in an almost equipotent inhibitor (56) to 6
(Ki = 140 nM), in disparity to previous observations of HCV NS3
protease inhibitors developed by us.38 Altogether, the flat SAR
shown by the R0 modifications discussed above indicated no impor-
tant contributions from this part. This finding was further sup-
ported by comparable and low Ki-values for both the truncated
urea is pointing towards the C-terminal of helix
a18 and is placed
in S6.37
A third binding mode (C, Fig. 6) was constructed by rotating the
initially proposed pose A 180° around the NH bond of the pyrazine
amine (Figs. 5 and 6). This move maintains the key interactions to
A157 and C159 and places the anilide moiety towards the exit at
the end of the C-terminal of helix
a18, and bulk water, with the
ortho, meta, and para positions being solvent exposed. The cyclo-
hexylethyl moiety is now inserted in between F438 of the Phe-loop
and F531 and W532 of helix
a14. Finally, the urea is directed
towards the catalytic site and is placed in S3.
Based on the presumptions that the ortho, meta, and para posi-
tions of the anilide should be water exposed, that the acid is not
required, and that hydrogen bonds to the backbone of bE2 should
be present, several alternative binding modes were discarded
(e.g., binding mode D and E, Supporting information).
inhibitors having
(Ki = 120 nM) and the corresponding methylester 57 (Ki = 120
a
carboxylic acid in ortho position 68
M).
l

A. K. Belfrage et al. / Bioorg. Med. Chem. 24 (2016) 2603–2620
2607
Table 1
Inhibitory potencies (Ki) of inhibtors 6, 46–58, 68–69
O
H
N
H
N
H
N
N
R´
O
N
O
Cl
Compound
R0
Full-length
NS3 1a
Full-length
NS3 1a R155K
Ki SD (nM)
Ki SD (nM)
CF3
H
N
O
S
6a
140 20
30
2
O
O
O
O
O
O
S
S
N
46
47
170 30
260 60
nd
60
H
CF3
CF3
H
N
8
O
O
F3C
H
O
N
S
O
48
130 20
nd
O
CF3
H
N
O
O
49
50
230 50
nd
nd
S
S
O
O
O
H
N
120 40
O
CF3
CF3
CF3
H
N
O
O
O
S
S
S
O
O
O
O
51
52
150 30
nd
nd
Br
H
N
O
120 20
H
N
O
53
54
120 20
30
20
4
2
N
N
O
CF3
H
N
S
O
O
110 20
F
(continued on next page)

2608
A. K. Belfrage et al. / Bioorg. Med. Chem. 24 (2016) 2603–2620
Table 1 (continued)
Compound
R0
Full-length
Full-length
NS3 1a
Ki SD (nM)
NS3 1a R155K
Ki SD (nM)
O
S
O
O
S
N
H
55
56
100 20
nd
nd
CF3
CF3
N
CF3
O
O
O
260 150
O
O
O
57
68
120 30
120 30
15
17
2
4
OH
O
O
O
S
58
140 30
>10,000
24
nd
3
N
H
CF3
O
H
N
H
N
OH
N
69
O
N
O
Bn
Cl
a
Previously reported in Gising et al.26; SD, standard deviation; nd, not determined.
We hypothesized that increased flexibility of the R0-substituent
could facilitate further interactions with the enzyme. Therefore,
compound 58 (Ki = 140 nM) with a methylene linker was tried
(Table 1). However, in comparison to the carboxylic acid and ester
68/57 (Ki-values = 120 nM) no additional beneficial effect could be
seen. Overall, this led us to questioning the importance of the
evaluated showing slightly improved potencies (65, Ki = 70 nM vs
6, Ki = 140 nM and 66, Ki = 150 nM vs 67, Ki = 270 nM). The lower
inhibitory potency for 70 (Ki = 450 nM) indicated either a beneficial
interaction from the benzyl group or that a hydroxyl moiety in that
position is suboptimal. However, it cannot be ruled out that the
lower potency is related to the removal of the C5-Cl in 66, since
we were not able to solely remove the benzyl group. Again, the flat
SAR in this region of the inhibitors is indicative of a minor interac-
tion with the enzyme target. However, the relatively low Ki-values
for the inhibitors in this series, compared with 1 (15 nM) and
the hexapeptide analogue of the N-terminal cleavage product43
(N-1725, 91 nM), makes these inhibitors interesting for further
evaluation.
R0-part, and hence
evaluated, which resulted in a drastic decrease in activity (69,
Ki >10 M). The drop in activity observed by replacing the anilide
a
compound without an R0-moiety was
l
with the corresponding carboxylic acid either show that the R0-aryl
is required for the binding, or that an acidic group is not tolerated
in this part. It could also suggest that the hydrogen bond donor of
the acetamide linker could be involved in polar interactions with
the protein.
Thirteen inhibitors, selected from Tables 1 and 2, were evalu-
ated against the known drug-resistant enzyme variant R155K.23
R155 is positioned in the S2 pocket and is part of a stabilizing salt
bridge with D168. An amino acid substitution, e.g., R155K, disrupts
the salt bridge, which could lead to drug-resistance for inhibitors
with interactions in this area, i.e., inhibitors containing extended
P2-substituents.44 We were very pleased to see that this series in
general gave lower Ki-values against the drug resistant variant as
compared with the wild-type, with inhibitory potencies between
15 and 90 nM. However, due to the difference in kinetic parame-
ters for the wild-type and mutant variant (see Supporting informa-
tion), it is not directly translatable into improved potency. The
retained potency of this class of inhibitors against the R155K NS3
enzyme compared with inhibitors 1, 2, 4, and 5 (Fig. 1) is indicative
of a reduced interaction with the extended S2 site. Inhibitor 60,
containing a reversed acyl sulfonamide in R0, was slightly more
potent against the R155K enzyme (30 nM) compared with 59 con-
taining the original R0-moiety (90 nM). Above all, the most intrigu-
ing finding was the potency of the R0-truncated inhibitors 57
(Ki = 15 nM) and 68 (Ki = 17 nM) (Table 2), and especially in
An interesting property of aryl acyl sulfonamides is the possibil-
ity to reverse the acyl sulfonamide functionality with retained or
slightly altered hydrogen bond pattern of the oxygens of the sul-
fone and the carbonyl. Therefore, compounds 59–64 were prepared
and evaluated as presented in Table 2. The varied regioisomers of
reversed acyl sulfonamides (60, Ki = 180 nM, 61, Ki = 190 nM and
62, Ki = 170 nM) showed slightly lower Ki-values compared with
the original acyl sulfonamide (59, Ki = 310 nM). A better R6-group
(phenethyl) was combined with the reversed acyl sulfonamide
(64, Ki = 170 nM) showing similar potency as the original acyl sul-
fonamide (63, Ki = 220 nM). These results indicated that a distinct
binding to active site and/or the prime side are lacking even in
these compounds.
During the course of this investigation we decided to look into
non-aryl moiety alternatives to the aryl R0-substituent, which
could be advantageous for drug properties.39,40 We speculated that
a spirocyclobutyl group could be useful,41 with the inherent oppor-
tunity to introduce different functionalities to the ring structure in
forthcoming SAR studies.27,28,42 Thus, 65 and 66 (Table 2) were

A. K. Belfrage et al. / Bioorg. Med. Chem. 24 (2016) 2603–2620
2609
Table 2
Inhibitory potencies (Ki) of inhibitors 59–67, 70, and reference compounds
O
H
N
H
N
H
N
N
R´
O
N
O
R6
R5
Compound
R0
R5
R6
Full-length
NS3 1a
Full-length
NS3 1a R155K
Ki SD (nM)
Ki SD (nM)
CF3
CF3
H
N
O
S
59a
Cl
Cl
310 110
90 10
O
O
H
N
O
S
O
60
180 30
30
4
4
O
O
O
O
O
S
N
61
62
Cl
Cl
190 36
170 40
60
nd
H
CF3
CF3
H
N
S
O
O
CF3
CF3
H
O
N
S
63a
Cl
Cl
220 60
170 90
nd
O
O
H
N
O
S
O
64
40 10
O
O
O
O
S
S
S
N
H
65
66
70
Cl
Cl
H
70 10
20
nd
nd
3
CF3
CF3
OBn
O
O
O
N
H
150 30
450 70
OBn
O
O
O
N
H
CF3
CF3
OH
O
H
N
S
67a
Cl
270 80
70
7
O
O
1
15b
91 21
nd
82a
nd
9
N-1725c
5
1
a
b
c
Previously reported in Gising et al.26
Previously reported in Dahl et al.33
Ac-Asp-D-Gla-Leu-Ile-Cha-Cys-OH, SD, standard deviation; nd, not determined.
comparison to the clinical inhibitors 1 (82 nM) and 5 (9 nM). These
inhibitors were found to be the most active among the evaluated
compounds against the R155K enzyme variant.
The flat SAR of the R0-part of the inhibitors challenged our
design and previous docking model (Fig. 2 and pose A in Figs. 4
and 5). As the pyrazinone inhibitors have not yet been

2610
A. K. Belfrage et al. / Bioorg. Med. Chem. 24 (2016) 2603–2620
O
O
O
OH
O
O
H
N
H
N
H
N
H
N
H
N
H
N
a
N
N
O
N
O
O
N
O
75 %
Cl
Cl
57
68
O
O
H
H
H
H
a
N
N
OBn
N
N
OH
N
N
98 %
O
N
O
O
N
O
Cl
Cl
44
69
O
O
O
O
H
N
H
N
H
N
S
S
b
N
N
H
O
O
N
O
O
59 %
Figure 4. The previously proposed binding mode A of 6 (orange) superpositioned
with the ligand of 4A92 (green) with the protease shown in gray; the helicase in
turquoise; the catalytic triad is highlighted in red. Hydrogen bonds to bE2 are
indicated by yellow dashes.
OBn
O
Cl
66
O
O
O
H
N
H
N
H
N
length NS3 in the assay, which has been the case in this study.
Until a co-crystal structure of this series is produced, compound
analogue evaluation to challenge the different hypothetical poses
remains the best option. However, one should also bear in mind
that the NS3 protease with its comparatively shallow pockets con-
stitute a difficult target for docking studies. Indeed, this is reflected
by the many alternative potential binding poses derived in this
study.
N
N
H
N
OH
70
Scheme 4. Reagents and conditions: (a) K2CO3, CH3CN/H2O, MW 100 °C, 15 min;
(b) Pd/C (10%), H2, MeOH, 1 atm, rt, 59%.
As discussed above, an advantage of this class of inhibitors is
the preserved potency against the R155K enzyme. This is sup-
ported by both the alternative binding modes B and C, suggesting
no interactions with the S2 pocket. Thus, inhibitor binding should
be less affected by amino acid substitutions in this region.
Three compounds in the series (54, 57 and 68) were evaluated
in a cell based replicon assay (Table 3).35 They all showed detect-
able inhibitory potencies, with a slight preference for the inhibitor
co-crystalized with the NS3 protein, and that they are very differ-
ent to the more well-known inhibitors (Fig. 1) an impartial docking
study was required in this stage, resulting in a number of alterna-
tive models (e.g., B and C, Figs. 5 and 6 and Supporting informa-
tion). Except for the ortho substituted inhibitors (e.g., 6), which
could be docked into model A (Fig. 4) close to the binding poses
proved by other advanced inhibitors (e.g., 5),45 none of the alterna-
tive suggested binding poses occupy the S1–S10 region. Based on
the presumption that the equipotency of the differently substi-
tuted compounds is a result of a common productive pose of the
shared scaffold (i.e., hydrogen bond interactions between the inhi-
bitors and the backbone of bE2; R155, A157 and C159), the origi-
nally proposed pose A could be considered less probable due to
restrictions in the S1 binding pocket, hosting the aniline moiety
(or only valid for the ortho analogues). Binding mode B (Fig. 5) ful-
fill the criteria for a flat SAR not too much affected by the substitu-
tion regiochemistry at the anilide. However, no significant
hydrophobic interactions are made with the target protein. There-
fore, the binding mode is not fully compatible with the need for a
cyclohexyl ethyl group or other hydrophobic moieties in this
region of the molecule.26 Out of the remaining alternatives, pose
C is making the best use of the hydrophobic cyclohexyl moiety,
containing an acyl sulfonamide (54, 7.3
lM) compared with the
truncated analogues, i.e., 57 (22 M) and 68 (49
l
lM). Possibly
the increased lipophilic character of 54 compared with 57 and 68
could influence cell-permeability positively. The potency range
was somewhat expected since a drop in potency going from bind-
ing studies to cell-based replicon studies is often seen for HCV pro-
tease inhibitors.46 This is true also for the reference inhibitor 1
(Ki = 15 nM, EC50 = 0.65 lM).
6. Conclusion
Dissimilar to the so-called product-based inhibitors47,48 origi-
nating from peptides containing a crucial C-terminal carboxylic
acid and in later design, a bioisosteric acyl sulfonamide (2, 4 and
5, Fig. 1) this series had a different starting point; that is the b-
sheet mimicking 2(1H)-pyrazinone (Fig. 3). Results herein demon-
strate that an acidic C-terminal is not decisive for the binding of
these inhibitors. Thus, the SAR studies indicate that the aryl sulfon-
amide probably protrudes out in the solvent. This allowed design
of smaller inhibitors, which not only retained potency for the
wild-type but were particularly effective against the drug resistant
R155K enzyme variant (57 Ki = 15 nM and 68 Ki = 17 nM). Based on
molecular modeling experiments it was suggested that this class of
inhibitors probably binds to the HCV NS3 protease in a different
manner as compared to the well-known, advanced inhibitors
approved or in clinical studies. Therefore, the pyrazinone based
HCV NS3 protease inhibitors could be useful for developing coming
generation drug candidates that should be active against drug-
resistant variants and less prone to resistance development.
embedding this in between the Phe-loop and helix a14 of the heli-
case (Fig. 6). This pocket is created by a conformational change in
the F438 v1 angle from gauche to anti and in the F531 v1 angle
from anti to gauche. If this pose is valid, it could be expected that
the inhibitory potency would be dependent on the use of full-
Table 3
EC50 values of inhibitors 54, 57 and 68 and the reference compounds 1 and 5
a
a
Compound
EC50
(
lM)
CC50
38
(lM)
54
57
68
1
7.3
22
49
>100
>100
>1
0.65
5
0.011
>1
a
Evaluated in a HCV replicon assay.35, mean value of two experiments.

A. K. Belfrage et al. / Bioorg. Med. Chem. 24 (2016) 2603–2620
2611
A
B
Cl
Cl
N
O
R
NH
N
O
N
N
O
O
N
N
N
O
O
NH2
NH2
N
O
N
H
R
O
O
H
H
+HN
NH2
+HN
NH2
H
H
O
O
H
N
O
H
N
O
O
H
N
O
H
N
O
N
H
N
H
N
N
H
N
H
N
R155
R155
H
H
O
HN
O
HN
A157
A157
SH
SH
C159
C159
C
Cl
N
HN
O
N
R
O
N
H
R = OH, OMe, or NSO2Ar
O
O
O
NH2
NH2
O
N
H
+HN
O
H
N
H
N
O
N
N
H
N
R155
H
H
O
HN
A157
SH
C159
Figure 5. A schematic 2D illustration of the three poses (A–C) considered. Hydrogen bond interactions to the backbone of bE2 (R155, A157, and C159) are shown by dashed
lines. For pose C the hydrophobic pocket is depicted by a solid line.
7.1.1. General procedure for preparation of compounds 12–14.
Method A
7. Experimental section
7.1. Chemistry
The aminobenzoic acid was dissolved in dioxane followed by
addition of 1 M NaOH aq di-tert-butyl dicarbonate was dissolved
in dioxane and the resulting solution was added, dropwise, to the
reaction mixture, which was allowed to stir at room temperature.
The progress of the reaction was analysed by analytical LC/MS and
found to be slow. After 24 h extra di-tert-butyl dicarbonate was
added in order to complete the reaction. After another 24–48 h
stirring the reaction was worked up. After evaporation of parts of
the solvent, 0.1 M NaOH aq was added and the reaction mixture
was washed with ether. The product precipitated from the alkaline
water phase by dropwise addition of 1 M HCl to pH around 6. The
difference of pKa-values of the product and the starting material
was used to obtain good separation.
NMR spectra were recorded on a Varian Mercury Plus at 25 °C
for 1H at 399.9 MHz and for 13C NMR at 100.5 MHz. All micro-
wave-assisted syntheses were carried out in a Smith synthesizer
performed in sealed vials dedicated for microwave processing. Col-
umn flash chromatography was performed using silica gel 60 (par-
ticle
size
0.040–0.063 mm,
Sigma-Aldrich).
Thin-layer
chromatography was performed with aluminum sheets coated
with silica gel 60-F254 (0.2 mm E. Merck), using UV-light for visual-
ization. Analytical HPLC-UV/MS was performed on a Dionex Ulti-
Mate 3000 HPLC system with a Bruker amaZon SL ion trap mass
spectrometer and detection by UV (DAD) and MS (ESI+), using a
Phenomenex Kinetex C18 column (50 ꢁ 3.0 mm, 2.6
lM particle
7.1.2. General procedure for synthesis of compounds 15–20,
23–24, 27 and 30. Method B
size, 100 Å pore size) and a flow rate of 1.5 mL/min. A gradient of
H2O/CH3CN/0.05% HCOOH was used. Preparative RP-HPLC was per-
formed by UV-triggered (254 nM) fraction collection with a Gilson
HPLC system using an Agilent PrepHT Zorbax SB C8 column
All solid chemicals used were dried in vacuum over P2O5 over-
night. The acid derivative and CDI were dissolved in dry THF under
N2 atmosphere and the mixture was allowed to stir at 66–68 °C for
2 h. The sulfonamide and DBU dissolved in THF were added to the
reaction mixture and stirring was continued at room temperature
(4 h-overnight).
Method B1: The solvent was removed in vacuo, water was added
and pH was adjusted to 2 by addition of 1 M HCl aq. The aqueous
phase was extracted with EtOAc (2 ꢁ 40 ml), dried with MgSO4, fil-
tered and evaporated in vacuo. For most of the compounds, a silica
gel column was first run, followed by purification on aluminum
oxide.
(150 ꢁ 21.2 mm,
5 lM particle size) and a H2O/CH3CN/0.05%
HCOOH gradient at a flow rate of 10 mL/min. High resolution
molecular mass (HRMS) was determined on a Micromass Q-Tof2
mass spectrometer equipped with an electrospray ion source. All
products were >95% pure according to HPLC-UV at 254 nM. Optical
rotation was obtained on a Rudolph Research Analytical Autopol II
Automatic Polarimeter and the concentration c is given as g/100 ml
in the specified solvent. Reactants and reagents, including 7–9,
10–11, 26, 32–34 that were commercially available were used
without further purification. Compounds 6,26 15,49 29,27,28
41–44,26 45,31 59,26 6326 and 6726 are known compounds.
Compounds 12–14, 16–25, 27–28, 30–31, 35–40, 46–58, 60–62,
64–66 and 68–70 are all new compounds and have been fully
characterized.
Method B2: The solvent was removed in vacuo. The crude mate-
rial was dissolved in EtOAc or DCM (25 ml), washed with 5% citric
acid aq (2 ꢁ 15 ml) and brine (15 ml), dried with MgSO4, filtered
and evaporated in vacuo. Purification by silica column chromatog-

2612
A. K. Belfrage et al. / Bioorg. Med. Chem. 24 (2016) 2603–2620
7.1.5. General procedure for synthesis of compounds 28 and 38–
40. Method E
The R0-building block was dissolved in EtOAc. Pd/C (10%) was
added and the reaction mixture was put under a hydrogen atmo-
sphere, at 1 atm, and was allowed to stir at room temperature
for 2–2.5 h. The reaction mixture was filtered through a celite plug
and the solvent was evaporated to give the product without further
purification.
7.1.6. General procedure for preparation of the final inhibitors
6, 46–67. Method F
A 2–5 ml microwave vial was charged with the benzyl/methyl
ester of the pyrazinone (41–44) K2CO3, CH3CN, and H2O. The vial
was capped under air and irradiated by MW to 100–120 °C for
15–20 min.30 1 M HCl was added and the mixture was extracted
with EtOAc. The organic layers were dried over MgSO4 and
evaporated.
Method F1: The crude acid and the amine (free or as the
hydrochloride salt) were dissolved in pyridine and cooled to
ꢀ15 °C under N2. POCl3 was added and the reaction mixture was
stirred at ꢀ15 °C to room temperature for 0.5–2 days. In most of
the cases, extra POCl3 (reaction on ice/aceton bath) and/or amine
was added to consume the pyrazinone-carboxylic acid. H2O
(20 ml) was added and the pH adjusted to 1 using 6.0 M HCl aq fol-
lowed by extraction with EtOAc (2–3 ꢁ 20–30 ml). The organic
layer was dried over MgSO4 and evaporated. Purification by silica
column flash chromatography gave the product.
Method F2: To the crude acid, the amine (hydrochloride salt)
and HATU dissolved in dry DCM, DIEA was added and pH con-
trolled to be 10 or higher. The reaction mixture was allowed to stir
at 45 °C for 2–4.5 h. After addition of DCM and washing with 0.1 M
NaHSO4 aq the organic layer was evaporated. Purification on silica
column gave the final compound.
7.1.7. Compound 12. 4-Bromo-2-((tert-butoxycarbonyl)amino)
benzoic acid
12 was prepared according to general method A. 7 (2.16 g,
0.01 mol), 1 M NaOH (15 ml, 0.015 mol), dioxane (15 ml), di-tert-
butyl dicarbonate (3.27 g, 0.015 mol) in dioxane (15 ml). Stirred
at room temperature overnight. Di-tert-butyl dicarbonate (1.09 g,
0.005 mol) and 1 M NaOH (5 ml) were added. Stirred at room tem-
perature overnight and then worked up. The product precipitated
at pH 6.4 (1.586 g, 50%). 1H NMR (CDCl3) d 10.02 (s, 1H), 8.74 (d,
J = 1.8 Hz, 1H), 7.95 (d, J = 8.6 Hz, 1H), 7.18 (dd, J = 8.6, 1.8 Hz,
1H), 1.55 (s, 9H). 13C NMR (CDCl3) d 172.5, 152.6, 143.9, 133.1,
131.2, 124.9, 122.0, 111.9, 81.6, 28.4. MS calcd for C12H14BrNO4
[M+H+]: 316.0 found: 316.1.
Figure 6. Binding mode C illustrated by compound 68 (green). The protease is
shown in gray; the helicase in turquoise; in the top view, the catalytic triad is
highlighted in red. The hydrophobic pocket in between the Phe-loop and helix
is shown as a turquoise surface.
a14
raphy gave the product as a DBU salt. The material was washed
with 0.1 N NaHSO4 aq to give the pure product.
7.1.3. General procedure for synthesis of compounds 35–37.
Method C
Nitrobenzenesulfonamide, 4-pyrrolidino pyridine and pyridine
were mixed and put on an ice bath under N2 atmosphere. 4-(Triflu-
oromethyl)benzoyl chloride was dissolved in toluene and added
through a syringe to the reaction mixture. The reaction was
allowed to stir at room temperature. The reaction was quenched
with water, and the solvents were evaporated to yield a yellow
oil. Purification by silica column flash chromatography and/or
extractions gave the product.
7.1.8. Compound 13. 2-((tert-Butoxycarbonyl)amino)-4-fluoro-
benzoic acid
13 was prepared according to general method A using 8
(776 mg 0.005 mol), 1 M NaOH (7.5 ml, 0.0075 mol), dioxane
(7.5 ml), di-tert-butyl dicarbonate (1.636 g, 0.0075 mol) in dioxane
(7.5 ml). Stirred at room temperature overnight and then another
portion of di-tert-butyl dicarbonate (546 mg, 0.0025 mol) was
added. Stirred at room temperature for two days followed by work
up. The product precipitated at pH 4.3, (793 mg, 66%). 1H NMR
(CDCl3) d 10.14 (s, 1H), 8.29 (dd, J = 12.2, 2.6 Hz, 1H), 8.13 (dd,
J = 9.0, 6.6 Hz, 1H), 6.73 (ddd, J = 9.0, 7.4, 2.6 Hz, 1H), 1.55 (s, 9H).
13C NMR (CDCl3) d 172.4, 167.4 (d, J = 254.3 Hz), 152.6, 145.5 (d,
J = 13.5 Hz), 134.6 (d, J = 11.2 Hz), 109.5 (d, J = 2.5 Hz), 109.1 (d,
7.1.4. General procedure for boc deprotection of compounds
15–25, and 31. Method D
The R0-building block was dissolved in 4 M HCl in dioxane and
stirred at room temperature. When the reaction was complete
(LC–MS analysis) the solvent was evaporated in vacuo. The result-
ing products as hydrochloride salts were dried under vacuum and
used in the subsequent coupling reactions without further
purification.
J = 22.7 Hz), 106.2 (d, J = 28.7 Hz), 81.6, 28.4. MS calcd for C12H14
-
FNO4 [M+H+]: 256.1 found: 256.1.

A. K. Belfrage et al. / Bioorg. Med. Chem. 24 (2016) 2603–2620
2613
7.1.9. Compound 14. 3-((tert-Butoxycarbonyl)amino)-5-(trifluo
romethyl)benzoic acid
amide (1.44 g, 0.008 mol), DBU (1.3 ml, 0.008 mol). Stirred at room
temperature overnight. Purification on silica gel (DCM/MeOH 9:1)
and aluminium oxide (DCM/HCOOH 100:0 to 99:1) gave 19 in 74%
yield (0.149 g). 1H NMR (CD3OD) d 8.10 (dd, J = 8.4, 1.2 Hz, 1H),
7.92 (dm, J = 8.3 Hz, 2H), 7.81 (m, 1H), 7.41 (ddd, J = 8.4, 7.3,
1.6 Hz, 1H), 7.35 (dm, J = 8.3 Hz, 2H), 7.01 (ddd, J = 7.9, 7.3,
1.2 Hz, 1H), 2.42 (s, 3H), 1.46 (s, 9H). 13C NMR (CD3OD) d 154.5,
144.7, 141.4, 140.0, 133.8, 131.0, 130.2, 128.9, 127.2, 122.7,
122.2, 120.4, 81.3, 28.6, 21.5.
14 was prepared according to general method A using 9
(250 mg, 0.001 mol), 1 M NaOH (1.5 ml, 0.0015 mol), dioxane
(1.5 ml), di-tert-butyl dicarbonate (0.327 g, 0.0015 mol) in dioxane
(1.5 ml). The reaction mixture was allowed to stir for two days. Di-
tert-butyl dicarbonate (0.109 g, 0.5 mmol) dissolved in dioxane
(0.5 ml) was added and the reaction mixture stirred for another
one day and worked up. The product precipitated at pH 6,
(188 mg, 62%). 1H NMR (CD3OD) d 8.25 (s, 1H), 8.07 (s, 1H), 7.86
(s, 1H), 1.54 (s, 9H). 13C NMR (CD3OD) d 168.2, 154.8, 142.2,
134.0, 132.4 (q, J = 32.0 Hz), 125.1 (q, J = 273.4 Hz), 123.6, 120.6
(q, J = 4.1 Hz), 119.4 (q, J = 3.6 Hz), 81.7, 28.6. MS calcd for
7.1.14. Compound 20. tert-Butyl(5-bromo-2-(((4-(trifluorometh
yl)phenyl)sulfonyl)carbamoyl)phenyl)carbamate
Prepared according to general method B2. 12 (316 mg,
1.0 mmol), CDI (324 mg, 2.0 mmol) in THF (20 ml), 66 °C. 4-(Triflu-
oromethyl)benzenesulfonamide (450 mg, 2.0 mmol), dissolved in
C
13H14F3NO4 [M+H+]: 306.1 found: 306.2.
7.1.10. Compound 16. tert-Butyl(3-(((4-(trifluoromethyl)phen
yl)sulfonyl)carbamoyl)phenyl)carbamate
THF (2 ml), DBU (450 ll, 3.0 mmol). Stirred at room temperature
for 4 h. Purification on silica column (DCM/MeOH 9:1 to 11:3). In
order to remove the DBU salt, the product was dissolved in EtOAc
and washed with 0.1 N NaHSO4. The organic layer was evaporated
to give the title compound in 78% yield (409 mg). 1H NMR (CD3OD)
d 8.40 (d, J = 1.9 Hz, 1H), 8.28 (d, J = 8.4 Hz, 2H), 7.92 (d, J = 8.4 Hz,
2H), 7.60 (d, J = 8.6 Hz, 1H), 7.23 (dd, J = 8.5, 2.0 Hz, 1H), 1.45 (s,
9H). 13C NMR (CD3OD) d 168.5, 153.6, 144.1, 142.8, 136.1 (q,
J = 32.8 Hz), 131.6, 130.2, 129.8, 127.4 (q, J = 190.0 Hz), 127.1 (q,
J = 3.9 Hz), 125.8, 123.6, 117.5, 82.2, 28.4. MS calcd for
Prepared according to method B1 described in general procedure.
11 (0.593 g, 0.0025 mol), CDI (0.811 g, 0.005 mol), THF (60 ml),
4-(Trifluoromethyl)benzenesulfonamide (1.16 g, 0.005 mol), DBU
(0.75 ml, 0.005 mol). Stirred at room temperature overnight.
Purification on silica gel (DCM/MeOH 9:1) and aluminum oxide
(DCM/HCOOH 100:0 to 99:1) gave 16 in 66% yield (0.734 g). 1H
NMR (CD3OD) d 8.26 (dm, J = 8.3 Hz, 2H), 7.91 (dm, J = 8.3 Hz, 2H),
7.86 (m, 1H), 7.60 (m, 1H), 7.46 (ddd, J = 7.8, 1.7, 1.1 Hz, 1H), 7.35
(m, 1H), 1.51 (s, 9H). 13C NMR (CD3OD) d 168.9, 164.6, 155.2,
145.5, 141.1, 135.6 (d, J = 32.8 Hz), 134.7, 130.1, 130.1, 127.0 (d,
J = 3.8 Hz), 124.9 (d, J = 272.1 Hz), 124.1, 123.4, 81.3, 28.6. HRMS
calcd for C19H19F3N2O5S [M+H+]: 445.1045; found 445.1049.
C
19H18BrF3N2O5S [M+H+]: 523.0 found: 523.1.
7.1.15. Compound 21. tert-Butyl(2-(((4-(trifluoromethyl)phen
yl)sulfonyl)carbamoyl)-5-vinylphenyl)carbamate
20 (83.7 mg, 0.160 mmol), 2,4,6-trivinylcycloboroxane pyridine
complex (71.2 mg, 0.16 mmol), Pd(OAc)2 (3.9 mg, 0.016 mmol), HP
(tBu)3BF4 (9.3 mg, 0.0.032 mmol), K2CO3 (133 mg, 0.960 mmol),
H2O (1 ml) and DME (3 ml) were added to a microwave vial, under
N2 atmosphere, and irradiated with MW to 100 °C for 15 min. The
crude material was filtered and the solvents were evaporated in
vacuo. 4 ml H2O was added and acidified to pHꢂ1 using 6 M HCl.
EtOAc (8 ml) was added and extracted with the aqueous phase.
The organic layer was separated and evaporated. Purification on
a silica column (DCM/MeOH 93:7) gave the title compound in
66% yield (49.6 mg). 1H NMR (CD3OD) d 8.27 (d, J = 1.8 Hz, 1H),
8.15 (d, J = 8.2 Hz, 2H), 8.04 (d, J = 8.3 Hz, 1H), 7.77 (d, J = 8.2 Hz,
2H), 7.00 (dd, J = 1.8, 8.3 Hz, 1H), 6.69 (dd, J = 11.0, 17.7 Hz, 1H),
5.83 (d, J = 17.7 Hz, 1H), 5.31 (d, J = 11.0 Hz, 1H), 1.41 (s, 9H). 13C
NMR (CD3OD) d 174.4, 154.6, 148.5, 142.9, 142.0, 137.8, 134.2 (q,
J = 33.5 Hz), 132.8, 128.5, 126.7 (q, J = 3.6 Hz), 125.1 (q,
J = 272.5 Hz), 122.8, 119.9, 117.3, 116.3, 81.1, 28.6. MS calcd for
7.1.11. Compound 17. tert-Butyl(2-(((2-(trifluoromethyl)phen
yl)sulfonyl)carbamoyl)phenyl)carbamate
Prepared according to general method B1. 10 (1.0 g, 0.004 mol),
CDI (1.387 g, 0.008 mol), THF (60 ml), 2-(trifluoromethyl)benzene-
sulfonamide (1.9 g, 0.008 mol), DBU (1.3 ml, 0.008 mol). Stirred at
room temperature overnight. Purification on silica gel (DCM/MeOH
9:1) and aluminium oxide (DCM/HCOOH 100:0 to 99:1) gave 17 in
19% yield (0.350 g). 1H NMR (CD3OD) d 8.42 (dd, J = 8.0, 1.3 Hz, 1H),
8.14 (dd, J = 8.4, 1.2 Hz, 1H), 8.05 (dd, J = 7.9, 1.8 Hz, 1H), 7.83 (dm,
J = 7.6 Hz, 1H), 7.77-7.58 (m, 2H), 7.33 (ddd, J = 8.4, 7.3, 1.7 Hz, 1H),
6.94 (ddd, J = 7.9, 7.2, 1.3 Hz, 1H), 1.47 (s, 9H). 13C NMR (CD3OD) d
174.4, 154.7, 143.2, 141.6, 133.1, 133.0, 132.9, 132.3, 128.9 (q,
J = 6.3 Hz), 128.8 (q, J = 6.3 Hz), 128.5 (q, J = 33.2 Hz), 124.6 (q,
J = 273.7 Hz), 124.0, 122.1, 119.6, 80.8, 28.6. HRMS calcd for
C
19H19F3N2O5S [M+H+]: 445.1045; found 445.1037.
7.1.12. Compound 18. tert-Butyl(2-(((3-(trifluoromethyl)phen
yl)sulfonyl)carbamoyl)phenyl)carbamate
C
21H21F3N2O5S [M+H+]: 471.1 found: 471.2.
Prepared following general method B1. 10 (1.0 g, 0.004 mol),
CDI (1.387 g, 0.008 mol), THF (60 ml), 3-(trifluoromethyl)benzene-
sulfonamide (1.9 g, 0.008 mol), DBU (1.3 ml, 0.008 mol). Stirred at
room temperature overnight. Purification on silica gel (DCM/MeOH
97:3 to 93:3 followed by acetone/i-hexane 1:4 to 2:3) gave 18 in
47% yield (0.833 g). 1H NMR ((CD3)2SO) d 8.16–8.05 (m, 3H), 8.00
(dd, J = 7.8, 1.7 Hz, 1H), 7.83 (dm, J = 7.6 Hz, 1H), 7.69 (m, 1H),
7.31 (ddd, J = 8.6, 7.2, 1.7 Hz, 1H), 6.91 (ddd, J = 7.8, 7.2, 1.1 Hz,
1H), 1.44 (s, 9H). 13C NMR ((CD3)2SO) d 171.8, 152.4, 146.7,
140.4, 131.3, 130.9, 130.9, 129.4, 128.6 (q, J = 31.8 Hz), 127.0 (q,
J = 3.9 Hz), 124.0 (d, J = 272.7 Hz), 123.5 (q, J = 4.0 Hz), 123.0,
120.6, 117.6, 79.1, 28.0. HRMS calcd for C19H19F3N2O5S [M+H+]:
445.1045; found 445.1042.
7.1.16. Compound 22. tert-Butyl(5-(pyrimidin-5-yl)-2-(((4-(trifl
uoromethyl)phenyl)sulfonyl)carbamoyl)phenyl)carbamate
20 (78.5 mg, 0.15 mmol), pyrimidine boronic acid (55.4 mg,
0.45 mmol), Pd(OAc)2 (3.56 mg, 0.015 mmol), HP(tBu)3BF4
(8.7 mg, 0,03 mmol), K2CO3 (124 mg, 0,9 mmol), H2O (0.95 ml)
and DME (2.85 ml) were added to a microwave vial, under N2
atmosphere, and was heated to 100 °C for 2 h. The crude material
was filtered and the solvents were evaporated in vacuo. 4 ml H2O
was added and acidified to pH <2 using 1 M HCl. EtOAc (5 ml)
was added and extracted with the aqueous phase. The organic
layer was separated and evaporated. Purification on a silica column
(i-hexane/EtOAc/HCOOH 60:40:2) gave the title compound in 48%
yield (37 mg). 1H NMR (CD3OD) d 9.15 (s, 1H), 9.05 (s, 2H), 8.49 (d,
J = 1.7 Hz, 1H), 8.28 (d, J = 8.4 Hz, 2H), 7.95 (d, J = 8.3 Hz, 1H), 7.89
(d, J = 8.4 Hz, 2H), 7.36 (dd, J = 1.8, 8.3 Hz, 1H), 1.46 (s, 9H). 13C
NMR (CD3OD) d 170.0, 158.5, 156.2, 154.1, 145.3, 142.5, 140.0,
135.5 (q, J = 31.7 Hz), 134.8, 131.9, 130.0, 126.9 (q, J = 3.8 Hz),
7.1.13. Compound 19. tert-Butyl(2-(tosylcarbamoyl)phenyl)
carbamate
Prepared following general method B1. 10 (1.0 g, 0.004 mol),
CDI (1.367 g, 0.008 mol), THF (60 ml), 4-methylbenzenesulfon-

2614
A. K. Belfrage et al. / Bioorg. Med. Chem. 24 (2016) 2603–2620
124.7 (q, J = 270.5 Hz), 121.0, 120.4, 118.8, 81.9, 28.5; MS calcd for
23H21F3N4O5S [M+H+]: 523.1 found: 523.1.
(CD3OD) d 171.5, 148.5, 144.9, 142.7, 135.7 (q, J = 32.8 Hz), 131.6,
129.9, 127.8 (q, J = 3.2 Hz), 124.8 (q, J = 271.9 Hz), 124.4, 43.5.
HRMS calcd for C15H11F3N2O5S [M+H+]: 389.0419; found 389.0418.
C
7.1.17. Compound 23. tert-Butyl(5-fluoro-2-(((4-(trifluorometh-
yl)phenyl)sulfonyl)carbamoyl)phenyl)carbamate
Prepared according to the general procedure method B2. 13
(255 mg, 1.0 mmol), CDI (324 mg, 2.0 mmol) in THF (20 ml),
66 °C. 4-(Trifluoromethyl)benzenesulfonamide (450 mg, 2 mmol),
7.1.21. Compound 28. 2-(4-Aminophenyl)-N-((4-(trifluorometh-
yl)phenyl)sulfonyl)acetamide
Prepared following method E in general procedure. 27 (0.08 g,
0.21 mmol) and Pd/C (10%) (22 mg) in EtOAc (10 ml). Filtration
and evaporation gave 28 in 92% yield. 1H NMR (CDCl3) d 8.12
(dm, J = 8.2 Hz, 2H), 7.77 (dm, J = 8.2 Hz, 2H), 6.90 (dm, J = 8.3 Hz,
2H), 6.62 (dm, J = 8.3 Hz, 1H), 3.47 (s, 2H). (EtOAc: 4.12 (q,
J = 7.2 Hz), 2.05 (s), 1.26 (t, J = 7.1 Hz)). 13C NMR (CDCl3) d 169.6,
146.4, 142.0, 135.6 (q, J = 33.2 Hz), 130.5, 129.1, 126.2 (q,
J = 3.8 Hz), 125.9 (q, J = 258.3 Hz), 121.6, 115.9, 43.1.; (EtOAc:
dissolved in THF (2 ml), DBU (450 ll, 3 mmol). Stirred at room
temperature for 4 h. Purification on silica column (DCM/MeOH
92:8). In order to transfer the DBU salt to the acid form, the product
was dissolved in EtOAc and washed with 0.1 N NaHSO4.The organic
layer was evaporated to give the title compound in 74% yield
(340 mg). 1H NMR (CD3OD) d 8.28 (d, J = 8.2 Hz, 2H), 7.99 (dd,
J = 12.1, 2.6 Hz, 1H), 7.95 (d, J = 8.2 Hz, 2H), 7.82 (dd, J = 9.0,
6.3 Hz, 1H), 6.83 (ddd, J = 8.9, 7.7, 2.6 Hz, 1H), 1.46 (s, 9H). 13C
NMR (CD3OD) d 168.7, 167.4 (d, J = 251.4 Hz), 153.8, 144.8, 144.6
(d, J = 2.3 Hz), 144.6 (d, J = 2.7 Hz), rotamer, 136.0 (q, J = 33.1 Hz),
133.2 (d, J = 11.1 Hz), 130.4, 127.2 (q, J = 3.8 Hz), 124.8 (q,
J = 271.6 Hz), 115.0 (d, J = 2.7 Hz), 109.9 (d, J = 22.9 Hz), 107.5 (d,
J = 28.5 Hz), 82.3, 28.4. MS calcd for C19H18F4N2O5S [M+H+]: 463.1
found: 463.1.
60.6, 21.2, 14.3). HRMS calcd for
753.2449; found 753.2455.
C
34H40ClF3N6O6S [M+H+]:
7.1.22. Compound 30. 3-(Benzyloxy)-1-(((tert-butoxycarbonyl)-
amino)cyclobutane-1-carboxylic acid
7.1.22.1. Mixture of isomers (isomer A:B 1:1.2).
LiOH
(0.069 g, 2.88 mmol) dissolved in water (0.5 ml) was added to
2928 (cis/trans) (0.10 g, 0.286 mmol) dissolved in THF (6 ml). The
reaction mixture was allowed to stir at rt overnight. 10 ml water
was added and the mixture was acidified with 1 M HCl to pH 1, fol-
lowed by extraction with EtOAc (2 ꢁ 10 ml). The organic layer was
dried with MgSO4, filtered and evaporated to yield the product (cis
and trans isomers) as a yellow/brown oil, which was not further
purified (0.092 g, quant.). 1H NMR (CDCl3) 7.38–7.27 (m, 5H+5H;
isomer A+B), 4.46 (s, 2H, isomer B), 4.44 (s, 2H, isomer A), 4.30–
4.22 (m, 1H), 4.14–4.22 (m, 2H), 3.05–2.91 (m, 2H), 2.70–2.57
(m, 4H), 2.35–2.23 (m, 2H), 1.43 (s, 9H+9H; isomer A+B). 13C
NMR (CDCl3) d 178.4, 177.1, 157.1, 155.2, 138.0, 137.7, 128.6,
128.6, 128.0, 128.0, 127.9, 127.9, 81.2, 80.9, 70.7, 70.6, 68.5, 68.4,
53.0, 51.9, 40.9, 39.0, 28.5, 28.4.
7.1.18. Compound 24. tert-Butyl(3-(trifluoromethyl)-5-(((4-(tri
fluoromethyl)phenyl)sulfonyl)carbamoyl)phenyl)carbamate
Prepared following method B2 in general procedure. 14
(219 mg, 0.72 mmol), CDI (233 mg, 1.43 mmol) in THF (20 ml),
66 °C.
4-(Trifluoromethyl)benzenesulfonamide
(322 mg,
1.43 mmol), dissolved in THF (2 ml), DBU (324
ll, 2.16 mmol). Stir-
red at room temperature for 4 h. After evaporation the crude mate-
rial was dissolved in DCM and washed repeated times with 0.1 N
NaHSO4. The organic phase was evaporated and purified on silica
column (DCM/MeOH 88:12) to give the title compound in a yield
of 80% (294 mg).1H NMR (CD3OD) d 8.15 (d, J = 8.9 Hz, 2H), 8.14
(s, 1H), 7.98 (s, 1H), 7.92 (s, 1H), 7.75 (d, J = 8.9 Hz, 2H), 1.50 (s,
9H). 13C NMR (CD3OD) d 173.1, 154.9, 148.9, 141.5, 140.3, 134.0
(q, J = 32.1 Hz), 131.8 (q, J = 32.3 Hz), 128.6, 126.6 (q, J = 3.8 Hz),
125.3 (q, J = 272.4 Hz), 125.1 (q, J = 272.4 Hz), 123.2, 120.4 (q,
J = 3.9 Hz), 118.3, 81.4, 28.6. MS calcd for C20H18F6N2O5S [M+H+]:
513.1 found: 513.2.
7.1.23. Compound 31. tert-Butyl(3-(benzyloxy)-1-(((4-(trifluo-
romethyl)phenyl)sulfonyl)carbamoyl)cyclobutyl)carbamate
7.1.23.1. Mixture of isomers (isomer A:B 1:2).
Prepared fol-
lowing method B1 described in general procedure. 30 (0.092 g,
0.286 mmol), CDI (0.094 g, 0.580 mmol), THF (6 ml), 4-trifluo-
robenzenesulfonamide (0.131 g, 0.582 mmol), DBU (0.086 ml,
0.578 mmol). Stirred at rt overnight. Purification on silica gel (pen-
tane/EtOAc 2:1-1:2) gave 31 as yellow crystals containing the cis
and trans isomers in 90% yield (0.136 g). 1H NMR (CDCl3) d 8.22–
8.13 (m, 2H+2H, isomer A+B), 7.82–7.75 (m, 2H+2H, isomer A+B),
7.38–7.27 (m, 5H+5H isomer A+B), 4.46 (s, 2H, isomer B), 4.38 (s,
2H, isomer A), 4.25–4.17 (m, 1H, isomer B), 4.02–3.91 (m, 1H, iso-
mer A), 3.01–2.90 (m, 2H, isomer A), 2.78–2.65 (m, 2H, isomer B),
2.40–2.50 (m, 2H, isomer B), 2.06–1.99 (m, 2H, isomer A), 1.50–
1.35 (m, 9H+9H; isomer A+B).13C NMR (CDCl3) (Isomer A+B) d
170.5, 170.5, 155.8, 155.7, 142.2, 142.1, 137.6, 137.0, 135.55 (q,
J = 33.4 Hz), 135.57 (q, J = 32.9 Hz), 129.2, 129.1, 128.74, 128.65,
128.3, 128.2, 128.1, 128.0, 126.2 (q, J = 3.8 Hz), 126.2 (q,
J = 3.8 Hz), 123.28 (q, J = 272.1 Hz), 123.25 (q, J = 273.0 Hz), 82.6,
81.9, 71.03, 70.93, 68.1, 66.7, 55.3, 53.6, 40.1, 37.7, 28.3, 28.2.
HRMS calcd for C24H27F3N2O6S [M+Na+]: 551.1440 found 551.1432.
7.1.19. Compound 25. tert-Butyl(2-(methyl((4-(trifluoromethyl)
phenyl)sulfonyl)carbamoyl)phenyl)carbamate
To a suspension of 15 (0.25 g, 0.563 mmol) and Cs2CO3 (0.275 g,
0.844 mmol) in dry DMF (15 ml), MeI (0.168 g, 0.001 mol) was
added. The reaction mixture was allowed to stir at 65 °C for 60 h.
The solvent was evaporated and purification by silica column chro-
matography (i-hexane/EtOAc 9:1, followed by a second purifica-
tion using the same eluent) gave the product as a white solid in
47% yield (0.12 g). 1H NMR (CD3OD) d 8.28 (dm, J = 8.2 Hz, 2H),
7.92 (dm, J = 8.2 Hz, 2H), 7.48–7.40 (m, 2H), 7.23–7.16 (m, 1H),
3.40 (s, 3H), 1.20 (s, 9H). 13C NMR (CD3OD) d 172.0, 155.3, 144.1,
138.0, 135.9 (q, J = 32.9 Hz), 132.9, 130.7, 129.1, 127.9, 127.2 (q,
J = 3.8 Hz), 125.3 (q, J = 272.2 Hz), 124.9, 124.1, 81.1, 37.3, 28.5.
HRMS calcd for C20H21F3N2O5S [M+H+]: 459.1202 found; 459.1203.
7.1.20. Compound 27. 2-(4-Nitrophenyl)-N-((4-(trifluoromethyl)
phenyl)sulfonyl)acetamide
7.1.24. Compound 35. N-((2-Nitrophenyl)sulfonyl)-4-(trifluoro-
methyl)benzamide
Prepared according to the general procedure method C, using 32
(500 mg, 2.47 mmol), 4-pyrrolidino-pyridine (660 mg, 4.45 mmol),
Prepared according to the general procedure, method B1. 2-(4-
Nitrophenyl)acetic acid (0.5 g, 0.003 mol), CDI (0.671 g, 0.004 mol),
THF (20 ml), 4-(Trifluoromethyl) benzenesulfonamide (0.746 g,
0.003 mol), DBU (0.622 ml, 0.004 mol). Stirred at rt overnight.
Purification on silica gel (EtOAc/i-hexane 1:2 to 1:1 followed by
DCM/MeOH 9:1) gave 27 in 26% yield (0.28 g). 1H NMR (CD3OD)
d 8.15 (dm, J = 8.2 Hz, 2H), 8.11 (d, J = 8.8 Hz, 2H), 7.86 (dm,
J = 8.2 Hz, 2H), 7.40 (dm, J = 8.8 Hz, 2H), 3.70 (s, 2H). 13C NMR
pyridine (10 ml) and benzoyl chloride (441 ll, 2.97 mmol) in
3.5 ml toluene. The reaction mixture was allowed to stir at rt for
3 h. Purification by silica column flash chromatography (DCM/
MeOH/HCOOH 100:0:0 to 93:7:3). In order to remove pyridine,
the material was dissolved in EtOAc (40 ml) and washed with 5%

A. K. Belfrage et al. / Bioorg. Med. Chem. 24 (2016) 2603–2620
2615
citric acid (3 ꢁ 40 ml). The organic phase was dried (NaSO4), fil-
tered and evaporated to give the product in 62% yield (575 mg).
1H NMR ((CD3)2SO) d 8.10 (dd, J = 1.6, 0.7 Hz, 1H), 8.07 (dm,
J = 8.1 Hz, 2H), 7.71 (dm, J = 8.1 Hz, 2H), 7.69-7.59 (m, 3H) (Con-
tains EtOAc). 13C NMR (CD3OD) d 173.5, 149.8, 142.7, 137.7,
133.4 (q, J = 32.1 Hz), 133.3, 132.1, 132.0, 130.4, 125.7 (q,
J = 3.8 Hz), 125.6 (q, J = 271.4 Hz), 124.5. HRMS calcd for
gave 39 as an off-white solid (244 mg, 65%). 1H NMR ((CD3)2SO)
d 8.08 (dm, J = 8.2 Hz, 2H), 7.68 (dm, J = 8.2 Hz, 2H), 7.07 (m, 1H),
7.04–6.94 (m, 2H), 6.58 (ddd, J = 7.7, 2.3, 1.3 Hz, 1H), 5.32 (s, 2H).
Contains EtOAc. 13C NMR ((CD3)2SO) d 167.9, 148.2, 146.4, 143.2,
129.8 (q, J = 31.1 Hz), 128.9, 128.0, 124.4 (q, J = 3.9 Hz), 124.4 (q,
J = 272.0 Hz), 115.3, 114.0, 112.1. HRMS calcd for C14H11F3N2O3S
[M+H+]: 345.0512; found 345.0515.
C
14H9F3N2O5S [M+H+]: 375.0263; found 375.0265.
7.1.29. Compound 40. N-((4-Aminophenyl)sulfonyl)-4-(trifluoro
methyl)benzamide
7.1.25. Compound 36. N-((3-Nitrophenyl)sulfonyl)-4-(trifluoro
methyl)benzamide
Prepared according to the general procedure method E using 37
(412 mg, 1.10 mmol) and Pd/C (410 mg) in EtOAc (15 ml), 2 h. Fil-
tration and evaporation gave 40 as a white powder in 66% yield
(251 mg). 1H NMR (CD3OD) d 8.10 (d, J = 8.2 Hz, 2H), 7.75–7.69
(m, 2H), 7.63 (d, J = 8.2 Hz, 2H), 6.70-6.62 (m, 2H). (Contains
EtOAc). 13C NMR (CD3OD) d 173.0, 170.0, 153.9, 140.7, 134.0 (q,
J = 32.3 Hz), 130.8, 130.1, 126.1 (q, J = 3.8 Hz), 125.4 (q,
Prepared according to the general procedure method C, using
33
(1.03 g,
5.08 mmol),
4-pyrrolidino-pyridine
(1.32 g,
l,
8.91 mmol), pyridine (15 ml) and benzoyl chloride (882
l
5.93 mmol) in 2 ml toluene. The reaction mixture was allowed
to stir at rt overnight. Purification by silica column flash chro-
matography (DCM/MeOH/HCOOH 100:0:0 to 93:7:3). To remove
impurities the material was dissolved in EtOAc (50 ml) and
washed with 5% citric acid (3 ꢁ 50 ml) and sat. NaHCO3 (aq)
(3 ꢁ 30 ml). The organic phase was dried (NaSO4), filtered and
evaporated to give the product in 51% yield (977 mg). 1H NMR
(CD3OD) d 8.84 (m, 1H), 8.40-8.32 (m, 2H), 8.14 (dm, J = 7.8 Hz,
2H), 7.73 (ddd, J = 8.3, 7.7, 0.5 Hz, 1H), 7.64 (dm, J = 7.8 Hz, 2H).
13C NMR (CD3OD) d 173.6, 149.1, 147.4, 142.6, 134.2, 133.5 (q,
J = 32.1 Hz), 130.8, 130.3, 126.6, 125.7 (q, J = 3.8 Hz), 125.5 (q,
J = 271.4 Hz), 114.0. HRMS calcd for
C
14H11F3N2O3S [M+H+]:
345.0512; found 345.0518.
7.1.30. Compound 46. 3-(2-(3-(3-(tert-Butyl)ureido)-5-chloro-6-
(2-cyclohexylethyl)-2-oxopyrazin-1(2H)-yl)acetamido)-N-((4-
(trifluoromethyl)phenyl)sulfonyl)benzamide
The title compound was prepared according to method F1. 41
(58 mg, 0.115 mmol), K2CO3 (24 mg, 0.173 mmol), CH3CN (2 ml),
H2O (1 ml). Irradiated by MW to 100 °C for 15 min. 1 M HCl
(20 ml), EtOAc (2 ꢁ 20 ml). The crude acid was dissolved in pyr-
idine (2 ml), POCl3 (20 mg, 0.128 mmol), ꢀ15 °C, 5 min. The
hydrochloride salt of the amine (16 treated by method D)
(66 mg, 0.109 mmol), rt, 3 h. Extra additions of POCl3: after 1 h:
POCl3 (18 mg, 0.115 mmol), 2 h: POCl3 (9 mg, 0.058 mmol). Purifi-
cation by silica column flash chromatography. EtOAc/i-hexane/
HCOOH 20:80:3 to 40:60:3, second column: DCM/MeOH 100:0
to 90:10, gave 46 in 36% yield, 31 mg as white solid. 1H NMR
(CD3OD) d 8.16 (dm, J = 8.0 Hz, 2H), 8.12 (m, 1H), 7.78–7.75 (m,
3H), 7.70 (ddd, J = 8.1, 2.3, 1.1 Hz, 1H), 7.29 (m, 1H), 4.92 (s,
2H), 2.70 (m, 2H), 1.79–1.57 (m, 6H), 1.42 (s, 9H), 1.37–1.07
(m, 5H), 0.93 (m, 2H); 13C NMR (CD3OD) d 174.0, 166.3, 153.9,
152.3, 148.7, 144.9, 139.0, 138.8, 134.1 (d, J = 32.4 Hz), 132.1,
129.6, 128.7, 126.6 (d, J = 3.9 Hz), 126.1, 125.1 (d, J = 271.8 Hz),
124.2, 123.0, 121.7, 52.2, 52.1, 38.9, 36.0, 34.1, 29.2, 28.2, 27.6,
27.3. HRMS calcd for C33H38ClF3N6O6S [M+H+]: 739.2292; found
739.2299.
J = 271.4 Hz), 123.5; HRMS calcd for
C
14H9F3N2O5S [M+H+]:
375.0263; found 375.0270.
7.1.26. Compound 37. N-((4-Nitrophenyl)sulfonyl)-4-(trifluoro
methyl)benzamide
Prepared following method C, using 34 (1.02 g, 5.03 mmol), 4-
pyrrolidino-pyridine (1.32 g, 8.91 mmol), pyridine (15 ml) and
benzoyl chloride (882 ll, 5.93 mmol) in 2 ml toluene. The reac-
tion mixture was allowed to stir at rt overnight. The solvent
was evaporated and the material was dissolved in 50 ml EtOAc
and washed with 5% citric acid (3 ꢁ 50 ml), sat. NaHCO3 (aq)
(3 ꢁ 40 ml) and 1 M NaOH (3 ꢁ 30 ml). The organic phase was
dried (MgSO4), filtered and evaporated. To remove the sulfon-
amide residues, the material was re-dissolved in 50 ml EtOAc
and washed with 1 M NaOH (3 ꢁ 30 ml). The organic phase was
dried, filtered and evaporated to give the product in 45% yield
(445 mg). 1H NMR ((CD3)2SO) d 8.26 (dm, J = 9.0 Hz, 2H), 8.10–
8.02 (m, 4H), 7.68 (dm, J = 8.1 Hz, 2H). (Contains EtOAc). 13C
NMR (CD3OD)
d
173.1, 173.0, 150.5, 141.8, 133.5 (q,
J = 32.4 Hz), 130.3, 129.6, 125.7 (q, J = 3.8 Hz), 125.3 (q,
J = 271.7 Hz), 124.4. HRMS calcd for
345.0512; found 345.0515.
7.1.31. Compound 47. 4-(2-(3-(3-(tert-Butyl)ureido)-5-chloro-6-
(2-cyclohexylethyl)-2-oxopyrazin-1(2H)-yl)acetamido)-N-((4-
(trifluoromethyl)phenyl)sulfonyl)benzamide
C
14H9F3N2O5S [M+H+]:
The title compound was prepared according to method F1. 41
(40 mg, 0.080 mmol), K2CO3 (16 mg, 0.119 mmol), CH3CN (2 ml),
H2O (1 ml). Irradiated by MW to 100 °C for 15 min. 1.0 M HCl
(20 ml), EtOAc (2 ꢁ 20 ml). The crude acid was dissolved in pyri-
dine (2 ml), POCl3 (12 mg, 0.08 mmol), ꢀ15 °C, 15 min. 4531
(66 mg, 0.109 mmol), rt, 1 h. Extra addition of POCl3: after 30 min:
POCl3 (12 mg, 0,080 mmol). Purification by silica column flash
chromatography. EtOAc/i-hexane/HCOOH 20:80:0.5 to 50:50:0.5,
second column: DCM/MeOH 95:5 to 90:10, gave 47 in 43% yield,
23 mg as white solid. 1H NMR (CD3OD) d 8.21 (dm, J = 8.3 Hz,
2H), 7.91–7.86 (m, 2H), 7.84 (dm, J = 8.3 Hz, 2H), 7.67–7.58 (m,
2H), 4.93 (s, 2H), 2.72 (m, 2H), 1.83–1.57 (m, 5H), 1.52–1.44 (m,
2H), 1.43 (s, 9H), 1.38–1.10 (m, 4H), 0.94 (m, 2H). 13C NMR
((CD3)2SO) d 165.1, 165.0, 150.8, 150.7, 150.4, 150.4, 143.4, 142.5,
132.6 (q, J = 31.1 Hz), 130.5, 129.8, 128.6, 126.2 (q, J = 3.4 Hz),
123.5 (q, J = 272.9 Hz), 120.7, 118.2, 50.1, 48.6, 36.6, 34.5, 32.4,
28.6, 26.6, 26.0, 25.6. HRMS calcd for C33H38ClF3N6O6S [M+H+]:
739.2292; found 739.2297.
7.1.27. Compound 38. N-((2-Aminophenyl)sulfonyl)-4-(trifluoro
methyl)benzamide
Prepared according to the general procedure method E. 35
(649 mg, 1.73 mmol) and Pd/C (10%) (650 mg) in EtOAc (15 ml),
2.5 h. Filtration and evaporation gave 38 as a colorless oil in 87%
yield (521 mg). 1H NMR ((CD3)2SO) d 8.08 (dm, J = 8.2 Hz, 2H),
7.68 (dm, J = 8.2 Hz, 2H), 7.58 (dd, J = 7.9, 1.7 Hz, 1H), 7.05 (ddd,
J = 8.1, 7.1, 1.7 Hz, 1H), 6.59 (dd, J = 8.1, 1.0 Hz, 1H), 6.49 (ddd,
J = 7.9, 7.1, 1.0 Hz, 1H). 13C NMR ((CD3)2SO) d 168.3, 145.8, 143.3,
130.8, 129.8 (q, J = 31.1 Hz),129.0, 128.9, 127.3, 124.4 (q,
J = 3.7 Hz), 124.4 (q, J = 272.1 Hz), 115.7, 114.1; HRMS calcd for
C
14H11F3N2O3S [M+H+]: 345.0512; found 345.0519.
7.1.28. Compound 39. N-((3-Aminophenyl)sulfonyl)-4-(trifluo
romethyl)benzamide
Prepared following method E. 36 (409 mg, 1.09 mmol) and Pd/C
(10%) (400 mg) in EtOAc (15 ml), 2 h. Filtration and evaporation

2616
A. K. Belfrage et al. / Bioorg. Med. Chem. 24 (2016) 2603–2620
7.1.32. Compound 48. 2-(2-(3-(3-(tert-Butyl)ureido)-5-chloro-
6-(2-cyclohexylethyl)-2-oxopyrazin-1(2H)-yl)acetamido)-N-((2-
(trifluoromethyl)phenyl)sulfonyl)benzamide
140.8, 133.5, 132.7, 132.3, 130.3, 127.5, 124.5, 124.2, 123.5,
121.4, 52.0, 51.1, 38.7, 36.0, 34.0, 29.3, 28.2, 27.6, 27.2, 21.5; HRMS
calcd for C33H41ClN6O6S [M+H+]: 685.2575; found 685.2568.
The title compound was prepared according to method F1. 41
(49 mg, 0.097 mmol), K2CO3 (20 mg, 0.146 mmol), CH3CN (2 ml),
H2O (1 ml). Irradiated by MW to 100 °C for 15 min. 1 M HCl
(20 ml), EtOAc (2 ꢁ 20 ml). The crude acid was dissolved in pyri-
dine (2 ml), POCl3 (16 mg, 0.104 mmol), ꢀ15 °C, 15 min. The
hydrochloride salt of the amine (17 treated by method D)
(55 mg, 0.144 mmol), rt, 2 days. Extra additions of POCl3: after
16 h, 18 h and 20 h: POCl3 (15 mg, 0.097 mmol). Purification by sil-
ica column flash chromatography. EtOAc/pentane/HCOOH 20:80:3
to 40:60:3, followed by a final purification by preparative HPLC
gave 48 in 10% yield, 7 mg. 1H NMR (CD3OD) d 8.49 (dd, J = 7.3,
1.7 Hz, 1H), 8.11 (dd, J = 8.2, 1.2 Hz, 1H), 7.95 (dd, J = 7.4, 1.8 Hz,
1H), 7.92-7.82 (m, 3H), 7.70 (dd, J = 8.0, 1.5 Hz, 1H), 7.55 (ddd,
J = 8.2, 7.5, 1.5 Hz, 1H), 7.23 (m, 1H), 4.86 (s, 2H), 2.71 (m, 2H),
1.76–1.57 (m, 5H), 1.45 (s, 9H), 1.39 (m, 2H), 1.34–1.10 (m, 4H),
0.90 (m, 2H). 13C NMR (CD3OD) d 166.4, 153.9, 152.5, 145.2,
139.2, 139.0, 135.2, 135.1, 134.7, 133.5, 131.7, 130.2, 129.3 (q,
J = 6.3 Hz), 129.3 (q, J = 6.2 Hz), 129.0 (q, J = 33.1 Hz), 125.4, 124.3
(q, J = 273.3 Hz), 123.5, 123.2, 123.2, 52.1, 50.0, 38.5, 36.0, 4.1,
29.2, 28.1, 27.5, 27.2. HRMS calcd for C33H38ClF3N6O6S [M+H+]:
739.2292; found 739.2303.
7.1.35. Compound 51. 4-Bromo-2-(2-(3-(3-(tert-butyl)ureido)-
5-chloro-6-(2-cyclohexylethyl)-2-oxopyrazin-1(2H)-yl)acetamido)-
N-((4-(trifluoromethyl)phenyl)sulfonyl)benzamide
The title compound was prepared according to method F1. 41
(50 mg, 0.100 mmol), K2CO3 (21 mg, 0.150 mmol), CH3CN (3 ml),
H2O (1.5 ml). Irradiated by MW to 100 °C for 15 min. 1 M HCl
(10 ml), EtOAc (2 ꢁ 10 ml). The crude acid was dissolved in pyri-
dine (1 ml). The hydrochloride salt of the amine (20 treated by
method D) (63 mg, 0.150 mmol) was added followed by POCl3
(17 mg, 0.110 mmol), ꢀ15 °C, 10 min, rt, 18 h. Extra additions of
POCl3: after 1 h: POCl3 (17 mg, 0.110 mmol). H2O (10 ml). Extracted
with EtOAc (2 ꢁ 10 ml). Purification by silica column flash chro-
matography (EtOAc/i-hexane/HCOOH 20:80:3) followed by a sec-
ond column (DCM/MeOH 95:5 to 90:10) gave 51 in 32% yield,
26 mg as white solid. 1H NMR (CD3OD) d 8.59 (s, 1H), 8.20 (d,
J = 8.3 Hz, 2H), 7.90 (d, J = 7.4 Hz, 1H), 7.71 (d, J = 8.4 Hz, 2H),
7.05 (d, J = 8.4 Hz, 1H), 4.95 (s, 2H), 2.74–2.66 (br s, 2H), 1.70–
1.51 (m, 5H), 1.41 (s, 9H), 1.39–1.31 (m, 2H), 1.27–1.03 (m, 4H),
0.92–0.75 (m, 2H). 13C NMR (CD3OD) d 174.0, 166.3, 153.84,
153.76 (rotamer), 152.2, 148.5, 144.8, 141.7, 134.1 (q,
J = 32.5 Hz), 134.0, 132.3, 128.7, 127.5, 126.9, 126.7 (q, J = 3.9 Hz),
125.1 (q, J = 271.8 Hz), 123.6, 123.4, 123.0, 52.1, 52.0 (rotamer),
51.0, 38.7, 36.0, 34.0, 29.23, 29.21 (rotamer), 28.3, 27.5, 27.2;
7.1.33. Compound 49. 2-(2-(3-(3-(tert-Butyl)ureido)-5-chloro-
6-(2-cyclohexylethyl)-2-oxopyrazin-1(2H)-yl)acetamido)-N-((3-
(trifluoromethyl)phenyl)sulfonyl)benzamide
HRMS calcd for
817.1401.
C
33H37BrClF3N6O6S [M+H+]: 817.1398; found
The title compound was prepared according to method F1. 41
(90 mg, 0.179 mmol), K2CO3 (37 mg, 0.269 mmol), CH3CN (2 ml),
H2O (1 ml). Irradiated by MW to 100 °C for 15 min. 1.0 M HCl
(20 ml), EtOAc (2 ꢁ 20 ml). The crude acid was dissolved in pyri-
dine (2 ml), POCl3 (30 mg, 0.196 mmol), ꢀ15 °C, 15 min. The
hydrochloride salt of the amine (18 treated by method D)
(103 mg, 0.271 mmol), rt, 2 days. Extra additions of POCl3: after
2 h, 3 h and 20 h: POCl3 (27 mg, 0.179 mmol). Purification by silica
column flash chromatography three times (EtOAc/pentane 1:4 to
1:1, DCM/EtOAc/MeOH 80:20:2 to 80:20:5, EtOAc/pentane 3:7 to
4:1). Final purification by preparative HPLC gave 49 in 4% yield,
6 mg. 1H NMR (CD3OD) d 8.39–8.32 (m, 2H), 8.03–7.97 (m, 2H),
7.84 (m, 1H), 7.76 (dm, J = 8.0 Hz, 1H), 7.55 (ddd, J = 8.1, 7.4,
1.4 Hz, 1H), 7.25 (m, 1H), 4.87 (s, 2H), 2.69 (m, 2H), 1.77–1.57
(m, 5H), 1.44 (s, 9H), 1.43–1.38 (m, 2H), 1.37–1.11 (m, 4H), 0.93
(m, 2H). HRMS calcd for C33H38ClF3N6O6S[M+H+]: 739.2292; found
739.2286.
7.1.36. Compound 52. 2-(2-(3-(3-(tert-Butyl)ureido)-5-chloro-6-
(2-cyclohexylethyl)-2-oxopyrazin-1(2H)-yl)acetamido)-N-((4-
(trifluoromethyl)phenyl)sulfonyl)-4-vinylbenzamide
The title compound was prepared following general method F2.
41 (50 mg, 0.100 mmol), K2CO3 (21 mg, 0.150 mmol), CH3CN
(3 ml), H2O (1.5 ml). Irradiated by MW to 100 °C for 15 min. 1 M
HCl (10 ml), EtOAc (2 ꢁ 10 ml). The resulting acid, the hydrochlo-
ride salt of the amine (21 treated by method D) (56 mg,
0,138 mmol) and HATU (46 mg, 0.120 mmol) in DCM (5 ml). DIEA
(83 mg, 0.640 mmol). 45 °C for 2.5 h. DCM (15 ml), 0.1 M NaHSO4
(10 ml). Purification on silica column (DCM/MeOH 93:7) followed
by a second column (EtOAc/i-hexane/HCOOH 20:80:3 to 30:70:3)
and a final purification (DCM/MeOH 93:7) to remove som trace
impurities, yielding 51 mg of the title compound (67%). 1H NMR
(CD3OD) d 8.45 (s, 1H), 8.19 (d, J = 8.3 Hz, 2H), 8.04 (d, J = 7.7 Hz,
1H), 7.74 (d, J = 8.3 Hz, 2H), 7.05 (d, J = 8.3 Hz, 1H), 6.66 (q,
J = 11.1, 17.7 Hz, 1H), 5.80 (d, J = 17.6 Hz, 1H), 5.30 (d, J = 11.1 Hz,
1H), 4.94 (s, 2H), 2.72 (t, J = 8.2 Hz, 2H), 1.72–1.51 (m, 5H), 1.48–
1.33 (m, 10H), 1.32–1.19 (m, 2H), 1.17–1.03 (m, 3H), 0.93–0.78
(m, 2H). 13C NMR (CD3OD) d 175.1, 166.0, 153.7, 152.1, 148.5,
144.7, 143.1, 141.1, 137.4, 134.1 (q, J = 32.5 Hz), 133.1, 132.4,
128.4, 126.7 (q, J = 3.8 Hz), 124.9 (q, J = 271.3 Hz), 123.5, 123.3,
121.7, 118.8, 116.8, 52.0, 51.3, 38.7, 36.0, 34.0, 29.2, 28.3, 27.5,
27.2; HRMS calcd for C35H40ClF3N6O6S [M+H+]: 765.2449; found
765.2446.
7.1.34. Compound 50. 2-(2-(3-(3-(tert-Butyl)ureido)-5-chloro-
6-(2-cyclohexylethyl)-2-oxopyrazin-1(2H)-yl)acetamido)-N-
tosylbenzamide
The title compound was prepared according to method F1. 41
(49 mg, 0.097 mmol), K2CO3 (20 mg, 0.146 mmol), CH3CN (2 ml),
H2O (1 ml). Irradiated by MW to 100 °C for 20 min. 1 M HCl
(30 ml), EtOAc (2 ꢁ 20 ml). The crude acid was dissolved in pyri-
dine (2 ml), POCl3 (16 mg, 0.104 mmol), ꢀ15 °C, 15 min. The
hydrochloride salt of the amine (19 treated by method D)
(47 mg, 0.145 mmol), rt, 3 h. Extra additions of POCl3: after 1 h
and 2.75 h: POCl3 (7.5 mg, 0.049 mmol). The reaction mixture
was placed in the ultrasonic bath for 1 min. Purification by silica
column flash chromatography (EtOAc/i-hexane/HCOOH 20:80:0.5
to 50:50:0.5; Second column: DCM/MeOH 97:3 to 90:10) gave 50
in 89% yield, 59 mg as white solid. 1H NMR (CD3OD) d 8.32 (dm,
J = 8.3 Hz, 1H), 8.15 (dd, J = 7.9, 1.6 Hz, 1H), 7.87 (dm, J = 8.2 Hz,
2H), 7.38 (ddd, J = 8.6, 7.9, 1.7 Hz, 1H), 7.25 (dm, J = 8.2 Hz, 2H),
7.04 (m, 1H), 4.81 (s, 2H), 2.71 (m, 2H), 2.34 (s, 3H), 1.74–1.54
(m, 5H), 1.38 (s, 9H), 1.32–1.06 (m, 5H), 0.96–0.81 (m, 2H). 13C
NMR (CD3OD) d 174.9, 165.8, 153.6, 152.1, 144.7, 143.7, 141.8,
7.1.37. Compound 53. 2-(2-(3-(3-(tert-Butyl)ureido)-5-chloro-6-(2-
cyclohexylethyl)-2-oxopyrazin-1(2H)-yl)acetamido)-4-(pyrimidin-
5-yl)-N-((4-(trifluoromethyl)phenyl)sulfonyl)benzamide
The title compound was prepared following general method F2.
41 (25 mg, 0.05 mmol), K2CO3 (10.4 mg, 0.075 mmol), CH3CN
(2 ml), H2O (1 ml). Irradiated by MW to 100 °C for 15 min. 1.0 M
HCl (10 ml), EtOAc (2 ꢁ 10 ml). The resulting acid, the hydrochlo-
ride salt of the amine (22 treated by method D) (30.4 mg,
0.072 mmol) and HATU (23 mg, 0.060 mmol) in DCM (4 ml). DIEA
(41 mg, 0.320 mmol). 45 °C for 2 h. DCM (5 ml), 0.1 M NaHSO4

A. K. Belfrage et al. / Bioorg. Med. Chem. 24 (2016) 2603–2620
2617
(5 ml). The organic layer was washed with brine (5 ml). Purfication
on silica column (DCM/MeOH 93:7 to 90:10) gave the title com-
pound in 54% yield (22 mg). 1H NMR (CD3OD) d 9.10 (s, 1H), 8.99
(s, 2H), 8.74 (s, 1H), 8.26 (d, J = 8.3 Hz, 1H), 8.23 (d, J = 8.3 Hz,
2H), 7.75 (d, J = 8.3 Hz, 2H), 7.32 (d, J = 8.3 Hz, 1H), 5.01 (s, 2H),
2.71 (t, J = 8.4 Hz, 2H), 1.71–1.49 (m, 6H), 1.47–1.32 (m, 11H),
1.32–1.00 (m, 3H), 0.93–0.76 (m, 2H). 13C NMR (CD3OD) d 174.0,
166.5, 158.4, 156.1, 153.4, 153.8 (rotamer), 152.3, 148.9, 144.9,
141.6, 138.5, 135.0, 133.9 (q, J = 33.1 Hz), 133.7, 132.2, 129.0,
126.5 (q, J = 3.9 Hz), 125.1 (q, J = 264.6 Hz), 124.9, 123.2, 122.2,
119.0, 52.1, 52.0 (rotamer), 50.8, 38.7, 36.0, 34.0, 29.2, 29.2 (rota-
mer), 28.3, 27.5, 27.2; HRMS calcd for C37H40ClF3N8O6S [M+H+]:
817.2510; found 817.2513.
7.1.40. Compound 56. 2-(2-(3-(3-(tert-Butyl)ureido)-5-chloro-
6-(2-cyclohexylethyl)-2-oxopyrazin-1(2H)-yl)acetamido)-N-
methyl-N-((4-(trifluoromethyl)phenyl)sulfonyl)benzamide
The title compound was prepared according to method F1. 41
(40 mg, 0.080 mmol), K2CO3 (16 mg, 0.119 mmol), CH3CN (2 ml),
H2O (1 ml). Irradiated by MW to 100 °C for 15 min. 1 M HCl
(20 ml), EtOAc (2 ꢁ 20 ml). The crude acid was dissolved in pyri-
dine (2 ml), POCl3 (12 mg, 0.078 mmol), ꢀ15 °C, 15 min. The
hydrochloride salt of the amine (25 treated by method D)
(43 mg, 0.109 mmol), rt, 1 h. Extra additions of POCl3: after 30 min
and 45 min: POCl3 (12 mg, 0.080 mmol). The reaction mixture
was placed in the ultrasonic bath for 1 min. Purification by silica
column flash chromatography. EtOAc/i-hexane/HCOOH 20:80:0.5
to 50:50:0.5, second column: DCM/MeOH 95:5 to 90:10, gave 56
in 35% yield, 19 mg as white solid. 1H NMR (CDCl3) d 8.09 (dm,
J = 8.3 Hz, 2H), 7.80 (dm, J = 8.3, 2H), 7.72 (dd, J = 8.2, 1.2 Hz, 1H),
7.44 (ddd, J = 8.2, 7.6, 1.6 Hz, 1H), 7.34 (dd, J = 7.9, 1.6 Hz, 1 H),
7.16 (dm, J = 7.6 Hz, 1H), 4.72 (s, 2H), 3.33 (s, 3H), 2.76 (m, 2H),
1.89–1.57 (m, 5H), 1.41 (s, 9H), 1.39–1.12 (m, 6H), 1.05–0.84 (m,
2H). 13C NMR (CDCl3) d 170.5, 164.6, 152.3, 150.9, 146.0, 143.8,
142.0, 135.9, 135.4 (q, J = 33.0 Hz), 132.7, 130.3, 129.3, 128.7,
127.9, 126.2 (q, J = 4.0 Hz), 124.8, 123.5, 123.2 (q, J = 273.2 Hz),
51.3, 49.4, 37.6, 36.0, 35.4, 33.1, 29.0, 27.1, 26.6, 26.3. HRMS calcd
for C34H40ClF3N6O6S [M+H+]: 753.2449; found 753.2448.
7.1.38. Compound 54. 2-(2-(3-(3-(tert-Butyl)ureido)-5-chloro-6-
(2-cyclohexylethyl)-2-oxopyrazin-1(2H)-yl)acetamido)-4-fluoro-
N-((4-(trifluoromethyl)phenyl)sulfonyl)benzamide
The title compound was prepared according to method F1. 41
(69 mg, 0.140 mmol), K2CO3 (29 mg, 0.21 mmol), CH3CN (3 ml),
H2O (1.5 ml). Irradiated by MW to 100 °C for 15 min. 1 M HCl
(10 ml), EtOAc (2 ꢁ 10 ml). The crude acid was dissolved in pyri-
dine (1 ml). The hydrochloride salt of the amine (23 treated by
method D) (76 mg, 0.210 mmol) was added followed by POCl3
(24 mg, 0.154 mmol), ꢀ15 °C, 10 min, room temperature, 5 h. Extra
addition of POCl3: after 2.5 h: POCl3 (24 mg, 0.154 mmol). H2O
(10 ml). Extracted with EtOAc (2 ꢁ 10 ml). Purification by silica
column flash chromatography (EtOAc/i-hexane/HCOOH 20:80:3
to 40:60:3 followed by a second column (DCM/MeOH 95:5) gave
54 in 48% yield, 51 mg as white solid. 1H NMR (CD3OD) d 8.20 (d,
J = 8.4 Hz, 2H), 8.15 (d, J = 11.7 Hz, 1H), 8.12-8.03 (br s, 1H), 7.70
(d, J = 8.4 Hz, 2H), 6.65 (dd, J = 8.5, 8.5 Hz, 1H), 4.96 (s, 2H), 2.75–
2.66 (br s, 2H), 1.69–1.51 (m, 5H), 1.40 (s, 9H), 1.38–1.30 (m,
3H), 1.26–1.03 (m, 3H), 0.90–0.76 (m, 2H). 13C NMR (CD3OD) d
174.1, 166.3, 166.1 (d, J = 249.2 Hz), 153.7, 152.2, 148.5, 144.8,
142.8 (d, J = 12.2 Hz), 135.1 (d, J = 10.3 Hz), 134.1 (q, J = 32.5 Hz),
132.2, 128.6, 126.7 (q, J = 3.8 Hz), 125.0 (q, J = 271.9 Hz), 123.5,
120.2, 110.6 (d, J = 22.1 Hz), 107.7 (d, J = 28.0 Hz), 52.0, 51.2, 38.7,
36.0, 34.0, 29.2, 28.3, 27.5, 27.2; HRMS calcd for C33H37ClF4N6O6S
[M+H+]: 757.2198; found 757.2195.
7.1.41. Compound 57. Methyl 2-(2-(3-(3-(tert-butyl)ureido)-5-
chloro-6-(2-cyclohexylethyl)-2-oxopyrazin-1(2H)-yl)acetamido)
benzoate
The title compound was prepared as described in general
method F2: 41 (0.040 mg, 0.795 mmol) K2CO3 (0.016 g,
0.119 mmol), CH3CN (2 ml), and H2O (1 ml). 100 °C for 15 min.
1 M HCl (20 ml), EtOAc (20 ml).
Methyl 2-aminobenzoate (0.018 g, 0.116 mmol), HATU (0.035 g,
0.93 mmol), DCM (2 ml), DIEA (0.041 ml, 0.232 ml). 45 °C, 3 h. DCM
(15 ml), 0.1 M NaHSO4 (20 ml). Purification on silica gel (iso-hex-
ane/EtOAc 4:1) gave 57 in 57% yield (24 mg). 1H NMR (CDCl3) d
11.42 (s, NH), 8.63 (s, NH), 8.59 (dd, J = 8.6, 1.2 Hz, 1H), 8.02 (dd,
J = 8.2, 1.7 Hz, 1H), 7.93 (s, NH), 7.55 (ddd, J = 8.6, 7.3, 1.7 Hz,
1H), 7.13 (ddd, J = 8.2, 7.3, 1.2 Hz, 1H), 4.89 (s, 2H), 3.89 (s, 3H),
2.69 (m, 2H), 1.78–1.58 (m, 6H), 1.43 (s, 9H), 1.29–1.12 (m, 5H),
0.96 (m, 2H). 13C NMR (CDCl3) d 169.0, 164.1, 151.7, 151.1, 144.1,
140.5, 135.0, 131.0, 129.2, 123.6, 122.9, 120.5, 115.5, 52.7, 51.1,
49.1, 38.0, 35.4, 33.0, 29.0, 27.3, 26.5, 26.3. HRMS calcd for
7.1.39. Compound 55. 3-(2-(3-(3-(tert-Butyl)ureido)-5-chloro-6-
(2-cyclohexylethyl)-2-oxopyrazin-1(2H)-yl)acetamido)-5-
(trifluoromethyl)-N-((4-(trifluoromethyl)phenyl)sulfonyl)
benzamide
C
27H36ClN5O5 [M+H+]: 546.2405; found 546.2483.
The title compound was prepared according to method F1. 41
(50 mg, 0.100 mmol), K2CO3 (21 mg, 0.150 mmol), CH3CN (3 ml),
H2O (1.5 ml). Irradiated by MW to 100 °C for 15 min. 1 M HCl
(10 ml), EtOAc (2 ꢁ 10 ml). The crude acid was dissolved in pyri-
dine (1 ml). The hydrochloride salt of the amine (24 treated by
method D) (63 mg, 0.150 mmol) was added followed by POCl3
(20 mg, 0.131 mmol), ꢀ15 °C, 10 min, rt, 4 h. Extra additions of
POCl3: after 2 h: POCl3 (10 mg, 0.066 mmol). H2O (10 ml). Extracted
with EtOAc (2 ꢁ 15 ml). Purification by silica column flash chro-
matography (EtOAc/i-hexane/HCOOH 20:80:3 to 30:70:3) followed
by a second column (DCM/MeOH 90:10) gave 55 in 22% yield,
19 mg as white solid. 1H NMR (CD3OD) d 8.26 (s, 1H), 8.16 (d,
J = 8.2 Hz, 2H), 8.10 (s, 1H), 8.01 (s, 1H), 7.74 (d, J = 8.4 Hz, 2H),
4.94 (s, 2H), 2.75–2.68 (m, 2H), 1.77–1.56 (m, 5H), 1.47–1.07 (m,
6H), 1.42 (s, 9H), 0.98–0.85 (m, 2H). 13C NMR (CD3OD) d 172.4,
166.7, 153.9, 152.4, 149.2, 145.0, 141.0, 139.9, 133.7 (q,
J = 30.8 Hz), 132.1, 131.9 (q, J = 33.0 Hz), 129.1, 126.4 (q,
J = 3.9 Hz), 125.2 (q, J = 263.8 Hz), 124.3 (q, J = 5.5 Hz), 123.0,
122.1 (q, J = 3.0 Hz), 119.6, 52.0, 38.8, 36.0, 34.1, 29.2, 28.2, 27.6,
27.2; HRMS calcd for C34H37ClF6N6O6S [M+H+]: 807.2166; found
807.2167.
7.1.42. Compound 58. 2-(3-(3-(tert-Butyl)ureido)-5-chloro-6-
(2-cyclohexylethyl)-2-oxopyrazin-1(2H)-yl)-N-(4-(2-oxo-2-(4-
(trifluoromethyl)phenylsulfonamido)ethyl)phenyl)acetamide
The title compound was prepared according to method F1. 41
(40 mg, 0.08 mmol), K2CO3 (16 mg, 0.119 mmol), CH3CN (2 ml),
H2O (1 ml). Irradiated by MW to 100 °C for 15 min. 1 M HCl
(20 ml), EtOAc (2 ꢁ 20 ml). The crude acid was dissolved in pyri-
dine (2 ml), POCl3 (12 mg, 0.078 mmol), ꢀ15 °C, 15 min. 28
(39 mg, 0.109 mmol), rt, 2 h. Extra addition of POCl3 and 28: after
1 h POCl3 (12 mg, 0.080 mmol), 28 (10 mg, 0.028 mmol). Purifica-
tion by silica column flash chromatography. EtOAc/i-hexane/
HCOOH 20:80:0.5 to 50:50:0.5, second column: DCM/MeOH
100:0 to 95:5, gave 58 in 27% yield, 55 mg as white solid. 1H
NMR (CD3OD) d 8.00 (dd, J = 8.1, 0.9 Hz, 2H), 7.75–7.69 (m, 2H),
7.40 (dd, J = 8.7, 2.2 Hz, 2H), 7.20–7.13 (m, 2H), 4.91 (s, 2H), 3.46
(s, 2H), 2.71 (m, 2H), 1.85–1.57 (m, 5H), 1.53–1.44 (m, 2H), 1.43
(s, 9H), 1.39–1.09 (m, 4H), 1.07–0.85 (m, 2H). 13C NMR (CD3OD)
d
166.2, 157.2, 153.9, 152.4, 147.7, 145.0, 137.8, 134.3 (q,
J = 32.4 Hz), 133.6, 132.1, 130.9, 128.8, 126.6 (q, J = 3.8 Hz), 125.0
(q, J = 271.8 Hz), 123.0, 121.1, 52.1, 45.9, 43.0, 38.9, 35.9, 34.1,

2618
A. K. Belfrage et al. / Bioorg. Med. Chem. 24 (2016) 2603–2620
29.2, 28.2, 27.6, 27.3; HRMS calcd for C34H40ClF3N6O6S [M+H+]:
753.2449; found 753.2455.
7.60–7.43 (m, 3H), 7.42–7.28 (m, 2H), 7.10 (m, 1H), 4.67 (s, 2H),
4.60 (s, 2H), 1.46 (s, 9H). 13C NMR ((CD3)2SO) d 167.8, 164.3,
150.9, 150.8, 144.6, 140.3 (q, J = 34.9 Hz), 133.4, 133.3, 131.4,
131.1, 128.9, 128.6, 128.0, 127.9, 127.6, 127.0, 126.6, 126.2,
125.7, 125.7, 124.8 (q, J = 268.6 Hz), 124.5 (q, J = 3.8 Hz), 124.0,
7.1.43. Compound 60. N-((2-(2-(3-(3-(tert-Butyl)ureido)-5-chloro-
6-(naphthalen-1-ylmethyl)-2-oxopyrazin-1(2H)-yl)acetamido)
phenyl)sulfonyl)-4-(trifluoromethyl)benzamide
123.4, 123.0, 117.9, 55.9, 50.2, 29.6, 28.6. HRMS calcd for C36H32
-
The title compound was prepared according to method F1. 42
(120 mg, 0.226 mmol), K2CO3 (47 mg, 0.339 mmol), CH3CN (3 ml),
H2O (1.5 ml). Irradiated by MW to 100 °C for 15 min. 1 M HCl
(25 ml), EtOAc (2 ꢁ 25 ml). The crude acid was dissolved in pyri-
dine (2 ml). 38 (78 mg, 0.226 mmol) was added followed by POCl3
(38 mg, 0.247 mmol), ꢀ15 °C to rt, 2 h. Extracted with EtOAc
(3 ꢁ 30 ml). Purification by silica column flash chromatography
(DCM/MeOH 100:0 to 90:10) gave 60 in 42% yield, 73 mg as white
solid. 1H NMR (CD3OD) d 8.09 (dm, J = 8.2 Hz, 1H), 8.03 (dm,
J = 8.3 Hz, 2H), 7.93 (dd, J = 8.0, 1.5 Hz, 1H), 7.87–7.82 (m, 2H),
7.72 (dm, J = 8.3 Hz, 1H), 7.53 (dm, J = 8.5 Hz, 2H), 7.50–7.45 (m,
2H), 7.40 (m, 1H), 7.27 (dd, J = 8.3, 7.1 Hz, 1H), 7.16 (m, 1H), 7.06
(m, 1H), 4.72 (s, 2H), 4.66 (s, 2H), 1.35 (s, 9H). 13C NMR (CD3OD)
d 173.5, 166.0, 153.7, 152.8, 149.8, 145.9, 141.8, 138.6, 136.9,
136.0, 135.3, 134.8, 133.6 (q, J = 32.0 Hz), 133.3, 132.7, 132.1,
130.5, 129.8, 129.0, 128.9, 127.8, 127.2, 126.6, 126.0, 125.8 (q,
J = 3.9 Hz), 125.5 (q, J = 271.7 Hz), 125.2, 124.0, 52.0, 50.8, 33.5,
29.2; HRMS calcd for C36H32ClF3N6O6S [M+H+]: 769.1823; found
769.1821.
ClF3N6O6S [M+H+]: 769.1823; found 769.1826.
7.1.46. Compound 64. N-((2-(2-(3-(3-(tert-Butyl)ureido)-5-chloro-
2-oxo6-phenethylpyrazin-1(2H)-yl)acetamido)phenyl)sulfonyl)-
4-(trifluoromethyl)benzamide
The title compound was prepared according to method F1. 43
(150 mg, 0.367 mmol), K2CO3 (61 mg, 0.440 mmol), CH3CN (3 ml),
H2O (1.5 ml). Irradiated by MW to 100 °C for 15 min. 1 M HCl
(25 ml), EtOAc (2 ꢁ 25 ml). The crude acid was dissolved in pyri-
dine (2 ml). 38 (101 mg, 0.293 mmol) was added followed by POCl3
(50 mg, 0.323 mmol), ꢀ15 °C, 3 h. Extracted with EtOAc
(3 ꢁ 30 ml). Purification by silica column flash chromatography
(DCM/MeOH 100:0 to 90:10) gave 64 in 61% yield, 95 mg as white
solid. 1H NMR (CD3OD) d 8.19 (dm, J = 8.4 Hz, 1H), 8.01 (dd, J = 8.1,
1.5 Hz, 1H), 7.97 (dm, J = 8.3 Hz, 2H), 7.70 (dm, J = 8.3 Hz, 2H), 7.60
(ddd, J = 8.4, 7.3, 1.5 Hz, 1H), 7.32 (m, 1H), 7.27–7.12 (m, 5H), 4.90
(s, 2H), 3.07 (dd, J = 9.3, 6.6 Hz, 2H), 2.92 (dd, J = 9.3, 6.6 Hz, 2H),
1.35 (s, 9H). 13C NMR (CD3OD) d 166.3, 164.5, 153.8, 152.5,
145.4, 141.2, 137.1, 134.5, 134.3, 131.1, 130.9 (d, J = 33.6 Hz),
130.7, 130.3, 130.2, 129.7, 129.6, 127.6, 126.2 (d, J = 3.9 Hz) 126.1
(d, J = 276.9 Hz), 125.9, 124.7, 124.2, 52.0, 50.0, 34.6, 33.0, 29.1.
7.1.44. Compound 61. N-((3-(2-(3-(3-(tert-Butyl)ureido)-5-chloro-
6-(naphthalen-1-ylmethyl)-2-oxopyrazin-1(2H)-yl)acetamido)
phenyl)sulfonyl)-4-(trifluoromethyl)benzamide
HRMS calcd for
733.1826.
C
33H32ClF3N6O6S [M+H+]: 733.1823; found
The title compound was prepared according to method F1. 42
(156 mg, 0.293 mmol), K2CO3 (61 mg, 0.440 mmol), CH3CN (3 ml),
H2O (1.5 ml). Irradiated by MW to 100 °C for 15 min. 1 M HCl
(25 ml), EtOAc (2 ꢁ 25 ml). The crude acid was dissolved in pyri-
dine (2 ml). 39 (101 mg, 0.293 mmol) was added followed by POCl3
(50 mg, 0.323 mmol), ꢀ15 °C to rt, 3 h. Extra additions of POCl3 and
39: after 2 h: POCl3 (23 mg, 0.147 mmol), 39 (51 mg, 0.147 mmol).
Extracted with EtOAc (3 ꢁ 30 ml). Purification by silica column
flash chromatography (DCM/MeOH 100:0 to 90:10) gave 61 in
41% yield, 93 mg as white solid. 1H NMR (CD3OD) d 8.13 (dm,
J = 8.1 Hz, 2H), 8.09 (m, 1H), 7.99 (m, 1H), 7.83 (m, 1H), 7.73–
7.65 (m, 2H), 7.56 (dm, J = 8.1 Hz, 2H), 7.49–7.41 (m, 3H), 7.34–
7.27 (m, 2H), 7.03 (dm, J = 7.1 Hz, 1H), 4.63 (s, 2H), 4.53 (s, 2H),
1.44 (s, 9H). 13C NMR (CD3OD) d 172.8, 166.0, 153.9, 152.5, 150.0,
145.9, 145.2, 141.9, 139.3, 138.5, 135.4, 133.8 (q, J = 32.3 Hz),
132.6, 131.8, 130.7, 130.0, 130.0, 129.1, 127.8, 126.7, 125.8 (q,
J = 3.7 Hz), 125.5 (q, J = 271.8 Hz), 125.5, 125.3, 123.8, 123.6,
123.3, 118.9, 61.5, 52.2, 33.1, 29.2; HRMS calcd for
7.1.47. Compound 65. 3-(Benzyloxy)-1-(2-(3-(3-(tert-butyl)
ureido)-5-chloro-6-(2-cyclohexylethyl)-2-oxopyrazin-1(2H)-yl)
acetamido)-N-((4-(trifluoromethyl)phenyl)sulfonyl)cyclobutane-
1-carboxamide. Mixture of isomers
The title compound was prepared following general method
F2. 41 (71 mg, 0.141 mmol), K2CO3 (29 mg, 0.212 mmol), CH3CN
(3 ml), H2O (1.5 ml). Irradiated by MW to 100 °C for 20 min.
1 M HCl (10 ml), EtOAc (20 ml). The resulting acid, the hydrochlo-
ride salt of the amine (31 treated by method D) (97 mg,
0,210 mmol) and HATU (64 mg, 0.168 mmol) in DCM (1 ml). DIEA
(0.112 ml, 0.643 mmol). Rt for 2 h. DCM (10 ml), 0.1 M NaHSO4
(10 ml). Purified four times on silica column (DCM/MeOH 98:2–
95:5)/iso-hexane/EtOAc/HCOOH 80:20:3–60:40:3. In order to
remove residues of impurities, the product was finally purified
on
a preparative RP-HPLC, eluent: NH4OAc in CH3CN/H2O,
(0.013 g, 11%) as a mixture of cis and trans isomers in NH+4 salt
form. 1H NMR ((CD3)2SO) d 8.42 (s, NH), 8.39 (s, NH), 8.33 (s,
NH), 7.97 (m, 2H+2H; 2 isomers), 7.80 (m, 2H+ 2H; 2 isomers),
7.36 -7.20 (m, 5H+5H; 2 isomers), 4.71 (s, 2H; 1 isomer), 4.70
(s, 2H, 1 isomer), 4.35 (s, 2H, 1 isomer), 4.34 (s, 2H; 1 isomer),
4.07 (p, J = 7.3 Hz, 1H; 1 isomer), 3.89 (m, 1H; 1 isomer), 2.72–
2.61 (m, 2H, 1 isomer), 2.57–2.49 (m, 2H+2H), 2.35–2.27 (m,
4H; 1 isomer), 2.14–2.03 (m, 2H) 1.75–1.54 (m, 10H), 1.34 (s,
9H+9H), 1.36–1.04 (m, 12H: 2 isomers), 0.95–0.81 (m, 2H+2H; 2
isomers). 13C NMR ((CD3)2SO) d 168.2, 167.8, 166.23, 166.16,
151.3 (2 carbons), 150.88, 150.86, 143.9, 143.8, 138.7, 138.6,
138.50, 138.47, 130.8, 130.7, 128.63(2 carbons), 128.58 (2 car-
bons), 128.1(2 carbons), 127.9, 127.8, 125.95, 125.95 (d,
J = 4.3 Hz), 125.89, 125.85 (d, J = 4.6 Hz), 123.99 (d, J = 286.4 Hz)
(2 carbons), 121.13 (2 carbons), 69.73, 69.69, 67.8, 67.7, 53.4,
53.3, 50.6 (2 carbons), 47.7, 47.6, 38.48, 38.46, 37.2, 37.1, 34.84,
34.79, 32.81, 32.75, 29.0 (2 carbons), 27.1, 27.0, 26.49, 26.46,
26.2, 26.1. HRMS (ES) m/z calcd for C38H46ClF3N6O7S [M+H+]:
823.2868; found 823.2857.
C
36H32ClF3N6O6S [M+H+]: 769.1823; found 769.1827.
7.1.45. Compound 62. N-((4-(2-(3-(3-(tert-Butyl)ureido)-5-chloro-
6-(naphthalen-1-ylmethyl)-2-oxopyrazin-1(2H)-yl)acetamido)
phenyl)sulfonyl)-4-(trifluoromethyl)benzamide
The title compound was prepared according to method F1. 42
(60 mg, 0.113 mmol), K2CO3 (23 mg, 0.170 mmol), CH3CN (2 ml),
H2O (1 ml). Irradiated by MW to 100 °C for 15 min. 1 M HCl
(20 ml), EtOAc (2 ꢁ 20 ml). The crude acid was dissolved in pyri-
dine (2 ml), POCl3 (19 mg, 0.0,124 mmol), ꢀ15 °C, 15 min. 40
(58 mg, 0.169 mmol), rt, 18 h. Extra additions of POCl3: after 16 h:
POCl3 (9 mg, 0.057 mmol). Extracted with EtOAc (3 ꢁ 20 ml).
Purification by silica column flash chromatography (EtOAc/i-hex-
ane/HCOOH 20:80:3 to 50:50:3 followed by a second column
(DCM/MeOH 100:0 to 95:5) gave 62 in 40% yield, 35 mg as white
solid. 1H NMR (CD3OD) d 8.93 (s, 1H), 8.11–8.01 (m, 3H), 7.93
(dd, J = 9.1, 2.1 Hz, 2H), 7.84 (dd, J = 8.0, 1.6 Hz, 1H), 7.73 (m, 3H),

A. K. Belfrage et al. / Bioorg. Med. Chem. 24 (2016) 2603–2620
2619
7.1.48. Compound 66. 1-(2-(6-Benzyl-3-(3-(tert-butyl)ureido)-5-
chloro-2-oxopyrazin-1(2H)-yl)acetamido)-3-(benzyloxy)-N-((4-
(trifluoromethyl)phenyl)sulfonyl)cyclobutane-1-carboxamide
The title compound was prepared following general method F2.
44 (34 mg, 0.071 mmol), K2CO3 (15 mg, 0.107 mmol), CH3CN
(2 ml), H2O (1 ml). Irradiated by MW to 100 °C for 15 min. 1 M
HCl (20 ml), EtOAc (2 ꢁ 20 ml). The resulting acid, the hydrochlo-
ride salt of the amine (31 treated by method D) (50 mg,
0,107 mmol) and HATU (33 mg, 0.086 mmol) in DCM (2 ml). DIEA
(0.075 ml, 0.428 mmol). 45 °C for 3 h. DCM (15 ml), 0.1 M NaHSO4
(20 ml). Purified on silica column (iso-hexane/EtOAc/HCOOH
80:20:0.5–0:50:0.5 followed by a second purification DCM/MeOH
98:2–9:1) to give the compound in 68% yield (0.039 g) as a mixture
of cis and trans isomers. (Isomer A: 6 mg, Isomer B: 12 mg, Isomer
A/B: 21 mg).
127.76, 127.72, 127.04, 124.16, 51.23, 46.49, 35.31, 28.91. HRMS
calcd for C18H21ClN4O4 [M+H+]: 393.1330; found 393.1349.
7.1.51. Compound 70. 1-(2-(6-Benzyl-3-(3-(tert-butyl)ureido)-2-
oxopyrazin-1(2H)-yl)acetamido)-3-hydroxy-N-((4-(trifluoro-
methyl)phenyl)sulfonyl)cyclobutane-1-carboxamide
66 (0.010 g, 0.012 mmol) was dissolved in MeOH (2 ml). 10%
Pd/C (0.05 g) was added and the reaction mixture was placed
under H2 (1 atm) and allowed to stir at rt for 6 h. The mixture
was filtered through celite and the solvent was evaporated to give
the title compound in 59% yield (5 mg). Mixture of isomers (Isomer
A:B 1.3:1.0) 1H NMR (CD3CN) d 8.11–8.00 (m, 2H+2H; isomer A+B),
7.86–7.75 (m, 2H+2H; isomer A+B), 7.38–7.18 (m, 5H+5H; isomer
A+B), 6.83 (br s, 1H+1H; isomer A+B), 4.50 (s, 2H, one isomer),
4.48 (s, 2H; one isomer), 4.25–4.13 (m, 1H+1H; isomer A+B), 4.09
(s, 2H; one isomer), 4.05 (s, 2H; one isomer), 3.91–3.82 (m, 2H
+2H; isomer A+B), 2.64–2.58 (m, 2H, one isomer), 2.50–2.29 (m,
1H+2H; isomer A+B), 1.38 (s, 9H), 1.25 (s, 9H). 13C NMR (CD3CN)
d 169.4, 169.0, 165.4, 164.5, 156.4, 155.9, 152.8, 152.0, 146.4,
145.8, 144.1, 142.9, 138.83, 138.79, 134.7 (d, J = 32.4 Hz), 134.4
(d, J = 32.0 Hz), 134.3, 134.2, 130.29, 130.27, 129.8, 129.7, 129.5,
129.5, 128.90 (d, J = 277.1 Hz), 128.87 (d, J = 274.1 Hz), 128.2,
128.1, 126.78 (d, J = 3.7 Hz), 126.79 (d, J = 3.7 Hz), 115.5, 115.2,
59.1, 59.0, 54.2, 54.1, 51.3, 50.2, 48.35 (2C), 44.7, 43.3, 38.7, 36.2,
29.2, 29.0. HRMS (ES) m/z calcd for C30H33F3N6O7S [M+H+]:
679.2162; found 679.2175.
Isomer A: 1H NMR (CD3CN) d 8.59 (s, NH), 8.11 (s, NH), 8.05 (m,
2H), 7.81 (m, 2H), 7.38–7.19 (m, 10H), 4.44 (s, 2H), 4.36 (s, 2H),
4.16 (s, 2H), 3.89 (p, J = 7.0 Hz, 1H), 2.73–2.65 (m, 2H), 1.97–1.89
(partly covered by solvent peak—confirmed by gCOSY experiment)
(m, 2H), 1.42 (s, 9H). 13C NMR (CD3CN) d 173.3, 166.6, 152.5, 152.3,
145.6, 143.8, 139.4, 136.3, 135.1 (q, J = 30.1 Hz), 129.98, 129.93,
129.7, 129.3, 128.9, 128.9, 128.8, 128.6, 128.4, 126.9 (q,
J = 3.8 Hz), 123.2 (d, J = 274.1 Hz), 70.8, 67.7, 54.0, 51.7, 50.4,
40.9, 35.6, 29.0.
Isomer B (analysed as a mixture B/A 10:1): 1H NMR (CD3CN) d
8.63 (s, NH), 8.15 (s, NH), 8.04 (m, 2H), 7.79 (m, 2H), 7.42–7.13
(m, 10H), 4.46 (s, 2H), 4.37 (s, 2H), 4.14 (s, 2H), 4.00 (p,
J = 7.1 Hz, 1H), 1.42 (s, 10H). 13C NMR (CD3CN) d 171.6, 167.3,
152.4, 152.4, 145.6, 143.5, 139.3, 136.3, 134.2 (q, J = 31.1 Hz),
130.0, 129.9, 129.3, 129.3, 128.9, 128.9, 128.8, 128.7, 128.3, 127.0
(q, J = 3.9 Hz), 123.7 (q, J = 280.6 Hz), 70.8, 68.3, 54.9, 51.7, 50.3,
39.0, 35.7, 29.1.
7.2. Computational methods
All calculations were performed within the Schrödinger Small
Molecule Drug Discovery Suite (Schrödinger Release 2015-4). Crys-
tal structures (4A92 and 4B74) of full-length NS3 were down-
loaded from the PDB and prepared using Protein Preparation
Wizard.50 To prepare structure 4B74 for docking, the C-terminal
625-631 was deleted and S624 was terminated by a N-methyl
amine. Also, the allosteric ligand 1LH was deleted. The OPLS3 force
field was used throughout this work,51 in combination with the
GB/SA continuum solvation model for water.52 Grids for docking
were prepared using Glide (Glide, version 6.9, Schrödinger, LLC,
New York, NY, 2015) and subsequent docking in SP mode was per-
formed using the same program.53 Docking poses were used as
starting points for subsequent molecular mechanics conforma-
tional searches in MacroModel (MacroModel, version 11.0,
Schrödinger, LLC, New York, NY, 2015). During these searches, var-
ious flat bottomed distance constraints from the ligand to the pro-
tein backbone of bE2 (R155, A157, and C159) were used to keep the
hydrogen bond pattern of the starting pose. The distance constraint
HRMS (ES) m/z calcd for C37H38ClF3N6O7S [M+H+]: 803.2242;
found 803.2237.
7.1.49. Compound 68. 2-(2-(3-(3-(tert-Butyl)ureido)-5-chloro-6-
(2-cyclohexylethyl)-2-oxopyrazin-1(2H)-yl)acetamido)benzoic
acid
57 (11 mg, 0.020 mmol) was dissolved in CH3CN (1 ml). K2CO3
(0.004 g, 0.030 mmol) in H2O (0.5 ml) was added and the reaction
mixture was heated in the MW (100 °C, 15 min). 1 M HCl (20 ml)
was added and the water phase was extracted two times with
20 ml EtOAc. The combined organic phases were dried (MgSO4), fil-
tered and evaporated. Purified on a silica gel column (DCM/MeOH
95:5) to yield the title compound as a white solid (8 mg, 75%). 1H
NMR (CD3OD) d 8.51 (dd, J = 8.4, 1.2 Hz, 1H), 8.08 (m, 1H), 7.51
(m, 1H), 7.15 (m, 1H), 4.96 (s, 2H), 2.75 (m, 2H), 1.82-1.49 (m,
5H), 1.43 (s, 9H), 1.48–1.09 (m, 6H), 0.92 (q, J = 11.3 Hz, 2H). 13C
NMR (CD3OD) d 169.7, 164.8, 151.6, 151.0, 147.6, 143.7, 134.8,
131.2, 130.2, 124.8, 123.0, 119.5, 110.0, 50.6, 48.9, 47.9, 37.3,
34.6, 32.6, 27.9, 26.8, 26.1, 25.8. HRMS (ES) m/z calcd for
used was 2.1 0.3 Å using a force constant of 100 kcal molꢀ1 Åꢀ2
.
Torsions selected for the conformational searches included the
rotatable bonds in the ligand investigated and side-chains of inter-
acting amino acids. The number of steps in the Monte Carlo multi-
ple minima (MCMM) were usually set to 1000 but in many cases, a
single binding mode was investigated using several starting
geometries and both crystal structures 4A92 and 4B74. Induced
fit docking (Induced Fit Docking protocol; Glide version 6.9,
Schrödinger, LLC, New York, NY, 2015; Prime version 4.2, Schrödin-
ger, LLC, New York, NY, 2015) was performed on compounds 57
and 68 using 4A92.
C
26H34ClN5O5 [M+H+]: 532.2327; found 532.2319.
7.1.50. Compound 69. 2-(6-Benzyl-3-(3-(tert-butyl)ureido)-5-
chloro-2-oxopyrazin-1(2H)-yl)acetate
The title compound was prepared according to the first part of
method F. 44 (0.040 g, 0.083 mmol), CH3CN (2 ml), K2CO3
(0.017 g, 0.124 mmol), H2O (1 ml). 100 °C, 15 min. 1 M HCl
(10 ml), EtOAc (2 ꢁ 10 ml). H2O (10 ml) was added and a solid
was formed. The material was centrifugated for 2 min. The water
layer was removed and the remaining solid was kept under vac-
uum overnight yielding the title compound in 98% yield (32 mg)
with no further purification. 1H NMR (CDCl3) d 7.37–7.26 (m,
3H), 7.16–7.09 (m, 2H), 4.58 (s, 2H), 4.06 (s, 2H), 1.43 (s, 9H). 13C
NMR (CDCl3) d 168.32, 151.83, 150.84, 144.37, 134.89, 129.44,
Acknowledgments
We acknowledge Gunnar Lindeberg for assistance in the purifi-
cation of the co-factor, NS4A protein, used in the biochemical
assay. UHD was supported by the Swedish Research Council –
Sweden (VR) (no. D0571301). We thank Katrien Geerts, Rega

2620
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Supplementary data
Supplementary data (1H, 13C NMR, LC-UV/MS of target com-
pounds, kinetic parameters of tested enzyme variants, binding
curves corrected for auto-fluorescence and additional molecular
modeling pictures of binding poses) associated with this article
can be found, in the online version, at http://dx.doi.org/10.1016/j.
bmc.2016.03.066.
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