From the first selective non-peptide AT2 receptor agonist to structurally related antagonists

Article abstract of DOI:10.1021/jm2015099

A para substitution pattern of the phenyl ring is a characteristic feature of the first reported selective AT₂ receptor agonist M024/C21 (1) and all the nonpeptidic AT₂ receptor agonists described so far. Two series of compounds structurally related to 1 but with a meta substitution pattern have now been synthesized and biologically evaluated for their affinity to the AT₁ and AT₂ receptors. A high AT ₂/AT₁ receptor selectivity was obtained with all 41 compounds synthesized, and the majority exhibited K_i ranging from 2 to 100 nM. Five compounds were evaluated for their functional activity at the AT₂ receptor, applying a neurite outgrowth assay in NG108-15 cells. Notably, four of the five compounds, with representatives from both series, acted as potent AT₂ receptor antagonists. These compounds were found to be considerably more effective than PD 123,319, the standard AT₂ receptor antagonist used in most laboratories. No AT₂ receptor antagonists were previously reported among the derivatives with a para substitution pattern. Hence, by a minor modification of the agonist 1 it could be transformed into the antagonist, compound 38. These compounds should serve as valuable tools in the assessment of the role of the AT₂ receptor in more complex physiological models.

Full text of DOI:10.1021/jm2015099

Article
pubs.acs.org/jmc
From the First Selective Non-Peptide AT2 Receptor Agonist to
Structurally Related Antagonists
A. M. S. Murugaiah,† Xiongyu Wu,† Charlotta Wallinder,† A. K. Mahalingam,† Yiqian Wan,†
Christian Skold,† Milad Botros,§ Marie-Odile Guimond,‡ Advait Joshi,† Fred Nyberg,§
̈
Nicole Gallo-Payet,*,‡ Anders Hallberg,*,† and Mathias Alterman*,†
†Department of Medicinal Chemistry, BMC, Uppsala University, P.O. Box 574, SE-751 23 Uppsala, Sweden
‡Service of Endocrinology and Department of Physiology and Biophysics, Faculty of Medicine, University of Sherbrooke, Sherbrooke
J1H 5N4, Quebec, Canada
§Department of Biological Research on Drug Dependence, BMC, Uppsala University, P.O. Box 591, SE-751 23 Uppsala, Sweden
S
* Supporting Information
ABSTRACT: A para substitution pattern of the phenyl ring is a
characteristic feature of the first reported selective AT2 receptor
agonist M024/C21 (1) and all the nonpeptidic AT2 receptor
agonists described so far. Two series of compounds structurally
related to 1 but with a meta substitution pattern have now been
synthesized and biologically evaluated for their affinity to the AT1
and AT2 receptors. A high AT2/AT1 receptor selectivity was
obtained with all 41 compounds synthesized, and the majority
exhibited Ki ranging from 2 to 100 nM. Five compounds were
evaluated for their functional activity at the AT2 receptor,
applying a neurite outgrowth assay in NG108-15 cells. Notably,
four of the five compounds, with representatives from both series,
acted as potent AT2 receptor antagonists. These compounds were
found to be considerably more effective than PD 123,319, the
standard AT2 receptor antagonist used in most laboratories. No AT2 receptor antagonists were previously reported among the
derivatives with a para substitution pattern. Hence, by a minor modification of the agonist 1 it could be transformed into the
antagonist, compound 38. These compounds should serve as valuable tools in the assessment of the role of the AT2 receptor in
more complex physiological models.
vasodilatation.1,3,17−20 Recently, the AT2 receptor has emerged
as a potential target for pharmaceutical agents. In 2004, we
INTRODUCTION
The octapeptide angiotensin II (Ang II) is the main effector of
the renin−angiotensin system (RAS) and exerts its effects via
activation of two receptors, AT1 and AT2. Activation of the AT1
■
disclosed the first druglike selective AT2 receptor agonist
M024/C21 (1),56 which displayed reasonable bioavailability
after oral administration (Figure 1).21 It demonstrated
receptor is associated with the well-known functions of the RAS
remarkable beneficial effects on heart function after myocardial
such as regulation of blood pressure and fluid/electrolyte
infarction in rats, and its potential use for the treatment of heart
balance. In contrast, the physiological role of the AT2 receptor
failure was recently discussed and reviewed.22−24 Compound 1
is the most selective AT2 receptor agonist reported to date and
represents a unique tool to delineate the specific roles of the
AT2 receptor in different cellular and animal models.25,26
Furthermore, stimulation of the AT2 receptor with intra-
cerebroventricular administered Nα-nicotinoyl-Tyr-(Nα-Cbz-
Arg)-Lys-His-Pro-Ile (CGP-42112A), a selective but peptidic
AT2 receptor agonist,27,28 is known to exhibit a neuroprotective
effect in a model of stroke in conscious rats.13
and the impact of its stimulation are less well understood.1,2
This may be due to the low expression of the AT2 receptor in
healthy adults where it is localized to specific tissues such as the
heart, ovary, kidney, and some areas of the brain.1,3 Prior to
birth, the AT2 receptor is the predominant Ang II receptor,
suggesting that it plays a role in fetal development.4,5 Notably,
the AT2 receptor is up-regulated in adults during certain
pathological conditions such as vascular, skin, and axonal injury,
renal damage, myocardial infarction, and brain ischemia.6−11 It
is postulated to be involved in wound healing and tissue repair
and to act anti-inflammatory.7,11−15 The AT2 receptor mediates
cell differentiation, apoptosis, and alkaline secretion in the
duodenal mucosa16 and frequently exerts opposing effects to
the AT1 receptor; for example, it promotes antiproliferation and
We have synthesized a series of compounds related to 1 but
with a meta rather than a para substitution pattern at the phenyl
Received: November 8, 2011
Published: January 16, 2012
© 2012 American Chemical Society
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receptor antagonist L-162,389 (4) (Figure 2).32 Notably, the
only difference between 3 and 4 is the methyl group in the alkyl
chain.
By systematic modification of the N-heterocyclic moiety of 2,
improved AT2 receptor selectivity was achieved and the
imidazole derivative 1 was eventually obtained.21 Considerable
efforts have been devoted to identifying selective AT2 receptor
agonists,33−38 and several series of analogues of 1 were
synthesized and biologically evaluated.39−42 Among those
analogues, compound 5 was found to act as an AT2 receptor
agonist despite the fact that the molecule is lacking a methyl
group in the alkyl chain (cf. the AT1 receptor agonist 3 vs the
AT1 receptor antagonist 4, Figure 2).39 The main structural
features of the compound class originating from 1 are the
imidazole ring, the sulfonyl carbamate substituent, and the
isobutyl chain all attached to a bicyclic scaffold. Structural
modifications of both the sulfonyl carbamate substituent and
the isobutyl group had in general a negative impact on the
binding affinity. In contrast, diverse substituents in the benzylic
position were often accepted and both substituted nitrogen-
containing aromatic heterocycles and linear amides were
proven to produce selective AT2 receptor ligands with high
affinities and potent agonistic effects. Furthermore, substituting
the phenyl ring for a furan ring rendered the receptor selective
compound 6 (Figure 2) with similar affinity and retained
functional activity, though the substitution pattern in the furan
ring more resembles a “meta” rather than a “para”
configuration.40 Hence, these findings encouraged us to assess
the effects of switching the substituted methylene group from
Figure 1.
ring. Herein we report that by this migration of substituents, as
exemplified by the transformation of 1 to 38, the AT2 receptor
agonism could be converted into AT2 receptor antagonism
(Figure 1).
DESIGN
■
The first druglike selective AT2 receptor agonist 1 was derived
from the nonselective AT1/AT2 receptor ligand L-162,313 (2)
(Figure 2).21 Ligand 2 was identified as an AT1 receptor agonist
2 decades ago29,30 and was thereafter shown by us to also act as
an agonist at the AT2 receptor.31 Compound 2 is a thiophene
analogue of the nonselective AT1 receptor agonist L-162,782
(3), which is structurally very similar to the nonselective AT1
Figure 2.
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a
Scheme 1
a
Reagents: (a) Pd(PPh3)4, NaOH, toluene, EtOH; (b) CBr4, PPh3, DMF; (c) heterocycle, 1,4-dioxane (10−13, 16, 19); (d) heterocycle, DMSO,
base (14, 15, 17, 18, 20−23); (e) BCl3, DCM (24−27, 29−37); (f) TFA, anisole (28); (g) n-butyl chloroformate, pyrrolidinopyridine, pyridine (38,
40, 44−51); (h) n-butyl chloroformate, Na2CO3, DCM, H2O (39, 41−43).
a
Scheme 2
a
Reagents: (a) 2-bromoimidazole, DMSO, NaOH; (b) boronic acid, Pd(PPh3)4, NaOH, toluene, EtOH (53, phenylboronic acid; 54, 3-
thiophenylboronic acid; and 55, 2-thiophenylboronic acid); (c) BCl3, DCM; (d) n-butyl chloroformate, Na2CO3, DCM, H2O.
the para position to the meta position of the phenyl ring. We
were intrigued to see whether these analogues would retain
high affinity and, if so, how the functional activity at the AT2
receptor would be affected. To address this question, two series
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a
Scheme 3
a
Reagents: (a) TFA, anisole; (g) n-butyl chloroformate, pyrrolidinopyridine, pyridine.
a
Scheme 4
a
Reagents: (a) heterocycle, dioxane (64, imidazole; 65, 2-ethylimidazole; and 66, benzimidazole); (b) boronic acid 7, Pd(PPh3)4, Na2CO3, toluene,
EtOH; (c) BCl3, DCM (70); (d) TFA, anisole (71, 72); (e) n-butyl chloroformate, Na2CO3, DCM, H2O (73, 74); (f) n-butyl chloroformate,
pyrrolidinopyridine, pyridine (75).
of compounds were synthesized and biologically evaluated. PD
123,319, used as a standard AT2 receptor antagonist with an
IC50 of 34 nM, is depicted for structural comparison in Figure
2.27,43
obtain the benzyl alcohol 8 in a modest yield. The benzyl
alcohol was successfully converted to the corresponding
bromide 9 in a high yield, a key intermediate in the synthesis
of series 1. The bromide 9 was reacted with the selected
heterocycles to give the desired compounds 10−23 in
moderate to high yields (51−99%). Compounds 17 and 18
were obtained simultaneously upon reacting 9 with imidazo-
pyridine, and the two regioisomers were separated to afford the
desired compounds in 19% and 49% yield, respectively. The
final compounds 38−51 were obtained after deprotection of
the tert-butyl group45 and subsequent coupling of the free
sulfonamide with n-butyl chloroformate in modest to good
yield (Scheme 1).
RESULTS
■
Chemistry. The synthesis of all compounds commenced
with the thiopheneboronic acid 7, which was prepared in
essence as previously described by Kevin et al.44 The synthesis
of compounds 38−51, series 1 (Scheme 1), were executed via a
Suzuki coupling of thiopheneboronic acid 7 and 3-bromobenzyl
alcohol with palladium tetrakis as catalyst and NaOH as base to
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a
Scheme 5
a
Reagents: (a) (i) NaBH4, MeOH, (ii) H2O, DCM, HCl(aq); (b) NaH, THF, CS2, MeI; (c) Bu3SnH, THF, AIBN; (d) boronic acid 7, Pd(PPh3)4,
Na2CO3, toluene, EtOH; (e) BCl3, DCM; (f) n-butyl chloroformate, Na2CO3, DCM, H2O.
a
Scheme 6
a
Reagents: (a) Pd(OAc)2, PPh3, K2CO3, THF/DME/EtOH/H2O; (b) amine, NaBH4, MeOH; (c) acid chloride, NEt3, DMAP, DCM; (d)
ammonium formate, MeCN; (e) TFA, anisole; (f) n-butyl chloroformate, NEt3, pyrrolidinopyridine, DCM. R1 and R2: See Table 2 for details.
Compounds 59−61 in series 1 were derived from the
alkylation of 2-bromoimidazole with compound 9 to obtain the
intermediate 52 in excellent yield (Scheme 2). The N-alkylated
2-bromoimidazole 52 was then coupled with three different
arylboronic acids in Suzuki reactions with palladium tetrakis as
catalyst and NaOH as base to form intermediates 53, 54, and
55, respectively, in modest to good yields. Subsequent
deprotection and reaction with n-butyl chloroformate rendered
the final compounds 59−61 in 55−87% yield.
The chloroimidazole derivative (63) was obtained from
compound 52. Most likely the bromide was substituted by
pyridine which subsequently was substituted by a chloride,
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Table 1. Binding Affinity of Series 1 to the AT2 Receptor
a
b
Ki values are an average from three determinations. Standard deviations are less than 15% in all cases. All compounds were evaluated for binding
toward the AT1 receptor, and all compounds exhibited Ki > 10 000 nM.
generated when the intermediate 62 was treated with pyridine
and n-butyl chloroformate (Scheme 3).
palladium tetrakis as catalyst and sodium carbonate as base in
excellent yields. Compounds 67−69 were treated with TFA or
BCl3 to deprotect the sulfonamide and subsequently with n-
butyl chloroformate to obtain the final compounds 73−75.
The final compound in the first series, 81 (Scheme 5), was
achieved by reduction of the carbonyl group of 64. Initially the
carbonyl was reduced to the corresponding alcohol with
sodium borohydride. Reaction of the alcohol 76 with sodium
Compounds 73−75 (Scheme 4), with a carbonyl-extended
chain between the phenyl and the imidazole, were synthesized
by N-alkylation of the appropriate heterocycle with 2-
bromophenacyl bromide giving compounds 64−66 in moder-
ate yields. The formed bromides were coupled with the
thiopheneboronic acid 7 under Suzuki conditions with
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hydride, carbon disulfide, and methyl iodide formed the S-
methyl carbonodithioate 77 in excellent yield (99%). Treat-
ment of compound 77 with Bu3SnH and AIBN rendered
compound 78 in modest yield. The boronic acid 7 was coupled
with 78 in a Suzuki reaction with palladium tetrakis as
palladium source and Na2CO3 as base to yield compound 79.
Deprotection of 79 with BCl3 and subsequent reaction with n-
butyl chloroformate gave the final compound 81 in good yield.
The synthesis of series 2, as outlined in Scheme 6, originated
with the coupling of thiopheneboronic acid 7 with 3-
bromobenzaldehyde in a Suzuki reaction with palladium acetate
as precatalyst, PPh3 as ligand, and K2CO3 as base to obtain the
aldehyde 82. The final compounds 86−104 were then prepared
in a combinatorial fashion where the key intermediate (82) was
dissolved in methanol and added to sample vials. A diverse set
of amines were dispensed to the vials, and after the samples
were stirred for 30 min, NaBH4 was added to achieve the
reductive amination with full conversion. The workup was
performed by solid−liquid extraction through a diatomaceous
earth plug in a polypropylene column with ethyl acetate as
eluent. The secondary amines (83a−f) were used in the third
step without further purification. The crude products 83a−f
were dissolved in dichloromethane, and triethylamine and
DMAP were added. Acid chlorides were added to the reaction
vials, and the mixtures were stirred for 2 h. The same solid−
liquid extraction workup, as in the previous step, was used again
to obtain the crude amides (84b−s) in high yields and high
purity. The formyl compound 84a was synthesized by refluxing
the secondary amine 83a with ammonium formate. In the final
steps the tert-butyl protecting group was removed from the
sulfonamide with TFA and the free sulfonamide was
subsequently reacted with n-butyl chloroformate to yield the
final products 86−104 in 41−86% overall yield after
purification on preparative HPLC.
Table 2. Binding Affinity of Series 2 to the AT2 Receptor
Binding Assays. The final products were evaluated in
radioligand assays by displacement of [125I]Ang II from AT1
receptors in rat liver membranes and from AT2 receptors in pig
uterus membrane as previously described.46,47 The selective
AT1 receptor antagonist losartan and the natural ligand Ang II
were used as reference substances. The results are summarized
in Tables 1 and 2.
In series 1, containing substituted heterocycles (Table 1), all
compounds showed affinity to the AT2 receptor and did not
bind to the AT1 receptor (Ki > 10 000 nM). Compound 38, the
meta substituted analogue of 1, exhibited an affinity of 19 nM
to the AT2 receptor. Introducing a 2-chloro substituent on the
imidazole ring resulted in higher affinity (63, Ki = 9.2 nM).
Alkyl substituents in the 2-position of the imidazole ring gave
compounds with similar affinities, whereas a methyl group
rendered decreased affinity (39, Ki = 29 nM) but prolongation
of the chain to an ethyl or n-butyl resulted in increased affinities
(40, Ki = 8.0 nM or 41, Ki = 10 nM, respectively). An acetyl
substituent in this position was accepted with no significant
effect on binding affinity compared to 38. Introducing
aromatic/heteroaromatic rings as substituents in the 2-position
of the imidazole ring gave compounds with lower affinity (43,
59−61), but the decrease in affinity was not as profound with
heteroaromatic substituents compared to what was encoun-
tered with a phenyl substituent. Replacing the imidazole with
bicyclic heteroaromatic rings gave compounds (44−46) that
showed reduced affinity for the AT2 receptor. Notably,
replacing imidazole with pyrazole, that is, moving the nitrogen
atom one position in the ring, diminished the affinity 5-fold (38
a
Ki values are an average from three determinations. Standard
b
deviations are less than 15% in all cases. All compounds were
evaluated for binding toward the AT1 receptor, and all compounds
exhibited Ki > 10 000 nM.
vs 47). Introducing a trifluoromethyl group in the 3-position of
the pyrazole resulted in further decline in affinity (48).
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Insertion of a carbonyl group to extend the distance between
the phenyl ring and the imidazole ring had no effect on binding
affinity (cf. 73 vs 38, 74 vs 40, and 75 vs 44), and reduction of
the carbonyl in compound 73, rendered the etylenimidazole 81
with lower affinity. Finally, the aromatic heterocycles were
replaced with saturated nitrogen-containing heterocycles as in
49−51. Compounds 49 and 50 exhibited low affinity to the
AT2 receptor, but the hydantoin 51 showed that some affinity
could be regained.
in reduced Ang II-induced neurite outgrowth, verifying
blockage of the AT2 receptor. Agonistic effect was verified
through co-incubation with the selective AT2 receptor
antagonist PD 123,319 (Figure 2), which reduced neurite
outgrowth, verifying that the effect was mediated through the
AT2 receptor. Treatment with PD 123,319 alone did not alter
the morphology compared to untreated cells. The neurite
outgrowth results are shown in Figures 3 and 4.
In the second series of compounds, tertiary amide-based
substituents in the 3-position of the phenyl ring were
investigated (Table 2). As in the first series none of the
compounds showed AT1 receptor affinity. The formamide
based compound 86 exhibited affinity to the AT2 receptor with
a Ki of 55 nM. Extending the R2 chain, exemplified by the
amide and carbamate substituents of compounds 87−89, did
not substantially affect the binding affinities. However, insertion
of a carbonyl group in 89 to afford 90 (Ki = 1.6 nM) resulted in
a significantly improved binding affinity, and introduction of a
thiophene in the amide substituent was also well-tolerated by
the AT2 receptor (91, Ki = 13 nM). Notably, the sulfonamide
substituent in 92 completely abolished the binding affinity,
which was surprising considering the relatively small structural
difference compared to 87.
Varying R1 did not improve the AT2 receptor affinity in the
compounds with acetamide-based substituents (87 and 93−
97). Altering the alkyl group, that is, a methyl to an ethyl, had
no impact on binding affinity (cf. 87 vs 93), and replacing the
terminal hydrogen atoms for fluorine (93 vs 94) decreased the
affinity, which also was the case when the size of R1 was made
larger by introducing aromatic rings (95−97). A 2-fold
improvement in affinity was observed with compounds with
aromatic groups in R1 and larger size groups in R2 (98−100 vs
95−97). Notably, the introduction of a thiophene ring in R2
and benzyl as R1 did lower the affinity (101 vs 96) while a
better affinity was observed with ethyl as R1 (102 vs 101). The
sulfonamide unit in 92 rendered the compound inactive, but by
extension of R2 with a thiophene ring and by increase of the
size of R1 to an ethyl, some of the affinity was recovered (103,
Ki = 133 nM). Attachment of a cyclopropyl ring to R2 in
compound 94 also reduced the affinity (104).
Figure 3. Effect of compounds 38, 63, and 75 on neurite outgrowth in
NG108-15 cells. The cells were plated at a cell density of 3.6 × 104
cells/Petri dish (35 mm) and were cultured for 3 days in the absence
(−) or presence (+) of 100 nM Ang II, 10 nM 38, 10 nM 63, or 10
nM 75 alone or in combination with 10 μM antagonist PD 123,319 or
100 nM Ang II. Cells with at least one neurite longer than a cell body
were counted as positive for neurite outgrowth. The number of cells
with neurites was expressed as the percentage of the total number in
the micrographs (at least 290 cells according to the experiment). The
results are significant according to two-way ANOVA: ∗∗∗, p < 0.001.
In Vitro Morphological Effects of 38, 63, 75, 90, and
100 in NG108-15 Cells. A set of five high-affinity and
structurally diverse compounds (38, 63, 75, 90, and 100) were
selected for evaluation of their functional activity at the AT2
receptor, applying a neurite outgrowth assay in NG108-15 cells.
We have previously shown that NG108-15 cells in their
undifferentiated state express only the AT2 receptor and that a
3-day treatment with Ang II or the selective peptidic AT2
receptor agonist CGP-42112A induces neurite outgrowth.28,48
The signaling pathways involve a sustained increase in Rap1/B-
Raf/p42/p44mapk activity and activation of the nitric oxide/
guanylyl cyclase/cGMP pathway.49−51 Cells were plated as
described in the Experimental Section, and adequate test
concentrations for each compound were determined by testing
a dilution series of each compound ranging from 1 pM to 1 μM.
For all the compounds it was only at the highest concentration
that any evidence of cell death was observed. Cells were then
treated in the absence or presence of Ang II (100 nM), 38 (10
nM), 63 (10 nM), 75 (10 nM), 90 (1 nM), and 100 (10 nM).
After 3 days of treatment, cells were examined under a phase-
contrast microscope and micrographs were taken. Antagonistic
effect was verified through co-incubation with Ang II resulting
Figure 4. Effect of compounds 90 and 100 on neurite outgrowth in
NG108-15 cells. The cells were plated at a cell density of 3.6 × 104
cells/Petri dish (35 mm) and were cultured for 3 days in the absence
(−) or presence (+) of 100 nM Ang II, 1 nM 90, or 10 nM 100 alone
or in combination with 10 μM antagonist PD 123,319 or 100 nM Ang
II. Cells with at least one neurite longer than a cell body were counted
as positive for neurite outgrowth. The number of cells with neurites
was expressed as the percentage of the total number in the
micrographs (at least 290 cells according to the experiment). The
results are significant according to two-way ANOVA: ∗∗∗, p < 0.001;
∗∗, p < 0.01; ∗, p < 0.05.
The meta-analogue of 1, compound 38 (10 nM), did not
stimulate neurite outgrowth upon cell treatment, and pretreat-
ment with 10 nM 38 reduced Ang II-induced neurite
outgrowth from 17.5% to 11.5%, (Figure 3). These results
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indicate that 38 exhibits antagonistic properties at the AT2
receptor in contrast to the agonism exerted by 1. Two more
analogues from series 1 (63 and 75) were evaluated for
functional activity, and none of the compounds induced neurite
outgrowth, while pretreatment of the cells with either
compound reduced Ang II-induced neurite outgrowth. Again,
the antagonistic effect was achieved at the low concentration of
10 nM for both compounds (Figure 3). From series 2
compounds 90 and 100 were selected for determination of
functional activity, and interestingly 90 (1 nM) showed
agonistic activity while 100 (10 nM) followed the pattern
from series 1 and exhibited antagonism (see Figure 4). All
results were significant according to two-way analysis of
variance (ANOVA).
receptor binding sites by these new antagonists could induce
partial inhibition of the signaling pathway and therefore inhibit
Ang II-induced neurite outgrowth. Nevertheless, since the
functional experiments were performed on intact cells, the
possibility of interactions with other growth stimulating systems
cannot be excluded.
Of the five compounds evaluated for functional activity at the
AT2 receptor, four (38, 63, 75, and 100) acted as antagonists
and inhibited Ang II-induced neurite outgrowth while one of
them, compound 90, functioned as a potent agonist.
Compound 90 differs structurally from the four antagonists
by having the longer and flexible α-keto ester substituent,
compared to more bulky substituents in the antagonists (38,
63, 75, and 100). A possible explanation of why compound 90
acts as an agonist despite the 1,3-substitution pattern is that
either the flexibility or the oxoacetate function of the
substituent can compensate for the substitution pattern.
Thereby it can facilitate the interactions with the receptor
required for activation that the more bulky substituents cannot
achieve. It is less likely that the oxoaceto group stabilizes the
active conformation of the receptor by serving as an
electrophilic trap for a suitable amino acid in the side chain
or that hydration of the oxoacetate should promote such
stabilization.
A general trend so far is that the antagonists exhibit lower
affinity (10−50 times lower) toward the AT2 receptor
compared to the agonists. Hence, the affinities are reduced
when the same substituent is moved from the para-position to
the meta-position. As a reference, the analogue to 86 in which
the same substituent is positioned in the para-position of the
phenyl ring has almost 20-fold higher AT2 receptor affinity and
presents a Ki of 3.1 nM.40 Further optimization of the
substituent in the meta-position could possibly lead to high
affinity antagonists. The potential clinical significance of
selective druglike AT2 receptor antagonist still remains to be
elucidated.
The SAR in the two series is not very clear. However, from
series 1 it can be concluded that a nitrogen in the 3-position of
the heterocycle is important for good binding, since all
compounds lacking a nitrogen atom in this position (47−50)
have about 10 times higher Ki, for example, 47 versus 38 and 50
versus 51. It can also be concluded that the carbonyl group is
important for good affinity when the linker between the phenyl
and the imidazole is extended, since the ethylenimidazole has 5
times higher Ki compared to the corresponding carbonyl
containing compound (81 vs 73). In the second series, with
linear amides as substituents, the free rotation around the
bonds between the central phenyl ring of the compounds and
the nitrogen atom in the amide-based substituent makes a clear
SAR hard to obtain. Depending on the properties of R1 and R2,
the binding mode could be inverted with respect to which
interaction with the AT2 receptor that is the most favorable in
total, considering both substituents. But it seems that the
binding affinity outcome is a function of a combination of R1
and R2, since (a) the thiophene did increase the affinity when
R1 is a methyl group (91 vs 87), (b) extending R1 to an ethyl
group did not affect the binding affinity in the acetamide-based
substituents (93 vs 87), and (c) the thiophene substituent did
decrease the affinity when R1 is equal to a benzyl group (96 vs
101) while the affinity improved 300 times when the thiophene
in R2 was kept and R1 was exchanged from benzyl to ethyl (101
vs 102). It also seems like the amide in series 2 is not
interchangeable with a sulfonamide in the interaction with the
DISCUSSION
■
The only nonpeptidic AT2 receptor antagonist available is PD
123,319 (Figure 2), which serves as the standard substance in
most studies on the AT2 receptor.26 Despite the good binding
of PD 123,319 to the AT2 receptor (an IC50 of 34 nM), 10 μM
was required to obtain a satisfactory inhibition of the Ang II
stimulated neurite outgrowth in NG108-15 cells (1 μM PD
123,319 can be used to get inhibition of neurite outgrowth but
with a high variability in the results), suggesting that the efficacy
is far from optimal. Hence, due to the lack of other potent and
effective AT2 selective antagonists, there is a need for new more
effective and robust antagonists.
There are several examples of subtle structural modifications
in non-peptide ligands to peptide activated receptors that result
in reversed functional response.52,53 In the case of the
nonpeptidic ligands binding to the AT1 receptor, the functional
activity switches from antagonism to agonism when the isobutyl
chain is exchanged to a n-propyl chain (3 vs 4; see Figure 2).32
The same transformation of the selective non-peptide AT2
receptor agonist 1 does not alter the functional activity; the n-
propyl analogue of 1 retains its agonistic properties (5 in Figure
2).39 In the present study it has been demonstrated that the
functional switch of the AT2 receptor ligands is related to the
substitution pattern of the phenyl group rather than small
variations in the lipophilic side chain. Thus, a transfer of
substituent from the para-position (1) to the meta-position
(38) interconverts the functional activity from agonism to
antagonism. The meta substitution pattern thus seems to
render ligands that retain affinity to the AT2 receptor but lack
the capacity to adopt conformations that allow interactions with
the receptor that are required for its activation. Compared to
the commercially available nonpeptidic AT2 antagonist, PD
123,319, these new antagonists exerted good inhibition of Ang
II-induced neurite outgrowth at a 1000-fold lower concen-
tration. While the antagonist PD 123,319 was used at 10 μM to
obtain a satisfactory inhibition of neurite outgrowth, the new
antagonists exhibited good inhibition at 10 nM, that is,
compounds 38, 63, 75, and 100. Even if these results are
somewhat surprising at first glance, because of the low
concentration, they were repeatedly observed and in this
study, only neurite outgrowth was assessed, which is the final
effect of AT2 receptor in these cells. However, it is known that a
partial inhibition of the signaling pathway is sufficient to
completely impede neurite outgrowth. It has been observed, for
example, that the TrkA inhibitor α-cyano-(3,5-di-tert-butyl-4-
hydroxy)thiocinnamide (AG879)54 only partially inhibits the
p42/p44mapk pathway but that its effect on neurite outgrowth is
total.55 In a similar manner, a partial occupation of AT2
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Article
purification by column chromatography (EtOAc/hexane, 10:90) gave
9 as a white solid (273 mg, 0.612 mmol, 95%). H NMR (CDCl3) δ:
AT2 receptor, even though these two groups often are
considered to be bioisosteric.
1
0.97(d, J = 6.3 Hz, 6H), 0.98 (s, 12H), 1.84−2.00 (m, 1H), 2.69 (d, J =
7.1 Hz, 2H), 4.18 (br s, 1H), 4.54 (s, 2H), 6.78(s, 1H), 7.37−7.44
(2H, m), 7.50−7.56 (m, 1H), 7.69 (br s, 1H). 13C NMR (CDCl3) δ:
22.2, 29.5, 30.5, 33.3, 39.2, 54.4, 128.6, 128.8, 128.97, 129.02, 129.7,
135.5, 136.8, 138.3, 142.1, 148.5. IR (cm−1) ν: 3296, 2969, 2870, 1586,
1452, 1303. Anal. Calcd for C19H26BrNO2S2: C, 51.3; H, 5.9; N, 3.2.
Found: C, 51.4; H, 6.0; N, 3.2.
General Procedure for the Synthesis of Compounds 10−13,
16, and 19. To a solution of 9 in 1,4-dioxane (2 mL), the appropriate
heterocycle was added. The reaction mixture was stirred at 80 °C until
the reaction was completed. The reaction mixture was concentrated
under vacuum, and the crude product was purified by column
chromatography to afford the products 10−13, 16, and 19.
SUMMARY
■
In conclusion, 41 new selective and high affinity AT2 receptor
ligands have been synthesized and biologically evaluated. Five
of these ligands were evaluated in a functional assay relying on
neurite outgrowth. One ligand acted as an agonist and four
ligands as antagonists at the AT2 receptor. The functional
response was found to be determined by the position of the
substituents of the central phenyl ring, where a meta-
substitution pattern could provide antagonists while the
previously reported agonists have a para-substitution pattern
as a characteristic feature. No antagonists were identified in the
latter series.21,31,39−42 The antagonist 38 is considerably more
effective in the assay used herein than PD 123,319 and should
exhibit similar pharmacokinetic properties to 1, thus consid-
erably facilitating studies in more complex animal models.
We believe that these new druglike antagonists reported
herein and the structurally similar agonist 1 previously reported
will serve as valuable tools in the continuing assessment of the
role of the AT2 receptor in physiological systems.
3-(3-Imidazol-1-ylmethylphenyl)-5-isobutylthiophene-2-(N-
tert-butyl)sulfonamide (10). Compound 10 was synthesized from 9
(58 mg, 0.13 mmol) following the general procedure with imidazole
(22 mg, 0.33 mmol) as heterocycle with a reaction time of 1 h. The
crude product was purified (MeOH/CH2Cl2, 5:95) to afford 10 as a
1
colorless syrup (38.4 mg, 89.0 μmol, 68%). H NMR (CDCl3) δ:
0.85−1.05 (m, 15H), 1.80−2.00 (m, 1H), 2.66 (d, J = 7.1 Hz, 2H),
4.38 (s, 1H), 5.19 (s, 2H), 6.72 (s, 1H), 6.99 (s, 1H), 7.10 (s, 1H),
7.22 (app d, J = 7.6 Hz, 1H), 7.41 (app t, J = 7.6 Hz, 1H), 7.47−7.55
(m, 2H), 7.82 (s, 1H). 13C NMR (CDCl3) δ: 22.1, 29.5, 30.5, 39.1,
51.0, 54.5, 119.3, 127.4, 128.4, 128.9, 128.7, 129.1, 135.6, 136.2, 136.7,
137.1, 142.3, 148.6. IR (cm−1) ν: 3287, 3063, 2961, 1439, 1311. Anal.
Calcd for C22H29N3O2S2: C, 61.2; H, 6.8; N, 9.7. Found: C, 61.0; H,
6.6; N, 9.8.
General Procedure for the Synthesis of Compounds 14, 15,
17, 18, 20−23, 52. To the appropriate heterocycle dissolved in
DMSO (1 mL) was added base (t-BuOK, KOH, or NaOH), and the
mixture was stirred for 40 min at ambient temperature. This solution
was added dropwise to a stirred solution of 9 in DMSO (1 mL). The
reaction mixture was stirred at ambient temperature until the reaction
was completed. CH2Cl2 (15 mL) was added, and the reaction mixture
was washed with water. The organic phase was dried and
concentration under vacuum, and the crude product was purified by
column chromatography to afford the products 14, 15, 17, 18, 20−23,
52.
3-[3-(2-Bromoimidazol-1-ylmethyl)phenyl]-5-isobutylthio-
phene-2-(N-tert-butyl)sulfonamide (52). Compound 52 was
synthesized from 9 (173.6 mg, 0.391 mmol) following the general
procedure with 2-bromoimidazole (68.9 mg, 0.469 mmol) as
heterocycle and NaOH (33 mg, 0.82 mmol) as base with a reaction
time of 1 h. The crude product was purified (MeOH/CH2Cl2, 4:96) to
afford 52 as a colorless syrup (197.3 mg, 0.386 mmol, 99%). 1H NMR
(CDCl3) δ: 0.94 (s, 9H), 0.96 (d, J = 6.8 Hz, 6H), 1.82−2.00 (m, 1H),
2.67 (d, J = 7.1 Hz, 2H), 4.03 (br s, 1H), 5.14 (s, 2H), 6.73 (s, 1H),
7.05 (s, 1H), 7.08 (s, 1H), 7.22 (d, J = 7.6 Hz, 1H), 7.42 (app t, J = 7.6
Hz, 1H), 7.45−7.57 (m, 2H). 13C NMR (CDCl3) δ: 22.1, 29.5, 30.5,
39.1, 51.1, 54.5, 119.6, 122.3, 127.4, 128.2, 128.7, 128.9, 129.1, 130.4,
135.7, 135.9, 136.7, 142.2, 148.6. IR (cm−1) ν: 3287, 3113, 2961, 2870,
1465, 1433, 1387, 1318. Anal. Calcd for C22H28BrN3O2S2: C, 51.8; H,
5.5; N, 8.2. Found: C, 51.7; H, 5.6; N, 8.1.
General Procedure for the Synthesis of Compounds 38, 40,
44−51. To a solution of the tert-butylsulfonamide (10, 12, 16−23) in
CH2Cl2 (2 mL) was added BCl3 (0.6 mL, 1.0 M in hexane). The
reaction mixture was stirred at ambient temperature for 1 h. The
reaction mixture was concentrated under vacuum. Water (5 mL) was
added. The aqueous portion was extracted with EtOAc, and the
combined organic phase was washed with water, brine, and water. The
organic phase was dried and concentrated under vacuum. To the crude
primary sulfonamides (24, 26, 30−37) were added pyrrolidienopyr-
idine (2 equiv) and pyridine (1.5 mL), and the solution was stirred for
30 min. n-Butyl chloroformate (10 equiv) was added, and the mixture
was stirred overnight at room temperature. Citric acid (10% aq) was
added, and the reaction mixture was extracted with EtOAc and
concentrated under vacuum. The crude product was purified by
EXPERIMENTAL SECTION
Chemistry. General Considerations. H and 13C NMR spectra
■
1
were recorded on a JEOL JNM-EX 270 spectrometer at 270.2 and
67.8 MHz, respectively. Chemical shifts are given as δ values (ppm)
downfield from tetramethylsilane. Infrared spectra were recorded on a
Perkin-Elmer model 1605 FT-IR with a compression cell and are
reported as λmax (cm−1). For neat solids the instrument was equipped
with a microfocus beam condenser with ZnSe lenses in a Diasqueeze
plus diamond compressor cell (Graseby Specac Inc., Woodstock, GA,
U.S.). Elemental analyses were performed by Mikro Kemi AB,
Uppsala, Sweden, or Analytische Laboratorien, Lindlar, Germany. All
compounds were confirmed to have >95% purity. Flash column
chromatography was performed on silica gel 60 (0.04−0.063 mm, E.
Merck). Thin-layer chromatography was performed on precoated silica
gel F-254 plates (0.25 mm, E. Merck) and was visualized with UV
light. Analytical RP-LC/MS was performed on a Gilson HPLC system
with a Zorbax SB-C8, 5 μm, 4.6 mm × 50 mm (Agilent Technologies)
column, with a Finnigan AQA quadropole mass spectrometer at a flow
rate of 1.5 mL/min (H2O/CH3CN/0.05% HCOOH). All the organic
phases were dried over MgSO4 unless otherwise stated. All chemicals
were purchased from commercial suppliers and used directly without
further purification.
3-(3-Hydroxymethylphenyl)-5-isobutylthiophene-2-(N-tert-
butyl)sulfonamide (8). A mixture of m-bromobenzyl alcohol (1.05 g,
5.80 mmol), 7 (2.41 g, 7.56 mmol), Pd(PPh3)4 (270 mg, 0.234 mmol),
NaOH (19.1 mL, 1.5 M aq), EtOH (5.0 mL), and toluene (30 mL)
was stirred under N2(g) at 90 °C for 4 h. After the mixture was cooled,
water (10 mL) was added. The aqueous portion was extracted with
ethyl acetate. The organic layer was concentrated and purified by
column chromatography (EtOAc/hexane, 30:70) to give 8 as a
1
colorless syrup (1.26 g, 3.31 mmol, 57%). H NMR (CDCl3) δ: 0.96
(d, J = 6.6 Hz, 6H), 0.98 (s, 9H), 1.82−2.00 (m, 1H), 2.66 (d, J = 7.1
Hz, 2H), 3.28 (br s, 1H), 4.67 (s, 2H), 4.81(br s, 1H), 6.76 (s, 1H),
7.30−7.50 (m, 3H), 7.64 (s, 1H). 13C NMR (CDCl3) δ: 22.1, 29.4,
30.4, 39.1, 54.4, 64.6, 127.1, 127.8, 128.5, 129.0, 134.9, 136.2, 141.2,
143.2, 148.2. IR (cm−1) ν: 3498, 3286, 2958, 2870, 1465, 1313. Anal.
Calcd for C19H27NO3S2: C, 59.8; H, 7.1; N, 3.7. Found: C, 60.1; H,
7.3; N, 3.9.
3-(3-Bromomethylphenyl)-5-isobutylthiophene-2-(N-tert-
butyl)sulfonamide (9). A mixture of 8 (246 mg, 0.644 mmol), CBr4
(534 mg, 1.61 mmol), and PPh3 (422 mg, 1.61 mmol) in DMF (5.0
mL) was stirred at room temperature overnight. Then water (10 mL)
was added and extracted with ethyl acetate. The organic layer was
washed with water twice to remove excess DMF. Concentration and
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column chromatography or by preparative LC−MS to obtain the
products 38, 40, 44−51.
123.2, 127.7, 128.8, 129.1, 129.3, 134.0, 134.1, 134.4, 135.7, 137.3,
141.9, 143.1, 143.9, 149.0, 191.4. IR (cm−1) ν: 3277, 3065, 2961, 2869,
1702, 1616, 1580, 1498, 1461, 1312. Anal. Calcd for C27H31N3O3S2: C,
63.6; H, 6.1; N, 8.2. Found: C, 63.4; H, 6.1; N, 8.2.
3-(3-Imidazol-1-ylmethylphenyl)-5-isobutylthiophene-2-(N-
butyloxycarbonyl)sulfonamide (38). Compound 38 was synthe-
sized from 10 (68.8 mg, 0.159 mmol) following the general procedure.
The crude product was purified with column chromatography
3-[3-(2-Benzoimidazol-1-ylacetyl)phenyl]-5-isobutylthio-
phene-2-(N-tert-butyl)sulfonamide (75). To the solution of 69
(54.4 mg, 0.107 mmol) in TFA (3 mL) were added six drops of
anisole. After being stirred at room temperature for 28 h, the solution
was concentrated. To the crude product were added pyrrolidinopyr-
idine (31.6 mg, 0.214 mmol) and pyridine (1.5 mL), and the mixture
was stirred at room temperature for about 30 min. n-Butyl
chloroformate (146 mg, 1.07 mmol) was then added to the solution
and stirred at room temperature overnight. Another portion of
pyrrolidinopyridine (31.6 mg, 0.214 mmol) and n-butyl chloroformate
(146 mg, 1.07 mmol) was added and stirred for another night. To the
mixture was added citric acid (10 mL 10% aq), and the mixture was
extracted with ethyl acetate, concentrated, and purified by column
chromatography (MeOH/CH2Cl2, 5:95) to give 75 (21 mg, 38 μmol,
1
(MeOH/CH2Cl2 6:94) to afford 38 (44.4 mg, 93.3 μmol, 59%). H
NMR (CDCl3) δ: 0.86 (t, J = 7.3 Hz, 3H), 0.97 (d, J = 6.6 Hz, 6H),
1.18−1.34 (m, 2H), 1.44−1.58 (m, 2H), 1.84−2.00 (m, 1H), 2.67 (d, J
= 7.1 Hz, 2H), 4.01 (t, J = 6.6 Hz, 2H), 4.93 (s, 2H), 6.69 (s, 1H),
6.76−7.10 (m, 3H), 7.17 (app t, J = 7.6 Hz, 1H), 7.32 (d, J = 7.6 Hz,
1H), 7.52 (s, 1H), 7.61 (br s, 1H), 12.9 (br s, 1H). 13C NMR (CDCl3)
δ: 13.7, 18.9, 22.2, 30.4, 30.7, 39.2, 51.1, 65.7, 119.6, 125.6, 126.5,
128.5, 128.8, 129.0, 129.3, 134.1, 134.7, 135.6, 136.2, 143.6, 149.7,
153.5. IR (cm−1) ν: 3130, 3057, 2958, 1740, 1656, 1450,1344. Anal.
Calcd for C23H29N3O4S2: C, 58.1; H, 6.2; N, 8.8. Found: C, 57.9; H,
6.1; N, 8.7.
3-[3-(2-Chloroimidazol-1-ylmethyl)phenyl]-5-isobutylthio-
phene-2-(N-butyloxycarbonyl)sulfonamide (63). To the solution
of 52 (44 mg, 86 μmol) in TFA (3.0 mL) were added six drops of
anisole. After being stirred at room temperature for 25 h, the solution
was concentrated and dried under vacuum. Then pyrrolidienopyridine
(25.6 mg, 0.173 mmol) and pyridine (1.5 mL) were added, and the
mixture was stirred at room temperature for about 30 min. To the
solution was added n-butyl chloroformate (118 mg, 0.864 mmol), and
the mixture was stirred for 36 h at room temperature. To the reaction
mixture was added EtOAc (25 mL), and the mixture was washed with
citric acid (10% aq) and water, concentrated, and purified on column
chromatography (MeOH/CH2Cl2, 5:95) to give 63 (25 mg, 49 μmol,
1
36%). H NMR (CDCl3) δ: 0.84 (t, J = 7.3 Hz, 3H), 1.01 (d, J = 6.6
Hz, 6H), 1.14−1.34 (m, 2H), 1.42−1.60 (m, 2H), 1.86−2.02 (m, 1H),
2.72 (d, J = 6.9 Hz, 2H), 4.07 (t, J = 6.4 Hz, 2H), 6.78 (s, 1H), 7.04−
7.30 (m, 3H), 7.37 (app t, J = 7.7 Hz, 1H), 7.45 (d, J = 7.7 Hz, 1H),
7.52−7.70 (m, 2H), 7.81 (s, 1H), 8.33 (s, 1H), 8.89 (br s, 1H). 13C
NMR (CDCl3) δ: 13.6, 18.8, 22.3, 30.5, 30.6, 39.3, 49.9, 66.1, 109.7,
119.6, 122.8, 123.7, 127.6, 128.9, 129.5, 133.4, 133.6, 133.9, 134.2,
135.3, 140.4, 143.4, 143.8, 150.7, 152.5, 190.7. IR (cm−1) ν: 3061,
2959, 2871, 1739, 1702, 1604, 1580, 1499, 1462, 1343, 1289. Anal.
Calcd for C28H31N3O5S2: C, 60.7; H, 5.6; N, 7.6. Found: C, 60.5; H,
5.6; N, 7.6.
1
57%). H NMR (CDCl3) δ: 0.87 (t, J = 7.3 Hz, 3H), 0.99 (d, J = 6.6
3-[3-Formyl]phenyl-5-isobutylthiophene-2-(N-tert-butyl)-
sulfonamide (82). A mixture of Pd(OAc)2 (85 mg, 0.38 mmol) and
PPh3 (0.4 g, 1.5 mmol) was stirred in DME (5 mL) under N2 (g).
After being stirred for 30 min, the suspension was introduced via
syringe into a nitrogen-flushed mixture of 7 (4.0 g, 13 mmol), 3-
bromobenzaldehyde (2.96 mL, 25.1 mmol), and K2CO3 (5.21 g, 37.7
mmol) in a solvent mixture of DME, ethanol, and water (28, 8, 12
mL). After being stirred for 12 h at reflux under N2 atmosphere, the
reaction mixture was diluted with 1 M NaOH solution (50 mL)
followed by ethyl acetate (150 mL). The organic layer was washed
with water, and brine, dried over anhydrous MgSO4, and concentrated
in vacuo and the residue subjected to flash chromatography (20% ethyl
acetate in petroleum ether) to afford 82 as a colorless solid (3.9 g, 10
Hz, 6H), 1.15−1.33 (m, 2H), 1.45−1.60 (m, 2H), 1.85−2.02 (m, 1H),
2.70 (d, J = 7.1 Hz, 2H), 4.04 (t, J = 6.6 Hz, 2H), 5.08 (s, 2H), 6.73 (s,
1H), 6.87 (d, J = 1.5 Hz, 1H), 6.90 (d, J = 1.5 Hz, 1H), 7.08−7.18 (m,
1H), 7.27−7.45 (m, 3H). 13C NMR (CDCl3) δ: 13.6, 18.7, 22.2, 30.4,
30.5, 39.2, 49.9, 66.8, 121.2, 127.4, 128.2, 128.4, 128.9, 129.2, 131.4,
131.9, 134.9, 135.3, 145.4, 150.7, 151.5. IR (cm−1) ν: 3126, 3043,
2959, 2871, 1740, 1608, 1587, 1474, 1389, 1344. Anal. Calcd for
C23H28ClN3O4S2: C, 54.2; H, 5.5; N, 8.2. Found: C, 53.9; H, 5.7; N,
8.1.
General Procedure for the Synthesis of Compounds 64−66.
To a solution of 3-boromophenacyl bromide (120 mg, 0.432 mmol) in
dioxane (2 mL), the appropriate heterocycle (2 equiv) was added. The
reaction mixture was stirred for 1 h at 80 °C. The reaction mixture was
concentrated and the crude product was purified on column
chromatography to give the corresponding products 64−66.
2-Benzoimidazol-1-yl-1-(3-bromophenyl)ethanone (66).
Compound 66 was synthesized following the general procedure with
benzoimidazole (102 mg, 0.864 mmol) as heterocycle. The crude
product was purified (MeOH/CH2Cl2, 6:94) to afford 66 (71.8 mg,
0.228 mmol, 53%). 1H NMR (CDCl3) δ: 5.38 (s, 2H), 7.02−7.12 (m,
1H), 7.12−7.26 (m, 2H), 7.33 (app t, J = 7.9 Hz, 1H), 7.60−7.86 (m,
4H), 8.02−8.12 (m, 1H). 13C NMR (CDCl3) δ: 50.3, 109.2, 120.5,
122.4, 123.3, 123.4, 126.4, 130.7, 131.0, 134.0, 135.7, 137.2, 143.4,
143.6, 190.1. IR (cm−1) ν: 3090, 3056, 2926, 1708, 1615, 1566, 1501,
1417, 1351. Anal. Calcd for C15H11BrN2O: C, 57.2; H, 3.5; N, 8.9.
Found: C, 56.9; H, 3.6; N, 8.8.
1
mmol, 82%). H NMR (CDCl3) δ: 0.98 (d, J = 6.6 Hz, 6H), 1.03 (s,
9H), 1.93 (m, 1H), 2.69 (d, J = 6.6 Hz, 2H), 4.22 (br s, 1H), 6.79 (s,
1H), 7.61 (t, J = 7.9 Hz, 1H), 7.88−7.98 (m, 2H), 8.04 (t, J = 1.7 Hz,
1H), 10.05 (s, 1H). 13C NMR (CDCl3) δ: 22.1, 29.6, 30.5, 39.1, 54.8,
128.9, 129.1, 129.4, 130.0, 135.2, 135.9, 136.3, 137.0, 141.8, 148.9,
192.0. IR (cm−1) ν: 2960, 1701, 1391, 1319, 1144, 1052. Anal. Calcd
for C19H25NO3S2: C, 60.1; H, 6.6, N, 3.7. Found C, 60.4; H, 6.7; N,
3.7.
General Procedure for the Synthesis of Compounds 86−
104. Step 1. To a solution of 82 (50 mg, 0.13 mmol) in
MeOH (1 mL) taken in a sample vial (5 mL size), amine (1.1
equiv, 0.14 mmol) was added. After the mixture was stirred for
30 min, NaBH4 (5 mg, 0.1 mmol) was added. The stirring
continued for 30 min. The mixture was acidified with dilute
HCl (5 M, 0.2 mL) and then neutralized with saturated
NaHCO3 solution (∼0.5 mL) and diluted with ethyl acetate
(10 mL). The contents were poured into diatomaceous earth
(solid−liquid extraction cartridge) in a polypropylene column
(packed for 1.5 cm, 24 mL size) and eluted with ethyl acetate
(15 mL). Concentration of the filtrate under vacuum afforded
the crude product.
3-[3-(2-Benzoimidazol-1-ylacetyl)phenyl]-5-isobutylthio-
phene-2-(N-tert-butyl)sulfonamide (69). A mixture of 66 (42.7
mg, 0.136 mmol), 7 (73.5 mg, 0.230 mmol), Pd(PPh3)4 (9.4 mg, 8.1
μmol), Na2CO3 (271 μL, 0.542 mmol, 2.0 M aq), EtOH (1 mL), and
toluene (7 mL) was stirred under N2 at 80 °C for 3 h. After the
mixture was cooled, water (10 mL) was added and extracted with ethyl
acetate. The organic phase was dried and concentrated under vacuum
and purified by column chromatography (MeOH/CH2Cl2, 5:95) to
Step 2. The preceding product was dissolved in dry CH2Cl2 (2 mL)
in a sample vial (5 mL size). Triethylamine (36 μL, 0.26 mmol) and
acid chloride (2 equiv, 0.16 mmol) were then added sequentially. The
sample vial was tightly closed, and the mixture was stirred for 2 h.
Water (1 mL) was added followed by ethyl acetate (5 mL), and the
mixture was filtered through diatomaceous earth (packed for 1.5 cm in
a column of 24 mL capacity) and eluted with CH2Cl2 (20 mL).
1
give 69 as a colorless syrup (62 mg, 0.122 mmol, 90%). H NMR
(CDCl3) δ: 0.99 (d, J = 6.6 Hz, 6H), 1.08 (s, 9H), 1.86−2.02 (m, 1H),
2.71 (d, J = 6.9 Hz, 2H), 4.22 (br s, 1H), 5.63 (s, 2H), 6.80 (s, 1H),
7.18−7.34 (m, 3H), 7.62 (app d, J = 7.7 Hz, 1H), 7.76−7.90 (m, 2H),
7.97 (d, J = 4.3 Hz, 1H), 8.02−8.09 (m, 1H), 8.35−8.45 (m, 1H). 13C
NMR (CDCl3) δ: 22.1, 29.7, 30.5, 39.1, 50.4, 54.8, 109.4, 120.3, 122.3,
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Concentration of the filtrate under vacuum afforded the crude
product.
Porcine (Pig) Myometrial Membrane AT2 Receptor-Binding
Assay. Myometrial membranes were prepared from porcine uteri
according to the method by Nielsen et al.47 A presumable interference
by binding to AT1 receptors was blocked by addition of 1 μM losartan.
Binding of [125I]Ang II to membranes was conducted in a final volume
of 0.5 mL containing 50 mM Tris-HCl (pH 7.4), 100 mM NaCl, 10
mM MgCl2, 1 mM EDTA, 0.025% bacitracin, 0.2% BSA, homogenate
corresponding to 10 mg of the original tissue weight, [125I]Ang II
(80000−85000 cpm, 0.03 nM), and variable concentrations of test
substance. Samples were incubated at 25 °C for 1.5 h, and binding was
terminated by filtration through Whatman GF/B glass-fiber filter
sheets, which had been presoaked overnight with 0.3% polyethylamine,
using a Brandel cell harvester. The filters were washed with 3 × 3 mL
of Tris-HCl (pH 7.4) and transferred into tubes. The radioactivity was
measured in a μ-counter. The characteristics of the Ang II binding AT2
receptor was determined by using six different concentrations (0.03−5
nmol/L) of the labeled [125I]Ang II. Nonspecific binding was
determined in the presence of 1 μM Ang II. The specific binding
was determined by subtracting the nonspecific binding from the total
bound [125I]Ang II. The apparent dissociation constant Ki values were
calculated from IC50 values using the Cheng−Prusoff equation (Kd =
0.73 0.06 nM, [L] = 0.057 nM). The binding data were best fitted
with a one-site fit. All determinations were performed in triplicate.
In Vitro Morphological Effects. General. The chemicals used in
the present study were obtained from the following sources:
Dulbecco’s modified Eagle’s medium (DMEM), heat-inactivated fetal
bovine serum (FBS), HAT supplement (hypoxanthine, aminopterin,
thymidine), gentamycin from Gibco BRL (Burlington, Ontario,
Canada), and [Val5]angiotensin II from Bachem (Marina Delphen,
CA, U.S.). PD 123,319 was obtained from RBI (Natick, MA, U.S.). All
other chemicals were of grade A purity.
Step 3. A mixture of the above product and anisole (∼2 drops) in
trifluoroacetic acid (1.5 mL) in a sample vial (5 mL size) was stirred at
room temperature overnight. After removal of the solvent under
vacuum, the residue was dissolved in acetonitrile (2 mL) and
evaporated (2×).
Step 4. To a mixture of the preceding product in dry CH2Cl2 (2
mL), pyrrolidinopyridine (2 mg, 0.01 mmol), triethylamine (54 μL,
0.39 mmol), and n-butyl chloroformate (34 μL, 0.26 mmol) were
sequentially added. The solution was stirred for 2 h and concentrated
under vacuum, and the crude product was purified by preparative LC−
MS to afford the products 86−104.
3-[3-(N-Ethoxyoxalyl,N-methyl)aminomethyl)]phenyl-5-iso-
butylthiophene-2-(N-butyloxycarbonyl)sulfonamide (90).
Compound 90 was synthesized from 82 following the general
procedure with methylamine and ethyloxalyl chloride as reagents.
The crude product in the final step was purified (45−75% aqueous
acetonitrile) to afford 90 as a colorless oil (52 mg, 95 μmol, 73%). 1H
NMR (CDCl3) δ: 0.87 (t, J = 7.3 Hz, 3H), 0.98 (dd, J = 2.0 Hz, J = 6.6
Hz, 6H), 1.18−1.41 (m, 5H), 1.53 (m, 2H), 1.94 (m, 1H), 2.70 (dd, J
= 1.7 Hz, J = 6.9 Hz, 2H), 2.91 and 3.04 (s, 3H, rotation isomers), 4.05
(t, J = 6.6 Hz, 2H), 4.35 (dq, J = 2.3 Hz, J = 6.9 Hz, 2H), 4.49 and 4.58
(s, 2H, rotation isomers), 6.79 (m, 1H), 7.26−7.44 (m, 3H), 7.49 and
7.62 (s, 1H, rotation isomers). 13C NMR (CDCl3) δ: 13.6, 13.9, 18.7,
22.2, 30.5, 32.2, 35.5, 39.3, 50.6, 53.5, 62.4, 62.7, 66.6, 66.7, 127.6,
128.2, 128.3, 128.4, 128.6, 128.7, 129.0, 129.5, 131.1, 134.6, 134.8,
135.2, 135.5, 145.3, 145.5, 150.4, 150.6, 151.5, 151.6, 161.7, 162.0,
162.5, 163.5. IR (cm−1) ν: 2960, 2159, 2032, 1977, 1747, 1658, 1446,
1347, 1219, 1157. Anal. Calcd for C25H34N2O7S2: C, 55.7; H, 6.4; N,
5.2; Found: C, 55.6; H, 6.4; N, 5.1.
Cell Culture. To study the in vitro morphological effects, NG108-
15 cells (initially provided by Drs. M. Emerit and M. Hamon;
INSERM, U. 238, Paris, France) were used as well as transfected
NG108-15/pcDNA3 cells. The transfected cell line has previously
been shown to have the same behavior as the native cell line.28 Both
cell lines were cultured (NG108-15 passage 18−28, NG108-15/
pcDNA3 passage 12−18) in Dulbecco’s modified Eagle’s medium
(DMEM, Gibco BRL, Burlington, Ontario, Canada) with 10% fetal
bovine serum (FBS, Gibco), HAT supplement (hypoxanthine,
aminopterin, thymidine from Gibco), and 50 mg/L gentamycin
(Gibco) at 37 °C in 75 cm2 Nunclon Δ flasks in a humidified
atmosphere of 93% air and 7% CO2, as previously described.28,48 The
transfected cell line was kept stable by addition of Geneticin (G-418,
200 μg/mL) to the medium.28 Subcultures were performed at
subconfluency. Under these conditions, cells express only the AT2
receptor subtype.28,48 Cells were stimulated during 3 days, once a day
(first stimulation 24 h after plating). Cells were cultured for 3
subsequent days under these conditions. For all experiments, cells
were plated at the same initial density of 3.6 × 104 cells/35 mm Petri
dish. To determine a good test concentration, compounds 38, 63, 75,
90, and 100 were tested at various concentrations ranging from 1 pM
to 1 μM. For all the compounds it was only at the highest
concentration that the tendency of cell death was observed. Cells were
treated without (control cells) or with [Val5]angiotensin II from
Bachem (Marina Delphen, CA, U.S.) (100 nM) or 38 (10 nM), 63
(10 nM), 75 (10 nM), 90 (1 nM), and 100 (10 nM) in the absence or
in the presence of PD 123,319 (RBI Natick, MA, U.S.) (10 μM), an
AT2 receptor antagonist introduced daily 30 min prior to Ang II, 38,
63, 75, 90, or 100. During the 3 days of treatment the transfected cell
line was cultured without Geneticin.
3-[3-(N-Benzoyl,N-{3-picolyl})aminomethyl)]phenyl-5-isobu-
tylthiophene-2-(N-butyloxycarbonyl)sulfonamide (100). Com-
pound 100 was synthesized from 82 following the general procedure
with 3-picolylamine and benzoyl chloride as reagents. The crude
product in the final step was purified (55−85% aqueous acetonitrile)
to afford 100 as a colorless solid (52 mg, 85 μmol, 65%). Mp 73−75
1
°C. H NMR (CDCl3) δ: 0.89 (t, 7.3 Hz, 3H), 0.97 (d, J = 6.6 Hz,
6H), 1.31 (m, 2H), 1.59 (m, 2H), 1.91 (m, 1H), 2.67 (d, J = 6.9 Hz,
2H), 4.11 (t, J = 6.6 Hz, 2H), 4.74 (br m, 4H), 6.46−7.08 (br m, 4H),
7.20 (br s, 2H), 7.33−7.6 (m, 6H), 7.64−8.33 (m, 2H). 13C NMR
(CDCl3) δ: 13.6, 18.9, 22.2, 30.5, 30.6, 39.2, 47.9, 50.2, 52.3, 54.5,
66.0, 123.6, 126.8, 127.8, 128.7, 129.5, 129.8, 132.2, 132.7, 134.8,
135.6, 136.1, 137.1, 137.9, 145.0, 146.4, 147.2, 149.4, 150.5, 151.7,
172.5. IR (cm−1) ν: 2960, 1740, 1635, 1412, 1345, 1157, 733. Anal.
Calcd for C33H37N3O5S2·H2O: C, 62.1; H, 6.2; N, 6.6; Found: C, 62.0;
H, 6.0; N, 6.2.
Rat Liver Membrane AT1 Receptor-Binding Assay. Rat liver
membranes were prepared according to the method of Dudley et al.46
Binding of [125I]Ang II to membranes was conducted in a final volume
of 0.5 mL containing 50 mM Tris-HCl (pH 7.4), 100 mM NaCl, 10
mM MgCl2, 1 mM EDTA, 0.025% bacitracin, 0.2% BSA (bovine
serum albumin), liver homogenate corresponding to 5 mg of the
original tissue weight, [125I]Ang II (80000−85000 cpm, 0.03 nM), and
variable concentrations of test substance. Samples were incubated at
25 °C for 2 h, and binding was terminated by filtration through
Whatman GF/B glass-fiber filter sheets, which had been presoaked
overnight with 0.3% polyethylamine, using a Brandel cell harvester.
The filters were washed with 3 × 3 mL of Tris-HCl (pH 7.4) and
transferred into tubes. The radioactivity was measured in a μ-counter.
The characteristics of the Ang II binding AT1 receptor was determined
by using six different concentrations (0.03−5 nmol/L) of the labeled
Determination of Cells with Neurites. Cells were examined
under a phase contrast microscope, and micrographs were taken after
3 days under the various experimental conditions. Cells with at least
one neurite longer than a cell body were counted as positive for
neurite outgrowth. The number of cells with neurites represents the
percentage of the total amount of cells in the micrographs. At least
three different experiments were conducted for each condition, each in
duplicate. At least five images were taken per Petri dish; hence, a total
of 250−400 cells from each of the duplicate dishes were examined.
[
125I]Ang II. Nonspecific binding was determined in the presence of 1
μM Ang II. The specific binding was determined by subtracting the
nonspecific binding from the total bound [125I]Ang II. The apparent
dissociation constant Ki values were calculated from IC50 values using
the Cheng−Prusoff equation (Kd = 1.7 0.1 nM, [L] = 0.057 nM).
The binding data were best fitted with a one-site fit. All determinations
were performed in triplicate.
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Journal of Medicinal Chemistry
Article
The data are represented as the mean SEM of the average number
of cells on a micrograph.
(10) Nio, Y.; Matsubara, H.; Murasawa, S.; Kanasaki, M.; Inada, M.
Regulation of Gene Transcription of Angiotensin II Receptor Subtypes
in Myocardial Infarction. J. Clin. Invest. 1995, 95, 46−54.
(11) Li, J.; Culman, J.; Hortnagl, H.; Zhao, Y.; Gerova, N.; Timm,
M.; Blume, A.; Zimmermann, M.; Seidel, K.; Dirnagl, U.; Unger, T.
Angiotensin AT2 Receptor Protects against Cerebral Ischemia-Induced
Neuronal Injury. FASEB J. 2005, 19, 617−619.
Data Analysis. The data are presented as the mean SEM of the
average number of cells on a micrograph. Statistical analyses of the
data were performed using the two-way ANOVA test. Homogeneity of
variance was assessed by Bartlett’s test, and p values were obtained
from Dunnett’s tables.
(12) Oishi, Y.; Ozono, R.; Yano, Y.; Teranishi, Y.; Akishita, M.;
Horiuchi, M.; Oshima, T.; Kambe, M. Cardioprotective Role of AT2
Receptor in Postinfarction Left Ventricular Remodeling. Hypertension
2003, 41, 814−818.
ASSOCIATED CONTENT
■
S
* Supporting Information
Synthesis procedures and characterization of all compounds
and procedures for the biological evaluations. This material is
available free of charge via the Internet at http://pubs.acs.org.
(13) McCarthy, C. A.; Vinh, A.; Callaway, J. K.; Widdop, R. E.
Angiotensin AT2 Receptor Stimulation Causes Neuroprotection in a
Conscious Rat Model of Stroke. Stroke 2009, 40, 1482−1489.
(14) Reinecke, K.; Lucius, R.; Reinecke, A.; Rickert, U.; Herdegen,
T.; Unger, T. Angiotensin II Accelerates Functional Recovery in the
Rat Sciatic Nerve in Vivo: Role of the AT2 Receptor and the
Transcription Factor NF-kappaB. FASEB J. 2003, 17, 2094−2096.
(15) Rompe, F.; Artuc, M.; Hallberg, A.; Alterman, M.; Stroder, K.;
Thone-Reineke, C.; Reichenbach, A.; Schacherl, J.; Dahlof, B.; Bader,
M.; Alenina, N.; Schwaninger, M.; Zuberbier, T.; Funke-Kaiser, H.;
Schmidt, C.; Schunck, W. H.; Unger, T.; Steckelings, U. M. Direct
Angiotensin II Type 2 Receptor Stimulation Acts Anti-Inflammatory
through Epoxyeicosatrienoic Acid and Inhibition of Nuclear Factor
kappaB. Hypertension 2010, 55, 924−931.
AUTHOR INFORMATION
■
Corresponding Author
*For N.G.-P.: phone, +1-819-5645243; e-mail, nicole.gallo-
payet@usherbrooke.ca.. For A.H.: phone, +46-18-4714284; e-
mail, anders.hallberg@orgfarm.uu.se.. For M.A.: phone, +46-18-
4714124; e-mail, mathias.alterman@orgfarm.uu.se..
Notes
The authors declare no competing financial interest.
(16) Johansson, B.; Holm, M.; Ewert, S.; Casselbrant, A.; Pettersson,
ACKNOWLEDGMENTS
■
A.; Fandriks, L. Angiotensin II Type 2 Receptor-Mediated Duodenal
̈
We gratefully acknowledge support from the Swedish Research
Council (VR), the Swedish Foundation for Strategic Reasearch
(SSF), Knut and Alice Wallenberg Foundation, and the Canada
Research Chairs Program. N.G.-P. is a recipient of a Canada
Research Chair in Endocrinology of the Adrenal Gland. N.G.-P.
is member of the FRSQ-funded Centre de Recherche Clinique
Etienne-le Bel. We thank Lucie Chouinard and the other
members of the laboratory for their technical assistance with
cell cultures.
Mucosal Alkaline Secretion in the Rat. Am. J. Physiol. 2001, 280,
G1254−G1260.
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Angiotensin II and Neuronal Differentiation: From Observations to
Mechanisms. J. Mol. Endocrinol. 2003, 31, 359−372.
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