N-(Methyloxycarbonyl)thiophene sulfonamides as high affinity AT2 receptor ligands

Article abstract of DOI:10.1016/j.bmc.2020.115859

A series of meta-substituted acetophenone derivatives, encompassing N-(alkyloxycarbonyl)thiophene sulfonamide fragments have been synthesized. Several selective AT2 receptor ligands were identified, among those a tert-butylimidazole derivative (20) with a

Full text of DOI:10.1016/j.bmc.2020.115859

Journal Pre-proofs
N-(Methyloxycarbonyl)thiophene sulfonamides as high affinity AT2 receptor
ligands
Johan Wannberg, Johan Gising, Jens Lindman, Jessica Salander, Hugo
Gutiérrez-de-Terán, Hanin Ablahad, Selin Hamid, Alfhild Grönbladh, Iresha
Spizzo, Tracey A Gaspari, Robert E Widdop, Anders Hallberg, Maria
Backlund, Anna Leśniak, Mathias Hallberg, Mats Larhed
PII:
S0968-0896(20)30689-1
https://doi.org/10.1016/j.bmc.2020.115859
BMC 115859
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
16 September 2020
29 October 2020
31 October 2020
Please cite this article as: J. Wannberg, J. Gising, J. Lindman, J. Salander, H. Gutiérrez-de-Terán, H. Ablahad, S.
Hamid, A. Grönbladh, I. Spizzo, T.A. Gaspari, R.E. Widdop, A. Hallberg, M. Backlund, A. Leśniak, M.
Hallberg, M. Larhed, N-(Methyloxycarbonyl)thiophene sulfonamides as high affinity AT2 receptor ligands,
Bioorganic & Medicinal Chemistry (2020), doi: https://doi.org/10.1016/j.bmc.2020.115859
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N-(Methyloxycarbonyl)thiophene sulfonamides as high
affinity AT2 receptor ligands
Johan Wannberg1, Johan Gising2, Jens Lindman2, Jessica Salander3, Hugo Gutiérrez-de-Terán3, Hanin Ablahad4,5, Selin
Hamid4,5, Alfhild Grönbladh4, Iresha Spizzo5, Tracey A Gaspari5, Robert E Widdop5, Anders Hallberg6, Maria Backlund7, Anna
Leśniak8, Mathias Hallberg4, Mats Larhed1*
1 Department of Medicinal Chemistry, Science for Life Laboratory, BMC, Uppsala University, Box 574, SE-751 23 Uppsala, Sweden
2 The Beijer Laboratory, Department of Medicinal Chemistry, Uppsala University, BMC, Box 591, 751 24 Uppsala, Sweden
3 Department of Cell and Molecular Biology, BMC, Box 596, Uppsala University, SE-751 24, Uppsala, Sweden
4 The Beijer Laboratory, Department of Pharmaceutical Biosciences, Uppsala University, BMC, Box 591, 751 24 Uppsala, Sweden
5 Department of Pharmacology and Biomedicine Discovery Institute, Monash University Clayton 3800 VIC Australia
6 Department of Medicinal Chemistry, Uppsala University, BMC, Box 574, 751 23 Uppsala, Sweden
7 Department of Pharmacy, Uppsala University, Uppsala, Sweden and Uppsala University Drug Optimization and Pharmaceutical Profiling Platform (UDOPP),
Science for Life Laboratory, Uppsala, Sweden
8 Department of Pharmacodynamics, Centre for Preclinical Research and Technology, Medical University of Warsaw, Banacha 1B Str., 02-097 Warsaw, Poland
R1
N
N
R2
R3
N
N
O
O
O
O O
S
O
O O
S
Me
R4
N
O
H
S
20
N
O
H
S
Generic structures of
new AT2R ligands
KiAT2R = 9.3 nM
t (HLM) = 62 min
½
t (MLM) = 16 min
½
t (HHep) = 194 min
½
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com
N-(Methyloxycarbonyl)thiophene sulfonamides as high affinity AT2 receptor ligands
Johan Wannberg1, Johan Gising2, Jens Lindman2, Jessica Salander3, Hugo Gutiérrez-de-Terán3, Hanin
Ablahad4,5, Selin Hamid4,5, Alfhild Grönbladh4, Iresha Spizzo5, Tracey A Gaspari5, Robert E Widdop5,
Anders Hallberg6, Maria Backlund7, Anna Leśniak8, Mathias Hallberg4, Mats Larhed1*
1 Department of Medicinal Chemistry, Science for Life Laboratory, BMC, Uppsala University, Box 574, SE-751 23 Uppsala, Sweden
2 The Beijer Laboratory, Department of Medicinal Chemistry, Uppsala University, BMC, Box 591, 751 24 Uppsala, Sweden
3 Department of Cell and Molecular Biology, BMC, Box 596, Uppsala University, SE-751 24, Uppsala, Sweden
4 The Beijer Laboratory, Department of Pharmaceutical Biosciences, Uppsala University, BMC, Box 591, 751 24 Uppsala, Sweden
5 Department of Pharmacology and Biomedicine Discovery Institute, Monash University Clayton 3800 VIC Australia
6 Department of Medicinal Chemistry, Uppsala University, BMC, Box 574, 751 23 Uppsala, Sweden
7 Department of Pharmacy, Uppsala University, Uppsala, Sweden and Uppsala University Drug Optimization and Pharmaceutical Profiling Platform
(UDOPP), Science for Life Laboratory, Uppsala, Sweden
8 Department of Pharmacodynamics, Centre for Preclinical Research and Technology, Medical University of Warsaw, Banacha 1B Str., 02-097 Warsaw, Poland
ARTICLE INFO
ABSTRACT
Article history:
Received
A
series
of
meta-substituted
acetophenone
derivatives,
encompassing
N-
(alkyloxycarbonyl)thiophene sulfonamide fragments have been synthesized. Several selective
Received in revised form
Accepted
AT2 receptor ligands were identified, among those a tert-butylimidazole derivative (20) with a
Ki of 9.3 nM, that demonstrates a high stability in human liver microsomes (t = 62 min) and in
½
Available online
human hepatocytes (t = 194 min). This methyloxycarbonylthiophene sulfonamide is a 20-fold
½
more potent binder to the AT2 receptor and is considerably more stable in human liver
microsomes, than a previously reported and broadly studied structurally related AT2R prototype
antagonist 3 (C38). Ligand 20 acts as an AT2R agonist and caused an AT2R mediated
concentration-dependent vasorelaxation of pre-contracted mouse aorta. Furthermore, in contrast
to imidazole derivative C38, the tert-butylimidazole derivative 20 is a poor inhibitor of
CYP3A4, CYP2D6 and CYP2C9. It is demonstrated herein that smaller alkyloxycarbonyl
groups make the ligands in this series of AT2 selective compounds less prone to degradation and
that a high AT2 receptor affinity can be retained after truncation of the alkyloxycarbonyl group.
Binding modes of the most potent AT2R ligands were explored by docking calculations
combined with molecular dynamics simulations.
Keywords:
Angiotensin II type 2 receptor
AT2R ligands
Sulfonyl carbamates
Liver microsomes
Carboxylic acid bioisosteres
20xx Elsevier Ltd. All rights reserved.
1. Introduction
attracted most interest and has now emerged as a new promising
target for drugs5,7–11 This receptor, in contrast to AT1R is
Angiotensin II (Ang II) is an important component of the
renin−angiotensin system (RAS). The octapeptide acts mainly
through the activation of two receptor subtypes; the AT1 receptor
(AT1R) and the AT2 receptor (AT2R). Stimulation of AT1R by Ang
II has a major impact on blood pressure regulation and the
fluid/electrolyte balance. Hence, suppression of the formation of Ang
II from angiotensinogen by inhibitors of the metalloprotease
angiotensin converting enzyme (ACE) or of the aspartyl protease
renin or alternatively, blockade of AT1R by angiotensin receptor
antagonists (ARBs) i.e. the sartans are major established strategies to
control blood pressure in hypertensive patients. The first ACE
inhibitor captopril,1,2 the first renin inhibitor aliskiren,3 and the first
ARB losartan 14,5 were introduced onto the market in 1978, 2007 and
1995, respectively.
sparsely expressed in healthy adults but is strongly up-regulated
after tissue damage12–14 such as neuronal injury15 and vascular
injury16 and following myocardial infarction17–19 and brain
ischemia20,21
The receptor very often mediates opposite actions to those
resulting from AT1R stimulation.22,23 In recent years a large
number of selective and potent AT2R agonists have been
reported.9,24–26 The first small-molecule AT2R agonist 2 (C21)
that was discovered by Hallberg’s group in our laboratories27 has
been studied extensively and demonstrated beneficial effects in a
series of experimental models.24,25 C21 is now in clinical trials
aimed for idiopathic pulmonary fibrosis. Recently, it was
observed that a migration of the methylene imidazole group from
the para to meta position converted the AT2R agonist 2 into an
AT2R antagonist, 3 (C38).28 One AT2R antagonist, the carboxylic
In recent years, several potential drug targets in RAS have
been assessed in detail6 and among those the AT2 receptor has

acid derivative 4 (EMA401, olodanrigan) reached Phase II
clinical trials but these trials were terminated in 2019 due to
safety reasons. EMA401 that was synthesized more than twenty
years ago by Parke-Davis, is structurally related to the old
prototype AT2R antagonists PD-123319 (EMA200) and PD-
synthesis, the binding affinities at the AT2 receptor and the
stability in liver human and mouse microsomes and of a series of
acetophenone
derivatives;
the
meta-substituted
imidazolylacetylphenyl N-alkyloxycarbonyl sulfonamides 7-23
and the N-acetylsulfonamide 24 (Scheme 2 and 3). High affinity,
receptor selective ligands exhibiting favorable in vitro ADME
profiles were identified.
126055 (EMA400) and has demonstrated
a good oral
bioavailability (33%) in rat.29 The antagonist 4, which is the S-
enantiomer of EMA400, has a very different chemical structure
as compared to 3 was aimed for the management of neuropathic
pain,30–32 hence both agonists and antagonists to AT2 receptor
have been examined as potential new pharmaceutical agents
(Figure 1).
2. Chemistry
The carboxylic acid derivative 5 was prepared by a
lithiation/borylation/esterification/Suzuki coupling sequence
where 5-isobutylthiophene-2-carboxylic acid was converted to
the 3-boronic acid-2-ethyl ester and then coupled to 1-(3-
bromobenzyl)-1H-imidazole to give 5 after ester hydrolysis. The
tetrazole compound 6 was prepared by Suzuki coupling of (2-
(tert-butylcarbamoyl)-5-isobutylthiophen-3-yl)boronic acid33 and
1-(3-bromobenzyl)-1H-imidazole followed by removal of the tBu
group and dehydration of the primary amide to the nitrile and
finally cycloaddition with sodium azide as shown in Scheme 1.
N
N
N
N
Cl
OH
N
O
O O
S
N
N
HO
OH
N
H
N
O
B
H
O
S
O
1. n-BuLi, B(iPr)3, THF
S
OH
1
2
C21
Losartan
2. SOCl2, EtOH
S
O
N
N
N
N
O
O
COOH
O
N
Br
1. Pd(dppf)Cl2, K2CO3
DME, H2O, 110 o
2. NaOH, EtOH, H2O
N
N
O
O
O O
S
C
S
OH
N
O
H
S
5
3
C38
4
EMA401
(Olodanrigan)
R1
N
R2
R3
N
N
N
HO
N
OH
Br
Pd(dppf)Cl2
K2CO3
B
N
O
O
O
S
NH
O
O O
S
S
NH
DME, H2O
120 oC, 1 h
R4
N
O
H
S
Generic structures of
new AT2R ligands
N
1. TFA, 70 oC, o.n.
2. TFAA, THF, pyridine
3. NaN3, NH4Cl, DMF
170 oC, 15 min
N
Figure 1. Ligands interacting with the AT1 receptor exemplified by the
antagonist losartan (1) and with the AT2 receptor exemplified by the agonist
C21 (2), the antagonists C38 (3) and Olodanrigan (4).
N
N
N
S
N
H
6
While comprehensive SAR has been established for AT2R
Scheme 1. Synthesis of the carboxylic acid and tetrazole
27,33–38
agonists related to 2 (C21),
very limited information is
analogs of C38
available on the impact of modifications of the AT2R antagonist
3 on binding affinities at the AT2R and on the metabolic stability
of 3 and analogous compounds thereof, neither from in vivo nor
in vitro experiments.28,39–41 It was previously shown by our
laboratories that 3 (C38) and other structurally related meta-
substituted compounds examined, act as AT2R antagonists in a
neurite outgrowth assay. Thus, pretreatment of NG108-15 cells
with the AT2R antagonists reduced C21 (2)-induced and Ang II-
induced neurite outgrowth.28,42 More recently, we reported, that
3 (C38) acts as a partial agonist in an assay relying on iNOS-
derived NO release in M1 phenotypic macrophages.43
The
MIDA
boronate,
2-(N-(tert-butyl)sulfamoyl)-5-
isobutylthiophen-3-yl)boronic acid MIDA ester, was prepared as
previously described39. The synthesis of butylsulfonylcarbamates,
7-19 was initiated by N-alkylation of the appropriate imidazoles
and benzimidazoles with 2,3′-dibromoacetophenone to deliver
the alkylated intermediates in moderate to high yields (Scheme
2). These were then applied to Suzuki couplings with the MIDA
boronate to deliver thienylphenyl intermediates. The compounds
were further treated with TFA to deprotect the N-tert-butyl
sulfonamide function and subsequently the primary sulfonamides
were reacted with butyl chloroformate to furnish the desired
We herein report two bioisosters of 3, the carboxylic acid 5
and the tetrazole derivative 6. Furthermore, we report the

compounds 7-19 in 3-58% yields over the last three steps
(Scheme 2).
N
O
B
O
O
O O
R
O
N
N
N
N
S
R =
NH
Br
R
O
S
N
N
8
N
N
7
9
10
Heterocycle
O
O
O O
S
K2CO3
DMF or MeCN
80-100 ºC, 1-12 h
PdCl2dppf (5 mol%)
K2CO3, DME, H2O
120 oC, 1 h
N
N
N
N
H
S
Br
Br
N
N
N
12
13
11
R
R
N
N
N
Cl
Br
O
O
N
O
N
N
15
Cl
OBu
TFA
16
14
O
O O
Et3N or DIEA
with or without DMAP
DCM, rt. 1-2 h
rt. on.
O O
S
S
N
N
N
N
OBu
NH2
H
S
N
N
N
S
17
18
19
7-19
four-step yields of 1.4-48%
Scheme 2. Synthesis of the compounds 7-19.
membrane assay using the peptide ligand sarile, that like Ang II
acts as a AT2R agonist.47
The primary sulfonamide containing the 2-tert-
butylimidazolyl moiety was treated with methyl chloroformate at
room temperature to give ligand 20 in 39% yield over three steps
Furthermore, we previously reported that a reduced ketone
function of compound 7 resulted in compound exhibiting a 5-fold
lower affinity, suggesting an importing role of the carbonyl
oxygen.25 We knew from studies of the C21 AT2R agonist series
that substituents at the imidazole ring system tended to limit the
interaction with CYP450 enzymes.35 This information prompted
us therefore to attach various alkyl substituents of different size
at the 2-position of the imidazole ring moiety.
and
a
primary
sulfonamide
containing
the
2-
cyclopropylimidazolyl moiety was treated with methyl- and ethyl
chloroformate to give the corresponding carbamates 21 and 22
(Scheme 3). Analogous to
a
previously described
methodology,39,44 compound 13 was heated in ethanol at 100 °C
for 30 min in a closed vessel to give 23 in 49% yield. Finally a
primary sulfonamide was treated with acetyl chloride to give the
acyl sulfonamide 24 in 57% yield.
3. Biological evaluation
In the present report, a binding assay using membrane
preparations from HEK-293 cells expressing human AT2R
(HEK293-hAT2R) and that relies on displacement of the non-
selective AT2R/AT1R ligand sarile ([125I][Sar1,Ile8]-angiotensin
II) using a seven-point dose-response curve in duplicate
measurements at each concentration was applied (Eurofins Cerep
SA, France). The imidazole compound 3 resulted in a Ki value of
270 nM39 Prior to initiating a more extensive medicinal chemistry
program we felt prompted to first assess two alternative and more
common acidic functions as alternatives to the butoxy sulfonyl
carbamate group of 3. However, neither the carboylic acid 5 nor
the tetrazole derivatives 6 displayed any notable affinity to
AT2R.
The prototype AT2R antagonist compound 3 was previously
reported to exhibit a Ki value of 19 nM and the acetophenone
derivative 7 comprising an imidazole heterocycle attached by a
methylene bridge to the meta position, a Ki of 22 nM.28 These
affinity data were obtained from a radioligand assay built on
displacement of [125I]Ang II from AT2 receptors in membrane
preparations from pig uterus myometrium.45,46 In the HEK293-
hAT2R assay applied in this report the acetophenone derivative 7
exhibited a Ki of 280 nM (Figure 2). Thus, an approximately 10-
fold lower affinity was encountered in the HEK293-hAT2R

R
Scheme 3. Synthesis of compounds not containing the butyl
sulfonyl carbamate group
R
N
N
N
N
O
Figure 2. Binding affinities (Ki) to the AT2 receptor (human HEK-293 cells)
Me/Et
and stability (t in minutes) in human liver microsomes (HLM) and mouse liver
½
Cl
O
O
O
microsomes (MLM) of the butyloxycarbonyl sulfonamides 3, 7, 9-13.
DIEA, DMAP
DCM, rt. 1.5-15 h
O
O O
S
O O
S
R2
N
Br
Cl
N
O
N
N
N
NH2
H
S
N
S
N
N
N
N
20, R = tBu, R2 = Me 39%
21, R = cyPr, R2 = Me 56%
22, R = cyPr, R2 = Et 49%
O
O
O
O
O
O
O
O
O O
S
O O
S
O
O O
S
O O
S
Bu
Bu
Bu
Bu
N
O
N
O
N
O
N
O
H
H
H
S
S
S
H
S
N
N
16
HLM = 12 min
MLM = 5.5 min
14
15
8
N
H
HLM = 7.1 min
MLM = 6.4 min
HLM= 6.7 min
MLM = 4.7 min
HLM = 7.8 min
MLM = 3.9 min
M
O
O
EtOH
Figure 3. The stability of the butyloxycarbonyl sulfonamides 8, 14-19 (t½ in
minutes) in human liver microsomes (HLM) and in mouse liver microsomes
(MLM).
100 ºC, 30 min
O
O
O O
S
O O
S
Bu
Et
N
O
N
O
H
H
S
S
23, 49%
13
seems obvious that considering the fast metabolism also in
mouse microsomes (MLM t½ < 6 min) that the compounds e.g.
12 and 13 are not suitable for experiments using mice in pain
models.
N
N
N
N
O
A series of ligands were synthesized to probe whether the
stability in microsomes could be improved with the
butyloxycarbonyl functionality intact but by replacing the
imidazole group with other functionalities. Neither of the
Cl
Me
O
O
DIEA, DCM
rt. 90 min
O
O O
S
O O
S
N
Me
compounds 8, 14-19, with
a possible exception of 19
NH2
H
S
S
demonstrated any significantly improved stability in human or
mouse liver microsomes (Figure 3).
24, 57%
As demonstrated in Figure 2 a significant improvement of the
binding affinity to the AT2 receptor could be achieved. Small
bulky groups like an isopropyl or a tert-butyl group in the 2-
position rendered the highest affinities. Compound 12 exhibited a
Ki value of 17 nM and compound 13 a Ki of 6.6 nM. Hence a 50-
As deduced from SAR studies of a series of AT2R agonists,
the n-butyl group seemed optimal for binding to the activated
form of the receptor.48 However, regarding AT2R antagonists,
structural variations of the lipophilic side chain was tolerated by
N
N
N
N
N
N
N
N
N
N
N
N
N
N
O
O
O
O
O
O
O
O
O
O O
S
O
O O
S
O O
S
O
O O
S
O O
S
O
O
O O
S
O O
S
Bu
Bu
Bu
Bu
Bu
Bu
Bu
N
O
N
O
N
O
N
O
N
O
N
O
N
O
H
H
H
S
S
S
H
H
S
S
H
H
S
S
3
7
9
10
11
13
12
KiAT2R = 270 nM
HLM = 12 min
MLM = 70 min
KiAT2R = 280 nM
HLM = 30 min
MLM > 334 min
KiAT2R = 48 nM
HLM = 5.2 min
MLM = 6.7 min
KiAT2R = 26 nM
HLM = 5.3 min
MLM = 5.6 min
KiAT2R = 39 nM
HLM = 5.5 min
MLM = 5.6 min
KiAT2R = 6.6 nM
HLM = 5.3 min
MLM = 5.2 min
KiAT2R = 17 nM
HLM = 6.3 min
MLM = 4.0 min
fold improvement of the affinity to the AT2 receptor could be
accomplished by this maneuver.
the receptor.39 Furthermore, the methoxycarbonyl analogue of 3
(C38) was reported to be considerably less prone to
decomposition in both mouse and human microsome assays
(MLM = 220 min; HLM = 77 min).39 As a consequence of these
findings we decided to retain the tert-butyl group attached at the
imidazole heterocycle to hopefully maintain a high affinity to
AT2R whilst reducing lipophilicity of the ligands by shorten the
n-alkyloxycarbonyl chain. A combination of a tert-butyl group
attached to the imidazole heterocycle of the parent acetophenone
derivative 7 with a Ki of 280 nM and a methyloxycarbonyl rather
than a butyloxycarbonyl group rendered 20 with a Ki of 9.3 nM.
An ethoxycarbonyl instead of a methyloxycarbonyl group
provided ligand 23 exhibiting a Ki of 7.6 nM (Figure 4). A
significant improvement of the metabolic stability was observed
for the methyloxycarbonyl group (20) compared to the
ethyloxycarbonyl (23). Ligand 20 exhibited a stability in the
Regarding stability of the compounds in liver microsomes,
whilst 4 (EMA401) and the AT2R agonist 2 (C21), in the assay
we applied exhibited good to fair in vitro stability in human liver
microsomes, t = 38 min and 22 min, respectively, the AT2R
½
antagonist 3 that is structurally related to 2, demonstrated a
considerably lower stability, t = 12 min.39 More encouraging, as
½
described herein the acetophenone derivative 7 that is essentially
equipotent to 3 regarding AT2R affinity displayed a substantial
improvement regarding the stability in human liver microsomes
(HLM) with a half-life of 30 min. Unfortunately, the 15-fold
more potent compound 12 encompassing the acetophenone
linker, suffers from very poor in vitro metabolic stability (HLM
t ≤ 6.3 min). Similarly, the tert-butyl derivative 13 that is a 40
½
times more potent than 7 as an AT2R binder decomposes fast in
HLM assay (t = 62 min) that was superior to what had been
½
the human liver microsome assay (HLM t ≤ 5.3 min). Thus, it
½
monitored with any other compound in the series and a high

stability in human hepatocytes (t = 194 min). Noteworthy, the
Figure 4. Binding affinities at the AT2 receptor (Ki) of human HEK-293
½
metabolic stability of 20 in liver microsomes from mice was
cells and stability (t in minutes) in human liver microsomes (HLM) and
½
much poorer (t = 16 min). A displacement of the tert-butyl
mouse liver microsomes (MLM) of the ethyloxycarbonyl and
½
group of the potent ethyloxycarbonyl compound 23 for a
cyclopropyl group resulted in 22 that was a significantly poorer
binder to AT2R than 23, although the metabolic stability of the
compound in the HLM assay (22: t = 36 min versus for 23: t =
methyloxycarbonyl sulfonamides 20-23 and of the acetylsulfonamide 24.
The following data were recorded; CYP3A4; IC50 ~120 nM,
CYP2D6; IC50 2 µM and remarkably CYP2C9; IC50 < 100 nM.
½
½
21 min) and in human hepatocytes (22: t = 79 min versus for 23:
½
t = 43 min) was improved. The cyclopropyl methyloxycarbonyl
½
sulfonamide 21 was the most inert compound in the HLM assay
of all derivatives assessed (t = 114 min). Furthermore, this
½
cyclopropyl compound (21) is also the most stable compound in
human hepatocytes, with a t of 280 min. The acetylsulfonamide
½
24 exhibited a more than 15-fold higher Ki value than the
methyloxycarbonyl derivative 20 and in addition, it exhibited a
lower stability in the HLM assay and in human hepatocytes t =
½
36 min and 40 min, respectively. However, the
acetylsulfonamide 24 is still more metabolically stable than 3 and
exhibits a considerably better Ki value than both 3 and the parent
acetophenone compound 7. (Figure 4).
The potent methoxycarbonyl derivative 20 neither inhibits
CYP3A4 (IC50 ~50 µM), CYP2D6 (IC50 > 50 µM) nor CYP2C9
(IC50 > 15 µM) significantly. This is in sharp contrast to 2 (C21),
which comprises a unsubstituted imidazole heterocycle as a
characteristic element and with a HLM half-life of 22 min.
Imidazole derivatives like 2 that are high affinity AT2R agonists
are often associated with a pronounced capacity to inhibit CYP
enzymes.35 The ability of the prototype AT2R antagonist 3 (C38)
to inhibit important CYP isoenzymes was therefore examined.
This
imidaz
ole
compo
und,
exhibit
N
N
N
N
O
O
ing
Ki
value
of 280
nM at
the
a
O
O
O O
S
O O
S
Me
Et
N
O
N
O
H
H
S
S
20
23
KiAT2R = 9.3 nM
HLM = 62 min
MLM = 16 min
KiAT2R = 7.6 nM
HLM = 21 min
MLM = 11 min
AT2
recepto
r
was
Figure 5. a) Concentration-dependant vasorelaxation evoked by 2 (C21)
and Ligand 20 in pre-contracted mouse aorta (top). b) Concentration-
dependant vasorelaxation evoked by C21 in pre-contracted mouse aorta in the
presence of Ligand 20 (bottom).
indeed
found
to act
as an
excepti
onally
potent
inhibit
or of
the
N
N
N
N
O
O
However, the imidazole derivative compound 3 was a somewhat
less efficient inhibitor of CYP2B6 and demonstrated an IC50 ~20
µM to be compared to IC50 > 50 µM) obtained with ligand 20. A
possible explanation of the lower CYP affinities could be that the
bulky tert-butyl group attached to the imidazole ring of ligand 20
prevents a productive coordination with the iron atom of this
class of oxidative enzymes.
O
O
O O
S
O O
Me
S
Et
N
O
N
O
H
H
S
S
21
22
three
KiAT2R = 110 nM
HLM = 114 min
MLM = 58 min
KiAT2R = 53 nM
HLM = 36 min
MLM = 35 min
commo
n drug-
metabo
lizing
CYP
enzym
es
assesse
d.
The apparent permeability (Papp) across human intestinal
epithelial Caco-2 cell monolayer of the AT2R antagonist 3 (C38)
and the 20 fold more efficient AT2R binder 20 were compared.
Regarding the monolayer permeability of the ligands 3 and 19,
apical to basolateral apparent permeabilities of 3.4 x 10-6 cm/s
and 0.4 x 10-6 cm/s, respectively, were observed. Furthermore,
efflux ratios of 7 for compound 3 and 76 for 20 were
encountered. Thus, a moderate permeability is expected for both
compounds and, due to efflux, ligand 20 is predicted to be less
N
N
O
O
O O
S
N
Me
H
S
24
KiAT2R = 170 nM
HLM = 36 min
MLM = 72 min

easily absorbed than 3 from the human small intestine mucosa as
deduced from this in vitro model. These data prompted us to
examine the clinical candidate 4, which we have previously re-
synthesized in our lab,49 with an AT2R Ki of 5.6 nM under our
assay conditions, regarding both stability in microsomes and
apparent permeability under experimental conditions identical to
Figure 5. The introduction of the carbonyl group of the ketone
function of 20 significantly improves the affinity of the ligands as
compared to 3 (C38). Furthermore, it is notable that the butoxy
group of metabolically unstable compound 13 and the methyloxy
group of the more stable 20 are both accommodated well in the
receptor which is reflected in their similar affinities.
those applied herein. The stability in HLM; t = 38 min, in MLM;
½
t = 90 min and in human hepatocytes t = 33 min. Furthermore,
½
½
an apparent permeability of 1.8 x 10-6 cm/s and efflux ratio of 32
were encountered. Thus, we were somewhat surprised to find that
4 (EMA401) with a relatively low apparent permeability in our in
vitro absorption model exhibits a good oral bioavalability.32
Ligand 20 exhibited a) a high affinity to AT2R (Ki = 9.3 nM),
b) did not significantly inhibit the CYP enzymes assessed and c)
demonstrated a good stability in the human liver microsome
(HLM) assay and was therefore selected for functional studies.
A brief preliminary study employing the spared nerve injury
(SNI) model in mice was undertaken and aimed to assess whether
ligand 20 behaves similar to the prototype AT2R antagonist PD-
123319 or the AT2R antagonist 4 (EMA401), the latter
extensively studied in animal models of neuropathic pain.30–32
The results obtained suggested that ligand 20, contrary to PD-
123319 did not exert any positive impact in the SNI model (data
not shown). This observation prompted us to utilize a
complementary model to allow us to determine if ligand 20 might
act as an agonist rather than an antagonist at the AT2 receptor.
Isolated aortic preparations from mice were previously used to
determine the vascular effects of the selective AT2R agonist 2
(C21).50 Compound C21 evoked relaxation in mouse aorta that
was blocked by the nitric oxide synthase inhibitor L-NAME,
suggesting that the effect of 2 (C21) was mediated by nitric
oxide.50 We report that ligand 20 presented here similarly to 2
(C21) caused a concentration-dependent vasorelaxation of pre-
contracted mouse aorta (Figure 5a). In separate experiments, pre-
incubation of ligand 20 at 1 and 10 µM did not inhibit 2 (C21)-
induced vasorelaxation (Figure 5b).
Modelling
After independent docking explorations in GLIDE, a common
binding pose was identified for the series of compounds, which
in each case was refined by MD equilibration. Figure 6 depicts
the proposed binding mode for the very potent compound (20)
that is compared to binding mode of 3 (C38). A model of the
binding mode of the prototype AT2R antagonist 3 when binding
to the receptor as deduced from docking redefined by molecular
dynamic simulations was recently suggested.40 The sulfonyl
carbamate moiety of the high affinity ligand 20 is anchored via
salt-bridge interactions with R1824.64 and K2155.42 and a hydrogen
bond of the carbonyl with T1253.33 (the Ballesteros-Weinstein
generic amino acid numbering scheme is indicated as
superscript51). The phenyl ring of ligand 20 is lining towards
W1002.60 and L1243.32
substituent to accommodate within a hydrophobic cluster
composed by residues Y511.39, Y1032.64, Y1042.65, Y1082.69
, allowing the tert-butyl imidazole
,
Figure 6. Compound 3 (C38) (top) and ligand 20 (bottom)
P3017.36, and I3047.39. The side chain carbonyl group that is
essential for high affinity to AT2R is bridging to R1824.64 and
Y1032.64 via a water molecule as clearly demonstrated in Figure
6. The isobutyl group attached to the thiophene ring is placed in a
deeper region of the transmembrane cavity, defined by residues
L1243.32, M1273.35, W2696.48, F2726.51, and F3087.43. Finally, the
methyloxy part on the sulfonyl carbamate is located between
transmembrane helices TM3-TM5 defined by the residues
Ile2115.38, Lys2155.42, and F1293.37. The binding mode proposed
explains to a big extent the SAR for this series, as depicted in
4. Conclusion
In summary, we have identified potent AT2 receptor selective
ligands, among those the tert-butylimidazole derivative 20
(AT2R; Ki = 9.3 nM, AT1R; Ki > 3000 nM) that exhibits a high
stability in human liver microsomes (t = 62 min) and in human
½
hepatocytes (t = 194 min). Hence, this AT2R ligand is a 20-fold
½
more potent binder to the receptor than the selective AT2R
prototype antagonist 3 and is considerably more stable in human
liver microsomes. Furthermore, it is shown that 20 is a very poor

inhibitor of the drug-metabolizing enzymes CYP3A4, CYP2D6
and CYP2C9 as compared to 3 (C38) that is an efficient inhibitor
of these oxidative enzymes. The tert-butoxy derivative 20 is
approximately equipotent to the former clinical candidate 4
(EMA401) regarding AT2R affinity and exhibits a superior
stability in human liver microsomes and hepatocytes but
demonstrates a mediocre apparent permeability (0.4 x 10-6 cm/s).
The Papp of 4 is similarly relatively moderate (1.8 x 10-6 cm/s) in
the in vitro model applied but 4 is nevertheless absorbed from the
intestine and exhibits an oral bioavailability of 33%. It is
demonstrated herein that the presence of an alkoxy chain that is
strongly preferred in AT2R agonists, e.g. 2, is not a requirement
in this series of ligands. Docking calculations combined with
molecular dynamics simulations allowed proposals of the
tentative binding modes of the most potent AT2R ligands. In
functional studies, it was also shown that ligand 20 acts as an
AT2R agonist and caused an AT2R mediated concentration-
dependent vasorelaxation of pre-contracted mouse aorta, while it
did not appear to exhibit AT2R antagonist properties, at least up
to 10 µM.
mmol), DME (10 mL) and water (5 mL) was heated overnight at
110 °C in a sealed vial purged with nitrogen. The reaction
mixture was diluted with 3 mL EtOAc and the organic layer
collected, dried with MgSO4, filtered and evaporated. The crude
product was purified by preparative RP-HPLC (5 – 100% MeCN
in water (0.05% formic acid)) to give ethyl 3-(3-((1H-imidazol-1-
yl)methyl)phenyl)-5-isobutylthiophene-2-carboxylate in 44%
1
yield (436 mg). H NMR (400 MHz, Chloroform-d) δ 7.63 (s,
1H), 7.45 – 7.33 (m, 2H), 7.29 – 7.24 (m, 1H), 7.17 – 7.08 (m,
2H), 7.00 – 6.93 (m, 1H), 6.75 – 6.69 (m, 1H), 5.15 (s, 2H), 4.19
(q, J = 7.1 Hz, 2H), 2.67 (dd, J = 7.1, 0.8 Hz, 2H), 2.01 – 1.87
(m, 1H), 1.23 (t, J = 7.1 Hz, 3H), 0.98 (d, J = 6.6 Hz, 6H). 13C
NMR (101 MHz, Chloroform-d) δ 161.9, 150.1, 147.6, 137.3,
136.9, 135.4, 129.7, 129.4, 129.3, 128.4, 128.3, 126.7, 125.0,
119.5, 60.8, 50.9, 39.5, 30.6, 22.3, 14.2. MS (ESI): m/z calc’d for
C21H24N2O2S: 369.1637 [M+H]+ ; found: 369.1627.
To
ethyl
3-(3-((1H-imidazol-1-yl)methyl)phenyl)-5-
isobutylthiophene-2-carboxylate (48.2 mg; 0.131 mmol) was
added NaOH (15.7 mg; 0.392 mmol), EtOH (2 mL) and water
(0.2 mL). The mixture was microwave heated at 70 °C for 1 h.
The reaction mixture was neutralized with HCl (2 M) and the
solvent evaporated. The residue was dissolved in acetonitrile and
the mixture filtered to remove residual sodium chloride. The
5. Experimental section
5.1. Chemistry
1
solvent was evaporated to give 5 in 85% yield (37.9 mg). H
All solvents and chemicals were used as purchased without
further purification. Microwave heating was performed in a
Biotage single-mode microwave reactor producing controlled
irradiation at 2450 MHz with a power of 0–400 W. The reaction
temperature was determined and controlled using the built-in
online IR-sensor. Microwave mediated reactions were performed
in septum sealed Biotage vials. Analytical TLC was performed
on silica gel 60 F-254 plates and visualized with UV light (λ =
254 nm). Automated flash column chromatography was
performed on Biotage Isolera or Grace Reveleris X2 instruments
using commercial silica cartridges. Analytical HPLC/ESI-MS
was performed using UV detection (214, 254 and 280 nm) and
electrospray ionization (ESI) MS on a C18 column (50x3.0 mm,
2.6 µm particle size, 100 Å pore size) with gradients of
acetonitrile in 0.05% aqueous HCOOH as mobile phase at a flow
rate of 1.5 mL/min. High resolution molecular masses (HRMS)
were determined on a mass spectrometer equipped with an ESI
source and 7-T hybrid linear ion trap (LTQ). Nuclear magnetic
NMR (400 MHz, Methanol-d4) δ 8.42 (s, 1H), 8.05 (s, 1H), 7.47
– 7.40 (m, 2H), 7.39 – 7.32 (m, 1H), 7.27 (s, 1H), 7.24 – 7.18 (m,
1H), 7.10 (s, 1H), 6.85 – 6.76 (m, 1H), 5.27 (s, 2H), 2.69 (dd, J =
7.1, 0.8 Hz, 2H), 2.00 – 1.86 (m, 1H), 0.98 (d, J = 6.6 Hz, 6H).
13C NMR (101 MHz, Methanol-d4) δ 168.6, 167.2, 149.4, 146.8,
138.7, 138.1, 136.7, 130.5, 130.4, 130.2, 129.5, 127.8, 127.2,
121.6, 52.2, 40.3, 31.8, 22.6. MS (ESI): m/z calc’d for
C19H20N2O2S: 341.1324 [M+H]+ ; found: 341.1318.
5.1.2. 5-(3-(3-((1H-imidazol-1-yl)methyl)phenyl)-5-
isobutylthiophen-2-yl)-1H-tetrazole (6)
N-tert-butyl-5-isobutyl-thiophene-2-carboxamide33 (500 mg;
2.09 mmol) was dissolved in dry THF (50 mL) and cooled to -78
°C. n-BuLi (2.5 M in hexanes; 2.1 mL; 5.2 mmol) was added
slowly under a N2 atmosphere. The mixture was stirred at -15 °C
for 4 h. The solution was cooled to -78 °C and triisopropyl borate
(0.725 mL; 3.14 mmol) was added slowly. The reaction mixture
was stirred at room temperature overnight. The reaction was
quenched with HCl (2M; 2 mL) and extracted with EtOAc. The
organic layer was dried with MgSO4, filtered and evaporated. To
the residue was added 1-[(3-bromophenyl)methyl]imidazole (353
mg; 1.49 mmol), Pd(dppf)Cl2·DCM (60.8 mg; 74.5 µmol),
K2CO3 (1030 mg; 7.45 mmol), DME (10 mL) and water (5 mL)
and the mixture was heated at 120 °C for 1 h in a sealed vial
under nitrogen. The reaction mixture was diluted with 9 mL
EtOAc and the organic layer dried and evaporated. The product
was isolated through automated silica flash chromatography (5-
10% methanol in dichloromethane) to give 3-(3-((1H-imidazol-1-
yl)methyl)phenyl)-N-(tert-butyl)-5-isobutylthiophene-2-
1
resonance (NMR) spectra were recorded at 400 MHz for H and
101 MHz for 13C. Chemical shifts (δ) are reported in ppm with
the residual solvent peak as internal standard (1H, CDCl3 at 7.26
ppm, 13C, CDCl3 at 77.16 ppm). Coupling constants J are
reported in hertz (Hz). All final compounds were ≥95% pure as
determined by HPLC (UV at 254 nm) and NMR.
5.1.1. 3-(3-((1H-imidazol-1-yl)methyl)phenyl)-5-
isobutylthiophene-2-carboxylic acid (5)
A solution of 5-isobutyl-thiophene-2-carboxylic acid (500 mg;
2.71 mmol) in dry THF (15 mL) was cooled to -78 °C and n-
BuLi (2.5 M in hexanes; 2.7 mL; 6.8 mmol) was added slowly
under nitrogen. The mixture was stirred at -78 °C for 1 h before
triisopropyl borate (0.94 mL; 4.1 mmol) was added slowly. The
reaction mixture was stirred at room temperature overnight. The
reaction was quenched with HCl (2 M; 8 mL) and extracted with
EtOAc. The organic layer was dried and evaporated. The crude
was dissolved in EtOH (6 mL) and SOCl2 (396 μL; 5.43 mmol)
was slowly added at room temperature. The mixture was heated
at 70 °C overnight. The reaction mixture was then evaporated to
carboxamide in 34% yield (200 mg).
3-(3-((1H-imidazol-1-yl)methyl)phenyl)-N-(tert-butyl)-5-
isobutylthiophene-2-carboxamide (371 mg; 0.937 mmol) was
stirred in neat TFA (10 mL) at 70 °C overnight and the reaction
mixture evaporated to dryness. The product was isolated through
automated flash chromatography (0–7% methanol in
dichloromethane) to give the primary amide in 55% yield (176
mg).
dryness
and
the
intermediate
ethyl
ester,
1-[(3-
1H NMR (400 MHz, Chloroform-d) δ 8.80 (s, 1H), 7.53 – 7.44
(m, 2H), 7.41 (s, 1H), 7.37 – 7.27 (m, 2H), 7.16 (s, 1H), 6.73 –
bromophenyl)methyl]imidazole (643 mg; 2.71 mmol),
Pd(dppf)Cl2 (99.3 mg; 0.136 mmol), K2CO3 (1880 mg; 13.6

6.67 (m, 1H), 5.33 (s, 2H), 2.66 (d, J = 7.0 Hz, 2H), 1.98 – 1.85
(m, 1H), 0.97 (d, J = 6.6 Hz, 6H).
separated and the aqueous layer extracted with 3 mL ethyl
acetate. The combined organic layer was dried (Isolute HM-N),
filtered and evaporated. The residue was carried forward to the
next step.
To
3-(3-((1H-imidazol-1-yl)methyl)phenyl)-5-
isobutylthiophene-2-carboxamide (176 mg; 0.517 mmol) in THF
(3 mL) was added pyridine (0.8 mL). The mixture was stirred at
room temperature for 30 min. Trifluoroacetic anhydride (87 µL;
0.62 mmol) was added at 0 °C and the reaction was stirred for 2 h
at room temperature. Ice was added to the solution, which was
extracted with EtOAc. The organic layer was dried with MgSO4,
filtered and evaporated and the residue purified by automated
flash chromatography (0 – 10% MeOH in DCM) to give 3-(3-
((1H-imidazol-1-yl)methyl)phenyl)-5-isobutylthiophene-2-
carbonitrile in 30% yield (50 mg). 1H NMR (400 MHz,
Chloroform-d) δ 7.86 (s, 1H), 7.64 (dd, J = 7.9, 1.6 Hz, 1H), 7.56
– 7.41 (m, 2H), 7.25 – 7.19 (m, 1H), 7.13 (s, 1H), 6.98 – 6.88 (m,
2H), 5.20 (s, 2H), 2.72 (d, J = 6.9 Hz, 2H), 2.01 – 1.88 (m, 1H),
0.98 (d, J = 6.6 Hz, 6H).
Deprotection: To the Suzuki product was added trifluoroacetic
acid (1 mL) and the mixture was stirred at 60 °C for 6 h. The
reaction mixture was co-evaporated with toluene to dryness. The
residue was partitioned between 3 mL dichloromethane and 2 mL
sat NaHCO3 (aq). The organic layer was loaded onto a plug of
silica. The plug was first eluted with dichloromethane
(discarded), then with 10% MeOH in dichloromethane. The
product containing fraction was evaporated and used as is in the
next step.
Coupling with butyl chloroformate: To a solution of the crude
primary sulfonamide in dichloromethane (2 mL) was added first
triethylamine (28 µL, 0.20 mmol) then butyl chloroformate (19
µL, 0.15 mmol) and the reaction mixture was stirred at room
temperature overnight. The reaction mixture was diluted with 2
mL dichloromethane, washed with 1 M HCl (2 mL), dried
(isolute HM-N) and loaded onto a silica plug. The plug was
eluted with 10% methanol in dichloromethane the collected
fraction was evaporated. The residue was purified by preparative
RP-HPLC (30-60% MeCN in water [0.1% TFA]). Product
fractions were freeze-dried to give 5.0 mg (8%, three step) of
To a 5 mL microwave vial was added 3-(3-((1H-imidazol-1-
yl)methyl)phenyl)-5-isobutylthiophene-2-carbonitrile (23.7 mg;
73.7 µmol), NaN3 (62.3 mg; 0.958 mmol), NH4Cl (51.3 mg;
0.959 mmol) and DMF (3 mL). The vial was capped and flushed
with N2. The mixture was microwave heated at 170 °C for 15
minutes. The reaction mixture was diluted with sat. NaHCO3 and
washed with ethyl acetate. The aqueous layer was acidified to pH
< 1 and extracted with chloroform. The organic layer was dried
with MgSO4, filtered and evaporated. The product was purified
by preparative RP-HPLC (C8 column, 10–80% MeCN in water
(0.05% formic acid). Product containing fractions were collected
and the solvent evaporated to give the product in 60% yield (16.0
butyl
((5-isobutyl-3-(3-(2-(2-methyl-1H-imidazol-1-
2,2,2-
yl)acetyl)phenyl)thiophen-2-yl)sulfonyl)carbamate
trifluoroacetate. 1H NMR (400 MHz, CDCl3) δ 8.41 (s, 1H), 8.01
(d, J = 7.8 Hz, 1H), 7.67 (d, J = 7.7 Hz, 1H), 7.57 (t, J = 7.7 Hz,
1H), 7.26 – 7.21 (m, 1H), 7.19 – 7.14 (m, 1H), 6.81 (s, 1H), 5.70
(s, 2H), 4.00 (t, J = 6.7 Hz, 2H), 3.13 (d, J = 7.6 Hz, 1H), 2.73 (d,
J = 7.1 Hz, 2H), 2.55 (s, 3H), 2.02 – 1.88 (m, 1H), 1.55 – 1.42
(m, 2H), 1.36 – 1.17 (m, 2H), 1.00 (d, J = 6.6 Hz, 6H), 0.86 (t, J
1
mg). H NMR (400 MHz, Chloroform-d) δ 8.74 (s, 1H), 7.49 –
7.39 (m, 3H), 7.30 – 7.26 (m, 2H), 7.20 (s, 1H), 6.84 (s, 1H),
5.27 (s, 2H), 2.73 (d, J = 7.0 Hz, 2H), 2.02 – 1.89 (m, 1H), 1.01
(d, J = 6.6 Hz, 6H). 13C NMR (126 MHz, Methanol-d4) δ 155.3,
147.1, 141.4, 137.6, 136.2, 134.1, 130.0, 129.8, 129.7, 128.5,
127.8, 123.5, 122.1, 121.8, 52.78, 39.7, 31.2, 22.6. MS (ESI):
m/z calc’d for C19H20N6S: 365.1548 [M+H]+ ; found:
365.1541.
+
= 7.4 Hz, 3H). MS (ESI): m/z calc’d for C25H32N3O5S2 :
518.1783 [M+H+]; found: 518.1792.
5.1.5. Butyl ((3-(3-(2-(2-ethyl-1H-imidazol-1-
yl)acetyl)phenyl)-5-isobutylthiophen-2-
yl)sulfonyl)carbamate (9)
The synthesis of 9 has been disclosed previously.28
5.1.3. Butyl ((3-(3-(2-(1H-imidazol-1-
yl)acetyl)phenyl)-5-isobutylthiophen-2-
yl)sulfonyl)carbamate (7)
The synthesis of 7 has been disclosed previously.28
5.1.6. Butyl ((5-isobutyl-3-(3-(2-(2-propyl-1H-
imidazol-1-yl)acetyl)phenyl)thiophen-2-
yl)sulfonyl)carbamate (10)
5.1.4. Butyl ((5-isobutyl-3-(3-(2-(2-methyl-1H-
imidazol-1-yl)acetyl)phenyl)thiophen-2-
yl)sulfonyl)carbamate 2,2,2-trifluoroacetate (8)
Alkylation: A mixture of 2-bromo-1-(3-bromophenyl)ethan-1-
one (125 mg, 0.450 mmol), 2-propyl-1H-imidazole (248 mg,
2.25 mmol) in DMF (2 mL) was heated at 80 °C for 2 h. The
reaction mixture was evaporated and the residue purified by
automated silica flash chromatography (1-7.5% methanol in
dichloromethane). Product fractions were concentrated under
reduced pressure and dried under vacuum to give 106 mg (77%
crude) of the alkylated imidazole.
Alkylation: A mixture of 2-bromo-1-(3-bromophenyl)ethan-1-
one (111 mg, 0.400 mmol), 2-methyl-1H-imidazole (164 mg,
2.00 mmol) in DMF (2 mL) was heated at 100 °C overnight. The
reaction mixture was evaporated and the residue purified by
automated silica flash chromatography (1-10% methanol in
dichloromethane). Product fractions were concentrated under
reduced pressure and dried under vacuum to give 50.4 mg (45%
crude) of the alkylated imidazole.
Suzuki coupling: The alkylation product (30.7 mg, 0.100
mmol),
(2-(N-(tert-butyl)sulfamoyl)-5-isobutylthiophen-3-
yl)boronic acid MIDA ester (51.6 mg, 0.12 mmol), K2CO3 (69.1
mg, 0.50 mmol), PdCl2(dppf) (3.7 mg, 5.0 µmol), DME (1.0 mL)
and water (0.20 mL). The vial was flushed with N2, sealed with a
screw-cap and the reaction was heated at 120 °C for 60 min.
Ethyl acetate (3 mL) and water (2 mL) was added to the cooled
reaction mixture and mixed thoroughly. The organic layer was
separated and the aqueous layer extracted with 3 mL ethyl
acetate. The combined organic layer was dried (Isolute HM-N),
Suzuki coupling: The alkylation product (27.9 mg, 0.100
mmol),
(2-(N-(tert-butyl)sulfamoyl)-5-isobutylthiophen-3-
yl)boronic acid MIDA ester (51.6 mg, 0.12 mmol), K2CO3 (69.1
mg, 0.50 mmol), PdCl2(dppf) (3.7 mg, 5.0 µmol), DME (1.0 mL)
and water (0.20 mL). The vial was flushed with N2, sealed with a
screw-cap and the reaction was heated at 120 °C for 60 min.
Ethyl acetate (3 mL) and water (2 mL) was added to the cooled
reaction mixture and mixed thoroughly. The organic layer was

filtered and evaporated. The residue was carried forward to the
next step.
(discarded), then with 10% MeOH in dichloromethane. The
product containing fraction was evaporated and used as is in the
next step.
Deprotection: To the Suzuki product was added trifluoroacetic
acid (1 mL) and the mixture was stirred at 40 °C overnight. The
reaction mixture was co-evaporated with toluene to dryness. The
residue was partitioned between 3 mL dichloromethane and 2 mL
sat NaHCO3 (aq). The organic layer was loaded onto a plug of
silica. The plug was first eluted with dichloromethane
(discarded), then with 10% MeOH in dichloromethane. The
product containing fraction was evaporated and used as is in the
next step.
Coupling with butyl chloroformate: To a solution of the crude
primary sulfonamide in dichloromethane (2 mL) was added first
triethylamine (17 µL, 0.12 mmol) then butyl chloroformate (13
µL, 0.10 mmol) and the reaction mixture was stirred at room
temperature for 2 h. The reaction mixture was diluted with 2 mL
dichloromethane, washed with 1 M HCl (2 mL), dried (isolute
HM-N) and loaded onto a silica plug. The plug was eluted with
10% methanol in dichloromethane the collected fraction was
evaporated. The residue was purified by preparative RP-HPLC
(30-60% MeCN in water [0.05% HCOOH]). Product fractions
were freeze-dried to give 5.2 mg (9%, three step) of butyl ((5-
isobutyl-3-(3-(2-(2-butyl-1H-imidazol-1-
Coupling with butyl chloroformate: To a solution of the crude
primary sulfonamide in dichloromethane (2 mL) was added first
triethylamine (28 µL, 0.20 mmol) then butyl chloroformate (19
µL, 0.15 mmol) and DMAP (1 mg) and the reaction mixture was
stirred at room temperature for 1 h. The reaction mixture was
diluted with 2 mL dichloromethane, washed with 1 M HCl (2
mL), dried (isolute HM-N) and evaporated. The residue was
purified by silica flash chromatography (5-10% MeOH in
dichloromethane). Product fractions were evaporated and dried
under vacuum to give 29.2 mg (54%, three step) of butyl ((5-
isobutyl-3-(3-(2-(2-propyl-1H-imidazol-1-
yl)acetyl)phenyl)thiophen-2-yl)sulfonyl)carbamate. 1H NMR
(400 MHz, CDCl3) δ 8.69 – 8.65 (m, 1H), 7.96 – 7.91 (m, 1H),
7.75 – 7.70 (m, 1H), 7.51 (t, J = 7.7 Hz, 1H), 7.07 – 7.01 (m,
2H), 6.79 (s, 1H), 5.55 (s, 2H), 3.95 (t, J = 6.8 Hz, 2H), 2.78 –
2.66 (m, 4H), 1.99 – 1.87 (m, 1H), 1.63 – 1.52 (m, 2H), 1.53 –
1.41 (m, 2H), 1.35 – 1.16 (m, 5H), 0.99 (d, J = 6.6 Hz, 6H), 0.89
+
– 0.78 (m, 6H). MS (ESI): m/z calc’d for C28H38N3O5S2 :
560.2253 [M+H+]; found: 560.2257.
yl)acetyl)phenyl)thiophen-2-yl)sulfonyl)carbamate. 1H NMR
(400 MHz, CDCl3) δ 8.71 (s, 1H), 7.91 (d, J = 7.9 Hz, 1H), 7.76
(d, J = 7.8 Hz, 1H), 7.42 (t, J = 7.8 Hz, 1H), 7.08 – 7.01 (m, 2H),
6.75 (s, 1H), 5.64 (s, 2H), 3.89 (t, J = 6.8 Hz, 2H), 2.75 – 2.59
(m, 4H), 1.97 – 1.82 (m, 1H), 1.65 – 1.54 (m, 2H), 1.51 – 1.38
(m, 2H), 1.27 – 1.16 (m, 2H), 0.96 (d, J = 6.6 Hz, 6H), 0.86 –
0.78 (m, 6H). 13C NMR (101 MHz, CDCl3) δ 191.2, 158.6, 148.5,
147.3, 140.3, 138.9, 136.3, 134.9, 133.1, 130.9, 128.8, 128.1,
127.3, 122.2, 120.7, 65.0, 53.2, 39.3, 31.2, 30.6, 26.6, 22.4, 21.3,
5.1.8. Butyl ((5-isobutyl-3-(3-(2-(2-isopropyl-1H-
imidazol-1-yl)acetyl)phenyl)thiophen-2-
yl)sulfonyl)carbamate 2,2,2-trifluoroacetate (12)
Alkylation: A mixture of 2-bromo-1-(3-bromophenyl)ethan-1-
one (125 mg, 0.45 mmol), 2-isopropyl-1H-imidazole (248 mg,
2.25 mmol) in DMF (2 mL) was heated at 80 °C for 2 h. The
reaction mixture was evaporated and the residue purified by
automated silica flash chromatography (1-7.5% methanol in
dichloromethane). Product fractions were concentrated under
reduced pressure and dried under vacuum to give 138 mg (83%
crude) of the alkylated imidazole.
+
19.2, 13.9, 13.6. MS (ESI): m/z calc’d for C27H36N3O5S2 :
546.2096 [M+H+]; found: 546.2092.
5.1.7. Butyl ((5-isobutyl-3-(3-(2-(2-butyl-1H-
imidazol-1-yl)acetyl)phenyl)thiophen-2-
yl)sulfonyl)carbamate (11)
Suzuki coupling: The alkylation product (30.7 mg, 0.100
mmol),
(2-(N-(tert-butyl)sulfamoyl)-5-isobutylthiophen-3-
Alkylation: A mixture of 2-bromo-1-(3-bromophenyl)ethan-1-
one (125 mg, 0.450 mmol), 2-butyl-1H-imidazole (279 mg, 2.25
mmol) in DMF (2 mL) was heated at 80 °C for 2 h. The reaction
mixture was evaporated and the residue purified by automated
yl)boronic acid MIDA ester (51.6 mg, 0.12 mmol), K2CO3 (69.1
mg, 0.50 mmol), PdCl2(dppf) (3.7 mg, 5.0 µmol), DME (1.0 mL)
and water (0.20 mL). The vial was flushed with N2, sealed with a
screw-cap and the reaction was heated at 120 °C for 60 min.
Ethyl acetate (3 mL) and water (2 mL) was added to the cooled
reaction mixture and mixed thoroughly. The organic layer was
separated and the aqueous layer extracted with 3 mL ethyl
acetate. The combined organic layer was dried (Isolute HM-N),
filtered and evaporated. The residue was carried forward to the
next step.
silica
flash
chromatography
(1-7.5%
methanol
in
dichloromethane). Product fractions were concentrated under
reduced pressure and dried under vacuum to give 99.3 mg (69%
crude) of the alkylated imidazole.
Suzuki coupling: The alkylation product (32.1 mg, 0.100
mmol),
(2-(N-(tert-butyl)sulfamoyl)-5-isobutylthiophen-3-
yl)boronic acid MIDA ester (51.6 mg, 0.12 mmol), K2CO3 (69.1
mg, 0.50 mmol), PdCl2(dppf) (3.7 mg, 5.0 µmol), DME (1.0 mL)
and water (0.20 mL). The vial was flushed with N2, sealed with a
screw-cap and the reaction was heated at 120 °C for 60 min.
Ethyl acetate (3 mL) and water (2 mL) was added to the cooled
reaction mixture and mixed thoroughly. The organic layer was
separated and the aqueous layer extracted with 3 mL ethyl
acetate. The combined organic layer was dried (Isolute HM-N),
filtered and evaporated. The residue was carried forward to the
next step.
Deprotection: To the Suzuki product was added trifluoroacetic
acid (3 mL) and the mixture was stirred at 60 °C for 6 h. The
reaction mixture was co-evaporated with toluene to dryness. The
residue was partitioned between 3 mL dichloromethane and 2 mL
sat NaHCO3 (aq). The organic layer was loaded onto a plug of
silica. The plug was first eluted with dichloromethane
(discarded), then with 10% MeOH in dichloromethane. The
product containing fraction was evaporated and used as is in the
next step.
Coupling with butyl chloroformate: To a solution of the crude
primary sulfonamide in dichloromethane (2 mL) was added first
triethylamine (28 µL, 0.20 mmol) then butyl chloroformate (19
µL, 0.15 mmol) and the reaction mixture was stirred at room
temperature overnight. The reaction mixture was diluted with 2
mL dichloromethane, washed with 1 M HCl (2 mL), dried
(isolute HM-N) and loaded onto a silica plug. The plug was
Deprotection: To the Suzuki product was added trifluoroacetic
acid (1 mL) and the mixture was stirred at 60 °C overnight. The
reaction mixture was co-evaporated with toluene to dryness. The
residue was partitioned between 3 mL dichloromethane and 2 mL
sat NaHCO3 (aq). The organic layer was loaded onto a plug of
silica. The plug was first eluted with dichloromethane

5.1.10. Butyl ((5-isobutyl-3-(3-(2-(2-chloro-1H-
imidazol-1-yl)acetyl)phenyl)thiophen-2-
yl)sulfonyl)carbamate (14)
eluted with 10% methanol in dichloromethane the collected
fraction was evaporated. The residue was purified by preparative
HPLC (30-60% MeCN in water [0.1% TFA]). Product fractions
were freeze-dried to give 38.3 mg (58%, three step) of butyl ((5-
isobutyl-3-(3-(2-(2-isopropyl-1H-imidazol-1-
Alkylation: A mixture of 2-bromo-1-(3-bromophenyl)ethan-1-
one (125 mg, 0.450 mmol), 2-chloro-1H-imidazole (46.1 mg,
0.45 mmol) and K2CO3 (124 mg, 0.90 mmol) in DMF (2 mL)
was heated at 80 °C for 2 h. The reaction mixture was evaporated
and the residue purified by automated silica flash
chromatography (1-7.5% methanol in dichloromethane). Product
fractions were concentrated under reduced pressure and dried
under vacuum to give 87.5 mg (65% crude) of the alkylated
imidazole.
yl)acetyl)phenyl)thiophen-2-yl)sulfonyl)carbamate
2,2,2-
trifluoroacetate. 1H NMR (400 MHz, CDCl3) δ 8.32 (s, 1H), 8.04
– 7.99 (m, 1H), 7.66 – 7.62 (m, 1H), 7.58 – 7.51 (m, 1H), 7.25 –
7.21 (s, 2H), 6.80 (s, 1H), 5.82 (s, 2H), 3.97 (t, J = 6.6 Hz, 2H),
3.09 2.99 (m, 1H), 2.70 (d, J = 7.1 Hz, 2H), 1.99 – 1.87 (m, 1H),
1.50 – 1.39 (m, 2H), 1.35 (d, J = 6.9 Hz, 6H), 1.26 – 1.15 (m,
2H), 0.98 (d, J = 6.6 Hz, 6H), 0.83 (t, J = 7.3 Hz, 3H). 13C NMR
(101 MHz, CDCl3) δ 190.0, 153.2, 152.0, 151.1, 144.4, 134.9,
134.8, 133.4, 132.0, 129.5, 129.4, 129.1, 128.4, 122.6, 119.0,
66.8, 53.7, 39.4, 30.7, 30.5, 25.8, 22.3, 20.5, 18.8, 13.7. MS
Suzuki coupling: The alkylation product (30.0 mg, 0.100
mmol),
(2-(N-(tert-butyl)sulfamoyl)-5-isobutylthiophen-3-
yl)boronic acid MIDA ester (51.6 mg, 0.12 mmol), K2CO3 (69.1
mg, 0.50 mmol), PdCl2(dppf) (3.7 mg, 5.0 µmol), DME (1.0 mL)
and water (0.20 mL). The vial was flushed with N2, sealed with a
screw-cap and the reaction was heated at 120 °C for 60 min.
Ethyl acetate (3 mL) and water (2 mL) was added to the cooled
reaction mixture and mixed thoroughly. The organic layer was
separated and the aqueous layer extracted with 3 mL ethyl
acetate. The combined organic layer was dried (Isolute HM-N),
filtered and evaporated. The residue was carried forward to the
next step.
+
(ESI): m/z calc’d for C27H36N3O5S2 : 546.2096 [M+H+]; found:
546.2099.
5.1.9. Butyl ((3-(3-(2-(2-(tert-butyl)-1H-imidazol-1-
yl)acetyl)phenyl)-5-isobutylthiophen-2-
yl)sulfonyl)carbamate (13)
To an 8 mL vial was added 1-(3-bromophenyl)-2-(2-(tert-
butyl)-1H-imidazol-1-yl)ethan-1-one (32.1 mg, 0.100 mmol), (2-
(N-(tert-butyl)sulfamoyl)-5-isobutylthiophen-3-yl)boronic acid
MIDA ester (51.6 mg, 0.12 mmol), K2CO3 (69.1 mg, 0.5 mmol),
PdCl2(dppf) (3.7 mg, 5.0 µmol), DME (1 mL) and water (0.2
mL). The vial was flushed with N2, sealed with a screw-cap and
the reaction was heated at 120 °C for 60 min. Ethyl acetate (3
mL) and water (2 mL) was added to the cooled reaction mixture
and mixed thoroughly. The organic layer was separated and the
aqueous layer extracted with 3 mL ethyl acetate. The combined
organic layer was dried (Isolute HM-N), filtered and evaporated.
To the residue was added trifluoroacetic acid (1.0 mL) and the
mixture was stirred at 60 °C overnight. The reaction mixture was
co-evaporated with toluene to dryness and the residue was
partitioned between 3 mL dichoromethane and 2 mL sat NaHCO3
(aq). The organic layer was dried (Isolute HM-N), filtered loaded
Deprotection: To the Suzuki product was added trifluoroacetic
acid (1 mL) and the mixture was stirred at 60 °C overnight. The
reaction mixture was co-evaporated with toluene to dryness. The
residue was partitioned between 3 mL dichloromethane and 2 mL
sat NaHCO3 (aq). The organic layer was loaded onto a plug of
silica. The plug was first eluted with dichloromethane
(discarded), then with 10% MeOH in dichloromethane. The
product containing fraction was evaporated and used as is in the
next step.
Coupling with butyl chloroformate: To a solution of the crude
primary sulfonamide in dichloromethane (2 mL) was added first
triethylamine (17 µL, 0.12 mmol) then butyl chloroformate (13
µL, 0.10 mmol) and the reaction mixture was stirred at room
temperature for 2 h. The reaction mixture was diluted with 2 mL
dichloromethane, washed with 1 M HCl (2 mL), dried (isolute
HM-N) and loaded onto a silica plug. The plug was eluted with
10% methanol in dichloromethane the collected fraction was
evaporated. The residue was purified by preparative RP-HPLC
(30-60% MeCN in water [0.05% HCOOH]). Product fractions
were freeze-dried to give 10.3 mg (19%, three step) of butyl ((5-
isobutyl-3-(3-(2-(2-chloro-1H-imidazol-1-
onto
a silica plug. The silica plug was washed with
dichloromethane (5 mL), then eluted with 10% methanol in
dichloromethane and the collected fraction was evaporated. The
residue was dissolved in dichloromethane (2 mL) and
triethylamine (17µL, 0.12 mmol) was added. The mixture was
cooled to 0 C and butyl chloroformate (13 µL, 0.10 mmol) was
added and the mixture allowed to warm and was stirred at room
temperature for 4 h. The reaction mixture was diluted with 2 mL
dichloromethane, washed with 1 M HCl (2 mL), dried (isolute
HM-N) and loaded onto a silica plug which was eluted with 10%
methanol in dichloromethane and the collected fraction was
evaporated. The residue was purified by preparative HPLC (30-
60% MeCN in water [0.05% HCOOH]). Product fractions were
freeze-dried to give 17.3 mg (31%, three steps) of butyl ((3-(3-(2-
(2-(tert-butyl)-1H-imidazol-1-yl)acetyl)phenyl)-5-
yl)acetyl)phenyl)thiophen-2-yl)sulfonyl)carbamate. 1H NMR
(400 MHz, CDCl3) δ 8.22 – 8.19 (m, 1H), 8.05 – 8.00 (m, 1H),
7.76 – 7.72 (m, 1H), 7.59 (t, J = 7.8 Hz, 1H), 7.03 (d, J = 1.5 Hz,
1H), 6.93 (d, J = 1.5 Hz, 1H), 6.81 (s, 1H), 5.40 (s, 2H), 4.08 (t, J
= 6.6 Hz, 2H), 2.74 (d, J = 7.1 Hz, 2H), 2.05 – 1.93 (m, 1H), 1.59
– 1.47 (m, 2H), 1.35 – 1.20 (m, 2H), 1.01 (d, J = 6.6 Hz, 6H),
0.88 (t, J = 7.4 Hz, 3H). MS (ESI): m/z calc’d for
1
isobutylthiophen-2-yl)sulfonyl)carbamate. H NMR (400 MHz,
+
C24H29ClN3O5S2 : 538.1237 [M+H+]; found: 538.1232.
CDCl3) δ 8.82 (s, 1H), 7.94– 7.90 (m, 1H), 7.77 – 7.70 (m, 1H),
7.49 (t, J = 7.8 Hz, 1H), 7.29 (s, 2H), 7.09 – 7.06 (m, 1H), 7.02 –
6.99 (m, 1H), 6.79 (s, 1H), 5.85 (s, 2H), 3.88 (t, J = 6.7 Hz, 2H),
2.67 (d, J = 7.0 Hz, 2H), 1.97 – 1.85 (m, 1H), 1.47 – 1.38 (m,
2H), 1.37 (s, 9H), 1.28 – 1.12 (m, 3H), 0.98 (d, J = 6.6 Hz, 6H),
0.82 (t, J = 7.4 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 191.7,
157.6, 153.3, 147.8, 140.7, 138.3, 136.0, 134.5, 133.1, 131.2,
129.0, 128.1, 127.1, 124.4, 120.8, 65.3, 55.3, 39.4, 33.8, 31.1,
30.6, 29.0, 22.5, 19.2, 13.9. MS (ESI): m/z calc’d for
5.1.11. Butyl ((5-isobutyl-3-(3-(2-(2-bromo-1H-
imidazol-1-yl)acetyl)phenyl)thiophen-2-
yl)sulfonyl)carbamate (15)
Alkylation: A mixture of 2-bromo-1-(3-bromophenyl)ethane-
1-one (125 mg, 0.450 mmol), 2-bromo-1H-imidazole (66.1 mg,
0.45 mmol) and K2CO3 (124 mg, 0.90 mmol) in DMF (2 mL)
was heated at 80 °C for 2 h. The reaction mixture was evaporated
and the residue purified by automated silica flash
chromatography (1-7.5% methanol in dichloromethane). Product
+
C28H38N3O5S2 : 560.2253 [M+H+]; found: 560.2239.

fractions were concentrated under reduced pressure and dried
under vacuum to give 115 mg (83% crude) of the alkylated
imidazole.
screw-cap and the reaction was heated at 120 °C for 60 min.
Ethyl acetate (3 mL) and water (2 mL) was added to the cooled
reaction mixture and mixed thoroughly. The organic layer was
separated and the aqueous layer extracted with 3 mL ethyl
acetate. The combined organic layer was dried (Isolute HM-N),
filtered and evaporated. The residue was carried forward to the
next step.
Suzuki coupling: The alkylation product (34.4 mg, 0.100
mmol),
(2-(N-(tert-butyl)sulfamoyl)-5-isobutylthiophen-3-
yl)boronic acid MIDA ester (51.6 mg, 0.12 mmol), K2CO3 (69.1
mg, 0.50 mmol), PdCl2(dppf) (3.7 mg, 5.0 µmol), DME (1.0 mL)
and water (0.20 mL). The vial was flushed with N2, sealed with a
screw-cap and the reaction was heated at 120 °C for 60 min.
Ethyl acetate (3 mL) and water (2 mL) was added to the cooled
reaction mixture and mixed thoroughly. The organic layer was
separated and the aqueous layer extracted with 3 mL ethyl
acetate. The combined organic layer was dried (Isolute HM-N),
filtered and evaporated. The residue was carried forward to the
next step.
Deprotection: To the Suzuki product was added trifluoroacetic
acid (1 mL) and the mixture was stirred at 40 °C overnight. The
reaction mixture was co-evaporated with toluene to dryness. The
residue was partitioned between 3 mL dichloromethane and 2 mL
sat NaHCO3 (aq). The organic layer was loaded onto a plug of
silica. The plug was first eluted with dichloromethane
(discarded), then with 10% MeOH in dichloromethane. The
product containing fraction was evaporated and used as is in the
next step.
Deprotection: To the Suzuki product was added trifluoroacetic
acid (1 mL) and the mixture was stirred at 60 °C overnight. The
reaction mixture was co-evaporated with toluene to dryness. The
residue was partitioned between 3 mL dichloromethane and 2 mL
sat NaHCO3 (aq). The organic layer was loaded onto a plug of
silica. The plug was first eluted with dichloromethane
(discarded), then with 10% MeOH in dichloromethane. The
product containing fraction was evaporated and used as is in the
next step.
Coupling with butyl chloroformate: To a solution of the crude
primary sulfonamide in dichloromethane (2 mL) was added first
triethylamine (17 µL, 0.12 mmol) then butyl chloroformate (13
µL, 0.10 mmol) and the reaction mixture was stirred at room
temperature for 2 h. The reaction mixture was diluted with 2 mL
dichloromethane, washed with 1 M HCl (2 mL), dried (isolute
HM-N) and loaded onto a silica plug. The plug was eluted with
10% methanol in dichloromethane the collected fraction was
evaporated. The residue was purified by preparative RP-HPLC
(30-80% MeCN in water [0.05% HCOOH]). Product fractions
were freeze-dried to give 1.6 mg (3%, three step) of butyl ((5-
isobutyl-3-(3-(2-(2-methyl-1H-benzo[d]imidazol-1-
Coupling with butyl chloroformate: To a solution of the crude
primary sulfonamide in dichloromethane (2 mL) was added first
triethylamine (17 µL, 0.12 mmol) then butyl chloroformate (13
µL, 0.10 mmol) and the reaction mixture was stirred at room
temperature for 2 h. The reaction mixture was diluted with 2 mL
dichloromethane, washed with 1 M HCl (2 mL), dried (isolute
HM-N) and loaded onto a silica plug. The plug was eluted with
10% methanol in dichloromethane the collected fraction was
evaporated. The residue was purified by preparative RP-HPLC
(30-60% MeCN in water [0.05% HCOOH]). Product fractions
were freeze-dried to give 4.4 mg (8%, three step) of butyl ((5-
isobutyl-3-(3-(2-(2-bromo-1H-imidazol-1-
yl)acetyl)phenyl)thiophen-2-yl)sulfonyl)carbamate. 1H NMR
(400 MHz, CDCl3) δ 8.54 – 8.48 (m, 1H), 8.06 (d, J = 7.8 Hz,
1H), 7.92 – 7.86 (m, 1H), 7.75 (d, J = 7.8 Hz, 1H), 7.62 (t, J =
7.8 Hz, 1H), 7.50 – 7.36 (m, 3H), 6.85 (s, 1H), 5.86 (s, 2H), 4.04
(t, J = 6.7 Hz, 2H), 2.79 – 2.73 (m, 5H), 2.03 – 1.92 (m, 1H),
1.55 – 1.42 (m, 2H), 1.30 – 1.19 (m, 2H), 1.02 (d, J = 6.6 Hz,
6H), 0.85 (t, J = 7.4 Hz, 3H). MS (ESI): m/z calc’d for
C29H34N3O5S2 +: 568.1940 [M+H+]; found: 568.1931.
yl)acetyl)phenyl)thiophen-2-yl)sulfonyl)carbamate. 1H NMR
(400 MHz, CDCl3) δ 8.22 – 8.19 (m, 1H), 8.03 (ddd, J = 7.8, 1.8,
1.2 Hz, 1H), 7.74 (ddd, J = 7.7, 1.8, 1.2 Hz, 1H), 7.59 (t, J = 7.7
Hz, 1H), 7.08 (d, J = 1.5 Hz, 1H), 7.00 (d, J = 1.5 Hz, 1H), 6.81
(s, 1H), 5.41 (s, 2H), 4.08 (t, J = 6.6 Hz, 2H), 2.74 (d, J = 7.1 Hz,
2H), 2.05 – 1.90 (m, 1H), 1.59 – 1.47 (m, 4H), 1.35 – 1.20 (m,
2H), 1.01 (d, J = 6.6 Hz, 6H), 0.88 (t, J = 7.4 Hz, 3H). MS (ESI):
m/z calc’d for C24H29BrN3O5S2 +: 582.0732 [M+H+]; found:
582.0731.
5.1.13. Butyl ((5-isobutyl-3-(3-(2-(2-ethyl-1H-
benzo[d]imidazol-1-yl)acetyl)phenyl)thiophen-2-
yl)sulfonyl)carbamate (17)
Alkylation: A mixture of 2-bromo-1-(3-bromophenyl)ethan-1-
one (125 mg, 0.450 mmol), 2-ethyl-1H-benzo[d]imidazole (65.7
mg, 0.45 mmol) and K2CO3 (124 mg, 0.90 mmol) in DMF (2
mL) was heated at 80 °C for 2 h. The reaction mixture was
evaporated and the residue purified by automated silica flash
chromatography (1-7.5% methanol in dichloromethane). Product
fractions were concentrated under reduced pressure and dried
under vacuum to give 73.5 mg (48% crude) of the alkylated
imidazole.
5.1.12. Butyl ((5-isobutyl-3-(3-(2-(2-methyl-1H-
benzo[d]imidazol-1-yl)acetyl)phenyl)thiophen-2-
yl)sulfonyl)carbamate (16)
Alkylation: A mixture of 2-bromo-1-(3-bromophenyl)ethan-1-
one (125 mg, 0.450 mmol), 2-methyl-1H-benzo[d]imidazole
(59.4 mg, 0.45 mmol) and K2CO3 (124 mg, 0.90 mmol) in DMF
(2 mL) was heated at 80 °C for 2 h. The reaction mixture was
evaporated and the residue purified by automated silica flash
chromatography (1-7.5% methanol in dichloromethane). Product
fractions were concentrated under reduced pressure and dried
under vacuum to give 80.7 mg (55% crude) of the alkylated
imidazole.
Suzuki coupling: The alkylation product (34.3 mg, 0.100
mmol),
(2-(N-(tert-butyl)sulfamoyl)-5-isobutylthiophen-3-
yl)boronic acid MIDA ester (51.6 mg, 0.12 mmol), K2CO3 (69.1
mg, 0.50 mmol), PdCl2(dppf) (3.7 mg, 5.0 µmol), DME (1.0 mL)
and water (0.20 mL). The vial was flushed with N2, sealed with a
screw-cap and the reaction was heated at 120 °C for 60 min.
Ethyl acetate (3 mL) and water (2 mL) was added to the cooled
reaction mixture and mixed thoroughly. The organic layer was
separated and the aqueous layer extracted with 3 mL ethyl
acetate. The combined organic layer was dried (Isolute HM-N),
filtered and evaporated. The residue was carried forward to the
next step.
Suzuki coupling: The alkylation product (32.9 mg, 0.100
mmol),
(2-(N-(tert-butyl)sulfamoyl)-5-isobutylthiophen-3-
yl)boronic acid MIDA ester (51.6 mg, 0.12 mmol), K2CO3 (69.1
mg, 0.50 mmol), PdCl2(dppf) (3.7 mg, 5.0 µmol), DME (1.0 mL)
and water (0.20 mL). The vial was flushed with N2, sealed with a
Deprotection: To the Suzuki product was added trifluoroacetic
acid (1 mL) and the mixture was stirred at 40 °C overnight. The

reaction mixture was co-evaporated with toluene to dryness. The
residue was partitioned between 3 mL dichloromethane and 2 mL
sat NaHCO3 (aq). The organic layer was loaded onto a plug of
silica. The plug was first eluted with dichloromethane
(discarded), then with 10% MeOH in dichloromethane. The
product containing fraction was evaporated and used as is in the
next step.
Coupling with butyl chloroformate: To a solution of the crude
primary sulfonamide in dichloromethane (2 mL) was added first
triethylamine (17 µL, 0.12 mmol) then butyl chloroformate (13
µL, 0.10 mmol) and DMAP (1 mg, cat.) and the reaction mixture
was stirred at room temperature. After 90 min another 5 µL butyl
chloroformate was added and the reaction stirred for a further
hour at room temperature. The reaction mixture was diluted with
2 mL dichloromethane, washed with 1 M HCl (2 mL), dried
(isolute HM-N) and loaded onto a silica plug. The plug was
eluted with 10% methanol in dichloromethane the collected
fraction was evaporated. The residue was purified by preparative
RP-HPLC (30-80% MeCN in water [0.05% HCOOH]). Product
fractions were freeze-dried to give 27.4 mg (46%, three step) of
butyl ((5-isobutyl-3-(3-(2-(2-isopropyl-1H-benzo[d]imidazol-1-
yl)acetyl)phenyl)thiophen-2-yl)sulfonyl)carbamate. 1H NMR
(400 MHz, CDCl3) δ 8.36 – 8.32 (m, 1H), 8.05 (dt, J = 7.8, 1.4
Hz, 1H), 7.79 – 7.71 (m, 2H), 7.59 (t, J = 7.8 Hz, 1H), 7.25 –
7.08 (m, 3H), 6.81 (s, 1H), 5.58 (s, 2H), 4.05 (t, J = 6.6 Hz, 2H),
3.05 – 2.95 (m, 1H), 2.74 (d, J = 7.1 Hz, 2H), 2.04 – 1.91 (m,
1H), 1.53 – 1.37 (m, 8H), 1.28 – 1.16 (m, 2H), 1.01 (d, J = 6.6
Hz, 6H), 0.83 (t, J = 7.4 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ
191.4, 160.3, 151.8, 151.6, 144.7, 140.5, 135.2, 134.8, 134.7,
134.1, 132.8, 129.8, 129.2, 129.1, 128.1, 123.0, 122.8, 118.7,
109.4, 66.8, 49.6, 39.5, 30.7, 30.6, 26.7, 22.4, 21.5, 18.9, 13.7.
Coupling with butyl chloroformate: To a solution of the crude
primary sulfonamide in dichloromethane (2 mL) was added first
triethylamine (17 µL, 0.12 mmol) then butyl chloroformate (13
µL, 0.10 mmol) and the reaction mixture was stirred at room
temperature. After 90 min another 5 µL butyl chloroformate was
added and the reaction stirred for a further hour at room
temperature. The reaction mixture was diluted with 2 mL
dichloromethane, washed with 1 M HCl (2 mL), dried (isolute
HM-N) and loaded onto a silica plug. The plug was eluted with
10% methanol in dichloromethane the collected fraction was
evaporated. The residue was purified by preparative RP-HPLC
(30-80% MeCN in water [0.05% HCOOH]). Product fractions
were freeze-dried to give 1.6 mg (3%, three step) of butyl ((5-
isobutyl-3-(3-(2-(2-ethyl-1H-benzo[d]imidazol-1-
yl)acetyl)phenyl)thiophen-2-yl)sulfonyl)carbamate. 1H NMR
(400 MHz, CDCl3) δ 8.36 (t, J = 1.7 Hz, 1H), 8.01 (dt, J = 7.8,
1.4 Hz, 1H), 7.79 – 7.71 (m, 2H), 7.57 (t, J = 7.8 Hz, 1H), 7.25 –
7.08 (m, 3H), 6.81 (s, 1H), 5.52 (s, 2H), 4.06 (t, J = 6.7 Hz, 2H),
2.81 – 2.68 (m, 4H), 2.03 – 1.91 (m, 1H), 1.55 – 1.36 (m, 5H),
1.29 – 1.18 (m, 2H), 1.02 (d, J = 6.6 Hz, 6H), 0.84 (t, J = 7.4 Hz,
+
MS (ESI): m/z calc’d for C31H38N3O5S2 : 596.2253 [M+H+];
found: 596.2254.
+
3H). MS (ESI): m/z calc’d for C30H36N3O5S2 : 582.2096 [M+H+];
5.1.15. Butyl ((5-isobutyl-3-(3-(2-(5,6-dimethyl-1H-
benzo[d]imidazol-1-yl)acetyl)phenyl)thiophen-2-
yl)sulfonyl)carbamate (19)
found: 582.2104.
Alkylation: A mixture of 2-bromo-1-(3-bromophenyl)ethan-1-
one (125 mg, 0.450 mmol), 5,6-dimethyl-1H-benzo[d]imidazole
(65.7 mg, 0.45 mmol) and K2CO3 (124 mg, 0.90 mmol) in DMF
(2 mL) was heated at 80 °C for 3 h. The reaction mixture was
evaporated and the residue purified by automated silica flash
chromatography (1-7.5% methanol in dichloromethane). Product
fractions were concentrated under reduced pressure and dried
under vacuum to give 74.0 mg (48% crude) of the alkylated
imidazole.
5.1.14. Butyl ((5-isobutyl-3-(3-(2-(2-isopropyl-1H-
benzo[d]imidazol-1-yl)acetyl)phenyl)thiophen-2-
yl)sulfonyl)carbamate (18)
Alkylation: A mixture of 2-bromo-1-(3-bromophenyl)ethan-1-
one (125 mg, 0.450 mmol), 2-isopropyl-1H-benzo[d]imidazole
(72.0 mg, 0.45 mmol) and K2CO3 (124 mg, 0.90 mmol) in DMF
(2 mL) was heated at 80 °C for 3 h. The reaction mixture was
evaporated and the residue purified by automated silica flash
chromatography (1-7.5% methanol in dichloromethane). Product
fractions were concentrated under reduced pressure and dried
under vacuum to give 51.2 mg (32% crude) of the alkylated
imidazole.
Suzuki coupling: The alkylation product (34.3 mg, 0.100
mmol),
(2-(N-(tert-butyl)sulfamoyl)-5-isobutylthiophen-3-
yl)boronic acid MIDA ester (51.6 mg, 0.12 mmol), K2CO3 (69.1
mg, 0.50 mmol), PdCl2(dppf) (3.7 mg, 5.0 µmol), DME (1.0 mL)
and water (0.20 mL). The vial was flushed with N2, sealed with a
screw-cap and the reaction was heated at 120 °C for 60 min.
Ethyl acetate (3 mL) and water (2 mL) was added to the cooled
reaction mixture and mixed thoroughly. The organic layer was
separated and the aqueous layer extracted with 3 mL ethyl
acetate. The combined organic layer was dried (Isolute HM-N),
filtered and evaporated. The residue was carried forward to the
next step.
Suzuki coupling: The alkylation product (35.7 mg, 0.100
mmol),
(2-(N-(tert-butyl)sulfamoyl)-5-isobutylthiophen-3-
yl)boronic acid MIDA ester (51.6 mg, 0.12 mmol), K2CO3 (69.1
mg, 0.50 mmol), PdCl2(dppf) (3.7 mg, 5.0 µmol), DME (1.0 mL)
and water (0.20 mL). The vial was flushed with N2, sealed with a
screw-cap and the reaction was heated at 120 °C for 60 min.
Ethyl acetate (3 mL) and water (2 mL) was added to the cooled
reaction mixture and mixed thoroughly. The organic layer was
separated and the aqueous layer extracted with 3 mL ethyl
acetate. The combined organic layer was dried (Isolute HM-N),
filtered and evaporated. The residue was carried forward to the
next step.
Deprotection: To the Suzuki product was added trifluoroacetic
acid (1 mL) and the mixture was stirred at 40 °C overnight. The
reaction mixture was co-evaporated with toluene to dryness. The
residue was partitioned between 3 mL dichloromethane and 2 mL
sat NaHCO3 (aq). The organic layer was loaded onto a plug of
silica. The plug was first eluted with dichloromethane
(discarded), then with 10% MeOH in dichloromethane. The
product containing fraction was evaporated and used as is in the
next step.
Deprotection: To the Suzuki product was added trifluoroacetic
acid (1 mL) and the mixture was stirred at 40 °C overnight. The
reaction mixture was co-evaporated with toluene to dryness. The
residue was partitioned between 3 mL dichloromethane and 2 mL
sat NaHCO3 (aq). The organic layer was loaded onto a plug of
silica. The plug was first eluted with dichloromethane
(discarded), then with 10% MeOH in dichloromethane. The
product containing fraction was evaporated and used as is in the
next step.
Coupling with butyl chloroformate: To a solution of the crude
primary sulfonamide in dichloromethane (2 mL) was added first
triethylamine (17 µL, 0.12 mmol) then butyl chloroformate (13

µL, 0.10 mmol) and the reaction mixture was stirred at room
temperature. After 90 min more triethylamine (17 µL, 0.12
mmol) and butyl chloroformate (13 µL, 0.10 mmol) was added
and the reaction stirred for a further 90 min at room temperature.
The reaction mixture was diluted with 2 mL dichloromethane,
washed with 1 M HCl (2 mL), dried (isolute HM-N) and loaded
onto a silica plug. The plug was eluted with 10% methanol in
dichloromethane the collected fraction was evaporated. The
residue was purified by preparative RP-HPLC (30-80% MeCN in
water [0.05% HCOOH]). Product fractions were freeze-dried to
give 5.0 mg (9%, three step) of butyl ((5-isobutyl-3-(3-(2-(5,6-
dimethyl-1H-benzo[d]imidazol-1-yl)acetyl)phenyl)thiophen-2-
butyl)-1H-imidazol-1-yl)acetyl)phenyl)-5-isobutylthiophen-2-
yl)sulfonyl)carbamate. H NMR (400 MHz, CDCl3) δ 8.90 (s,
1
1H), 7.92 – 7.87 (m, 1H), 7.77 – 7.72 (m, 1H), 7.46 (t, J = 7.8
Hz, 1H), 7.13 – 7.07 (m, 2H), 6.77 (s, 1H), 5.91 (s, 2H), 3.43 (s,
3H), 2.64 (d, J = 7.0 Hz, 2H), 1.97 – 1.83 (m, 1H), 1.36 (s, 9H),
0.96 (d, J = 6.6 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ 191.7,
159.3, 153.0, 147.2, 140.1, 138.8, 136.1, 134.7, 132.8, 131.3,
128.9, 128.0, 127.1, 124.8, 119.7, 55.6, 52.3, 39.3, 33.8, 30.6,
+
28.8, 22.4. MS (ESI): m/z calc’d for C25H32N3O5S2 : 518.1783
[M+H+]; found: 518.1796.
1
yl)sulfonyl)carbamate. H NMR (400 MHz, CDCl3) δ 8.37 (s,
5.1.17. Methyl ((3-(3-(2-(2-cyclopropyl-1H-
imidazol-1-yl)acetyl)phenyl)-5-isobutylthiophen-2-
yl)sulfonyl)carbamate (21)
1H), 7.85 (s, 1H), 7.71 – 7.63 (m, 2H), 7.43 (t, J = 7.7 Hz, 1H),
7.04 (s, 1H), 6.79 (s, 1H), 5.23 (s, 2H), 4.09 (t, J = 6.7 Hz, 2H),
2.75 (d, J = 7.1 Hz, 2H), 2.34 (s, 3H), 2.30 (s, 3H), 2.02 – 1.93
(m, 1H), 1.60 – 1.51 (m, 2H), 1.33 – 1.22 (m, 2H), 1.02 (d, J =
6.6 Hz, 6H), 0.87 (t, J = 7.4 Hz, 3H). MS (ESI): m/z calc’d for
To
a solution of 3-(3-(2-(2-cyclopropyl-1H-imidazol-1-
yl)acetyl)phenyl)-5-isobutylthiophene-2-sulfonamide (16.0 mg,
0.036 mmol) in dichloromethane (2 mL) was added first DIEA
(25 µL, 0.14 mmol) then methyl chloroformate (3.3 µL, 0.043
mmol) and the reaction mixture was stirred at room temperature
for 90 min. The reaction mixture was then evaporated and the
residue purified by automated silica flash chromatography (5-
20% methanol in dichloromethane). Product fractions were
concentrated under reduced pressure and dried under vacuum to
give 10.2 mg (56%) of methyl ((3-(3-(2-(2-cyclopropyl-1H-
imidazol-1-yl)acetyl)phenyl)-5-isobutylthiophen-2-
+
C30H36N3O5S2 : 582.2096 [M+H+]; found: 582.2090.
5.1.16. Methyl ((3-(3-(2-(2-(tert-butyl)-1H-
imidazol-1-yl)acetyl)phenyl)-5-isobutylthiophen-2-
yl)sulfonyl)carbamate (20)
A mixture of 2-bromo-1-(3-bromophenyl)ethane-1-one (278
mg, 1.00 mmol), 2-tert-butyl-1H-imidazole (149 mg, 1.2 mmol)
and K2CO3 (276 mg, 2.0 mol) in 3 mL acetonitrile was heated at
80 °C for 2 h. The reaction mixture was evaporated and the
residue purified by automated silica flash chromatography (1-
7.5% methanol in dichloromethane). Product fractions were
concentrated under reduced pressure and dried under vacuum to
give 233 mg (73%) of 1-(3-bromophenyl)-2-(2-(tert-butyl)-1H-
imidazol-1-yl)ethan-1-one.
yl)sulfonyl)carbamate. 1H NMR (400 MHz, CDCl3) δ 8.52 – 8.49
(m, 1H), 8.02 (d, J = 7.8 Hz, 1H), 7.69 (d, J = 7.7 Hz, 1H), 7.58
(t, J = 7.7 Hz, 1H), 7.23 – 7.15 (m, 2H), 6.82 (d, J = 0.8 Hz, 1H),
5.84 (s, 2H), 3.59 (s, 3H), 2.72 (d, J = 6.9 Hz, 2H), 2.03 – 1.79
(m, 2H), 1.26 – 1.09 (m, 4H), 1.00 (d, J = 6.6 Hz, 6H). MS (ESI):
+
m/z calc’d for C24H28N3O5S2 : 502.1470 [M+H+]; found:
502.1488.
To an 8 mL vial was added 1-(3-bromophenyl)-2-(2-(tert-
butyl)-1H-imidazol-1-yl)ethan-1-one (96.4 mg, 0.300 mmol), (2-
(N-(tert-butyl)sulfamoyl)-5-isobutylthiophen-3-yl)boronic acid
MIDA ester (129.1 mg, 0.30 mmol), K2CO3 (207 mg, 1.5 mmol),
PdCl2(dppf) (11.0 mg, 0.015 mmol), DME (3 mL) and water (1
mL). The vial was flushed with N2, sealed with a screw-cap and
the reaction was heated at 120 °C for 60 min. Ethyl acetate (3
mL) was added to the cooled reaction mixture. The organic layer
was separated and the aqueous layer extracted with 3 mL ethyl
acetate. The combined organic layer was dried (Isolute HM-N),
filtered and evaporated and purified by automated flash
chromatography (2.5-7.5% MeOH in dichloromethane). Product
fractions were concentrated under reduced pressure and dried
under vacuum.
5.1.18. Ethyl ((3-(3-(2-(2-cyclopropyl-1H-imidazol-
1-yl)acetyl)phenyl)-5-isobutylthiophen-2-
yl)sulfonyl)carbamate (22)
Alkylation: A mixture of 2-bromo-1-(3-bromophenyl)ethan-1-
one (69.5 mg, 0.25 mmol), 2-cyclopropyl-1H-imidazole (81.1
mg, 0.75 mmol), K2CO3 (69.1 mg, 0.50 mmol) in 2 mL DMF
was heated at 80 °C for 1 h. The reaction mixture was evaporated
and the residue purified by automated silica flash
chromatography (2-10% methanol in dichloromethane). Product
fractions were concentrated under reduced pressure and dried
under vacuum to give 64.9 mg (85% crude) of the alkylated
imidazole.
Suzuki coupling: The intermediate (64.9 mg, 0.21 mmol), (2-
(N-(tert-butyl)sulfamoyl)-5-isobutylthiophen-3-yl)boronic acid
MIDA ester (91.5 mg, 0.21 mmol), K2CO3 (147 mg, 1.1 mmol),
PdCl2(dppf) (7. 8 mg, 0.011 mmol), DME (3 mL) and water (1
mL). The vial was flushed with N2, sealed with a screw-cap and
the reaction was heated at 120 °C for 60 min.61 The organic layer
was evaporated and purified by automated flash chromatography
(5-15% MeOH in dichloromethane). Product fractions were
concentrated under reduced pressure and dried under vacuum to
give 92.3 mg (87% crude) of the Suzuki product.
To the residue was added trifluoroacetic acid (4 mL) and the
mixture was stirred at room temperature over the weekend, then
heated at 60 °C for 5 h. The reaction mixture was co-evaporated
with toluene to dryness and the residue was partitioned between 5
mL ethyl acetate and 5 mL sat NaHCO3 (aq). The organic layer
was dried (Isolute HM-N), filtered and evaporated.
A
solution of the crude primary sulfonamide in
dichloromethane (3 mL) was treated with first DIEA (0.16 mL,
0.91 mmol) and DMAP (2 mg, cat) then methyl chloroformate
(23µL, 0.30 mmol) and the reaction mixture was stirred at room
temperature overnight (15 h). The reaction mixture was washed
with sat. NH4Cl (aq) (3 mL) and brine (3 mL). The organic layer
was dried (Isolute HM-N), filtered and evaporated and the
residue purified by automated silica flash chromatography (2.5-
10% methanol in dichloromethane). Product fractions were
concentrated under reduced pressure and dried under vacuum to
give 61.2 mg (39%, three step) of methyl ((3-(3-(2-(2-(tert-
Deprotection: To the Suzuki product was added trifluoroacetic
acid (3 mL) and the mixture was stirred at 60 °C for 2 h. The
reaction mixture was co-evaporated with toluene to dryness. The
residue was partitioned between 10 mL EtOAc and 10 mL sat
NaHCO3 (aq). The organic layer was dried (Isolute HM-N),
filtered, evaporated and dried under vacuum to give 64.5 mg
(79%) of the primary sulfonamide.

Coupling with ethyl chloroformate: To a solution of the
primary sulfonamide (32.0 mg, 0.072 mmol) in dichloromethane
(2 mL) was added first DIEA (50 µL, 0.29 mmol) then Ethyl
chloroformate (8.3 µL, 0.086 mmol) and the reaction mixture
was stirred at room temperature for 1 h whereby product and
double acylation (m/z 588 = product + one extra ethyl carbamate)
was detected. Added 200 µL ethanol and heated at 60 °C for 2 h
in order to cleave off the extra carbamate. The reaction mixture
was then evaporated and the residue purified by automated silica
flash chromatography (5-15% methanol in dichloromethane).
Product fractions were concentrated under reduced pressure and
dried under vacuum to give 18.3 mg (49% crude) of ethyl ((3-(3-
(2-(2-cyclopropyl-1H-imidazol-1-yl)acetyl)phenyl)-5-
2H), 2.67 (d, J = 7.0 Hz, 2H), 1.99 – 1.86 (m, 1H), 1.81 (s, 3H),
1.35 (s, 9H), 0.98 (d, J = 7.0 Hz, 6H). 13C NMR (101 MHz,
CDCl3) δ 192.0, 173.8, 153.7, 149.4, 142.3, 135.7, 134.5, 133.6,
130.4, 129.1, 128.5, 127.6, 124.2, 122.6, 54.7, 39.4, 33.7, 30.7,
+
29.4, 24.8, 22.4. MS (ESI): m/z calc’d for C25H32N3O4S2 :
502.1834 [M+H+]; found: 502.1840.
5.2. Stability in liver microsomes
Human and mouse liver microsomes obtained from XenoTech
LLC, KS, USA were used to determine the metabolic stability.
Metabolic stability was determined in 0.5 mg/mL human or
mouse liver microsomes at a compound concentration of 1 µM in
100 mM potassium phosphate buffer (pH 7.4) in a total
incubation volume of 500 µL. The reaction was initiated by
addition of 1 mM NADPH. At various incubation times, i.e. at 0,
5, 10, 20, 40 and 60 min, a sample was withdrawn from the
incubation and the reaction was terminated by addition of ice-
cold acetonitrile containing Warfarin as internal standard. The
amount of parent compound remaining was analysed by LC-
MS/MS. The LC-MS/MS system used was an Acquity UPLC
coupled to a triple quadrupole mass spectrometer (Waters),
operating in multiple reaction monitoring (MRM) mode with
positive or negative electrospray ionization. Mass spectrometric
settings were optimized for each compound for one MRM
transition. Chromatographic separation was typically done on a
C18 Ethylene Bridged Hybrid (BEH) 1.7 µm column using a
general gradient of 1% to 90% of mobile phase consisting of A,
5% acetonitrile and 0.1% formic acid in purified water, and B,
0.1% formic acid in 100% acetonitrile, over a total running time
of 2 min. In a few cases, separation was done on a HSS T3 2x50
mm 2.1 µm column using a mobile phase consisting of A, 0.05%
heptafluorobutyric acid (HFBA) and 0.05% propionic acid (PA)
in water, and B, 0.05% HFBA and 0.05% PA in acetonitrile, with
a total running time of 2 min. In both cases, the flow rate was set
to 0.5 mL/min and 5 µL of the sample was injected. In vitro half-
1
isobutylthiophen-2-yl)sulfonyl)carbamate. H NMR (400 MHz,
CDCl3) δ 8.68 (s, 1H), 7.95 – 7.89 (m, 1H), 7.78 – 7.73 (m, 1H),
7.49 – 7.41 (m, 1H), 7.05 (s, 1H), 6.96 (s, 1H), 6.76 (s, 1H), 5.71
(s, 2H), 3.93 (q, J = 7.1 Hz, 2H), 2.65 (d, J = 7.2 Hz, 2H), 1.98 –
1.83 (m, 1H), 1.81 – 1.69 (m, 1H), 1.10 – 0.91 (m, 13H). 13C
NMR (101 MHz, CDCl3) δ 191.3, 157.7, 149.3, 147.9, 140.8,
138.0, 136.1, 134.8, 133.4, 130.7, 128.9, 128.3, 127.4, 122.3,
120.7, 61.3, 53.2, 39.3, 30.6, 22.4, 14.6, 7.7, 6.0. MS (ESI): m/z
+
calc’d for C25H30N3O5S2 : 516.1627 [M+H+]; found: 516.1623.
5.1.19. Ethyl ((3-(3-(2-(2-(tert-butyl)-1H-imidazol-
1-yl)acetyl)phenyl)-5-isobutylthiophen-2-
yl)sulfonyl)carbamate (23)
A solution of butyl ((3-(3-(2-(2-(tert-butyl)-1H-imidazol-1-
yl)acetyl)phenyl)-5-isobutylthiophen-2-yl)sulfonyl)carbamate
(13) (10.0 mg, 0.0179 mmol) in ethanol (0.7 mL) was heated at
100 °C for 30 min. The reaction mixture was evaporated and the
residue purified by automated silica flash chromatography (5-
10% methanol in dichloromethane. Product fractions were
concentrated under reduced pressure and dried under vacuum
overnight to give 4.7 mg (49%) of ethyl ((3-(3-(2-(2-(tert-butyl)-
1H-imidazol-1-yl)acetyl)phenyl)-5-isobutylthiophen-2-
1
yl)sulfonyl)carbamate. H NMR (400 MHz, CDCl3) δ 8.72 (s,
life (t ) and in vitro intrinsic clearance (Clint) were calculated
½
1H), 7.97 (d, J = 7.8, 1H), 7.73 (d, J = 7.8 Hz, 1H), 7.54 (t, J =
7.8 Hz, 1H), 7.15-7.08 (m, 2H), 6.81 (s, 1H), 5.87 (s, 2H), 3.98
(q, J = 7.1 Hz, 2H), 2.70 (d, J = 7.0 Hz, 2H), 2.00 – 1.87 (m, 1H),
1.40 (s, 9H), 1.10 (t, J = 7.1 Hz, 3H), 1.00 (d, J = 6.6 Hz, 6H).
13C NMR (101 MHz, CDCl3) δ 191.5, 155.1, 153.3, 149.4, 142.1,
136.1, 135.7, 134.6, 133.3, 130.8, 129.2, 128.4, 127.5, 124.4,
120.9, 61.9, 55.2, 39.4, 33.8, 30.7, 29.2, 22.4, 14.5. MS (ESI):
using previously published models.52,53
5.3. Metabolic stability in human hepatocytes
Metabolic stability assay was performed in cryopreserved
hepatocytes from one healthy donor.54 A simple approach for
restoration of differentiation and function in cryopreserved
human hepatocytes.) Thawed hepatocytes were resuspended in
pre-warmed Williams medium E. The incubation was carried out
on a heater-shaker at 37°C, using about 350 000 cells/ml and a
final compound concentration of 1 µM. Samples were withdrawn
at different time-points and the reaction was quenched by adding
ice-cold acetonitrile containing 50 nM Warfarin as internal
control. Samples were centrifuged at 3000 rpm for 15 min and
before analysis of parent compound remaining by LC-MS/MS as
described above.
+
m/z calc’d for C26H34N3O5S2 : 532.1940 [M+H+]; found:
532.1932.
5.1.20. N-((3-(3-(2-(2-(tert-butyl)-1H-imidazol-1-
yl)acetyl)phenyl)-5-isobutylthiophen-2-
yl)sulfonyl)acetamide (24)
To
a
solution of 3-(3-(2-(2-(tert-butyl)-1H-imidazol-1-
yl)acetyl)phenyl)-5-isobutylthiophene-2-sulfonamide (16.5 mg,
0.0359 mmol) in dichloromethane (1 mL) was added first DIEA
(25 µL, 0.14 mmol) then acetyl chloride (3.1 µL, 0.043 mmol)
and the reaction mixture was stirred at room temperature for 90
min. The reaction mixture was washed with sat NH4Cl (aq) (3
mL) and brine (3 mL). The organic layer was dried (Isolute HM-
N), filtered and evaporated and the residue purified by automated
5.4. CYP inhibition assay
The activity of human cytochrome P450 (CYP) was
determined in human liver microsomes by following the
formation of a metabolite of the probe substrate. Substrates and
their concentrations for the incubation were: CYP2B6,
Bupropion (80 µM); CYP2C9, Diclofenac (5 µM); CYP2D6
Bufuralol (2 µM); CYP3A4, Midazolam (2µM). The incubations
contained 0.1 mg/ml human liver microsomes in 0.1 M
phosphate buffer (pH 7.4) and 1 mM NDAPH. The potency of
compound on the CYP enzymes was tested at concentrations
silica
flash
chromatography
(5-10%
methanol
in
dichloromethane). Product fractions were concentrated under
reduced pressure and dried under vacuum to give 10.2 mg. (57%)
of N-((3-(3-(2-(2-(tert-butyl)-1H-imidazol-1-yl)acetyl)phenyl)-5-
1
isobutylthiophen-2-yl)sulfonyl)acetamide. H NMR (400 MHz,
CDCl3) δ 8.59 (s, 1H), 7.98 – 7.91 (m, 1H), 7.75 – 7.69 (m, 1H),
7.57 – 7.48 (m, 1H), 7.00 – 6.94 (m, 2H), 6.78 (s, 1H), 5.75 (s,

between 0.07 to 50 μM. The test compound and positive control
inhibitors were dissolved in DMSO, while substrate compounds
were dissolved in acetonitrile or methanol. The final solvent
concentration in all incubations was below 0.5%. In reversible
inhibition incubations, inhibitor or buffer control and substrate
were premixed with microsomes and buffer for 3 min before
addition of 1.0 mM NADPH, which initiated the reactions. The
final incubations were performed in at 37°C in a shaking
incubator block. Reactions were terminated by adding icecold
acetonitrile containing 100 nM warfarin as internal standard to
the incubations. Samples were thereafter placed on ice for at least
15 min before centrifugation at 3500 rpm, 4 °C for 20 min. The
supernatants were collected and analysed by LC-MS/MS as
described above.
U46619 (300nM) was added to attain a maximum contractile
response. When the response plateaued, tissues were washed with
Krebs solution a number of times until the tissue returned to
baseline. Tissues were then pre-contracted to 30-40% maximal
contraction of U44619, after which a concentration response
curve was performed to the vasorelaxant effects of either 2 (C21)
or ligand 20 in parallel experiments. A time control was also
obtained whereby a vessel was precontracted by U46619 for the
duration of the time taken to perform concentration response
curves. In separate experiments, ligand 20 was also tested as a
potential AT2R antagonist against 2 (C21). To this end, tissues
were pre-incubated with 20 for 30 min prior to U46619-induced
contraction and subsequent concentration response curves to 2
(C21).
5.5. Caco-2 cell permeability assay
5.8. Computational methods
5.8.1. Modelling experiments
Permeability measurements were performed as previously
described.55 Caco-2 cell monolayers (passage 94-105) were
grown on permeable filter supports and used for transport studies
on day 21 after seeding. Prior to start of the experiment, culture
medium was replaced with preheated Hank’s Balanced Salt
Solution (HBSS) buffered with HEPES to pH 7.4. Apparent
permeability was measured, at a compound concentration of 5
µM, in both apical-to-basolateral (A-B) and basolateral-to-apical
(B-A) direction at pH 7.4 using a shaking speed of 500 rpm.
Immediately after the start of the experiment, a sample was
removed from the donor compartment (C0), and subsequent
samples were taken from the receiver compartment at 15, 30 and
60 min. At the end of the experiment (t=60 min), a sample was
removed from the donor chamber (Cf) for mass balance
calculation. Each experimental condition was assayed in
triplicate. Compound concentration was determined by LC-
MS/MS as described above.
The crystal structure of the active-like human AT2R was
retrieved from the Protein Data Bank (PDB code 5UNG with
antagonist L-161,638.56,57 and was subject to preparation and
minor modifications with the Schrödinger suite (Schrödinger
Release 2017-3, Schrödinger, LSS, New York, NY, 2017),
including (i) deletion of the engineered B562RIL protein (fused
to the truncated N-terminus); (ii) addition of protons, assessment
of the rotamers for Asn/Gln/His residues, and protonated state for
titratable residues, resulting in all Asp, Gln, Lys, and Arg
residues assigned to their default charged state and all His
modelled as neutral with the proton on Nδ; (iii) addition of
missing side chains, modelling the most probable conformer
based on additional crystal structures of AT2 and the related AT1
receptor.
5.8.2. Ligand docking
Ligands from Figure 2 and 4 were built and their 3D
conformation were optimized using the Maestro graphical
interface and the LigPrep utility from the Schrödinger suite
(Schrödinger Release 2017-3: Maestro, Schrödinger, LSS, New
York, NY, 2017; Schrödinger Release 2017-3: LigPrep,
Schrödinger, LSS, New York, NY, 2017). This method also
allowed determination of their most probable protonation state at
physiological pH, with a net negative change localized on the
sulfonylcarbamate group in all cases. Docking was performed
with Glide SP using default settings (Schrödinger Release 2017-
3: Glide, Schrödinger, LSS, New York, NY, 2017).58–60 The
docking grid centroid was placed taken as reference the
coordinates of the co-crystallized ligand L-161,638, and
expanding the cubic grid box was set to 30 Å on each
dimensions. The selection of poses was done on the basis of a
double criterion, combining the highest possible scoring while
looking for the consensus among all ligands in the series.
5.6. Radioligand binding assays
All synthesized ligands were evaluated in a radioligand
binding assay performed by Eurofins Cerep SA, France, by
displacing [125I][Sar1Ile8]-angiotensin II from HEK-293 cells
expressing human AT2R. [Sar1Ile8]-angiotensin II (Sarile) acts as
a nonselective AT2R agonist.47 The affinity was determined using
a seven-point dose-response curve and the measurements were
performed in duplicates, the AT2R selective antagonist PD-
123,319 was used as reference compound. The compounds were
also evaluated for inhibition of [125I][Sar1Ile8]-angiotensin II
binding to human AT1R expressed in HEK-293 cells. For AT1R
the percent inhibition was determined at 10 µM, in duplicates,
with the endogenous ligand (angiotensin II) used as reference.
5.8.3. Membrane insertion and Molecular Dynamics
equilibration
5.7. Vasorelaxation
Each Ligand receptor complex obtained in the previous stage
was subject to an MD equilibration following the PyMedDyn
protocol, as implemented in a GPCR-ModSim web server.62,63
Briefly, the receptor-ligand complex was inserted in a pre-
equilibrated membrane consisting of 1-palmitoyl-2-oleoyl
phosphatidylcholine (POPC) lipids, with the transmembrane
(TM) bundle aligned to its vertical axis. The simulation box was
created with a hexagonal-prism geometry, which was soaked
with bulk water and energy-minimized using the OPLS-AA force
field for proteins and ligands, combined with the Berger
parameters for the lipids.62,64–66 It follows a molecular dynamics
Male FVB/N mice (approximately 10-12 weeks old) were
humanely killed by isoflurane inhalation and the thoracic aorta
was cut into 2-3 mm lengths and mounted for isometric force
recording in myographs (model 610M; DMT, Australia)
connected to a Powerlab 8/35 channel recorder (ADInstruments,
Australia). Vessels were maintained in physiological salt solution
comprising (in mM) NaCl 118, KCl 4.7, KH2·PO4 1.2,
MgSO4·7H2O 1.2, CaCl2 2.5, NaHCO3 25 and glucose 11.7) at 37
°C with carbogen (95% O2 and 5% CO2). The tension of the
vessels was gradually stretched to 0.5 g resting tension over
approximately 30 minutes. After an additional 15 min
equilibration at 0.5 g, the thromboxane A2 receptor agonist

doi:10.1016/j.coph.2015.01.004
equilibration using periodic boundary conditions (PBC) and the
NPT ensemble with the GROMACS simulation package.64
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The first phase consists of 2.5 ns with a gradual release of
harmonic restraints on protein (and ligand) heavy atoms. The
second phase consists of free MD for another 2.5 ns, except for
weak distance restraints between 24 pairs of interacting residues
corresponding to conserved positions within the TM bundle of
class-A GPCRs with a structural role.63,67 The final snapshot was
energy minimized and retained for analysis and figures.
doi:10.1097/01.hco.0000166596.44711.b4
Gallinat S, Yu M, Dorst A, Unger T, Herdegen T. Sciatic nerve
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nerves. Mol Brain Res. 1998;57(1):111-122. doi:10.1016/S0169-
328X(98)00079-5
Nakajima M, Hutchinson HG, Fujinaga M, et al. The angiotensin II
type 2 (AT2) receptor antagonizes the growth effects of the AT1
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16.
17.
18.
19.
Acknowledgments
We thank the Kjell and Märta Beijer Foundation and the
Swedish Brain Foundation for financial support, the SciLifeLab
Drug Discovery and Development Platform for support with
compound syntheses and ADME evaluations and the Swedish
National Infrastructure for Computing (SNIC) for computational
resources. This work was supported in part by the National
Health and Medical Research Council (NHMRC) of Australia
(GNT1127792) to REW. Centre for Preclinical Research and
Technology, Medical University of Warsaw infrastructure was
used in the conduction of the study.
20.
21.
22.
23.
Appendix A. Supplementary material
Supplementary data to this article can be found online at
https://doi.
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Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: