A Series of Analogues to the AT2R Prototype Antagonist C38 Allow Fine Tuning of the Previously Reported Antagonist Binding Mode

Article abstract of DOI:10.1002/open.201800282

We here report on our continued studies of ligands binding to the promising drug target angiotensin II type 2 receptor (AT₂R). Two series of compounds were synthesized and investigated. The first series explored the effects of adding small subs

Full text of DOI:10.1002/open.201800282

Full Papers
DOI: 10.1002/open.201800282
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A Series of Analogues to the AT2R Prototype Antagonist
C38 Allow Fine Tuning of the Previously Reported
Antagonist Binding Mode
Rebecka Isaksson,[a] Jens Lindman,[a] Johan Wannberg,[b] Jessica Sallander,[c]
Maria Backlund,[d] Dhaniel Baraldi,[e] Robert Widdop,[e] Mathias Hallberg,[f] Johan Åqvist,[c]
Hugo Gutierrez de Teran,[c] Johan Gising,[a] and Mats Larhed*[a]
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We here report on our continued studies of ligands binding to
the promising drug target angiotensin II type 2 receptor (AT2R).
Two series of compounds were synthesized and investigated.
The first series explored the effects of adding small substituents
to the phenyl ring of the known selective nonpeptide AT2R
antagonist C38, generating small but significant shifts in AT2R
affinity. One compound in the first series was equipotent to
C38 and showed similar kinetic solubility, and stability in both
human and mouse liver microsomes. The second series was
comprised of new bicyclic derivatives, amongst which one
ligand exhibited a five-fold improved affinity to AT2R as
compared to C38. The majority of the compounds in the
second series, including the most potent ligand, were inferior to
C38 with regard to stability in both human and mouse
microsomes. In contrast to our previously reported findings,
ligands with shorter carbamate alkyl chains only demonstrated
slightly improved stability in microsomes. Based on data
presented herein, a more adequate, tentative model of the
binding modes of ligand analogues to the prototype AT2R
antagonist C38 is proposed, as deduced from docking redefined
by molecular dynamic simulations.
1. Introduction
regulation; there are several drugs on the market for the
treatment of hypertension targeting proteins in RAAS. Although
a series of bioactive components are formed in RAAS, the
octapeptide angiotensin II (AngII) is considered to constitute
the major effector peptide.[1] Hence, inhibitors of the two
The renin-angiotensin-aldosterone system (RAAS) is well-known
for its role in fluid-electrolyte control and blood-pressure
[a] R. Isaksson, J. Lindman, Dr. J. Gising, Prof. Dr. M. Larhed
Department of Medicinal Chemistry
Uppsala University
SE-751 23, Uppsala, SWEDEN
E-mail: mats.larhed@ilk.uu.se
[b] Dr. J. Wannberg
SciLifeLab Drug Discovery & Development Platform, Medicinal Chemistry –
Lead Identification, Department of Medicinal Chemistry
Uppsala University
SE-751 23, Uppsala, SWEDEN
[c] Dr. J. Sallander, Prof. Dr. J. Åqvist, Dr. H. Gutierrez de Teran
Department of Cell and Molecular Biology
Uppsala University
SE-751 23, Uppsala, SWEDEN
[d] Dr. M. Backlund
SciLifeLab Drug Discovery & Development Platform, ADME of Therapeutics,
Department of Pharmacy
Uppsala University
SE-751 23 Uppsala, SWEDEN
[e] Dr. D. Baraldi, Prof. Dr. R. Widdop
Department of Pharmacology
Monash University
Clayton, Victoria, 3800 AUSTRALIA
[f] Prof. Dr. M. Hallberg
The Beijer Laboratory, Department of Pharmaceutical Biosciences
Uppsala University
SE-751 24, Uppsala, SWEDEN
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/open.201800282
© 2018 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This
is an open access article under the terms of the Creative Commons Attri-
bution Non-Commercial NoDerivs License, which permits use and distribu-
tion in any medium, provided the original work is properly cited, the use is
non-commercial and no modifications or adaptations are made.
Figure 1. The first selective nonpeptide AT2R agonist C21, the structurally
related AT2R antagonist C38, and the AT2R antagonist EMA401. [a] AT2R from
pig uterus membrane assay. [b] HEK-293 cells expressing human AT2R. [c]
See Ref. [19].
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Scheme 1. Synthesis of AT2R ligands 40–51. a) ZnCl2, isobutylmagnesium chloride, Pd(t-Bu3P)2, toluene, THF; b) 1. n-BuLi (4.5 eq.), triisopropyl borate, THF;
2. methyliminodiacetic acid, DMSO, toluene; c) imidazole, DCM (to 16–17) ; d) 1. TEA, MsCl, DCM; 2. imidazole, DMF (to 18–19, 22–23, 25–27); e) 1. thionyl
chloride, DCM; 2. imidazole DMF (to 20–21, 24); f) PdCl2(dppf), K2CO3, DME, H2O (to 28–33, 35–39); g) Pd(PPh3)4, K2CO3, EtOH, H2O, THF (to 34); h) 1. TFA;
2. butyl chloroformate, Na2CO3, DCM, H2O.
proteases important for formation of AngII (angiotensin con-
verting enzyme, ACE, and renin) or compounds acting as
antagonists at its receptor (i.e. the sartans) are well established
therapeutics. The angiotensin II type 1 receptor (AT1R) was long
believed to be the only mediator of the effects elicited by the
endogenous AngII. However, in the late 1980s the first evidence
of a second protein binding AngII appeared in the literature,
the angiotensin II type 2 receptor (AT2R).[2,3] This receptor
proved to be an enigmatic protein, which in recent years has
emerged as a promising new drug target.[4–6]
following tissue damage,[11,12] such as vascular[13] and neuronal
injury,[14] myocardial infarction[15–17] and brain ischemia.[18]
There are currently two AT2R ligands in clinical trials, for
different indications, again raising questions regarding the role
(s) of this protein. The AT2R antagonist EMA401 (Figure 1),
acquired by Novartis from Spinifex Pharmaceuticals Pty Ltd,
Australia, is in phase II clinical trials for peripheral neuropathic
pain.[19,20] The AT2R agonist C21 (Figure 1), developed in our
laboratory,[21] is entering phase II clinical trials as a potential
treatment for idiopathic pulmonary fibrosis. Recently published
reviews detail the discovery of C21,[22,23] and a large number of
structurally related AT2R ligands were subsequently
disclosed.[24–28] In 2012 we reported that shifting the imidazole
head group from the para position in the agonist C21 to meta
AT2R is predominantly expressed in fetal tissue, indicating
its important role in fetal development.[7,8] In adults AT2R is
mainly expressed in uterus, adrenal gland, smooth muscle,
heart, and kidney.[9,10] Notably, AT2R is strongly upregulated
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structure, as well as if replacing the phenyl for pyridine could
improve solubility. In the second series we explored a new head
group of C38 where the benzyl imidazole moiety was
exchanged for bicyclic amides. Previous work from our group
demonstrated that the affinity and selectivity of the ligands can
be retained by replacing the imidazole with amides;[29] with
these new bicyclic amides we continue our exploration of the
promising amide functionality by trying to ascertain the binding
conformation. A majority of the bicyclic compounds retained a
shortened sulfonyl carbamate chain, as we have recently found
this to be beneficial for metabolic stability.[35] The key building
block for the synthesis of both series was MIDA boronate 3, that
was synthesized according to the pathway previously published
by our group.[35] Microwave assisted Negishi coupling in a
sealed vial of 5-bromo-N-(tert-butyl)thiophene-2-sulfonamide
(1)] with in situ generated isobutylzinc chloride produced N-
(tert-butyl)-5-isobutylthiophene-2-sulfonamide (2) in 51%
yield.[36,37] Compound 2 was in turn converted to the MIDA
boronate (3) in 76% yield over 2 steps (Scheme 1). Substituted
benzylimidazoles and pyridinemethyl imidazoles 16–27 were
generated either via direct alkylation of 3-halo benzylbromides
(4–5, 10) with imidazole, or via chlorination or mesylation of the
3-bromo benzylalcohols (6–9, 11–15) and subsequent alkylation
with imidazole. Chlorination was initially tested for 3-bromo
benzylalcohols 6, 7, and 11, however generated no or low
yields. Mesylation was instead explored and yielded the desired
products 18, 19, and 23 in moderate to good yields (72%, 43%,
and 66% respectively). Once generated, the benzyl-/pyridine-
methyl imidazoles (16-27) were coupled with the MIDA
boronate 3 under Suzuki conditions in sealed vials either using
conventional heating (16–18) or microwave assisted (19–27),
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Scheme 2. Synthesis of compounds 57–64. a) Acetic anhydride, K2CO3, MeCN
(to 57–61); b) cyclopropanecarbonyl chloride, DIPEA, DCM (to 62); c) methyl
chloroformate, DIPEA, DCM (to 63); d) MsCl, DIPEA, DCM (to 64).
position switches the pharmacological profile, resulting in the
prototype antagonist C38 (Figure 1).[29,30]
The first crystal structure of AT1R binding antagonist
ZD7155 was published in 2015 by Zhang et al., enabling further
elucidation of the binding mode of AT1R ligands.[31,32] The crystal
structure allowed for comparison with our predicted inactive-
like homology model of both AT1R and AT2R, confirming the
general topology and residue location in the binding cavity of
our models. Minor structural changes resulting in an altered
pharmacological profile. which could be rationalized in our
AT2R homology model.[33] Zhang et al. recently published the
crystal structure of AT2R binding the selective AT2R antagonist
L-161,638, revealing a similar binding mode in comparison to
ZD7155 from the previously published AT1R crystal structure.[34]
The published structures of both AT1R and AT2R provide insight
in the structure-function relationship and allows design of new
selective ligands.
delivering the tert-butyl protected sulfonamides 28–39.[36,37]
A
tert-butyl deprotection followed by treatment with butyl
chloroformate resulted in the target compounds 40–51, gen-
erated in low to fair yields over 3 steps (3-28%). The low yields
obtained for some compounds (40, 42, 45, and 48 were isolated
in 3%, 3%, 4%, and 5% respectively) may relate to electronic
and/or steric properties effecting the Suzuki coupling efficiency.
The synthesis of the bicyclic compounds commenced with
the acylation of isoindoline (52, 54) and tetrahydroisoquinoline
(53, 55–56) yielding compounds 57–63 (Scheme 2). In addition,
isoquinoline 55 was mesylated to form compounds 64. Bicyclic
compounds 68–69 (Scheme 3) were generated by converting
bromophenylacetic acid (65) into secondary amides 66–67
using thionyl chloride and methylamine or ethylamine. These
were cyclized to the corresponding dihydroisoquinolinones 68–
69 via a Pictet-Spengler condensation/cyclization with parafor-
maldehyde, using Eaton's reagent (7.7 wt-% P2O5 in MeSO3H) to
replace the polyphosphoric acid traditionally used for this
cyclization.[38,39] The reaction was initiated with Eaton’s reagent
activating the aldehyde, followed by the N-alkylated amide
attacking the activated carbonyl carbon. Dehydration led to
alkylideneacetamide formation and after an intramolecular
electrophilic aromatic substitution, the dihydroisoquinolinones
68–69 were formed in 99% and 94% yield, respectively. Some
of the bicyclic amide 69 obtained was dimethylated to yield
compound 70. At this point the generated bicycles (57–64, 68–
We herein report the impact on AT2R affinity of chemical
modifications of the prototype AT2R antagonist C38, and
consequently adjust our previously reported AT2 receptor-
antagonist model to the new SAR data through computational
simulations using the newly published AT2R crystal structure.[34]
2. Results and Discussion
2.1. Chemistry
Two series of compounds were synthesized: the first series was
motivated by our ambition to explore the impact of adding
substituents to the central phenyl ring of the C38 core
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pound 94 and 96 that were isolated in 7% and 5% yield,
respectively.
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2.2. In Vitro Pharmacology
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Selectivity for AT2R over AT1R was retained for all compounds in
series 1, listed in Table 1. Introducing a fluoro substituent in
ortho (40) or meta (41) position relative to the methylene
imidazole group resulted in no change in affinity as compared
to C38. Interestingly, compound 42 with a fluoro atom in the
para position to the methylene imidazole exhibited a slight (3-
fold) reduction of affinity. This may relate to the altered
electronic properties of the molecule or possibly a steric
interaction. Replacing the fluoro substituent with methyl in the
ortho position to the methylene imidazole was well tolerated
(43). Adding a methyl to the meta or para position relative to
the imidazole (44 and 45) did, however, result in a similar
reduced affinity as was seen for compound 42, which further
indicates a possible steric interaction. A bromide (46) or the
larger trifluoromethoxy group (47) in the ortho position
generated compounds with similar affinity as C38. Interestingly,
a methoxy group (48) in the same position significantly
decreased affinity. This may relate to the slightly lower lip-
ophilicity of the methoxy as compared to the trifluoromethoxy
moiety, or possibly the electron-donating properties of the
substituent. Replacing the phenyl ring for pyridine and placing
the nitrogen of the pyridine in the ortho position relative to the
methylene imidazole moiety (49) furnished a slightly reduced
affinity while the equivalent meta and para pyridine analogues
(50, 51) exhibit similar affinity for AT2R as C38.
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Scheme 3. Synthesis for compounds 68–70. e) 1. Thionyl chloride, DMF,
toluene; 2. methylamine, H2O (to 66); f) 1. thionyl chloride, DMF, toluene;
2. ethylamine, H2O (to 67); g) paraformaldehyde, Eaton’s reagent; h) NaH,
MeI, DME.
70) were coupled with MIDA boronate
3 under Suzuki
conditions in sealed vials using conventional heating, to give
compounds 71–75, 77–82 (Scheme 4). Isoindoline 54 was also
coupled with MIDA boronate 3, generating compound 76.
Subsequent deprotection and reaction with butyl or ethyl
chloroformate produced the sulfonyl butyl carbamate products
83–86 and ethyl carbamate products 87–96 in moderate to
good overall yield (15%–82%), with the exception of com-
Scheme 4. Synthesis of AT2R ligands 83–96. i) PdCl2(dppf), K2CO3, DME, H2O; j) 1. TFA; 2. butyl chloroformate, TEA, DCM (to 83–87); k) 1. TFA; 2. ethyl
chloroformate, DMAP, DIPEA, DCM (to 88–96).
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C102 the affinity dropped almost 200-fold (420 nM in human
AT2R vs 2.2 nM in pig AT2R). The affinity for human AT2R in HEK-
293 cells for the three tested amides were all in the same range
as C38 in the same assay. Similar to the compounds in series 1,
the bicyclic derivatives in series 2 (Table 2) also displayed a high
Table 1. Analogues of C38 with substituents on the central phenyl ring,
synthesized via Scheme 1.
1
2
3
4
5
Cmpd
Structure
Ki hAT2R [nM][a]
[%] Inhibition of
selectivity for AT2R. Isoindoline 83 showed
a significant
hAT1R at 10 μM[b]
6
reduction in affinity compared to C93 (>1500 nM vs 110 nM),
suggesting a highly unfavorable orientation of the amide
group. The isoquinoline 84, with similar amide orientation, also
displayed reduced affinity. A more favorable amide orientation
was obtained in isoindoline 85 and tetrahydroisoquinolines 86
and 87. Compound 85 and 87 displayed similar affinity as C38,
while a 5-fold improvement of affinity was seen for compound
86 compared to C38 (and a 2-fold improvement compared to
amides C93 and C97). Having identified the more favorable
orientation of the amide, we explored compounds with a
shorter and less lipophilic sulfonamide carbamate chain, as well
as a few other moieties binding to the amide nitrogen.
Interestingly the sulfonyl ethyl carbamate (88) resulted in a
slightly decreased affinity as compared to the sulfonyl butyl
carbamate (85).
The affinity was further decreased when introducing a
bicyclic N-ethyl carbamate in combination with the sulfonamide
ethyl carbamate (89), displaying a 20-fold drop in affinity as
compared to compound 86. Comparing compound 90 and
compound 86, a slight decrease in affinity could also be
detected with the shorter carbamate chain (120 nM vs 56 nM).
This indicates the length of the sulfonyl carbamate chain may
be more significant in the bicyclic scaffold than in the C38-
scaffold, where our previous studies showed a large tolerability
in this moiety.[35] The cyclopropane carboxamide 91 had a
similar affinity as the amides C93/C97 and the bicyclic amide
90. Interestingly, the bicyclic methyl carbamate 92 was well
tolerated in the binding cavity, in contrast to the ethyl
carbamate 89 where an almost 5-fold decrease in affinity was
observed. The amide bioisosteric mesyl group was introduced
in compound 93, which rendered a reduced affinity to AT2R.
Lastly, we had synthesized a three compounds where the
carbonyl of the amide function was incorporated as a lactam,
locking the amide functionality in a different conformation
compared to the previously synthesized bicycles, resulting in
the lactam derivatives 94–96. These three compounds all
demonstrated a significantly reduced affinity to AT2R.
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270
7
C38
40
(19[c])
IC50 >10 000 nM[e]
IC50 =694 nM[d]
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IC50 >10 000 nM[e]
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800
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9.7
[a] Radioligand displacement from hAT2R in membranes from HEK-293 cells
overexpressing hAT2R (assay 1). N=6 for C38 and 40, N=2 for all other. [b]
Inhibition of radioligand binding from hAT1R expressed in HEK-293 cells (assay
1). [c] Radioligand displacement from AT2R in pig uterus membrane.[29] [d]
Radioligand displacement from hAT2R expressed in HEK-293 cells (assay 2). [e]
Radioligand displacement from hAT1R expressed in HEK-293 cells, ligands
were not active on hAT1R at the concentrations tested (assay 2). N=9.
To ensure quality we assessed three of the compounds and
the AT2R agonist C21 in an orthogonal second assay using
whole cells, performed in a different laboratory. The IC50 values
at both hAT2R and hAT1R were examined for compounds C38,
40, and 86. The AT2R agonist C21 exhibited an IC50 of 1.47 nM
at hAT2R in the orthogonal assay, correlating well with data
obtained from the standard assay (Ki =1.10 nM). Comparing
C38 and 40, the IC50 at hAT2R was improved 3-fold by
introducing the para-fluoro, resulting in an estimated 46-fold
selectivity for hAT2R over hAT1R (cf. 14-fold hAT1R/hAT2R
selectivity for C38). It is notable that C38 and 86 exhibited
similar IC50 values in the orthogonal assay (C38; IC50 =694 nM
and 86; IC50 =818 nM, respectively) although the affinities
As we have reported on previously, affinity to human AT2R
expressed in HEK-293 cells for C38 is reduced as compared to
affinity for AT2R in pig uterus membrane (270 nM vs 19 nM).[29,35]
When evaluating affinity of the previously published, and very
promising, amides C93, C97, and C102 (Table 2) for human
AT2R in HEK-293 cells we note a slight decrease in affinity for
compound C93 (110 nM in human AT2R vs 29 nM in pig AT2R).
For compound C97 the affinity did not change (110 nM in
human AT2R vs 83 nM in pig AT2R), but notably for compound
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Table 2. Bicyclic amides with N-ethoxycarbonyl or N-butoxycarbonyl sulfonamide moieties synthesized according to Scheme 2–4.
1
2
3
Cmpd Structure
Ki hAT2R
%-Inhibition of hAT1R at
Cmpd Structure
Ki hAT2R
%-Inhibition of hAT1R at
[nM][a]
10 μM[b]
[nM][a]
10 μM[b]
4
5
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7
8
9
110
C93
22%
23%
30%
88
89
90
870
7.7%
26%
NDI[g]
(29[c])
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110
C97
1300
120
(83[c])
420
C102
(2.2[c])
83
84
85
>1500[d]
15%
22%
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92
120
290
28%
15%
700
360
56
7.5%
34%
93
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760
19%
86
87
1300
1.5%
IC50 =818 nM[e] IC50 >10 000 nM[f]
270
–
45%
95
96
850
11%
15%
–
–
–
1100
[a] Radioligand displacement from hAT2R in membranes from HEK-293 cells overexpressing hAT2R (assay 1). N=2. [b] Inhibition of radioligand binding from
hAT1R expressed in HEK-293 cells (assay 1). [c] Radioligand displacement from AT2R in pig uterus membrane.[29] [d] Ki estimated to more than 1500 nM, IC50
was determined to be >3 000 nM. [e] Radioligand displacement from hAT2R expressed in HEK-293 cells (assay 2). [f] Radioligand displacement from hAT1R
expressed in HEK-293 cells, ligands were not active on hAT1R at the concentrations tested (assay 2). N=9 [g] NDI=no detectable inhibition.
differed considerably in the standard assay applied herein (C38;
Ki =270 nM and 86; Ki =56 nM, respectively).
2.3. Molecular Modelling
Using the published crystal structure of AT2R[34], a comprehen-
sive docking exploration with GLIDE revealed a common
binding pose for the compounds in the first series (Table 1),
which in each case was refined by MD equilibration.
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Figure 2 depicts the binding mode for the most potent
compound (41) in series 1, overlaid with the co-crystalized AT2R
antagonist L-161,638. The sulfonyl carbamate is anchored via
salt-bridge interactions with R1824.64 and K2155.42 and a hydro-
gen bond of the carbonyl with T1253.33 (the Ballesteros-
Weinstein generic amino acid numbering scheme is indicated
as superscript[40]). The phenyl ring is surrounded by W1002.60
and L1243.32, allowing the imidazole substituent to be accom-
modated within a hydrophobic cluster composed by residues
Y511.39, Y1032.64, Y1042.65, Y1082.69, P3017.36 and I3047.39. The
isobutyl group is placed in a deeper region of the trans-
1
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membrane cavity, defined by residues L1243.32
, ,
M1273.35
W2696.48, F2726.51, and F3087.43. Finally, the ethyl substituent on
the sulfonyl carbamate is located in the cavity between trans-
membrane helices TM3-TM5 defined by the residues Pro1774.59
,
Met2145.41, Lys2155.42, and F1293.37
.
The binding mode proposed explains to a big extent the
SAR for the compounds in the first series (Figure 3), which are
structurally related to the prototype antagonist C38. The
Figure 2. Compound 41 (blue) biding to the AT2 receptor (gray), overlaid
with the co-crystalized ligand L-161,638 (PDB 5UNG, gray sticks).
Figure 3. The docked ligands (40–45) in the most common pose on the modeled conformation of the AT2R. The ligands are color coded based on the binding
affinities, blue – high, green – moderate, and orange – low binding affinity. The N-terminal, EL3 and parts of TM6-TM7 of the AT2R are not shown for better
clarity.
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experimental affinities (Table 1) show that introduction of a
fluoro substituent in ortho position (40) relative to the
methylene imidazole substituent is well tolerated, as is
introducing a methyl group in the ortho position to the
imidazole (43). This is consistent with the modelling of these
substituents located in a cavity pointing towards the extrac-
ellular side (Figure 3a and Figure 3d). A meta-fluoro substitution
(41) slightly improves the affinity, probably due to favorable
electrostatic interactions with Arg1824.64 (Figure 3b).
Interestingly, compound 42 with a fluoro atom in the para
position to the imidazole exhibited a slight (3-fold) reduction of
affinity. The interaction with Arg1824.64 cannot occur for
compound 42 (Figure 3c), due to an electrostatic repulsion to
the carbonyl of the sulfonyl carbamate. Adding a methyl to the
meta or para position relative to the methylene imidazole (44
and 45) also result in a similar reduced affinity as was seen for
compound 42, which correlates with a sub-optimal fitting in the
site between Arg1824.64 and Trp1002.61 as indicated in Figure 3e
and 3f.
were not studied in the antagonist binding model presented
herein.
1
2
3
4
5
6
7
8
9
2.4. Stability in Liver Microsomes and Kinetic Solubility
Table 3 lists compounds that were evaluated for metabolic
stability in human and mouse liver microsomes (HLM/MLM). For
a selection of compounds the kinetic solubility was also
determined. The previously reported AT2R ligands C38 and C93
were also, for comparison, evaluated in the same assays.[29] The
parent compound C38 exhibited a moderate stability in both
human (12 min) and mouse (70 min) liver microsomes. All
analogues related to the imidazole derivative C38 displayed a
similar trend, with the compounds being more prone to
undergo metabolism in human microsomes. Introduction of a
fluoro atom onto phenyl rings is a well-known strategy in
medicinal chemistry to block phase I metabolism (oxidation), a
potential problem suggested for C38.[41,42] Adding a fluoro atom
encouragingly displayed a retained affinity for AT2R (40, 41,
Table 1). Unfortunately, the metabolic stability in human liver
microsomes was not improved for any of the fluorinated
compounds (40, 41, and 42). In mouse liver microsomes the
metabolic stability was only retained for the ortho fluorinated
compound (40) (Table 3). This implies that the phenyl ring is
likely not the main site for oxidative metabolism of C38 in
human liver microsomes. The solubility of fluoro-analogue 40
was similar to C38 but was interestingly reduced for compound
41. Methylation of the phenyl ring produced slightly more
lipophilic ligands (43-46), for which the affinity could only be
retained for compound 43. The metabolic stability was
unsurprisingly reduced for these compounds, as they are likely
better substrates for benzylic oxidation than C38. Methyl
analogues 43–45 also displayed reduced solubility, likely related
to the added lipophilicity.
The bromo and trifluoromethoxy derivatives 46 and 47
displayed a retained stability profile in both mouse and human
liver microsomes (Table 3). For methoxy compound 48 the
metabolic stability was reduced. Introducing the bromo,
trifluoromethoxy and methoxy resulted in reduced solubility
compared to C38. Attempts to improve solubility of C38 by
exchanging the phenyl ring for a pyridine gave unsatisfying
results. Although exhibiting similar affinity as C38 the solubility
was reduced for all three pyridine compounds 49–51. Moreover,
the metabolic stability of the pyridines in mouse liver micro-
some assay was also reduced as compared to C38.
The solubility and stability in human liver microsomes of
the amide C93 were similar to C38. Notably, in mouse liver
microsomes the metabolic stability of C93 was significantly
reduced as compared to C38. All of the bicyclic amides in the
series demonstrated very low stability in mouse microsomes,
with two exceptions (compound 88 and 94). For example, the
bicyclic amides 85, 86, 87, and 89 rapidly decompose and are
similarly unstable in human microsomes. The solubility was also
reduced compared to the non-cyclized amide C93. Neither the
bicyclic acetyl amide 83 nor 84, regioisomers of 85 and 86,
respectively, demonstrated any improved metabolic profile.
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15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
The pharmacological profile of the compounds in the
second series has not been assessed due to lack of reliable
functional biological models. The related amides reported by
our group in 2012 displayed both agonistic and antagonist
properties indicating a complex pharmacological relationship
for ligands deviating from the imidazole head group[29]. Hence,
tentative binding modes of the ligands in the second series
Table 3. Compounds evaluated for stability in mouse and human liver
microsome assay. The kinetic solubility was also determined for a selection
of compounds.
Cmpd
HLM t1= [min][a]
MLM t1= [min][a]
Kinetic solubility [μM][b]
2
2
C38
C93
40
41
42
43
44
45
46
47
48
49
50
51
83
84
85
86
87
88
89
90
91
92
93
94
96
12
15
12
70
5.8
88
41
48
39
35
11
99
103
31
6.5
18
90
72
81
7.3
7.8
11
7.0
5.2
5.1
10
5.3
7.6
17
52
ND[c]
37
24
46
21
52
32
50
58
7.6
15
13
44
6.9
3.8
1.8
2.9
2.2
21
2.1
14
7.4
12
ND[c]
ND[c]
30
6.4
2.4
8.7
5.4
7.8
2.3
32
13
13
22
7.8
45
31
35
58
76
47
60
4.6
23
3.9
ND[c]
61
25
78
[a] The metabolic stability was determined in 0.5 mg/mL human or mouse
liver microsomes for compounds at a concentration of 1 μM in potassium
phosphate buffer. [b] The kinetic solubility was determined at a final
compound concentration of 100 μM in potassium phosphate buffer with
1% DMSO. [c] ND=Not determined.
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detection. Analytical HPLC/ESI-MS was performed using electro-
spray ionization (ESI) and a C18 column. High resolution molecular
masses (HRMS) were determined on a mass spectrometer equipped
with an ESI source and 7-T hybrid linear ion trap (LTQ). NMR spectra
were recorded at 400 MHz for 1H and 101 MHz for 13C.
Comparing the butyloxycarbonyl sulfonamide 85, one of the
most metabolically unstable compounds in this report, with the
corresponding ethoxycarbonyl compound 88 reveals only a
slightly improved stability in mouse liver microsomes for the
latter. A larger increase was expected in accordance with
previously reported data.[35] A larger impact on stability in
human microsomes with a shortened carbamate alkyl chain can
be noted when comparing 86 and with 90. The very low
stability of the bicyclic amide 86 is unfortunate as compound
86 exhibits the highest affinity of all compounds assessed.
Exchanging the acetyl of compound 90 gave the equipotent
acetyl propyl 91 and carbamate 92, neither of which showed
any improved solubility or metabolic stability. The bioisosteric
sulfonamide 93 exhibit a metabolic stability of more than
20 min in human microsomes. The same was seen for
compound 96, however both 93 and 96 are poor AT2R binders.
The solubility was only comparable to C93 for compounds 90
and 96.
1
2
3
4
5
6
7
8
9
Synthesis
Butyl
((3-(2-acetyl-1,2,3,4-tetrahydroisoquinolin-6-yl)-5-isobutylthiop-
hen-2-yl)sulfonyl)carbamate (86)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
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56
57
A 20 mL vial containing ZnCl2 (1.7 eq.) was dried in a vacuum oven
°
at 120 C overnight. The vial was capped and evacuated twice with
vacuum/N2(g). After cooling to room temperature the ZnCl2 was
dissolved in dry THF (5 mL). Isobutylmagnesium chloride (2 M in
THF; 1.5 eq.) was added dropwise. After 10 min of stirring, a
solution of 5-bromo-N-(tert-butyl)thiophene-2-sulfonamide (1,
7.3 mmol, 1.0 eq.) and Pd(t-Bu3P)2 (0.015 eq.) in dry toluene (5 mL)
°
was added. The mixture was microwave heated at 130 C for 15 min
after which it was partitioned between DCM and sat. aq. NH4Cl
(3:2). The aqueous layer was extracted with DCM and the
combined organic layers were washed with brine, dried with
MgSO4 and concentrated under reduced pressure. The remaining
residue was purified using silica gel flash chromatography (isohex-
anes with 10% (v/v) EtOAc). N-(tert-Butyl)-5-isobutylthiophene-2-
sulfonamide (2) was isolated in 51% yield.[35,43]
3. Conclusion
In summary, two series of new AT2R ligands were synthesized
and evaluated. In the first series, small structural changes were
introduced to the central phenyl ring of the known AT2R
antagonist C38. These were well-tolerated with half of the
compounds synthesized exhibiting similar or slightly improved
affinity to AT2R compared to C38. A common binding pose was
identified for the compounds in the first series, a pose that
could ascribe the reduced affinity for 3 out of 4 low-affinity
compounds to a sub-optimal fit between Arg182 on helix TM4
and Trp100 on helix TM2. The highest affinity in the first series
was displayed by the meta fluoro derivative 41 and the ortho
bromo substituted derivative 46, of which only the latter
displayed retained metabolic stability in mouse liver micro-
somes. In the second series where the imidazole heterocycle of
C38 was replaced by bicyclic amides, the most favorable amide
orientation was identified and explored. Compound 86 dis-
played the highest affinity to AT2R of all compounds assessed
(Ki =56 nM at AT2R). Unfortunately, all compounds in the
second series exhibited a low metabolic stability both in human
and mouse liver microsomes. The stability could be slightly
improved by reducing the sulfonyl carbamate chain length (cf.
compound 85 vs 88, and compound 86 vs 90).
N-(tert-Butyl)-5-isobutylthiophene-2-sulfonamide
(2,
8.2 mmol,
1.0 eq.) was dissolved in dry THF (70 mL) and transferred to a dry
°
three-necked round bottom flask. The flask was cooled to À 78 C
and evacuated thrice with vacuum/N2(g). To this was added n-
butyllithium (2.5 M in hexane; 4.5 eq.) dropwise after which the
°
mixture was stirred for 1 h at À 78 C. The reaction was sub-
°
sequently stirred at 0 C for 1 h, after which it was again cooled to
°
À 78 C and triisopropyl borate (2.5 eq.) was added. After 15 min the
°
flask was again stirred at 0 C for 3 h. The mixture was quenched
with 2 M HCl (aq.) and partially evaporated before it was diluted
with water and the product was extracted with DCM. The combined
organic layers were dried with MgSO4 and the solvent was removed
under reduced pressure. The crude residue was dissolved in DMSO
(2 mL) and toluene (30 mL), methyliminodiacetic acid (1.3 eq.) was
added and the mixture was refluxed for 3 h. The mixture was
diluted with EtOAc and washed with 0.1 M HCl (aq.). The organic
phase was dried with MgSO4 and the solvent was removed under
reduced pressure. The residue obtained was dissolved in minimal
amounts of acetone and equal amounts of diethyl ether after which
hexane (100 mL) was added using a dropping funnel. The solid
formed was filtered off and submitted to the same precipitation
procedure once more. N-(tert-Butyl)-5-isobutyl-3-(6-methyl-4,8-di-
oxo-1,3,6,2-dioxazaborocan-2-yl)thiophene-2-sulfonamide (3) was
collected in 76% yield.[35]
6-Bromo-1,2,3,4-tetrahydroisoquinoline (55, 0.44 mmol, 1.0 eq.) and
K2CO3 (2.3 eq.) were dissolved in MeCN (4 mL). Acetic anhydride
(1.4 eq.) was added and the mixture was stirred overnight. The
solvent was removed under reduced pressure and the product was
purified by silica gel column chromatography (DCM with 5% (v/v)
Experimental Section
General Chemistry
All chemicals and solvents were purchased from Sigma Aldrich,
Fisher Scientific, FluoroChem, and Enamine, and were used without
further purification. Microwave heating was performed in a Biotage
Initiator+ single-mode microwave reactor. Automated flash column
chromatography was performed on Biotage Isolera or Grace
Reveleris instruments using commercial silica cartridges. Manual
flash chromatography was performed on silica gel 60. Preparative
reverse-phase HPLC was performed using a C18 column with UV
MeOH).
1-(6-Bromo-3,4-dihydroisoquinolin-2(1H)-yl)ethan-1-one
(60) was isolated in 97% yield as 2 amide rotamers in 60:40 ratio at
1
room temperature. H NMR (400 MHz, DMSO-d6, T=373 K) δ 7.45–
7.28 (m, 2H), 7.21–7.09 (m, 1H), 4.57 (s, 2H), 3.65 (t, J=6.0 Hz, 2H),
2.84 (m, 2H), 2.07 (s, 3H). 13C NMR (101 MHz, DMSO-d6, T=353 K) δ
168.2, 130.5, 130.2, 130.1, 128.6, 128.0, 118.8, 42.7, 38.9, 28.9, 20.8.
MIDA-boronate (3, 0.20 mmol, 1.0 eq.), K2CO3 (5.0 eq.), 1-(6-bromo-
3,4-dihydroisoquinolin-2(1H)-yl)ethan-1-one (60) (1.0 eq.), and
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PdCl2(dppf) (0.05 eq.) were dissolved in DME (1 mL) and water
(0.2 mL) in a 2–5 mL vial. The vial was flushed with N2 and the
previously[45–47]. Cells were grown to approximately 80% confluence
before being re-plated into 48 well plates at 1×105 cells/well and
1
2
3
4
5
6
7
8
9
°
°
mixture was heated at 120 C for 1 h. The reaction mixture was
grown for 48 h at 37 C for a whole cell competition binding assay.
125
1
8
°
diluted with EtOAc and the layers were separated. The organic layer
was purified by automated silica flash chromatography (isohexane
with 50–100% (v/v) EtOAc). The fractions containing product were
collected and the solvent removed under reduced pressure. The
crude 3-(2-acetyl-1,2,3,4-tetrahydroisoquinolin-6-yl)-N-(tert-butyl)-5-
isobutylthiophene-2-sulfonamide (74) was stirred in TFA (99.9%;
[
I]-Sar Ile Ang II at 50,000 cpm, incubated for 45 min at 37 C, in
the absence or presence of unlabeled ligands, prepared in binding
buffer (DMEM, 0.1% BSA), were used in the competition assays at
concentrations ranging from 1 pM to 10 μM. For each experiment,
each ligand concentration was tested in triplicate, and each
experiment was repeated at least 3 separate times. Non-specific
binding (NSB) was defined in the presence of the unlabeled Ang II
(10 μM). The ability of each ligand to inhibit specific binding of
°
65 eq.) at 40 C overnight. The TFA was removed and the remaining
crude material was dissolved in DCM (2 mL). To this was added
triethylamine (2.1 eq.) and the mixture was stirred for 10 min at
room temperature, after which butyl chloroformate (0.7 eq.) was
added and the mixture was stirred at room temperature for 1 h.
The reaction mixture was washed with 2 M HCl (aq.) and brine and
dried with MgSO4. The solvent was evaporated and the product
was purified by preparative RP-HPLC (20-100% MeCN in water
(0.05% formic acid)). Butyl ((3-(2-acetyl-1,2,3,4-tetrahydroisoquino-
lin-6-yl)-5-isobutylthiophen-2-yl)sulfonyl)carbamate (86) was ob-
tained in 28% yield over 2 steps as a mixture of 2 amide rotamers
in 60:40 ratio. 1H NMR (400 MHz, Chloroform-d) δ 7.93 (overlapping;
s, 2H), 7.35–7.28 (overlapping; m, 2H), 7.26–7.22 (overlapping; m,
2H), 7.19–7.12 (overlapping; m, 2H), 6.743 (minor; s, 1H), 6.737
(major; s, 1H), 4.74 (major; s, 2H), 4.65 (minor; s, 2H), 4.07 (minor; t,
J=6.6, 2H), 4.06 (major; t, J=6.6, 2H), 3.81 (minor; t, J=5.9 Hz, 2H),
3.69 (major; t, J=5.9 Hz, 3H), 2.93 (major; t, J=5.9 Hz, 3H), 2.85
(minor; t, J=6.0 Hz, 2H), 2.70 (d, J=7.1 Hz, 4H), 2.18 (minor; s, 3H),
2.17 (major; s, 3H), 1.93 (overlapping; m, 2H), 1.52 (overlapping; m,
4H), 1.27 (overlapping; m, 4H), 0.99 (overlapping; d, J=6.6 Hz, 12H),
0.89 (overlapping; t, J=7.4 Hz, 6H). 13C NMR (101 MHz, Chloroform-
d) δ 169.8, 169.7, 151.7, 151.6, 150.4, 146.4, 146.3, 135.2, 134.2,
134.1, 133.2, 132.9, 132.6, 130.9, 129.64, 129.60, 129.5, 129.1, 127.3,
127.2, 126.7, 126.2, 67.0, 66.9, 48.1, 44.1, 44.0, 39.5, 30.7, 30.6, 29.5,
28.6, 22.4, 22.0, 21.7, 18.9, 13.7. MS (ESI): m/z calc’d for C24H32N2O5S2:
491.1674 [MÀ H]À ; found: 491.1664
[
125I]-Sar1Ile8Ang II was measured on a gamma counter with all
10
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14
15
16
17
18
19
20
21
22
23
24
25
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28
29
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32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
counts corrected for NSB. Non-linear regression of the data using
one-site fit model was performed and IC50 values, representing the
concentration at which each ligand displaced 50% of
[
125I]-
Sar1Ile8Ang II binding, were calculated as affinity estimates for each
ligand at AT1R and AT2R, using GraphPad Prism 6 (GraphPad
Software Inc., San Diego, CA, USA).
Kinetic Solubility
The kinetic solubility was investigated for compounds 40–41, 43,
46–48, 50–51, 85–92, 94, 96. Kinetic solubility was measured at a
final compound concentration of 100 μM and 1% DMSO in 100 mM
°
potassium phosphate buffer (pH 7.4) and incubated at 37 C for at
least 20 h. After incubation, the samples are centrifuged at 3000xg
°
at 37 C for 30 min to pellet insoluble material and an aliquot of the
supernatant was taken for quantification of compound concen-
tration by LC-MS/MS analysis. The LC-MS/MS system 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 2×50 mm 2.1 μm
column using a mobile phase consisting of A, 0.05% heptafluor-
obutyric 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.
Further details on reaction conditions is available for all reactions in
1
the supporting information. H NMR spectra were generated for all
final compounds. Purity and elemental analyses were performed on
all final compounds. 13C spectra were generated for a majority of
the final compounds. All available spectral analysis is reported in
the supplementary information.
Binding Assays
Assay 1 (Ki Determination)
Stability in Liver Microsomes
All synthesized ligands were evaluated in a radioligand assay by
displacing [125I][Sar1Ile8]-angiotensin II from human AT2R in HEK-293
cells membrane preparations. [Sar1Ile8]-angiotensin II (Sarile) acts as
a nonselective AT2R agonist.[44] The affinity was determined using a
seven-point dose-response curve, each point performed in dupli-
cates. All dose-response curves are available in the Supplementary
Information. Each new assay was validated using a selection of
known ligands in accordance with Eurofins Cerep standard
protocol. The compounds were also evaluated for inhibition of [125I]
[Sar1Ile8]-angiotensin II binding to human AT1R in HEK-293 cell
membranes. For AT1R the percent inhibition was determined at
10 μM, in duplicates, with the endogenous ligand (angiotensin II)
used as reference.
Human and mouse liver microsomes were used to assess the
metabolic stability for all compounds (95 excluded). 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 analyzed by LC-MS/MS as described above (Kinetic Solubility).
1
In vitro half-life (t = ) and in vitro intrinsic clearance (Clint) were
2
calculated using previously published models.[48,49] Extraction ratio
(E), i.e. the ratio of the hepatic clearance of a drug to the hepatic
blood flow, can be generally classified as high (>0.7), intermediate
(0.3–0.7) or low (<0.3), according to the fraction of drug removed
during one pass through the liver. For human and mouse liver
Assay 2 (IC50 Determination)
The IC50 values of C38, 40, 86 and C21 were assessed in whole cells
assay using HEK293 cells expressing AT1R or AT2R as described
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1
microsomes, E of 0.3 and 0.7 would correspond to a t = of 126 min
and 23 min, and 193 min and 35 min, respectively.
Acknowledgements
2
1
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7
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16
We thank the SciLifeLab Drug Discovery and Development Plat-
form for support with compound synthesis and ADME evaluations,
the Swedish National Infrastructure for Computing (SNIC) for
synthetic and computational resources, the Kjell and Märta Beijer
Foundation and the Swedish Research Council for financial
support.
Molecular Modelling of the AT2 Receptor
The crystal structure of the human AT2R was retrieved from the
Protein Data Bank (PDB code 5UNG with antagonist L-161,638)[31,34]
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 proto-
nated 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.
Conflict of Interest
The authors declare no conflict of interest.
Keywords: AT2 receptor · angiotensin II · medicinal chemistry ·
molecular docking · prototype antagonist
17 Ligand Docking
18
Ligands from Tables 1 were built and optimized their 3D conforma-
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
tion 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).[50–52] The docking grid was
placed taking as reference the coordinates of the co-crystallized
ligand L-161,638, and expanding the cubic grid box to 30 Å on
each dimensions. The selection of poses was done on the basis of a
double criteria, combining the highest possible scoring while
looking for the consensus among all ligands in the series.
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Manuscript received: December 6, 2018
Revised manuscript received: January 2, 2019
Version of record online: ■■■, ■■■■
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