Design, synthesis, and biological evaluation, of the first selective nonpeptide AT2 receptor agonist

Article abstract of DOI:10.1021/jm049715t

The first druglike selective angiotensin II AT₂ receptor agonist (21) with a K_i value of 0.4 nM for the AT₂ receptor and a Ki > 10 μM for the AT₁ receptor is reported. Compound 21, with a bioavailability of 20-3

Full text of DOI:10.1021/jm049715t

J. Med. Chem. 2004, 47, 5995-6008
5995
Design, Synthesis, and Biological Evaluation of the First Selective Nonpeptide
AT2 Receptor Agonist
Yiqian Wan,† Charlotta Wallinder,† Bianca Plouffe,‡,§ He´le`ne Beaudry,‡ A. K. Mahalingam,† Xiongyu Wu,†
Berndt Johansson,| Mathias Holm,‡ Milad Botoros, Anders Karle´n,† Anders Pettersson,| Fred Nyberg,
Lars Fa¨ndriks,| 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 Gastrosurgical Research, Sahlgrenska Academy at Gothenburg
University, SE-413 45 Gothenburg, Sweden, and Department of Biological Research on Drug Dependence, BMC,
Uppsala University, P.O. Box 591, SE-751 23 Uppsala, Sweden
Received April 13, 2004
The first druglike selective angiotensin II AT2 receptor agonist (21) with a Ki value of 0.4 nM
for the AT2 receptor and a Ki >10 µM for the AT1 receptor is reported. Compound 21, with a
bioavailability of 20-30% after oral administration and a half-life estimated to 4 h in rat,
induces outgrowth of neurite cells, stimulates p42/p44mapk, enhances in vivo duodenal alkaline
secretion in Sprague-Dawley rats, and lowers the mean arterial blood pressure in anesthetized,
spontaneously hypertensive rats. Thus, the peptidomimetic 21 exerts a similar biological
response as the endogenous peptide angiotensin II after selective activation of the AT2 receptor.
Compound 21, derived from the prototype nonselective AT1/AT2 receptor agonist L-162,313
will serve as a valuable research tool, enabling studies of the function of the AT2 receptor in
more detail.
Introduction
cell proliferation, induction of programmed cell death,
extracellular matrix remodeling, and axonal regenera-
tion.(for reviews see refs 4-9) Recently, we demon-
strated that activation of the AT2 receptor stimulates
alkaline secretion by the duodenal mucosa in rats.10
The octapeptide angiotensin II (Ang II), the active
component of the renin-angiotensin system, mediates
its action via two major receptors, namely, AT1 and AT2.
While the AT1 receptor is closely associated with the
regulation of blood pressure, fluid and electrolyte bal-
ance, and thirst, the role of the AT2 receptor has been
more difficult to elucidate due to its low level of
expression.1 However, one of the most remarkable
features of the AT2 receptor is its high level of expres-
sion in most fetal tissues, including the brain. The
receptor ratio AT2/AT1 decreases dramatically after
birth,2,3 suggesting an involvement of the AT2 receptor
in fetal development. Indeed, in cells of neuronal origin,
activation of the AT2 receptor was shown to induce
neurite outgrowth and elongation, modulate neuronal
excitability, and promote cellular migration in the adult.
The expression of the AT2 receptor is mostly limited to
certain tissues, such as the adrenal gland and specific
areas of the brain, but importantly is up-regulated in
pathological conditions such as heart failure, renal
failure, myocardial infarction, brain lesions, vascular
injury, and wound healing. In the adult, activation of
the AT2 receptor results in vasodilatation, inhibition of
The precise nature of the signaling pathways acti-
vated by AT2 receptor stimulation is still controversial.
This seven-transmembrane domain receptor is not
coupled to any of the classical, well-established, second
messengers, such as cAMP or inositol phosphates, and
i
its coupling to a GR protein, reported by several authors,
is not viewed in consensus.(for reviews see refs 4-9)
However, various mediators, which could individually
exert opposite effects, such as cGMP, tyrosine or serine/
threonine phosphatases, and the extracellular signal-
regulated kinases ERK1/ERK2 (p42/p44mapk), have been
associated with activation of the AT2 receptor, depend-
ing on the cell types and experimental conditions used.
More precisely, a sustained increase in p42/p44mapk
activity is found associated with neuronal differentia-
tion.11,12
The AT2 receptor often mediates opposing effects to
the AT1 receptor. While there are several AT1 receptor
antagonists in the clinic (e.g., losartan, Figure 2), few
selective AT2 receptor agonists are available as research
tools and are all peptides [e.g. CGP-42112 and (p-amino-
Phe6)Ang II].13-15
* Corresponding authors. M.A.: Tel, +46-18-4714905; E-mail,
mathias.alterman@orgfarm.uu.se. A.H.:
Tel, +46-18-4714284;
Fax, +46-18-4714474; E-mail, anders.hallberg@orgfarm.uu.se.
N.G.-P.: Tel, +1-819-5645243; Fax, +1-819-5645292; E-mail,
nicole.gallo-payet@usherbrooke.ca.
Therefore metabolically stable, druglike AT2 receptor
agonists are highly desirable as research tools and as
potential therapeutics. We herein report that the non-
selective AT1 and AT2 receptor agonist L-162,313 (1)16-18
indeed can be modified to yield the highly selective AT2
receptor agonist 21 (Figure 1).
† Department of Medicinal Chemistry, Uppsala University.
‡ Service of Endocrinology, University of Sherbrooke.
§ Department of Physiology and Biophysics, University of Sher-
brooke.
| Department of Gastrosurgical Research, Sahlgrenska Academy at
Gothenburg University.
Compound 21, which has a bioavailability of 20-30%
after oral administration in rat, is to the best of our
Department of Biological Research on Drug Dependence, Uppsala
University.
10.1021/jm049715t CCC: $27.50 © 2004 American Chemical Society
Published on Web 10/15/2004

5996 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 24
Wan et al.
Figure 1.
Figure 2.
knowledge the first selective nonpeptide AT2 receptor
agonist reported. Compound 21 (a) induces neurite
outgrowth, (b) stimulates MAPK, (c) increases duodenal
mucosal alkalization in the rat, and (d) decreases mean
arterial blood pressure in spontaneously hypertensive
rats (SHR), effects that are all suppressed in the
presence of the selective AT2 receptor antagonist PD
123,319 (Figure 2).19
We decided initially to keep both the butyloxycarbonyl
sulfonamide and the isobutyl groups unaltered and first
focus our research efforts on the bicyclic nitrogen
heterocycle.
The compounds were prepared as shown in Schemes
1 and 2. The thiopheneboronic acid 2, used as the
starting material for all the compounds, was prepared,
in essence, as described by Kevin et al.20,22 Thus,
thiophene-2-sulfonyl chloride was first converted to the
N-tert-butylsulfonamide. Subsequent alkylation followed
by selective 3-lithiation/boronation delivered the key
intermediate 2. The aryl bromides, 3-7, were obtained
by N-alkylation of the appropriate nitrogen-containing
heterocycle with 4-bromobenzyl bromide, utilizing KOH
as base. The aryl bromides 3-7 were thereafter reacted
with the boronic acid 2 under Suzuki coupling conditions
with Pd(PPh3)4 as the source of palladium and KOH as
Results
Chemistry. Previous studies have demonstrated that
replacement of the isobutyl group of 1 yielded com-
pounds with low AT2 receptor selectivity.20 Note that
replacement of the isobutyl group with a m-methoxy-
benzyl group in a structurally related series provided
the first selective nonpeptide AT1 receptor agonist
L-163,491 (Figure 2).21
Scheme 1a
a Conditions: (a) Pd(PPh3)4, NaOH (aq), ethanol/toluene, thermal or microwave heating; (b) TFA or BCl3 in CH2Cl2; (c) butylchloroformate
with pyrrolidinopyridine, pyridine or K2CO3 in CH2Cl2/water (3:1).

The First Selective Nonpeptide AT2 Receptor Agonist
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 24 5997
Scheme 2a
a Conditions: (a) Pd(PPh3)4, NaOH (aq), ethanol/toluene, microwave heating; (b) Pd(PPh3)4, Zn(CN)2, DMF, microwave heating; (c)
imidazole, CuI, Cs2CO3, 1,2-diaminocyclohexane, DMF, microwave heating; (d) TFA or BCl3 in CH2Cl2; (e) butylchloroformate,
pyrrolidinopyridine, pyridine.
base to give compounds 10-14 (Scheme 1). Analogously,
the sulfonamides 15 and 16 were obtained after Suzuki
coupling with 4-bromophenylacetonitrile (8) and 4-
bromobenzonitrile (9), respectively. The biaryl com-
pounds 10-16 were isolated in moderate to good yields.
Compound 14 was alternatively prepared by a fast
microwave-promoted Suzuki reaction.23,24 Deprotection
by TFA or BCl3, a milder deprotecting reagent,25 deliv-
ered the primary sulfonamide, and subsequent reactions
with butyl chloroformate in pyridine with 4-pyrrolidin-
1-yl-pyridine as nucleophilic catalyst afforded the target
compounds 17-19 and 21-23 (Scheme 1). To obtain the
methylimidazole compound 20, a minor modification
was required, since an acylimidazolium salt seemed to
have been formed using 4-pyrrolidin-1-ylpyridine in
pyridine. To circumvent this obstacle, the reaction
was performed in a CH2Cl2/water (3:1) mixture with
Na2CO3 as base, which provided the compound 20 in a
good yield.
To obtain the compounds 27 and 28, the boronic acid
2 was first treated with 4-bromobenzyl bromide under
Suzuki coupling conditions with Pd(PPh3)4 and K2CO3
as base to give the aryl bromide 24, isolated in 80% yield
(Scheme 2). A small-scale microwave-mediated version
of the Suzuki benzylation reaction required 30 min at
110 °C and provided a yield of 78%. The weaker carbon-
ate base was used rather than KOH to prevent the
benzylic bromide from being converted to the corre-
sponding benzyl alcohol. The cyanide group was intro-
duced by a microwave-promoted palladium-catalyzed
cyanation reaction.26 The reaction was complete after
2 min at 180 °C and provided the nitrile 25 in 75% yield.
To introduce the imidazole ring, we aimed to use
Buchwald-Hartwig conditions27,28 with microwave heat-
ing. This reaction was first explored using 4-bromotolu-
ene as a model substrate in DMF to optimize the
reaction conditions. To our surprise, 4-tolylimidazole
was not obtained. Instead, N,N-dimethyltolyl amide was
the predominant product. The carbonyl moiety was
proven to be derived from DMF, serving as an excellent
carbon monoxide source.29,30 Only with electron-deficient
aryl bromides could the arylimidazoles be obtained.31
Therefore, the imidazole was instead attached to the
aromatic ring by a microwave-promoted Ullman cou-
pling.32,33 The reaction was sluggish and yielded only
27% of compound 26 after 40 min at 180 °C. Finally 25
and 26 were deprotected with BCl3 to give the primary
sulfonamide, and subsequent reaction with butyl chlo-
roformate gave the target compounds 27 and 28.
Binding Assays. Compounds 17-23, 27, and 28
were evaluated in radioligand-binding assays by dis-
placement of [125I]Ang II from AT1 receptors in rat liver
membranes and from AT2 receptors in pig uterus
membranes in essence as described previously (Table
1).34,35 The natural substrate Ang II, the selective AT1
receptor antagonist losartan,36 and the selective AT2
receptor antagonist PD 123,31919 were used as reference
substances.
The Ki values for 1 in Table 1 were determined to be
3.9 nM for AT1 and 2.8 nM for the AT2 receptors (lit.17
IC50 ) 1.1 and 2.0 nM, respectively). As apparent from
Table 1, removal of the alkyl groups from the bicyclic
heterocycle resulted in a dramatic loss of affinity to the
AT1 receptor, while the AT2 receptor affinity was only
slightly affected (entry 2, compound 17). A similar
response was encountered after replacement of the
imidazopyridine with a 2-ethyl-1H-benzimidazole (entry
3, compound 18). Unexpectedly, the AT1 affinity was
increased by the removal of the ethyl group from the
benzimidazole (entry 4, compound 19). Notably, with
2-methylimidazole or imidazole (entries 5 and 6) as
heterocycles, the affinity to the AT1 receptor remained
poor, but a 10-fold improvement of the AT2 receptor
affinity was observed. On the other hand, further
simplification of the structure, i.e. changing from an
imidazole to a nitrile (entry 7, compound 22) led to a
100-fold improved AT1 affinity, while the affinity to the
AT2 receptor now dropped more than 100-fold. Thus,
this alteration delivered a nonselective ligand. Removal
of the benzylic methylene group of 22 (entry 8) resulted
in the inactive arylnitrile 23. Furthermore, employing
a benzylthiophene instead of a phenylthiophene scaffold,
in combination with attachment of the imidazole direct
on the aromatic ring, had a deleterious effect on the

5998 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 24
Wan et al.
Table 1.
Figure 3. Effect of 21 and angiotensin II (Ang II) on neurite
outgrowth in NG108-15 cells. NG108-15 cells were plated at
a density of 3 × 104 cells per dish in 35 mm Petri dishes and
were cultured for 3 days in the absence (control, A), or in
the presence of 0.1 µM compound 21 (B, D), 0.1 µM Ang II
(C), 1 µM PD 123,319 (E) or 21, and 0.1 µM PD 123,319 (F).
Panels A, B, E, and F are seen at the same magnification, and
panels C and D at higher magnification (D is a magnification
of B).
various concentrations ranging from 1 pM to 1 µM.
Except for the higher concentration of 1 µM, none of the
other doses induced cell death. In addition, cells stimu-
lated for 3 days with concentrations of compound 21
higher than 0.1 nM induced neurite outgrowth. As
shown by phase-contrast microscopy, nontreated control
cells had rounded cell bodies, without or with some thin
processes (Figure 3A). After a 3-day treatment with 21
(0.1 µM), most cells extended one or two neurite
processes, while the cell body retained a rounded
appearance (Figure 3B). In comparison to Ang II (0.1
µM) (Figure 3C), compound 21 induces the same mor-
phological changes, with the longer processes exhibiting
a growth cone at the tip38 (Figure 3D). This effect was
mediated through the AT2 receptor, since coincubation
of 21 with the AT2 receptor antagonist PD 123,319 (1
µM) virtually halted neurite elongation (Figure 3F),
while alone PD 123,319 did not alter the morphology of
the untreated cells (Figure 3E).
To further explore if compound 21 shares the same
signaling transduction as Ang II,12,39 cells were pre-
incubated for 3 days with 10 µM PD 98,059: a dose that
abolished MAPK activity. Alone, PD 98,059 induced a
rounding up appearance of the cell body (compare
Figure 4C to Figure 3A) and, when coincubated with
21, decreased length and number of neurites was also
observed (Figure 4 D). Cells were also preincubated with
LY-83,583 (0.5 µM), an inhibitor of soluble guanylyl
cyclase (sGC), or with KT 5823 (1 µM), an inhibitor of
cGMP-dependent protein kinases, which were added
every day, 30 min before compound 21. KT 5823- or LY-
83,583-treated cells had the same morphological ap-
pearance as the untreated control cells (Figure 4E,G).
However, cells coincubated with 21 and LY-83,583 or
a Ki values are an average from three determinations. Standard
deviations are less than 15% in all cases.
affinities (entry 10). Compound 21, with the highest
affinity and receptor selectivity, was chosen for further
in vitro and in vivo studies.
In Vitro Morphological Effects Induced by 21 in
NG 108-15 Cells. To study the effects of compound 21
on differentiation, we used NG108-15 cells. In their
undifferentiated state, neuroblastoma × glioma hybrid
NG108-15 cells have a rounded shape and divide
actively. We have shown previously that these cells
express only the AT2 receptor37,38 and that a three-day
treatment with Ang II or the selective peptidic AT2
receptor agonist CGP-42112 induces neurite outgrowth.38
The mechanisms involve a sustained increase in p42/
p44mapk activity12 and activation of the nitric oxide/
guanylyl cyclase/cGMP pathway39(for review, see ref 9).
For all experimental conditions, cells were plated at
the same initial density (30 × 104 cells/35 mm Petri
dish) and were treated without or with Ang II or
compound 21. After 3 days of culture, cells were
examined under a phase-contrast microscope and mi-
crographs were taken. We first tested compound 21 at

The First Selective Nonpeptide AT2 Receptor Agonist
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 24 5999
Figure 4. Effect of various inhibitors on 21-induced neurite
outgrowth in NG108-15 cells. NG108-15 cells were plated at
a density of 3 × 104 cells per dish in 35 mm Petri dishes and
were cultured for 3 days in the absence (control, A, C, E, and
G) or in the presence of 0.1 µM 21 (B, D, F, H). Cells were
cultured in the presence of 10 µM PD 98,059 (C, D), 0.5 µM
LY-83,583 (E, F), or 1 µM KT 5823 (G, H). All panels are seen
at the same magnification.
Figure 5. Effect of various inhibitors on 21-induced neurite
outgrowth in NG108-15 cells. Cells with at least one neurite
longer than a cell body were counted as positive for neurite
outgrowth The number of cells with neurites represent the
percentage of the total number of cells in the micrographs
(from 50 to 100 cells according to the experiment). (A) Control,
with Ang II (0.1 µM) stimulation, Ang II (0.1 µM) in the
presence of PD 123,319 (0.1 µM), 21 (0.1 µM), and 21 (0.1 µM)
in the presence of PD 123,319 (0.1 µM). (B) control, Ang II
(0.1 µM), Ang II (0.1 µM) in the presence of PD 98,059 (10
µM), Ang II (0.1 µM) in the presence of LY-83,583 (0.5 µM),
Ang II (0.1 µM) in the presence of KT 5823 (1.0 µM), 21 (0.1
µM), 21 (0.1 µM) in the presence of PD 98,059 (10 µM), 21
(0.1 µM) in the presence of LY-83,583 (0.5 µM), and 21 (0.1
µM) in the presence of KT 5823 (1.0 µM).
KT 5823 have only one or two thin processes (Figures
4F,H). Quantification of these results (Figure 5) indi-
cated that a 3-day treatment with 21 increased the
number of cells with neurites (from 5.5 ( 0.39% in
control cells to 15.6 ( 0.7% in Ang II-treated cells
and 19.9 ( 1.10% in the compound 21-treated cells).
This effect was abolished in cells coincubated with PD
123,319 (Figure 5A), PD 98,059, LY-83,583, and KT
5823 (Figure 5B).
Compound 21 also induced tyrosine phosphorylation
of p42/p44mapk, as determined by using an antibody
directed against the tyrosine phosphorylated form of
p42/p44mapk. This effect is observed within 30 min of
compound 21 application (2.2 ( 0.4-fold increase over
control) and then decreases to basal level after 60 min.
The stimulation observed after 30 min was abolished
in cells preincubated with 10 µM PD 123,319 (Figure
6).
Intravenous doses of compound 21 raised the duode-
nal alkaline secretion dose-dependently with a threshold
for response at an infusion rate of 0.03 mg kg-1 h-1
(Figure 7). The secretory response to the highest dose
was significantly inhibited by the AT2-receptor antago-
nist PD 123,319 (0.3 mg kg-1 h-1) (Figure 8, left panel).
Compound 21 also stimulated secretion when exposed
topically to the mucosa by administering it into the
intraluminal perfusate at a concentration of 100 µM.
In addition, this response was sensitive to PD 123,319
(150 µM intraluminally) (Figure 8, right panel). Mean
arterial pressure was not significantly influenced during
the secretion experiments in the S-D rat strain (data
not shown). As AT2-related hypotensive effects have
been reported, particularly in SHR,41 a limited number
of experiments were performed in this rat strain. Basal
In Vivo Assays. The in vivo experiments were
performed on anaesthetized nonfasted male Sprague-
Dawley rats (S-D) and spontaneously hypertensive rats
(SHR). A femoral artery and one or two veins were
catheterized for subsequent blood pressure measure-
ments and drug infusions, respectively. Duodenal mu-
-
cosal alkaline secretion (HCO3 secretion) was mea-
sured by a pH-stat titration technique.40 Alkaline
secretion to the perfusate was continuously titrated to
pH 7.4 with 0.02 M aqueous hydrochloric acid controlled
by a pH-stat device.

6000 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 24
Wan et al.
at the higher dose levels did not reach statistical
significance as compared to vehicle. PD 123,319 had no
intrinsic effect on MAP (data not shown).
Pharmacokinetics. The oral absorption study was
performed on male Sprague-Dawley rats in triplicate
with three oral concentrations (30, 100, and 300 µg/kg)
and one intravenously (30 µg/kg). The orally adminis-
trated doses were given by gastric gavage, and the
intravenous doses were administrated as a single bolus
injection into the tail vein. All dose weights were
adjusted to take into account the weight of the animal
at the time of administration. A blood sample (200 µL)
was taken from the animals by venepuncture of the tail
vein at the following time points postdose: 30 min and
1, 2, 4, 6, 8, and 24 h. The concentration of compound
21 was analyzed with LC-MS/MS.
An approximate dose-proportional increase in the
systematic exposure of 21 was observed with increasing
dose levels. Total clearance of 21 from plasma was
nominally similar to hepatic plasma flow rate in rats.
The apparent volume of distribution was up to 2.9-fold
greater than total body water in rats, suggesting that
21 may be sequestered widely into the tissues. Plasma
concentrations of 21 declined with apparent terminal
half-lives ranging from 3 to 6 h following oral admin-
istration. Following intravenous administration, plasma
concentrations declined with apparent terminal half-
lives of 0.5-2.5 h. Estimates of absolute bioavailability
of 21 following oral administration ranged from 20 to
30%.
Figure 6. Effect of 21 on p42/p44mapk activation. NG108-15
cells were stimulated without or with 0.1 µM 21 for 30 and 60
min alone or in the presence of 1 µM PD 123,319. Upper panels
for each condition show Western blot analysis of phosphory-
lated p42/p44mapk and lower panels represent the same blots
reprobed for total p42/p44mapk. Results are representative of
at least three different experiments.
Discussion
The present results demonstrate that compound 21
induces neurite outgrowth, one of the first steps of
neuronal differentiation, as does Ang II or CGP-42112.38
In addition, 21 like Ang II stimulates MAPK, with a
maximal activity observed at 30 min, and as previously
shown,12 this sustained activation of the p42/p44mapk
pathway is essential to promote neurite outgrowth.
These effects are attributable to the AT2 receptor, since
coincubation with PD 123,319 abolishes the effect of 21.
These results indicate that compound 21 acts as an
agonist at the AT2 receptor and is the first nonpeptidic
AT2 agonist to our knowledge.
Moreover, inhibition of PKG with KT 5823 or sGC
with LY-83,583 indicates that cGMP participates in the
early steps of neurite elongation. All these results
corroborate previous observations made with Ang II or
the peptide agonist CGP-42112.12,38,39
The existence of AT2 receptors in the rat duodenal
mucosa has been reported recently.10 When the endog-
enous hormone Ang II or the nonpeptide receptor
agonist L-162,313 was administered in vivo in the
presence of the AT1 receptor antagonist losartan, duode-
nal mucosal alkaline secretion increases markedly.
These responses were inhibited by the AT2 receptor
antagonist PD 123,319,10,18 indicating mediation through
the AT2 receptor. Also the selective peptidic AT2 receptor
agonist CGP-42112 was shown to increase mucosal
alkaline secretion to the same extent, and the response
was sensitive to PD 123,319 but not to losartan. From
these experiments it is apparent that duodenal mucosal
alkaline secretion is accelerated by activation of the AT2
receptors located in the duodenal mucosa/submucosa,
Figure 7. The effect of increasing iv doses of compound 21
on rat duodenal mucosal alkaline secretion in vivo. Data are
expressed as the percent change from baseline and plotted as
mean ( SEM, with n ) 5 in each 21 group, n ) 4 in the time
controls (NaCl).
mean arterial pressure before drug administration
ranged between 158 and 203 mmHg (mean value 182
mmHg, n ) 41), and a wide dose range was studied
(0.008-4 mg/kg intravenously). Compound 21 at low
doses (0.008 and 0.05 mg/kg) decreased MAP, a response
that was attenuated in animals treated with PD
123,319 (0.3 mg kg-1 h-1 iv) (Figure 9). Changes in MAP

The First Selective Nonpeptide AT2 Receptor Agonist
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 24 6001
Figure 8. The effect of compound 21 on rat duodenal mucosal alkaline secretion in vivo at 0.3 mg kg-1 h-1 intravenously (left
panel) or at 100 µM intraluminally (right panel). The figure also shows sensitivity to the selective AT2 receptor antagonist PD
123,319 given intravenously at 0.3 mg kg-1 h-1 bolus (left panel) or intraluminally at 150 µM (right panel). Data are expressed
as the percent change from baseline and are plotted as mean (SEM, n ) 5 in each group.
Moderate changes in local blood flow cannot be excluded
but seem unlikely to influence duodenal mucosal alka-
line secretion.42 It was reported recently that local
bradykinin BK2-receptors, possibly by a paracrine
bradykinin action, are activated when the peptidic AT2
receptor agonist CGP-42112 stimulates rat duodenal
mucosal alkaline secretion.43 However, further studies
are needed to investigate in detail the mediation of the
increased alkaline secretion following AT2 receptor
stimulation.
In the spontaneously hypertensive rat (SHR), AT2
receptor stimulation with CGP-42112 induces a depres-
sor response during simultaneous AT1 receptor block-
ade.41 Selective AT2 receptor stimulation was also found
to mediate a hypotensive response in conscious normo-
tensive rats, an effect dependent on an intact L-
arginine-nitric oxide axis.44 Activation of AT2 receptors
Figure 9. The acute effect of compound 21 (0.05 mg/kg
by endogenous Ang II has been shown to mediate flow-
induced dilation of rat mesenteric resistance arteries,
and AT2 receptors modulate Ang II-induced contractions
of small mesenteric arteries in SHR. In Ang II-depend-
ent hypertension in rats, the hypotensive response to
AT1 receptor blockade has been shown to be attenuated
by AT2 receptor blockade (for review, see ref 45). It
follows that from the actions described for the AT2
receptors thus far, one would logically predict that a
specific AT2 receptor agonist should act hypotensive,
particularly so in SHR. This was confirmed by the
present experiments, where administration of 21 to
S-D rats had no significant influence on arterial
pressure, whereas a significant reduction occurred in
the µg/kg dose range (0.008-0.05 mg/kg µg/kg) in SHR
(N.B., the S-D rats were instrumented for secretion
studies, which may have influenced the results). The
hypotensive response to 21 in SHR was partially sensi-
tive to the selective AT2 receptor antagonist PD
123,319, indicating action through the AT2 receptor.
However, no effect on mean arterial pressure was
observed at higher doses (mg/kg range), suggesting a
bell-shaped dose-response curve. It may be that 21 acts
as a partial agonist on the vasculature. It should also
be noted that the effects observed were recorded only
some 30-40 min after administration of the compound
in anesthetized animals. Measurements of long term
intravenously) alone or together with the selective AT2 receptor
antagonist PD 123,319 (0.3 mg/kg h intravenously) on mean
arterial pressure in anesthetized spontaneously hypertensive
rats. Data are the absolute change from baseline pressure
(MAP) within 30 min and are plotted as mean (SEM; /
denotes a significant difference from the NaCl group.
and this model could therefore preferably be used to
validate the agonistic properties of compound 21. The
present investigation shows that both when infused
intravenously (0.3 mg kg-1 h-1) or given topically in the
luminal perfusate (100 µM), 21 markedly increased the
mucosal alkaline secretion. Such responses were absent
or greatly reduced in the presence of PD 123,319 (0.3
mg/kg iv bolus), strongly suggesting interaction with
AT2 receptors. Furthermore, increasing iv doses of 21
demonstrated a dose-response relationship, giving ad-
ditional support to the agonistic nature of the compound
in vivo.
It should be noted that the secretory effect could be
obtained by both a systemic and local route of admin-
istration of 21. The latter opens up the possibility of
luminal (per oral) administration and local mucosal
action with limited need for systemic concentrations of
the drug, in turn reducing the risk of side effects on
distant organs. The staining in the lamina propria of
the villi for both AT1 and AT2 receptors suggests
receptor localization in proximity to blood vessels.10

6002 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 24
Wan et al.
8.1 Hz, 2H), 7.26 (dd, J ) 8.1, 5.0 Hz, 1H), 7.18 (d, J ) 8.1 Hz,
blood pressure effects in conscious animals following
chronic administration are needed to more completely
characterize the hypotensive action of the compound.
2H), 5.44 (s, 2H).
1-(4-Bromobenzyl)-2-ethyl-1H-benzoimidazole
(4).
DMSO (20 mL, dried over 4Å molecular sieves) was added to
KOH (2.24 g, 40 mmol) and the mixture was stirred for 5 min.
2-Ethyl-1H-benzoimidazole (1.1 g, 7.5 mmol) was then added
and the mixture was stirred for 2 h. 4-Bromobenzyl bromide
(3.25 g, 13.0 mmol) was added and the mixture was cooled
briefly and stirred for another hour before water (20 mL) was
added. The mixture was extracted with ether and the combined
organic phase was washed with water. The organic phase was
dried over CaCl2 and the solvent was removed in vacuo. The
residue was chromatographed on silica gel with hexane as
eluent, yielding the titled compound (2.30 g, 7.3 mmol, 97%
yield). 1H NMR (CDCl3), δ, ppm: 7.80 (d, J ) 6.9 Hz, 1H),
7.43 (d, J ) 8.6 Hz, 2H), 7.25-7.20 (m, 3H), 6.90 (d, J ) 8.6
Hz, 2H), 5.29 (s, 2H), 2.87 (q, J ) 7.6 Hz, 2H), 1.43 (t, J ) 7.6
Hz, 3H). 13C NMR (CDCl3), δ, ppm: 156.1, 141.8, 135.0, 134.8,
132.2, 127.8, 122.6, 122.4, 121.9, 119.2, 109.3, 46.3, 20.8, 11.7.
Anal. (C16H15BrN2) C, H, N.
1-(4-Bromobenzyl)-1H-benzoimidazole (5).47 Dimethyl
sulfoxide (20 mL, dried over 4Å molecular sieves) was added
to potassium hydroxide (2.24 g, 40 mmol, crushed pellets) and
the mixture was stirred for 5 min. Benzimidazole (1.18 g, 0.01
mol) was then added and the mixture was stirred for a further
2 h. 4-Bromobenzyl bromide (5.0 g, 0.02 mol) was added, the
mixture was cooled briefly and stirred for another hour before
water (20 mL) was added. The mixture was extracted with
ether and the combined organic phase was washed with water.
The organic phase was dried over CaCl2 and the solvent was
removed in vacuo. The residue was chromatographed on silica
gel with CH2Cl2:Et2O (1:2) as eluent, yielding the title product
(2.36 g, 8.2 mmol, yield: 82%).47 1H NMR (CDCl3 + CD3OD),
δ, ppm: 8.04 (s, 1H), 7.83 (dd, J ) 6.5, 1.7 Hz, 1H), 7.45 (d, J
) 8.3 Hz, 2H), 7.28 (m, 3H), 7.04 (d, J ) 8.3 Hz, 2H), 5.32 (s,
2H).
Conclusion
Here we have reported the first druglike selective AT2
receptor agonist, compound 21, with 20-30% oral
bioavailability and a half-life estimated to be 4 h in rat,
which mimics Ang II in its actions on the AT2 receptor.
Thus, compound 21 induces outgrowth of neurite cells,
enhances alkaline secretion from the rat intestine, and
lowers the mean arterial blood pressure in spontane-
ously hypertensive rats. We believe that compound 21,
derived from the prototype nonselective AT1/AT2 recep-
tor agonist 1 will serve as a valuable research tool for
studies of the function of the AT2 receptor. Although
long-term clinical studies are lacking, numerous experi-
mental studies indicate that the clinical improvements
observed following treatment with selective AT1 antago-
nists to a large extent are due to activation of the
unopposed AT2 receptor and an increased plasma level
of Ang II.45 It follows that direct activation of AT2
receptors during, for example, hypertension and car-
diovascular remodeling is an attractive pharmaceutical
principle. The presently reported AT2-selective agonist
compound 21 could possibly be regarded as the first
member of such a novel drug class.
Experimental Section
Chemistry. General Considerations. H and 13C NMR
1
spectra 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
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 Pulse Diamond Compressor Cell
(Graseby Specac Inc., Woodstock, GA). Elemental analyses
were performed by Mikro Kemi AB, Uppsala, Sweden, or
Analytische Laboratorien, Lindlar, Germany. 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 ×
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). Temperature-controlled micro-
wave heating was carried out using an automatic Smith single-
mode synthesizer producing a radiation frequency of 2450 MHz
(Personal Chemistry AB, Uppsala, Sweden). All the organic
phases were dried over MgSO4, unless otherwise is stated. All
chemicals were purchased from commercial suppliers and used
directly without further purification.
3-(4-Bromobenzyl)-3H-imidazo[4,5-b]pyridine (3).46
Dimethyl sulfoxide (20 mL dried over 4 Å molecular sieves)
was added to potassium hydroxide (2.24 g, 40 mmol, crushed
pellets) and the mixture was stirred for 5 min. 3H-Imidazo-
[4,5-b]pyridine (1.0 g, 8.4 mmol) was then added and the
mixture was stirred for 2 h. 4-Bromobenzyl bromide (3.25 g,
13 mmol) was added and the mixture was cooled briefly and
stirred for another hour before water (20 mL) was added. The
mixture was extracted with ether and the combined organic
phase was washed with water. The organic phase was dried
over CaCl2 and the solvent was removed in vacuo. The residue
was chromatographed on silica gel with CH2Cl2:Et2O (1:2) as
eluent to yield the title compound (1.275 g, 4.4 mmol, 53%
yield).46 1H NMR (CDCl3), δ, ppm: 8.42 (dd, J ) 5.0, 1.5 Hz,
1H), 8.10 (dd, J ) 8.1, 1.5 Hz, 1H), 8.07 (s, 1H), 7.45 (d, J )
1-(4-Bromobenzyl)-2-methyl-1H-imidazole (6). DMSO
(40 mL, dried over 4 Å molecular sieves) was added to KOH
(3.42 g, 61 mmol) and the mixture was stirred for 5 min.
2-Methyl-1H-imidazole (1.0 g, 12.1 mmol) was then added and
the mixture was stirred for 2 h. 4-Bromobenzyl bromide (4.5
g, 18.3 mmol) was added and the mixture was cooled briefly
and stirred for another hour before water (20 mL) was added.
The mixture was extracted with ether and the combined
organic phase was washed with water. The organic phase was
dried over CaCl2 and the solvent was removed in vacuo. The
residue was chromatographed on silica gel with CHCl3:MeOH
(30:1) plus 0.05% formic acid as eluent, yielding the title
1
compound (2.58 g, 10.3 mmol, 85% yield). H NMR (CDCl3),
δ, ppm: 7.46 (d, J ) 8.3 Hz, 2H), 6.95 (s, 1H), 6.91 (d, J ) 8.3
Hz, 2H), 6.82 (s, 1H), 5.00 (s, 2H), 2.31 (s, 3H). 13C NMR
(CDCl3), δ, ppm: 145.0, 135.5, 132.2, 128.4, 127.7, 122.0, 119.9,
49.2, 13.2. IR (compression cell), cm-1: 3124, 3105, 2916. Anal.
(C11H11BrN2) C, H, N.
1-(4-Bromobenzyl)-1H-imidazole (7). DMSO (20 mL,
dried over 4 Å molecular sieves) was added to KOH (2.24 g,
0.04 mol) and the mixture was stirred for 5 min. Imidazole
(0.57 g, 8.4 mmol) was then added and the mixture was stirred
for 2 h. 4-Bromobenzyl bromide (3.25 g, 13.0 mmol) was added
and the mixture was cooled briefly and stirred for another hour
before water (20 mL) was added. The mixture was extracted
with ether and the combined organic phase was washed with
water. The organic phase was dried over CaCl2 and the solvent
was removed in vacuo. The residue was chromatographed on
silica gel with CHCl3:MeOH (30:1) plus 0.05% formic acid as
eluent, yielding the title compound (1.28 g, 5.4 mmol, 64%
yield). 1H NMR (CDCl3), δ, ppm: 7.58 (s, 1H), 7.49 (d, J ) 8.4
Hz, 2H), 7.10 (s, 1H), 7.02 (d, J ) 8.3 Hz, 2H), 6.88 (s, 1H),
5.09 (s, 2H). 13C NMR (CDCl3), δ, ppm: 137.5, 135.3, 132.3,
130.2, 129.0, 122.4, 119.3, 50.2. IR (compression cell), cm-1
3138, 3091, 2985. Anal. (C10H9BrN2) C, H, N.
:
3-(4-Imidazo[4,5-b]pyridin-3-yl-methylphenyl)-5-isobu-
tyl-N-tert-butylthiophene-2-sulfonamide (10). Compound
2 (151.0 mg, 0.473 mmol), 3 (90.0 mg, 0.314 mmol), toluene

The First Selective Nonpeptide AT2 Receptor Agonist
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 24 6003
(15 mL), ethanol (1.3 mL), NaOH (1.3 mL, 1 M), and Pd(PPh3)4
(10.9 mg, 9 µmol) were mixed together under a N2(g) atmo-
sphere. The mixture was heated to reflux and stirred for 3 h.
After dilution with EtOAc (20 mL), the reaction mixture was
washed with water and brine. The organic phase was dried
and concentrated under vacuum. The crude product was
purified by column chromatography (hexane:acetone 2:1) to
give the title compound in 76% yield (115.8 mg, 0.240 mmol).
1H NMR (CDCl3), δ, ppm: 8.43 (d, J ) 4.9 Hz, 1H), 8.12-8.10
(m, 2H), 7.58 (d, J ) 8.1 Hz, 2H), 7.38 (d, J ) 8.1 Hz, 2H),
7.28 (m, 1H), 6.71 (s, 1H), 5.53 (s, 2H), 4.09 (brs, 1H), 2.64 (d,
J ) 7.1 Hz, 2H), 1.90 (sep, J ) 6.7 Hz, 1H), 0.95 (d, J ) 6.7
Hz, 6H), 0.93 (s, 9H). 13C NMR (CDCl3), δ, ppm: 148.7, 147.0,
144.8, 143.8, 142.5, 136.8, 136.3, 135.2, 135.1, 129.8, 128.9,
128.2, 128.0, 118.8, 54.7, 47.0, 39.3, 30.6, 29.6, 22.3. IR
(compression cell), cm-1: 3095, 2954, 2872. Anal. (C25H30N4O2S2)
C, H, N.
3-(4-Imidazol-1-ylmethylphenyl)-5-isobutyl-N-tert-bu-
tylthiophene-2-sulfonamide (14). Compound 2 (200.5 mg,
0.628 mmol), 7 (98.8 mg, 0.416 mmol), toluene (15 mL), ethanol
(1.5 mL), NaOH (1.5 mL, 1 M), and Pd(PPh3)4 (14.5 mg, 12.5
µmol) were mixed together under a N2(g) atmosphere. The
mixture was heated to reflux and stirred for 3 h. After dilution
with EtOAc (50 mL), the reaction mixture was washed with
water and brine. The organic phase was dried and concen-
trated under vacuum. The crude product was purified by
column chromatography (CHCl3:MeOH 20:1) to give the title
compound in 63% yield (113.9 mg, 0.264 mmol).
Alternatively, a dried heavy-walled Pyrex tube was charged
with 2 (49.2 mg, 0.154 mmol), 7 (21.9 mg, 92.4 µmol), toluene
(1.56 mL), ethanol (0.22 mL), Na2CO3 (2.0 M, 0.22 mL), and
Pd(PPh3)4 (8 mg, 7 µmol). The reaction mixture was flushed
with nitrogen and the cap tightened thoroughly. The reaction
mixture was exposed to microwave heating (150 °C, 5 min).
The reaction mixture was diluted with EtOAc (15 mL), washed
with water and brine, and dried. The solvent was removed
and the residue was separated by column chromatography
(CH2Cl2:MeOH 20:1) to give the title compound in 75% yield
(30.0 mg, 0.0695 mmol). 1H NMR (CDCl3), δ, ppm: 7.60-7.57
(m, 3H), 7.22 (d, J ) 8.1 Hz, 2H), 7.08 (s, 1H), 6.90 (s, 1H),
6.73 (s, 1H), 5.15 (s, 2H), 4.35 (m, 1H), 2.66 (d, J ) 7.3 Hz,
2H), 1.90 (m, 1H), 0.98 (s, 9H), 0.96 (d, J ) 6.6 Hz, 6H).
13C NMR (CDCl3), δ, ppm: 148.7, 142.5, 137.5, 136.8, 136.5,
135.1, 130.0, 129.8, 129.0, 127.5, 119.3, 54.7, 50.6, 39.3, 30.6,
3-[4-(2-Ethylbenzoimidazol-1-ylmethyl)phenyl]-5-isobu-
tyl-N-tert-butylthiophene-2-sulfonamide (11). Compound
2 (151.0 mg, 0.473 mmol), 4 (131.4 mg, 0.417 mmol), toluene
(15 mL), ethanol (1.5 mL), NaOH (1.7 mL, 1 M), and Pd(PPh3)4
(14.5 mg, 12 µmol) were mixed together under a N2(g)
atmosphere. The mixture was heated to reflux and stirred for
3 h. After dilution with EtOAc (20 mL), the reaction mixture
was washed with water and brine. The organic phase was
dried and concentrated under vacuum. The crude product was
purified by column chromatography (hexane:acetone 4:1) to
give the title compound in 54% yield (114.8 mg, 0.225 mmol).
1H NMR (CDCl3), δ, ppm: 7.82 (d, J ) 7.6 Hz, 1H), 7.56 (d, J
) 8.3 Hz, 2H), 7.30-7.20 (m, 3H), 7.12 (d, J ) 8.3 Hz, 2H),
6.70 (s, 1H), 5.39 (s, 2H), 4.05 (s, 1H), 2.94 (q, J ) 7.4 Hz,
2H), 2.65 (d, J ) 6.9 Hz, 2H), 1.90 (m, 1H), 1.45 (t, J ) 7.4
Hz, 3H), 1.00 (s, 9H), 0.95 (d, J ) 6.9 Hz, 6H). 13C NMR
(CDCl3), δ, ppm: 155.9, 148.6, 142.3, 135.8, 134.8, 129.7, 129.1,
128.8, 126.4, 126.1, 122.8, 122.7, 122.5, 119.1, 109.5, 54.6, 47.0,
29.6, 22.3. IR (compression cell), cm-1
(C22H29N3O2S2·H2O) C, H, N.
: 3060, 2996. Anal.
3-(4-Cyanomethylphenyl)-5-isobutyl-N-tert-butyl-
thiophene-2-sulfonamide (15). Compound 2 (200.5 mg,
0.628 mmol), (4-bromophenyl)acetonitrile (81.7 mg, 0.416
mmol), toluene (15 mL), ethanol (1.5 mL), NaOH (1.5 mL, 1
M), and Pd(PPh3)4 (18.0 mg, 15.6 µmol) were mixed together
under a N2(g) atmosphere. The mixture was heated to reflux
and stirred for 3 h. After dilution with EtOAc (50 mL), the
reaction mixture was washed with water and brine. The
organic phase was dried and concentrated under vacuum. The
crude product was purified by column chromatography (hexa-
ne:acetone 4:1) to give the title compound in 57% yield (92.5
39.2, 30.5, 29.4, 22.1, 20.8, 11.8. IR (compression cell), cm-1
3056, 2980, 2868. Anal. (C28H35N3O2S2) C, H, N.
:
3-(4-Benzoimidazole-1-ylmethylphenyl)-5-isobutyl-N-
tert-butylthiophenesulfonamide (12). Compound 2 (88.8
mg, 0.278 mmol), 5 (53.2 mg, 0.185 mmol), toluene (10 mL),
ethanol (0.65 mL), NaOH (0.74 mL, 1 M) and Pd(PPh3)4 (6.4
mg, 5.5 µmol) were mixed together under a N2(g) atmosphere.
The mixture was heated to reflux and stirred for 3 h. After
dilution with EtOAc (20 mL), the reaction mixture was washed
with water and brine. The organic phase was dried and
concentrated under vacuum. The crude product was purified
by column chromatography (hexane:acetone 2:1) to give the
1
mg, 0.237 mmol). H NMR (CDCl3), δ, ppm: 7.65 (d, J ) 8.2
Hz, 2H), 7.42 (d, J ) 8.2 Hz, 2H), 6.75 (s, 1H), 4.05 (brs, 1H),
3.81 (s, 2H), 2.70 (d, J ) 7.1 Hz, 2H), 1.97 (m, 1H), 1.01 (s,
9H), 0.98 (d, J ) 6.9 Hz, 6H). 13C NMR (CDCl3), δ, ppm: 148.7,
142.3, 134.8, 130.1, 129.8, 128.8, 128.0, 119.9, 117.6, 54.6, 39.2,
30.6, 29.6, 23.5, 22.1. IR (compression cell), cm-1: 3291, 2996,
2243. Anal. (C20H26N2O2S2·1/2H2O) C, H, N.
1
title compound in 95% yield (84.7 mg, 0.176 mmol). H NMR
3-(4-Cyanophenyl)-5-isobutyl-N-tert-butylthiophene-2-
sulfonamide (16). Compound 2 (200.5 mg, 0.628 mmol),
4-bromobenzonitrile (75.8 mg, 0.416 mmol), toluene (15 mL),
ethanol (1.5 mL), NaOH (1.5 mL, 1 M), and Pd(PPh3)4 (18.0
mg, 15.6 µmol) were mixed together under a N2(g) atmosphere.
The mixture was heated to reflux and stirred for 3 h. After
dilution with EtOAc (50 mL), the reaction mixture was washed
with water and brine. The organic phase was dried and
concentrated under vacuum. The crude product was purified
by column chromatography (hexane:acetone 5:1) to give the
title compound in 64% yield (100.1 mg, 0.266 mmol). 1H NMR
(CDCl3), δ, ppm: 7.74 (m, 4H), 6.75 (s, 1H), 4.12 (brs, 1H),
2.68 (d, J ) 7.1 Hz, 2H), 1.91 (m, 1H), 1.03 (s, 9H), 0.97 (d, J
) 6.6 Hz, 6H). 13C NMR (CDCl3), δ, ppm: 149.2, 141.2, 139.5,
137.6, 132.0, 129.9, 128.6, 118.5, 112.0, 54.9, 39.1, 30.6, 29.6,
(CDCl3), δ, ppm: 8.26 (d, J ) 6.9 Hz, 1H), 7.89 (d, J ) 8.6 Hz,
2H), 7.60-7.20 (m, 6H), 6.70 (s, 1H), 5.55 (s, 2H), 4.12 (s, 1H),
2.67 (d, J ) 7.1 Hz, 2H), 1.90 (m, 1H), 0.94 (m, 15H). 13C NMR
(CDCl3), δ, ppm: 148.7, 143.9, 143.2, 142.5, 136.8, 135.8, 135.2,
133.8, 129.9, 129.0, 127.3, 123.4, 122.6, 120.6, 110.1, 54.7, 48.8,
39.3, 30.7, 29.6, 22.3. IR (compression cell), cm-1: 3063, 2964.
Anal. (C26H31N3O2S2) C, H, N.
3-[4-(2-Methylimidazol-1-ylmethyl)phenyl]-5-isobutyl-
N-tert-butylthiophene-2-sulfonamide (13). Compound 2
(479 mg, 1.50 mmol), 6 (251 mg, 1.00 mmol), toluene (12 mL),
ethanol (3 mL), NaOH (160 mg, 4 mmol as pellets), PPh3 (16
mg, 60 µmol), and Pd(OAC)2 (6.7 mg, 0.03 mmol) were mixed
together under a N2(g) atmosphere. The mixture was heated
to 80 °C and stirred for 2 h. After dilution with EtOAc (20
mL), the reaction mixture was washed with water and brine.
The organic phase was dried and concentrated under vacuum.
The crude product was purified by column chromatography
(CH2Cl2:MeOH 25:1) to give the title compound in 83% yield
22.1. IR (compression cell), cm-1
: 3260, 2985, 2224. Anal.
(C19H24N2O2S2·1/3H2O) C, H, N.
N-Butyloxycarbonyl-3-(4-imidazo[4,5-b]pyridin-3-
ylmethylphenyl)-5-isobutylthiophene-2-sulfonamide (17).
Compound 10 (150 mg, 0.311 mmol) and anisole (150 µL) were
dissolved in TFA (5 mL) and stirred overnight. The reaction
mixture was evaporated under vacuum and coevaporated with
acetonitrile twice. The residue was dissolved in pyridine (1.6
mL), and pyrrolidinopyridine (52.1 mg, 0.35 mmol) was added.
The solution was cooled on an ice bath and butyl chloroformate
(447 µL, 3.49 mmol) was added under a N2(g) atmosphere. The
reaction was stirred at ambient temperature overnight. The
1
(368.8 mg, 0.828 mmol). H NMR (CDCl3), δ, ppm: 7.59 (d, J
) 8.1 Hz, 2H), 7.13 (d, J ) 8.1 Hz, 2H), 6.98 (s, 1H), 6.86 (s,
1H), 6.73 (s, 1H), 5.09 (s, 2H), 4.06 (s, 1H), 2.67 (d, J ) 7.1
Hz, 2H), 2.34 (s, 3H), 1.91 (m, 1H), 0.99 (s, 9H), 0.97 (d, J )
6.6 Hz, 6H). 13C NMR (CDCl3), δ, ppm: 148.6, 144.8, 142.4,
136.7, 136.5, 134.8, 129.7, 128.9, 127.6, 126.7, 119.9, 54.6, 49.4,
39.2, 30.5, 29.5, 22.1, 13.1. IR (compression cell), cm-1: 3053,
2958. Anal. (C23H31N3O2S2) C, H, N.

6004 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 24
Wan et al.
solvent was removed under vacuum and coevaporated with
acetonitrile twice. The residue was dissolved in ethyl acetate
(50 mL) and washed with citric acid (10% aq) and water. The
organic phase was dried, evaporated, and purified by column
chromatography (hexane:acetone 3:2) to give the title com-
residue was dissolved in CH2Cl2 (2 mL) and H2O (0.7 mL), and
Na2CO3 (61 mg, 0.57 mmol) was added. The solution was
cooled on an ice bath and butyl chloroformate (23 µL, 0.18
mmol) was added under a N2(g) atmosphere. The reaction was
stirred at ambient temperature overnight. The reaction mix-
ture was diluted with CHCl3 (15 mL) and washed with water
and brine. The organic phase was dried, evaporated, and
purified by column chromatography (CHCl3:MeOH 25:1) to
1
pound in 90% yield (147.9 mg, 0.281 mmol). H NMR (CDCl3
+ CD3OD), δ, ppm: 8.42 (dd, J ) 4.8, 1.3 Hz, 1H), 8.39 (s,
1H), 8.10 (dd, J ) 8.1, 1.3 Hz, 1H), 7.47 (d, J ) 8.3 Hz, 2H),
7.37 (d, J ) 8.3 Hz, 2H), 7.34 (m, 1H), 6.77 (s, 1H), 5.58 (s,
2H), 3.94 (t, J ) 6.6 Hz, 2H), 2.70 (d, J ) 7.1 Hz, 2H), 1.92
(m, 1H), 1.42 (m, 2H), 1.17 (m, 2H), 0.97 (d, J ) 6.6 Hz, 6H),
0.82 (t, J ) 7.3 Hz, 3H). 13C NMR (CDCl3 + CD3OD), δ, ppm:
157.2, 152.3, 151.8, 147.3, 146.4, 145.7, 145.4, 137.2, 135.6,
135.1, 130.5, 130.4, 128.6, 128.4, 119.8, 66.9, 47.6, 39.8, 31.5,
31.3, 22.5, 19.5, 13.9. IR (compression cell), cm-1: 3033, 2960,
2871, 1734. Anal. (C26H30N4O4S2) C, H, N.
1
give the title compound in 72% yield (44.6 mg, 91 µmol). H
NMR (CDCl3), δ, ppm: 10.42 (brs, 1H) NH-signal, 7.62 (d, J
) 7.9 Hz, 2H), 7.05 (d, J ) 8.0 Hz, 2H), 6.71-6.69 (m, 3H),
5.08 (s, 2H), 3.90 (t, J ) 6.6 Hz, 2H), 2.65 (d, J ) 7.1 Hz, 2H),
2.54 (s, 3H), 1.91 (m, 1H), 1.44 (m, 2H), 1.19 (m, 2H), 0.97 (d,
J ) 6.6 Hz, 6H), 0.82 (t, J ) 7.3 Hz, 3H). 13C NMR (CDCl3), δ,
ppm: 155.7, 148.4, 144.1, 141.8, 136.3, 136.1, 133.0, 130.1,
128.4, 127.2, 121.4, 119.9, 65.0, 50.1, 39.2, 30.8, 30.4, 22.3, 18.9,
13.7, 11.1. IR (compression cell), cm-1: 3145, 2952, 1665. Anal.
(C24H31N3O4S2·H2O) C, H, N.
N-Butyloxycarbonyl-3-[4-(2-ethylbenzoimidazol-1-yl-
methyl)phenyl]-5-isobutylthiophene-2-sulfonamide (18).
Compound 11 (140 mg, 0.275 mmol) and anisole (150 µL) were
dissolved in TFA (5 mL) and stirred overnight. The reaction
mixture was evaporated under vacuum and coevaporated with
acetonitrile twice. The residue was dissolved in pyridine (1.6
mL), and pyrrolidinopyridine (47.7 mg, 0.32 mmol) was added.
The solution was cooled on an ice bath and butyl chloroformate
(440 µL, 3.43 mmol) was added under a N2(g) atmosphere. The
reaction was stirred at ambient temperature overnight. The
solvent was removed under vacuum and coevaporated with
acetonitrile twice. The residue was dissolved in ethyl acetate
(50 mL) and washed with citric acid (10% aq) and water. The
organic phase was dried, evaporated, and purified by column
chromatography (hexane:acetone 3:2) to give the title com-
pound in 31% yield (47.6 mg, 86 µmol). 1H NMR (CDCl3 + CD3-
OD), δ, ppm: 7.65 (m, 1H), 7.47 (d, J ) 8.3 Hz, 2H), 7.40 (m,
1H), 7.27 (m, 2H), 7.13 (d, J ) 8.3 Hz, 2H), 6.79 (s, 1H), 5.51
(s, 2H), 3.95 (t, J ) 6.6 Hz, 2H), 2.94 (q, J ) 7.6 Hz, 2H), 2.70
(d, J ) 6.9 Hz, 2H), 1.92 (m, 1H), 1.43 (m, 2H), 1.39 (t, J ) 7.6
Hz, 3H) 1.20 (m, 2H), 0.97 (d, J ) 6.6 Hz, 6H), 0.84 (t, J ) 7.3
Hz, 3H). 13C NMR (CDCl3 + CD3OD), δ, ppm: 157.5, 152.7,
151.6, 150.5, 146.2, 137.2, 135.2, 134.9, 130.6, 130.4, 127.0,
123.9, 123.7, 118.6, 111.1, 66.9, 47.0, 39.8, 31.5, 31.4, 22.5, 21.3,
19.6, 13.9, 11.9. IR (compression cell), cm-1: 2954, 2880, 1751.
Anal. (C29H35N3O4S2) C, H, N.
N-Butyloxycarbonyl-3-(4-imidazol-1-ylmethylphenyl)-
5-isobutylthiophene-2-sulfonamide (21). Compound 14
(113 mg, 0.262 mmol) and anisole (150 µL) were dissolved in
TFA (5 mL) and stirred overnight. The reaction mixture was
evaporated under vacuum and coevaporated with acetonitrile
twice. The residue was dissolved in pyridine (1.6 mL), and
pyrrolidinopyridine (40.5 mg, 0.262 mmol) was added. The
solution was cooled on an ice bath and butyl chloroformate
(364 µL, 2.50 mmol) was added under a N2(g) atmosphere. The
reaction was stirred at ambient temperature overnight. The
solvent was removed under vacuum and coevaporated with
acetonitrile twice. The residue was dissolved in ethyl acetate
(50 mL) and washed with citric acid (10% aq) and water. The
organic phase was dried, evaporated, and purified by column
chromatography (CH2Cl2:MeOH 10:1) to give the title com-
1
pound in 46% yield (57.8 mg, 0.122 mmol). H NMR (CDCl3),
δ, ppm: 9.71 (brs, 1H), 7.95 (s, 1H), 7.55 (d, J ) 8.1 Hz, 2H),
7.09 (d, J ) 8.1 Hz, 2H), 6.86 (m, 2H), 6.74 (s, 1H), 5.16 (s,
2H), 4.03 (t, J ) 6.6 Hz, 2H), 2.70 (d, J ) 7.1 Hz, 2H), 1.94
(m, 1H), 1.51 (m, 2H), 1.25 (m, 2H), 0.98 (d, J ) 6.6 Hz, 6H),
0.87 (t, J ) 7.4 Hz, 3H). 13C NMR (CDCl3), δ, ppm: 156.5,
152.6, 150.5, 144.1, 136.6, 134.8, 133.2, 130.0, 129.0, 127.5,
125.9, 119.7, 66.0, 51.3, 39.4, 30.8, 30.6, 22.4, 19.0, 13.8. IR
(compression cell), cm-1
(C23H29N3O4S2‚H2O) C, H, N.
: 3555, 3120, 2956, 1694. Anal.
N-Butyloxycarbonyl-3-(4-benzoimidazol-1-ylmethylphe-
nyl)-5-isobutylthiophene-2-sulfonamide (19). Compound
12 (92.2 mg, 0.191 mmol) and anisole (150 µL) were dissolved
in TFA (5 mL) and stirred overnight. The reaction mixture
was evaporated under vacuum and coevaporated with aceto-
nitrile twice. The residue was dissolved in pyridine (1.6 mL),
and pyrrolidinopyridine (32.6 mg, 0.220 mmol) was added. The
solution was cooled on an ice bath and butyl chloroformate
(301 µL, 2.07 mmol) was added under a N2(g) atmosphere. The
reaction was stirred at ambient temperature overnight. The
solvent was removed under vacuum and coevaporated with
acetonitrile twice. The residue was dissolved in ethyl acetate
(50 mL) and washed with citric acid (10% aq) and water. The
organic phase was dried, evaporated, and purified by column
chromatography (CH2Cl2:MeOH 30:1) to give the title com-
pound in 79% yield (78.8 mg, 0.150 mmol). 1H NMR (CDCl3 +
CD3OD), δ, ppm: 8.34 (s, 1H), 7.70 (m, 1H), 7.51 (d, J ) 8.3
Hz, 2H), 7.33 (d, J ) 8.3 Hz, 2H), 7.29 (m, 3H), 6.84 (s, 1H),
5.55 (s, 2H), 3.91 (t, J ) 6.6 Hz, 2H), 2.73 (d, J ) 7.0 Hz, 2H),
1.92 (m, 1H), 1.41 (m, 2H), 1.21 (m, 2H), 0.98 (d, J ) 6.6 Hz,
6H), 0.84 (t, J ) 7.3 Hz, 3H). 13C NMR (CDCl3 + CD3OD), δ,
ppm: 157.3, 153.3, 151.8, 151.7, 146.3, 144.9, 143.9, 143.8,
137.7, 135.5, 130.8, 128.4, 124.6, 123.8, 120.0, 112.0, 67.0, 48.0,
N-Butyloxycarbonyl-3-(4-cyanomethylphenyl)-5-isobu-
tylthiophene-2-sulfonamide (22). Compound 15 (60.0 mg,
0.154 mmol) was dissolved in CH2Cl2 (5 mL) under a N2(g)
atmosphere, BCl3 (2 mL, 1 M CH2Cl2) was added, and the
reaction mixture was stirred for 3 h. To the reaction mixture
was added Na2CO3 (10% aq, 50 mL) and the aqueous phase
was extracted with EtOAc. The combined organic phase was
washed with brine and water and then dried and evaporated.
The residue was dissolved in pyridine (2 mL), and pyrrolidi-
nopyridine (23.3 mg, 0.154 mmol) was added. The solution was
cooled on an ice bath and butyl chloroformate (195 µL, 1.34
mmol) was added under a N2(g) atmosphere. The reaction was
stirred at ambient temperature overnight. The solvent was
removed under vacuum and coevaporated with acetonitrile
twice. The residue was dissolved in ethyl acetate (50 mL)
and washed with citric acid (10% aq) and water. The organic
phase was dried, evaporated, and purified by column chroma-
tography (CHCl3:MeOH 45:1) to give the title compound in 72%
1
yield (47.9 mg, 0.110 mmol). H NMR (CDCl3), δ, ppm: 9.73
(brs, 1H), 7.45 (d, J ) 8.2 Hz, 2H), 7.38 (d, J ) 8.2 Hz, 2H),
6.76 (s, 1H), 4.07 (t, J ) 6.6 Hz, 2H), 3.81 (s, 2H), 2.72 (d, J )
7.1 Hz, 2H), 1.97 (m, 1H), 1.53 (m, 2H), 1.28 (m, 2H), 1.01 (d,
J ) 6.6 Hz, 6H), 0.89 (t, J ) 7.4 Hz, 3H). 13C NMR (CDCl3), δ,
ppm: 157.8, 152.0, 150.4, 145.9, 133.9, 130.6, 129.7, 129.4,
128.0, 117.8, 67.1, 39.4, 30.6, 30.5, 23.5, 22.3, 18.9, 13.7. IR
(compression cell), cm-1: 3217, 2960, 2872, 2251, 1747. Anal.
(C21H26N2O4S2‚1/3H2O) C, H, N.
N-Butyloxycarbonyl-3-(4-cyanophenyl)-5-isobutyl-
thiophene-2-sulfonamide (23). Compound 16 (90.0 mg,
0.239 mmol) was dissolved in CH2Cl2 (5 mL) under a N2(g)
atmosphere, BCl3 (2 mL, 1 M CH2Cl2) was added, and the
39.9, 31.8, 31.7, 22.5, 19.9, 13.9. IR (compression cell), cm-1
3033, 2944, 2872, 1734. Anal. (C27H31N3O4S2) C, H, N.
:
N-Butyloxycarbonyl-3-[4-(2-methylimidazol-1-ylmeth-
yl)phenyl]-5-isobutylthiophene-2-sulfonamide (20). Com-
pound 13 (56.6 mg, 0.127 mmol) was dissolved in CH2Cl2 (2
mL) under a N2(g) atmosphere and BCl3 (130 +100 + 100 µL,
1 M CH2Cl2) was sequentially added for 0.5 h to the stirred
reaction mixture. The solvent was evaporated under vacuum
and coevaporated three times with dichloromethane. The

The First Selective Nonpeptide AT2 Receptor Agonist
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 24 6005
reaction mixture was stirred for 3 h. To the reaction mixture
was added Na2CO3 (10% aq, 50 mL) and the aqueous phase
was extracted with EtOAc. The combined organic phase was
washed with brine and water and then dried and evaporated.
The residue was dissolved in pyridine (2 mL), and pyrrolidi-
nopyridine (34.4 mg, 0.239 mmol) was added. The solution was
cooled on an ice bath and butyl chloroformate (295 µL, 2.32
mmol) was added under a N2(g) atmosphere. The reaction was
stirred at ambient temperature overnight. The solvent was
removed under vacuum and coevaporated with acetonitrile
twice. The residue was dissolved in ethyl acetate (50 mL) and
washed with citric acid (10% aq) and water. The organic phase
was dried, evaporated, and purified by column chromatography
(CHCl3:MeOH 50:1) to give the title compound in 53% yield
imidazole (238 mg, 3.50 mmol), CuI (6.7 mg, 35 µmol), 1,2-
diaminocyclohexane (17.2 µL, 0.140 mmol), and Cs2CO3 (228
mg, 0.70 mmol) in DMF (1 mL). The reaction mixture was
flushed with nitrogen and the cap tightened thoroughly. The
reaction mixture was exposed to microwave heating (180 °C,
40 min). The reaction tube was cooled to room temperature
before the reaction mixture was diluted in EtOAc (60 mL) and
washed with water. The organic phase was dried, and the
solvent was evaporated. The crude product was purified on
column chromatography with CHCl3:MeOH (10:1) to give the
title compound in 27% yield (40.1 mg, 93 µmol). 1H NMR
(CDCl3 + CD3OD), δ, ppm: 8.15 (s, 1H), 7.41-7.35 (m, 6H),
6.41 (s, 1H), 4.21 (s, 2H), 2.55 (d, J ) 7.3 Hz, 2H), 1.85 (m,
1H), 1.23 (s, 9H), 0.93 (d, J ) 8.1 Hz, 6H). 13C NMR (CDCl3 +
CD3OD), δ, ppm: 151.3, 145.9, 139.4, 135.0, 134.9, 130.1,
128.5, 127.8, 121.5, 118.7, 113.5, 66.7, 39.1, 34.4, 30.6, 30.1,
22.3. Anal. (C22H29N3O2S2) C, H, N.
1
(53.1 mg, 0.126 mmol). H NMR (CDCl3), δ, ppm: 8.20 (brs,
1H), 7.65 (d, J ) 8.6 Hz, 2H), 7.59 (d, J ) 8.6 Hz, 2H), 6.76 (s,
1H), 4.09 (t, J ) 6.6 Hz, 2H), 2.73 (d, J ) 7.1 Hz, 2H), 1.99
(m, 1H), 1.53 (m, 2H), 1.23 (m, 2H), 0.98 (d, J ) 6.6 Hz, 6H),
0.87 (t, J ) 7.4 Hz, 3H). 13C NMR (CDCl3), δ, ppm: 157.9,
152.3, 150.5, 144.4, 138.8, 132.0, 130.0, 129.0, 118.5, 112.2,
67.0, 39.3, 30.6, 30.5, 22.3, 18.9, 13.7. IR (compression cell),
cm-1: 3203, 2959, 2872, 2228, 1749. Anal. (C20H24N2O4S2) C,
H, N.
3-(4-Bromophenylmethyl)-5-isobutyl-N-tert-butyl-
thiophene-2-sulfonamide (24). Compound 2 (159.6 mg, 0.5
mmol), 1-bromo-4-bromomethylbenzene (312.4 mg, 1.25 mmol),
toluene (15 mL), ethanol (1.3 mL), K2CO3 (2.0 mL, 1 M), and
Pd(PPh3)4 (17 mg, 15 µmol) were mixed together under a N2-
(g) atmosphere. The mixture was heated to reflux and stirred
for 3 h. After dilution with EtOAc (50 mL), the reaction
mixture was washed with water and brine. The organic phase
was dried and concentrated under vacuum. The crude product
was purified by column chromatography (hexane:acetone 4:1)
to give the title compound in 80% yield (178.2 mg, 0.401 mmol).
N-Butyloxycarbonyl-3-(4-cyanophenylmethyl)-5-iso-
butylthiophene-2-sulfonamide (27). Compound 25 (59.5
mg, 0.152 mmol) was dissolved in CH2Cl2 (5 mL) under a
N2(g) atmosphere, BCl3 (1 mL, 1 M CH2Cl2) was added, and
the reaction mixture was stirred for 1 h. To the reaction
mixture was added Na2CO3 (10% aq, 50 mL) and the aqueous
phase was extracted with EtOAc. The combined organic phase
was washed with brine and water and then dried and
evaporated. The residue was dissolved in pyridine (1 mL), and
pyrrolidinopyridine (21.9 mg, 0.148 mmol) was added. The
solution was cooled on an ice bath and butyl chloroformate
(188 µL, 1.52 mmol) was added under a N2(g) atmosphere. The
reaction was stirred at ambient temperature overnight. The
solvent was removed under vacuum and coevaporated with
acetonitrile twice. The residue was dissolved in ethyl acetate
(50 mL) and washed with citric acid (10% aq) and water. The
organic phase was dried, evaporated, and purified by column
chromatography (CHCl3:MeOH 40:1) to give the title com-
Alternatively, a dried heavy-walled Pyrex tube was charged
with 2 (32.7 mg, 0.103 mmol), 1-bromo-4-bromomethylbenzene
(64 mg, 0.26 mmol), toluene (1.56 mL), ethanol (0.22 mL),
Na2CO3 (2.0 M, 0.22 mL), and Pd(PPh3)4 (7 mg, 6 µmol)). The
reaction mixture was flushed with nitrogen and the cap
tightened thoroughly. The reaction mixture was exposed to
microwave heating (110 °C, 30 min). The reaction mixture was
diluted with EtOAc (15 mL), washed with water and brine,
and dried. The solvent was removed and the residue was
separated by column chromatography (hexane:acetone 8:1) to
1
pound in 75% yield (49.7 mg, 0.114 mmol). H NMR (CDCl3),
δ, ppm: 8.53 (brs, 1H), 7.54 (d, J ) 8.3 Hz, 2H), 7.26 (d, J )
8.3 Hz, 2H), 6.46 (s, 1H), 4.31 (s, 2H), 4.07 (t, J ) 6.6 Hz, 2H),
2.59 (d, J ) 7.3 Hz, 2H), 1.82 (m, 1H), 1.54 (m, 2H), 1.27 (m,
2H), 0.88 (d, J ) 6.6 Hz, 6H), 0.85 (d, J ) 7.3 Hz, 3H). 13C
NMR (CDCl3), δ, ppm: 156.5, 152.9, 150.7, 146.1, 144.8, 132.5,
129.7, 128.8, 118.9, 110.5, 67.1, 39.4, 34.7, 30.6, 30.5, 22.3, 18.9,
13.7. IR (compression cell), cm-1: 3223, 2959, 2871, 2228, 1748.
Anal. (C21H26N2O4S2‚3H2O) C, H, N.
1
give the title compound in 78% yield (35.3 mg, 79 µmol). H
N-Butyloxycarbonyl-3-(4-imidazol-1-ylphenylmethyl)-
5-isobutylthiophene-2-sulfonamide (28). Compound 26
(40.1 mg, 93 µmol) was dissolved in CH2Cl2 (5 mL) under a
N2(g) atmosphere, BCl3 (1 mL, 1 M CH2Cl2) was added, and
the reaction mixture was stirred for 1 h. To the reaction
mixture was Na2CO3 (10% aq, 50 mL) and the aqueous phase
was extracted with EtOAc. The combined organic phase was
washed with brine and water and then dried and evaporated.
The residue was dissolved in pyridine (1 mL), and pyrrolidi-
nopyridine (15.1 mg, 0.102 mmol) was added. The solution was
cooled on an ice bath and butyl chloroformate (126 µL, 0.93
mmol) was added under a N2(g) atmosphere. The reaction was
stirred at ambient temperature overnight. The solvent was
removed under vacuum and coevaporated with acetonitrile
twice. The residue was dissolved in ethyl acetate (50 mL) and
washed with citric acid (10% aq) and water. The organic phase
was dried, evaporated, and purified by column chromatography
(CHCl3:MeOH 12:1) to give the title compound in 54% yield
(23.8 mg, 50 µmol). 1H NMR (CDCl3 + CD3OD), δ, ppm: 8.15
(s, 1H), 7.41-7.35 (m, 6H), 6.47 (s, 1H), 4.32 (s, 2H), 4.10 (t, J
) 6.6 Hz, 2H), 2.62 (d, J ) 7.1 Hz, 2H), 1.85 (m, 1H), 1.59 (m,
2H), 1.34 (m, 2H), 0.93 (d, J ) 6.6 Hz, 6H), 0.89 (t, J ) 7.1
Hz, 3H). 13C NMR (CDCl3 + CD3OD), δ, ppm: 151.7, 151.1,
146.0, 139.6, 134.9, 134.8, 130.3, 128.4, 127.7, 121.6, 118.7,
113.5, 66.3, 39.1, 33.7, 30.5, 30.3, 21.9, 18.6, 13.4. IR (compres-
sion cell), cm-1: 3223, 2958, 2871, 1740. Anal. (C23H29N3O4S2·
1/2H2O) C, H, N.
NMR (CDCl3), δ, ppm: 7.42 (d, J ) 8.4 Hz, 2H), 7.12 (d, J )
8.4 Hz, 2H), 6.45 (s, 1H), 4.35 (brs, 1H), 4.12 (s, 2H), 2.59 (d,
J ) 7.3 Hz, 2H), 1.86 (m, 1H), 1.18 (s, 9H), 0.90 (d, J ) 7.1
Hz, 6H). 13C NMR (CDCl3), δ, ppm: 149.2, 143.2, 138.5, 135.6,
131.8, 130.8, 129.0, 120.5, 55.1, 39.4, 34.0, 30.7, 30.1, 22.3. IR
(compression cell), cm-1: 3274, 2961. Anal. (C19H26BrNO2S2)
C, H, N.
3-(4-Cyanophenylmethyl)-5-isobutyl-N-tert-butyl-
thiophene-2-sulfonamide (25). A dried heavy-walled Pyrex
tube was charged with 24 (89 mg 0.20 mmol), Zn(CN)2 (23.5
mg, 0.20 mmol), and Pd(PPh3)4 (6.9 mg, 6 µmol) in DMF (1
mL). The reaction mixture was flushed with nitrogen and the
cap tightened thoroughly. The reaction mixture was exposed
to microwave heating (180 °C, 2 min). The reaction tube was
cooled to room temperature before the reaction mixture was
diluted in EtOAc (60 mL) and washed with water. The organic
phase was dried, and the solvent was evaporated. The crude
product was purified by column chromatography (hexane:
acetone 5:1) to give the title compound in 76% yield (59.5 mg,
1
0.152 mmol). H NMR (CDCl3), δ, ppm: 7.60 (d, J ) 8.3 Hz,
2H), 7.34 (d, J ) 8.3 Hz, 2H), 6.43 (s, 1H), 4.43 (brs, 1H), 4.24
(s, 2H), 2.60 (d, J ) 7.3 Hz, 2H), 1.87 (m, 1H), 1.21 (s, 9H),
0.91 (d, J ) 7.3 Hz, 6H). 13C NMR (CDCl3), δ, ppm: 149.6,
145.1, 142.0, 136.2, 132.5, 129.8, 128.7, 119.0, 110.6, 55.3, 39.3,
34.6, 30.7, 30.1, 22.3. IR (compression cell), cm-1: 3297, 2965,
2231. Anal. (C20H26N2O2S2) C, H, N.
3-(4-Imidazol-1-ylphenylmethyl)-5-isobutyl-N-tert-
butylthiophene-2-sulfonamide (26). A dried heavy-walled
Pyrex tube was charged with 24 (156 mg 0.351 mmol),
Rat Liver Membrane AT1 Receptor Binding Assay. Rat
liver membranes were prepared according to the method of
Dudley et al.34 Binding of [125I]Ang II to membranes was

6006 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 24
Wan et al.
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 (80 000-85 000 cpm, 0.03 nM), and vari-
able 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 to tubes. The
radioactivity was measured in a γ-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 [125I]-
AngII. Nonspecific binding was determined in the presence of
1 µM Ang II. The specific binding was determined by subtract-
ing the nonspecific binding from the total bound [125I]AngII.
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.
described.37 Subcultures were performed at subconfluency.
Under these conditions, cells express only the AT2 receptor
subtype.37,38 According to experiments, cells were stimulated
for periods ranging from minutes (MAPK) to 3 days (neurite
elongation) (first stimulation 24 h after plating). Cells were
cultured for three subsequent days under these conditions. For
all experiments, cells were plated at the same initial density
of 3 × 104 cells/35 mm Perti dish. Cells were untreated (control
cells) or treated with Ang II (100 nM) or 21 (100 nM), in the
absence or in the presence of the various inhibitors: PD 123,-
319 (1 µM), an AT2 receptor antagonist; PD 98,059 (10 µM),
an inhibitor of MEK; LY-83,583 (0.5 µM), an inhibitor of
soluble guanylyl cyclase; or KT 5823 (1 µM), an inhibitor of
cGMP-dependent protein kinases (each introduced daily with
inhibitors applied 30 min prior to Ang II or 21).
Determination of Cells with Neurites. Cells were ex-
amined daily under a phase contrast microscope and micro-
graphs 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. At least
140 cells were counted in three independent experiments.39
Western Blotting for p42/p44mapk. After 3 days of culture,
cells were washed with Hanks buffered saline (HBS: 130 mM
NaCl, 3.5 mM KCl, 2.3 mM CaCl2·2H2O, 0.98 mM MgCl2·6H2O,
5 mM HEPES, 0.5 mM EGTA) and then incubated for 10 min
with 800 µL of stabilization buffer (100 nM staurosporine, 1
mM Na3VO4 in HBS) and finally lysed in 50 µL of lysis buffer
(50 mM HEPES pH 7.8, 100 nM staurosporine, 1mM Na3VO4,
1% Triton-X100, protease inhibitors). Samples were separated
on 10% SDS-polyacrylamide gels. Proteins were transferred
electrophoretically to PVDF membranes. Membranes were
blocked with 1% gelatin, 0.05% Tween 20 in TBS buffer (pH
7.5). After washing with TBS-Tween 20 (0.05%), membranes
were incubated overnight at 4 °C with anti-phosphorylated
p42/p44mapk (1:1000) or anti-p42/p44mapk (1:1000), diluted in
TBS-Tween 20 (0.05%) plus BSA (0.1%). After washing with
TBS-Tween 20, detection was accomplished using horseradish
peroxidase-conjugated anti-mouse or anti-rabbit IgG (1:2000)
and an enhanced chemiluminescence (ECL) detection system,
as described previously.12
Data Analysis. The data are presented as mean ( SEM of
the number of experiments indicated in the text, each per-
formed in duplicate or triplicate. Statistical analyses of the
data were performed using the one-way analysis of variance
(ANOVA) test. Homogeneity of variance was assessed by
Bartlett’s test, and p values were obtained from Dunnett’s
tables.
In Vivo Rat Studies. General. All in vivo experiments
were approved by the Animal Ethics Committee of Gothenburg
University. The secretion experiments were performed on
nonfasted male Sprague-Dawley rats (Mo¨llegård Breeding
Center Ltd., Ejby, Denmark). For induction of anaesthesia,
pentobarbital sodium, a barbiturate with a medium duration
of action, was injected intraperitoneally (60 mg/kg of body wt)
followed by continuous infusion (30 mg kg-1 h-1).
Spontaneously hypertensive rats (SHR, Charles River,
Germany) were anesthetized using 150 mg/kg Inactin
(Byk-Gulden, Germany) by a single bolus ip.
A catheter was inserted into the trachea to ensure free
airways. A femoral artery was catheterized for subsequent
blood pressure measurements and the continuous infusion of
the anaesthetic. One or two femoral veins were catheterized
for intravenous drug administration. The body temperature
was maintained at 38 °C with a heating pad and lamp, both
controlled thermostatically.
Porcine (Pig) Myometrial Membrane AT2 Receptor
Binding Assay. Myometrial membranes were prepared from
porcine uteri according to the method by Nielsen et al.35
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, homo-
genate corresponding to 10 mg of the original tissue weight,
[
125I]Ang II (80 000-85 000 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 to tubes. The
radioactivity was measured in a γ-counter. The characteristics
of the Ang II binding AT2 receptor were 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. IC50 was determined by Scatchard analysis of data
obtained with Ang II by using GraFit (Erithacus Software,
UK). The apparent dissociation constant Ki values were
calculated from IC50 values using the Cheng-Prusoff equation
(Kd ) 0.7 ( 0.1 nM, [L] ) 0.057 nM). The binding data were
best fitted with a one-site fit. All determinations were per-
formed in triplicates.
In Vitro Morphological Effects. General. The chemicals
used in the present study were obtained from the follow-
ing sources. Dulbecco’s modified Eagle’s medium (DMEM),
fetal bovine serum (FBS), HAT supplement (hypoxanthine,
aminopterin, thymidine), and gentamycin were from Gibco
BRL (Burlington, Canada); [Val5]angiotensin II was from
Bachem (Marina Delphen, CA); PD 123,319 was from RBI
(Natick, MA); LY-83,583 and KT 5823 were from Calbiochem-
Novabiochem (La Jolla, CA); PD 98,059, anti-phosphorylated
p42/p44mapk, and total p42/p44mapk antibodies were from New
England Biolabs (Beverly, MA); horseradish peroxidase-
conjugated anti-mouse and anti-rabbit antibodies were from
Amersham Pharmacia Biotech Inc. (NJ); and complete pro-
tease inhibitor, poly(vinylidene difluoride) (PVDF) membranes,
and the enhanced chemiluminescence (ECL) detection system
were from Roche Laboratories (Montreal, Canada). All other
chemicals were of grade A purity.
Blood Pressure Recording (All Experiments). Arterial
pressure was measured by a Statham P23Dc transducer
(Statham, Hato Rey, PR) connected to a PE-50 catheter in the
right femoral artery. Pressure data were integrated by a
microcomputer to mean arterial pressure (MAP) over 5 min.
Secretion (Only S-D Rats). Duodenal mucosal alkaline
(HCO3-) secretion was measured by a pH-stat titration
technique.40 After midline laparotomy, a segment of the
duodenum (with its proximal end 0.5 cm distal to the pylorus)
with intact vascular supply was isolated between two glass
Cell Culture. NG108-15 cells (provided by Drs. M. Emerit
and M. Hamon; INSERM, U. 238, Paris, France) were cultured
(passage 7-30) in DMEM with 10% fetal bovine serum (FBS,
Gibco BRL, Burlington, Canada), HAT supplement, and 50
mg/L gentamycin at 37 °C in 75 cm2 Nunclon Delta flasks in
a humidified atmosphere of 93% air and 7% CO2, as previously

The First Selective Nonpeptide AT2 Receptor Agonist
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 24 6007
tubes connected to a reservoir containing isotonic saline
maintained at 38 °C by a water jacket. Saline was recirculated
through the segment by means of a gas lift (pure air). The
common bile duct was catheterized ∼5 mm proximal to the
papilla of Vater to avoid contamination of the segment by bile
and pancreatic juice. Alkaline secretion to the perfusate was
continuously titrated to pH 7.4 with 0.02 M HCl controlled by
a pH-stat device.
Experimental Protocol. After surgery, the animals were
left undisturbed to recover, after which baseline values were
monitored during a 30 min control period. The study com-
pounds or vehicle were then administered and the animals
were monitored for an additional 45 min period.
Statistics. Secretion data obtained during the last 15 min
before administration of drug were regarded as basal condi-
tions. Net change was defined as the difference between basal
conditions and data obtained between 30 and 45 min following
onset of each drug administration. Values given in the figures
are means ( SEM. Comparisons between groups were made
by ANOVA and the Bonferroni post hoc test.
was calculated by extrapolation of the last measurable con-
centration to BLQ.
Acknowledgment. We thank Erik Helgstrand and
Ann Christin Kvarnstro¨m for valuable discussions. We
gratefully acknowledge support from the Swedish Re-
search Council (VR), the Swedish Foundation for Stra-
tegic Research (SSF), the Knut and Alice Wallenberg
Foundation (VRmedicine 8663), the Canadian Institutes
of Health Research to N.G.P. and MDP (MOP 37891),
and Vicore Pharma AB. N.G.P. is a holder of a Canada
Research Chair in Endocrinology of the Adrenal Gland.
We thank Biotage AB for providing the microwave
synthesizer.
Supporting Information Available: Analysis data for
4, 6, 7, and 10-28. This material is available free of charge
via the Internet at http://pubs.acs.org.
Oral Absorption Study. All materials and reagents used
throughout the study were of standard analytical grade or
equivalent. Twenty-four male Sprague-Dawley rats (age ca.
6-7 weeks at the time of dosing) were obtained from Harlan
UK Ltd, a recognized supplier of laboratory animals. All
animals were individually identified by tail markings with
indelible ink. The oral absorption study was performed in
triplicates with three oral concentrations (30, 100, and 300 µg/
kg) and one intravenously (30 µg/kg). The orally administrated
doses were given via gastric gavage and the intravenously were
administrated as a single bolus injection into the tail vein. All
dose weights were altered to take into account the weight of
the animal at the time of administration. The actual dose
received by each animal was calculated from the calculated
dose concentration, the animal weight, and the weight of dose
administered. A blood sample (200 µL) was taken from the
animals via venepuncture of the tail vein at the following time
points post dose: 30 min and 1, 2, 4, 6, 8, and 24 h. All blood
samples were collected into heparinized blood tubes and
centrifuged to prepare plasma, within 2 h of collection. The
plasma was transferred into plain tubes and frozen at ca -20
°C until analyzed. Plasma (50 µL) was transferred into an
Eppendorf tube. For calibration samples, 20 µL of an appropri-
ate calibration spiking solution prepared in acetonitrile:water
(1:1) was added, and for all study samples, 20 µL of acetoni-
trile:water (1:1) was added. To all samples, 150 µL of aceto-
nitrile was then added, vortex mixed for approximately 2 s,
and then centrifuge at 4000 rpm for 5 min at ∼4 °C. Aliquots
(50 µL) of supernatant were transferred to autosampler vials
containing 20 µL of deionized water and vortex mixed. The
samples were analyzed by LC-MS/MS using a Perkin-Elmer
Series 200 Micropump and Series 200 Autosampler (Perkin-
Elmer, Beaconsfield, UK) coupled to a Turbo IonSpray (TIS)
source of an Applied Biosystems API 365 triple quadropole
mass spectrometer (Applied Biosystems, Warrington, UK).
Compound 21 was analyzed on a Metachem Polaris (5 µm, 50
× 2 mm) column using a gradient HPLC method. The mobile
phases comprised of A, acetonitrile, and B, aqueous ammonium
acetate (10 mM). The flow from the column was not split prior
to entering the source of the mass spectrometer; however, the
first minute of flow was directed to waste, with the remaining
4.2 min directed into the mass spectrometer. The injection
volume was 40 µL and the autosampler needle was washed
with acetonitrile:water (1:1). The mass spectrometer was
operated in positive ion mode and compound 21 was analyzed
by selected reaction monitoring (SRM) using the specific
precursor to product ion transitions (476.3 f 293.2). Peak
areas were compared against a calibration line of peak area
versus concentration. The absolute bioavailability and dose
proportionality were calculated using AUC0-8h instead of
AUC0-∞, as AUC0-∞ could not be calculated in many cases.
Plasma concentrations below the limit of quantification of the
assay, BLQ (<1 ng/mL), were taken as zero for all calculations.
If the concentration reached BLQ before 8 h postdose, AUC0-8h
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JM049715T