A convenient transesterification method for synthesis of AT2 receptor ligands with improved stability in human liver microsomes

Article abstract of DOI:10.1016/j.bmcl.2017.11.042

A series of AT₂R ligands have been synthesized applying a quick, simple, and safe transesterification-type reaction whereby the sulfonyl carbamate alkyl tail of the selective AT₂R antagonist C38 was varied. Furthermore, a limited number of compounds where acyl sulfonamides and sulfonyl ureas served as carboxylic acid bioisosteres were synthesized and evaluated. By reducing the size of the alkyl chain of the sulfonyl carbamates, ligands 7a and 7b were identified with significantly improved in vitro metabolic stability in both human and mouse liver microsomes as compared to C38 while retaining the AT₂R binding affinity and AT₂R/AT₁R selectivity. Eight of the compounds synthesized exhibit an improved stability in human microsomes as compared to C38.

Full text of DOI:10.1016/j.bmcl.2017.11.042

Bioorganic & Medicinal Chemistry Letters 28 (2018) 519–522
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A convenient transesterification method for synthesis of AT2 receptor
ligands with improved stability in human liver microsomes
Johan Wannberg a, Rebecka Isaksson b, Ulf Bremberg a, Maria Backlund c, Jonas Sävmarker d,
Mathias Hallberg e, Mats Larhed a,
⇑
a Department of Medicinal Chemistry, Science for Life Laboratory, BMC, Uppsala University, P.O. Box 574, SE-751 23 Uppsala, Sweden
b Department of Medicinal Chemistry, Division of Organic Pharmaceutical Chemistry, BMC, Uppsala University, P.O. Box 574, SE-751 23 Uppsala, Sweden
c Department of Pharmacy, Uppsala University, Uppsala, Sweden and Uppsala University Drug Optimization and Pharmaceutical Profiling Platform (UDOPP), Science for Life
Laboratory, Uppsala, Sweden
d The Beijer Laboratory, Department of Medicinal Chemistry, BMC, Uppsala University, P.O. Box 574, SE-751 23 Uppsala, Sweden
e The Beijer Laboratory, Department of Pharmaceutical Biosciences, Division of Biological Research on Drug Dependence, BMC, Uppsala University, P.O. Box 591, SE-751 24
Uppsala, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of AT2R ligands have been synthesized applying a quick, simple, and safe transesterification-type
reaction whereby the sulfonyl carbamate alkyl tail of the selective AT2R antagonist C38 was varied.
Furthermore, a limited number of compounds where acyl sulfonamides and sulfonyl ureas served as car-
boxylic acid bioisosteres were synthesized and evaluated. By reducing the size of the alkyl chain of the
sulfonyl carbamates, ligands 7a and 7b were identified with significantly improved in vitro metabolic sta-
bility in both human and mouse liver microsomes as compared to C38 while retaining the AT2R binding
affinity and AT2R/AT1R selectivity. Eight of the compounds synthesized exhibit an improved stability in
human microsomes as compared to C38.
Received 12 September 2017
Revised 15 November 2017
Accepted 24 November 2017
Available online 24 November 2017
Keywords:
Angiotensin II type 2 receptor antagonists
AT2R antagonists
Sulfonyl carbamates
Transesterification
Ó 2017 Elsevier Ltd. All rights reserved.
Liver microsomes
The octapeptide hormone angiotensin II (Ang II) is the main
effector of the Renin-Angiotensin-Aldosterone system (RAAS).
Ang II mediates its effects through activation of two G-protein cou-
pled receptors (GPCRs), the angiotensin II type 1 (AT1R) and the
angiotensin II type 2 (AT2R) receptors. AT1R is involved in regula-
tion of blood pressure and electrolyte balance and is a well-estab-
lished drug target for the treatment of hypertension and heart
failure (angiotensin receptor blockers, ARBs). The first ARB, losar-
tan, was introduced into the market in 1995.1 The antihypertensive
angiotensin converting enzyme inhibitors (ACE inhibitors, e.g. cap-
topril introduced into the market 1978) act by suppressing the for-
mation of Ang II.2 In contrast to the well-investigated AT1R, less is
known about the role of the AT2R. It is abundant during fetal devel-
opment but only very low levels of AT2R can be detected in healthy
adults. However, in certain pathological conditions e.g. myocardial
infarction, heart and renal failure, and some brain injuries, a pro-
nounced upregulation of AT2R is frequently observed. Thus, the
receptor is upregulated in areas of tissue damage and it is postu-
lated that AT2R is important in tissue repair. The physiological
actions mediated by AT2R have been reviewed.3–10
The use of AT2R as a potential drug target has recently seen two
different approaches and produced compounds that have reached
clinical trials. The selective AT2R agonist C21/M024 (Vicore
Pharma) discovered by Anders Hallberg’s group at our laboratory11
has entered Phase I clinical trials for the indication idiopathic pul-
monary fibrosis. The malonic acid sulfonamide derivative MP-157,
a selective AT2R agonist from Mitsubishi Tanabe Pharma, is also in
Phase I clinical trials in Europe and aimed for the cardiovascular
system.12 The AT2R antagonist EMA401 (Spinifex/Novartis) has
completed a phase II clinical trial with positive results in patients
with postherpetic neuralgia,13 a form of chronic neuropathic
pain.14,15 AT2R antagonists as potential new chemical agents for
the treatment of peripheral neuropathic pain is based on the find-
ings that AT2R exhibits a higher expression in damaged nerve tis-
sue e.g. in the dorsal root ganglia (DRG). Furthermore, activation
of these AT2Rs by the endogenous ligand Ang II potentiates pain
signaling by increasing neurite length and density, and by nocicep-
tor sensitization by phosphorylation of nociceptor ion channels on
the DRG via AT2R secondary messenger pathways.16–20
⇑
Corresponding author.
E-mail address: mats.larhed@orgfarm.uu.se (M. Larhed).
https://doi.org/10.1016/j.bmcl.2017.11.042
0960-894X/Ó 2017 Elsevier Ltd. All rights reserved.

520
J. Wannberg et al. / Bioorganic & Medicinal Chemistry Letters 28 (2018) 519–522
1) nBuLi
B(OiPr)3
ZnCl
N
B
O
O
-78 °C to 0 °C
THF
O
O O
S
O
O
O
O
O
S
Pd(tBu3P)2 (1.5 mol%)
S
NH
NH
NH
S
S
S
THF, toluene
2) MIDA
Br
130 oC, 15 min
toluene/DMSO
reflux
3
2
1
93% yield over
62%
2 steps (4 g scale)
Br
imidazole
100 oC, DMF
N
N
Br
4 74%
Br
N
N
N
N
TFA
40 oC
PdCl2dppf (5 mol%)
O
O
+
O
4
3
O
S
S
K2CO3, DME, H2O
120 oC, 1 h
NH2
N
S
S
H
5 83%
6 89%
N
O
N
Cl
O
O
O
O
O
S
DIEA, DCM, r.t.
N
S
H
86%
C38
Scheme 1. Improved synthesis of the AT2R antagonist C38.
We published the first selective drug-like AT2R antagonists in
2012, among them compound C38 which in structure closely
relates to the AT2R agonist C21.21–23 Profiling C38 in various ADME
in vitro assays revealed a relatively short half-life of C38 in human
liver microsomes indicating poor metabolic stability.
A large number of structural modifications at several different
sites of the AT2R antagonist C38 were explored but no efforts to
alter the butylsulfonyl carbamate moiety were conducted. We
had previously in the AT2R agonist project found the n-butyl chain
superior in producing potent compounds in all series studied11,24
and thus the n-butyl chain was initially kept intact.25
The recent discovery that alkylsulfonyl carbamates can be inter-
converted to alternative alkylsulfonyl carbamates by a transester-
ification-type reaction by simply heating in an alkyl alcohol,26,27
gave us the incentive to explore this part of the C38 scaffold. In
addition, we were encouraged to explore the impact of using acyl
sulfonamides and sulfonyl ureas as conceivable replacements for
the sulfonyl carbamate group.
Scheme 2. Synthesis of new AT2R ligands.
C38 in enough quantity to allow compound profiling as well as
use as starting material for variations of the alkylsulfonyl carba-
mate motif (Scheme 1).
During the efforts of profiling the properties of C38, a larger
batch of the compound was required. This was achieved through
Essentially employing our previously developed transesterifica-
tion/transcarbamoylation method, C38 was heated in various alkyl
alcohols (straight and branched) of various sizes at 100 °C for 60
min (Scheme 2).26 The resulting products 7a–l (Table 1) were suc-
cessfully isolated in 26–85% yield,33 except for the reaction with t-
BuOH where only primary sulfonamide was isolated. The tert-
butylsulfonyl carbamate 7i was instead isolated by reacting the
primary sulfonamide with Boc anhydride. Also 2-methoxyethanol
requires a special permit for use and handling in Sweden and as
a consequence 2-methoxyethyl chloroformate was coupled with
6 to give 7m (Scheme 2, Table 1).
a
modified version of the previously published procedure
(Scheme 1). First, a Negishi coupling of 5-bromo-N-(tert-butyl)
thiophene-2-sulfonamide 1 with isobutylzinc under microwave
heating28 provided N-(tert-butyl)-5-isobutylthiophene-2-sulfon-
amide 2 in reasonable yield. This intermediate 2 was then con-
verted to the MIDA boronate 3 in excellent yield. Compared to
the corresponding boronic acid (semi-solid, stored in freezer) the
MIDA boronate 3 is much easier to handle and store (solid, stable
at ambient temperature under air).29 The MIDA boronate 3 was
subjected to a Suzuki coupling with 1-(3-bromobenzyl)-1H-imida-
zole 4 producing 5 in very good yield.30 Deprotection of the tert-
butyl sulfonamide 5 was performed in neat TFA to give the primary
sulfonamide 6 in quantitative yield and finally the primary sulfon-
amide was coupled with butyl chloroformate to give the desired
Heating C38 in primary or secondary alkylamines at 120–150 °C
allowed for the formation of sulfonyl ureas 7n (18%)33 and 7o (59%)
by aminolysis of the sulfonyl carbamate (Table 1). Acylsulfon-
amides 7p–7r were synthesized from primary sulfonamide 6 by

J. Wannberg et al. / Bioorganic & Medicinal Chemistry Letters 28 (2018) 519–522
521
Table 1
Receptor binding results and in vitro metabolic stability.
Compound
C38
R
AT2R Ki (nM)a
AT1R (%inh@10 mM)a
HLM t½ (min)b
MLM t½ (min)b
270
400
7
8
12
77
70
7a
220
7b
7c
300
290
5
61
11
180
82
10
O
O
7d
7e
390
160
À3
6.5
17
17
79
18
O
7f
440
16
43
47
O
O
7g
7h
130
230
14
8
7.0
4.9
38
13
O
7i
270
17
18
28
O
O
7j
140
180
13
7
9.4
5.6
59
31
7k
O
O
7l
730
480
400
3
6.6
27
20
27
CF3
7m
7n
10
15
120
56
O
O
N
H
7o
7p
2300
340
13
18
ndc
20
ndc
66
N
7q
7r
300
280
13
3
4.8
7.0
6.9
8.9
a
b
c
Radioligand binding assays performed by Eurofins Cerep SA, France.
In vitro half-life (t1/2) in human (HLM) and mouse liver microsomes (MLM) was determined as described in the Supplementary Information.
Not determined.
coupling with either acid chlorides or anhydrides (Scheme 2,
Table 1).
had been found with the previous binding assay which was based
on pig membrane preparations (270 nM vs 19 nM). The reason for
drop in potency when changing assay is not clear but it has been
noticed and discussed previously.32
Testing of the alkyl sulfonylcarbamate series for AT2R binding
revealed a relatively flat structure–activity relationship. The alkyl
chain could be shortened and branched without any significant
change in AT2R binding affinity. Importantly, a good selectivity
compared to AT1R was maintained as all compounds showed less
than 20% inhibition of [125I][Sar1,Ile8]-angiotensin II binding to
AT1R at 10 mM. The in vitro metabolic stability of the compounds
were subsequently determined using human liver microsomes
The AT2R binding assay previously used by our group (mem-
brane preparations from pig uterus myometrium)31 is no longer
available in house.21,24,25 We therefore needed to search for an
alternative source of binding data. The decision was made to
employ membranes from HEK-293 cells expressing human AT2R
(HEK293-hAT2R) with [125I][Sar1,Ile8]-angiotensin II as the radioli-
gand as assay using a seven-point dose–response curve in dupli-
cate measurements at each concentration (Eurofins Cerep SA,
France). Upon re-testing the compound C38 in the new assay, a
10–15-fold drop in potency (Ki) was observed as compared to what

522
J. Wannberg et al. / Bioorganic & Medicinal Chemistry Letters 28 (2018) 519–522
(HLM). From the obtained data we found a clear relationship
between size and lipophilicity of the carbamate chain and the
HLM stability of the compounds. Reducing logP/logD clearly, and
perhaps not surprisingly, increased the metabolic stability. The sta-
bility of the compounds were also assessed in mouse liver micro-
somes (MLM) to get an estimate on the likelihood of exposure
after administration of the compounds in murine models of neuro-
pathic pain. As seen in Table 1, this series of derivatives are in gen-
eral less prone to undergo metabolism in MLM than in HLM.
Notably though, the methyl (7a) and ethyl (7b) carbamates show
good metabolic stability in both human and mouse liver micro-
somes. This observation combined with the fact that 7a and 7b
are more or less equipotent to C38 with regard to AT2R binding
affinity and AT2R/AT1R selectivity make them valuable tools for
in vivo models of neuropathic pain in mouse.
Supplementary data
Supplementary data (experimental procedures, analytical data)
associated with this article can be found, in the online version, at
https://doi.org/10.1016/j.bmcl.2017.11.042.
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Acknowledgments
We thank the SciLifeLab Drug Discovery and Development plat-
form, Uppsala University and the Kjell and Märta Beijer Foundation
for support.