Specific nonpeptide photoprobes as tools for the structural study of the angiotensin II AT1 receptor

Article abstract of DOI:10.1021/jm991050l

The aim of this work was to obtain photoactivatable nonpeptide antagonists of the angiotensin II AT₁ receptor. Based on structure-function relationships, two chemical structures as well as appropriate synthetic schemes were chosen as a frame for the design of radiolabeled azido probes. The feasibility of the strategy was first assessed by the synthesis of two tritiated ligands 21 and 22 possessing a high affinity for the AT₁ receptor and a low nonspecific binding to membrane or cell preparations. We then prepared two unlabeled azido derivatives 7 and 14 which retained a fairly high affinity for the AT₁ receptor. The latter compound proved to be suitable for receptor irreversible labeling and was prepared in its tritiated form 28. This tritiated azido nonpeptide probe displayed a K(d) value of 11.8 nM and a low nonspecific binding. It was suitable for specific and efficient covalent labeling of the recombinant AT(1A) receptor stably expressed in CHO cells. The electrophoretic pattern of the specifically labeled entity was strictly identical to that of purified receptor photolabeled with a biotinylated peptidic photoactivatable probe. This new tool should be useful for the mapping of the nonpeptide receptor binding site. These potential applications are discussed in light of the current knowledge of molecular mechanisms of G-protein coupled receptor activation and inactivation.

Full text of DOI:10.1021/jm991050l

4572
J . Med. Chem. 1999, 42, 4572-4583
Sp ecific Non p ep tid e P h otop r obes a s Tools for th e Str u ctu r a l Stu d y of th e
An gioten sin II AT1 Recep tor
Sandrine Nouet,† Pierre R. Dodey,‡ Michel R. Bondoux,‡ Didier Pruneau,‡ J ean-Michel Luccarini,‡
Thierry Groblewski,† Rene´e Larguier,† Colette Lombard,† J acky Marie,† Patrice P. Renaut,‡ Ge´rard Leclerc,§ and
J ean-Claude Bonnafous*,†
Laboratoires Fournier S. A., 50 Rue de Dijon, 21121 Daix, France, Universite´ J oseph Fourier, DPM, EP 811 du CNRS,
Laboratoire de Chimie Organique, B.P. 138-38243 Meylan Cedex, France, and INSERM U 439, 70 Rue de Navacelles,
34090 Montpellier, France
Received October 23, 1998
The aim of this work was to obtain photoactivatable nonpeptide antagonists of the angiotensin
II AT1 receptor. Based on structure-function relationships, two chemical structures as well
as appropriate synthetic schemes were chosen as a frame for the design of radiolabeled azido
probes. The feasibility of the strategy was first assessed by the synthesis of two tritiated ligands
21 and 22 possessing a high affinity for the AT1 receptor and a low nonspecific binding to
membrane or cell preparations. We then prepared two unlabeled azido derivatives 7 and 14
which retained a fairly high affinity for the AT1 receptor. The latter compound proved to be
suitable for receptor irreversible labeling and was prepared in its tritiated form 28. This tritiated
azido nonpeptide probe displayed a Kd value of 11.8 nM and a low nonspecific binding. It was
suitable for specific and efficient covalent labeling of the recombinant AT1A receptor stably
expressed in CHO cells. The electrophoretic pattern of the specifically labeled entity was strictly
identical to that of purified receptor photolabeled with a biotinylated peptidic photoactivatable
probe. This new tool should be useful for the mapping of the nonpeptide receptor binding site.
These potential applications are discussed in light of the current knowledge of molecular
mechanisms of G-protein coupled receptor activation and inactivation.
In tr od u ction
present study was to design the first AT1 specific
photoactivatable nonpeptide derivative. The strategy
used for the development of these compounds was based
on previous structure-function relationships and the
prevision of appropriate synthetic schemes.18-22 We
investigated a new series of molecules potentially suit-
able for the introduction of an azido function selected
as the photoactivatable group and easy to be obtained
in a tritiated form. This series was designed according
to the nonpeptide AT1 receptor antagonist SKF
10856618,21,22 (Figure 1). SAR observed in this series is
featured as follows:22,23 (i) the N-1 phenylmethyl sub-
stituent bears an acidic functionality, (ii) the substituent
in position 2 is an alkyl straight chain, (iii) the sub-
stituent in position 5 encompasses an aromatic moiety
and an acidic function. Taking into account all these
requirements, we focused on the general structure A as
shown in Figure 1. This structure can accommodate an
azido group in two distinct regions, either on the N-1
phenylmethyl substituent or on the phenyl linked to the
C-5 imidazole nucleus substituent. Photoprobes corre-
sponding to this latter possibility should allow one to
check the validity of the previous proposal from Smith-
Kline Beecham21 that this portion of the molecule mimic
the phenyl of Phe(8) of AII. Indeed efficient photolabel-
ing of the AT1 receptor was obtained using azido-Phe(8)
angiotensin II-derived peptides15,24 and recently applied
to receptor mapping.17,25
Besides their potential therapeutic applications, non-
peptide derivatives1 constitute interesting tools for the
structural analysis of G-protein coupled receptors.
Ligand recognition and activation processes are closely
interrelated. The existence of receptor active and inac-
tive conformations has allowed one to provide mecha-
nistic explanations for receptor constitutive activities,
as well as inverse agonism properties found for many
synthetic ligands.2,3 As a consequence, the study of the
mechanisms underlying blockade of receptor activation
by nonpeptide antagonists, which in fact often behave
as inverse agonists (when the appropriate experimental
conditions are fulfilled), requires experiments which
provide unambiguous data about inactive receptor
conformations. The obvious limitations of mutagenesis
studies, which do not allow one to discriminate between
direct and indirect effects, are increased when the
evaluation of mutant receptor properties is carried out
through heterologous binding assays involving a tracer
ligand and an unlabeled ligand which display optimal
affinities for different receptor conformations.4,5 This
difficulty is overcome when radioactive nonpeptide
ligands are available. A more difficult but straightfor-
ward manner of investigating receptor topography
consists of its covalent labeling with appropriate syn-
thetic ligands,6-13 as previously carried out with peptidic
angiotensin II-derived probes.14-17 The goal of the
The experimentation involved the following steps: (i)
synthesis of two synthetic tritiated nonpeptide antago-
nists displaying a high affinity for the AT1 receptor and
a low nonspecific binding to membranes or intact cells
† INSERM U 439.
‡ Laboratoires Fournier S. A.
§ Universite´ J oseph Fourier.
10.1021/jm991050l CCC: $18.00 © 1999 American Chemical Society
Published on Web 10/09/1999

Nonpeptide Photoprobes of the Angiotensin II AT1 Receptor
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22 4573
Sch em e 1a
a
Reagents: (a) methyl 2-aminobenzoate, 2,6-dimethylpyridine,
toluene, reflux; (b) 2 N NaOH, MeOH, reflux.
Sch em e 2a
F igu r e 1. SAR of SKF 108566 family used for the design of
general structure A.
expressing this receptor,19 which is an essential pre-
requisite to decide the synthesis of photoactivatable
derivatives (these radioligands constitute by themselves
interesting tools for mapping studies); (ii) obtention of
two nonradioactive photoactivatable derivatives through
the introduction of an azido group into positions of the
molecules potentially interacting with different portions
of the receptor; (iii) evaluation of the ability of these
compounds to irreversibly block AII binding sites upon
irradiation; (iv) synthesis of the radioactive counterpart
of the ligand considered as suitable on the basis of these
experiments; (v) direct demonstration of the ability of
the tritiated azido nonpeptide derivative to covalently
label the AII receptor and characterization of the labeled
entity.
a
Reagents: (a) 2-chloro-benzenesulfonamide or 2-azidoben-
zenesulfonamide, EDCI, DMAP, CH2Cl2; (b) 2 N NaOH, MeOH,
H2O, reflux.
was then hydrolyzed in alcaline medium to yield com-
pound 5. The azido derivative 7 was similarly obtained
starting from 2-azido-benzenesulfonamide.26
The azido compound 14 was prepared in seven steps
with an overall yield of ca. 15% (Scheme 3). Commercial
2-amino-4-nitro-benzoic acid was esterified to 8 which
was protected as a trifluoroacetamide to afford 9 and
catalytically reduced to 10. Diazotization of 10 allowed
its conversion into the corresponding azido derivative
11, which was then smoothly deprotected with 7%
aqueous K2CO3 to afford the key intermediate 12.
Nucleophilic displacement of the chlorine atom of meth-
yl 4-[(2-butyl-5-chloromethyl-1H-imidazol-1-yl)-methyl]-
benzoate22 by 12 in the presence of 2,6-dimethylpyridine
afforded the diester 13 which was converted to the final
azido diacid 14 by alcaline hydrolysis.
The tritiated compound 21 was obtained according to
Scheme 4. Reduction of aldehyde 15 and chlorination
of the resulting alcohol 16 gave 17. Condensation of
benzyl anthranilate with 17 led to the orthogonally
protected diester 18 from which the desired monoacid
monoester 19 was obtained by selective acidic hydroly-
sis. Acylation of known 2-chloro-4-iodo-benzene sulfona-
The advantage of this strategy was to avoid undue
difficult synthesis of inefficient tritiated azido deriva-
tives that would have involved several steps using
labeled compounds.
Ch em istr y
The 1,2,5-trisubstituted imidazole 2 was prepared
according to Scheme 1. The first step consisted of the
nucleophilic displacement of the substituted 5-chloro-
methyl imidazole21 by methyl 2-aminobenzoate in tolu-
ene containing 1 equiv of 2,6-dimethylpyridine to yield
the expected ester intermediate 1. Compound 2 was
finally obtained by alcaline hydrolysis.
The chlorosulfonimide 5 was obtained according to
Scheme 2. The required imidazole 320 was coupled with
2-chloro-benzenesulfonamide to give the ester 4 which

4574 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22
Nouet et al.
Sch em e 3a
Ch a r t 1
Sch em e 5a
a
Reagents: (a) (CF3CO)2O, toluene, reflux; (b) Pd/C 10%, DMF,
1 atm H2; (c) NaNO2, 2 N HCl, 0 °C, then NaN3, 0 °C; (d) K2CO3,
MeOH/H2O; (e) 2,6-dimethylpyridine, toluene, reflux; (f) KOH,
MeOH/H2O, reflux.
a
Reagents: (a) [3H]2, Pd/C 10%, NEt3, MeOAc; (b) NaB[3H]4,
MeOH; (c) SOCl2, CHCl3; (d) 12, 2,6-dimethylpyridine, toluene,
reflux; (e) KOH, MeOH/H2O, reflux.
Sch em e 4a
the same procedure as described for the preparation of
13 gave the diester 27 in 45% yield, and final basic
hydrolysis led to the di-tritiated target 28 in 81% yield.
Biology
Biologica l P r op er ties of 2 a n d 5. 1. An ta gon ism
of AII Action on Ra bbit Aor ta Rin g. Compounds 2
and 5 (Chart 1) did not change the basal tension of the
arteries but inhibited AII-induced contraction in a
concentration-dependent manner, with IC50 values of 3.6
( 0.4 nM and 0.8 ( 0.2 nM, respectively (mean of six
experiments).
2. Bin d in g P r op er ties of th e Tr itia ted Com -
p ou n d s 21 a n d 22 (Ch a r t 1). Some binding properties
of 22 have been previously described.19 They empha-
sized a very low extent of nonspecific binding to rat liver
membranes and membranes from CHO cells expressing
the AT1 receptor, as compared to the commercially
available tritiated Losartan.28 We found quite similar
properties for 21 which displayed an improved affinity
for the AT1 receptor. The binding of 21 to rat liver
membranes, which exclusively contain the AT1 receptor
subtype,29,30 revealed a single class of high affinity
binding sites characterized by Kd values of 2.55 ( 0.35
nM and 2.75 ( 0.30 nM (mean of three experiments)
when nonspecific binding was evaluated using an excess
of either Sar1-AII or 5, respectively. These quite similar
values, systematically obtained within the same experi-
ment, for both assay conditions (see typical experiment
reported in Figure 2A) indicate the total absence of
“pseudo-specific” binding which could be observed with
tritiated Losartan.28 They are consistent with Ki deter-
mined for 5 in competition binding assays using rat liver
as receptor source and [125 I]-Sar1-AII as tracer ligand
(Table 1).
a
Reagents: (a) NaBH4, MeOH, 0 °C; (b) SOCl2, CH2Cl2, 0 °C;
(c) benzylanthranilate, 2,6-lutidine, toluene, reflux; (d) TFA, 0 °C;
(e) 2-chloro-4-iodobenzenesulfonamide, DMAP, EDCI, CH2Cl2; (f)
[3H]2, 1 atm, Pd/C 5%, MeOH.
mide27 with compound 19 in the presence of EDCI gave
20 which allowed us to obtain the desired monotritiated
compound 21 in a single step. Compound 22 (Chart 1)
was obtained in six steps according to the previously
published procedure.19
The di-tritiated analogue of 14 (28) was prepared as
indicated (Scheme 5). The chloroaldehyde 2322 was
tritiated in position 4 of the imidazole ring by catalytic
dehalogenation using Pd/C and tritium gas in 61% yield.
The second tritium atom was introduced thanks to the
reduction of the aldehyde 24 by NaB[3H]4 in 25 which
was treated by SOCl2 to afford the requisite synthon
26. Displacement of the chlorine atom with 12 using

Nonpeptide Photoprobes of the Angiotensin II AT1 Receptor
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22 4575
respective Kd values of 1.3 ( 0.2 nM and 3.5 ( 0.5 nM
(mean of four experiments). These results make the two
nonpeptide radioligands convenient tools for mapping
studies based on the evaluation of binding properties
of mutated receptors.
3. Selectivity for th e AT1 Recep tor . To assess their
selectivity for the AT1 receptor subtype, we checked the
ability of 2 and 5 to inhibit [125I]-Sar1-AII binding to
the AT2 receptor. The experiments were performed on
ewe lamb uterus membranes which contain both recep-
tor subtypes and which have been previously treated
with dithiothreitol to selectively abolish AII binding to
the AT1 receptor.31 When tested at concentrations up
to 10-5 M, 2 and 5 did not significantly reduce the
binding of [125I]-Sar1-AII to the ewe lamb AT2 receptor
(data not shown).
Biologica l P r op er ties of th e Non p ep tid ic Azid o
Der iva tives 14 a n d 7. The properties of the two
unlabeled azido compounds 14 and 7 were compared to
those of the parent derivatives 2 and 5, the binding
properties of which fulfilled essential requirements for
the overall strategy, as assessed by the characteristics
of their tritiated counterparts 22 and 21, respectively.
1. An ta gon ist P r op er ties. The introduction of an
azido substituent into the structures of 2 and 5 does
not alter significantly their antagonist properties (Table
1). Inhibition of AII-induced contraction of rabbit aorta
rings by azido compounds 14 and 7 were characterized
by IC50 values of 6.3 nM (confidence interval: 3.5-11.5
nM) and 3.5 nM (confidence interval: 1.1-11.2 nM),
respectively. These values are close to those found for
the parent compounds 2 and 5 (3.6 nM and 0.8 nM,
respectively; see ref 19 and Table 1). Moreover, all these
compounds were able to block AII-stimulated inositol
phosphate production in CHO cells expressing the AT1A
receptor (data not shown).
2. In h ibition of [125I]Sa r 1-AII Bin d in g to th e Ra t
Liver AT1 Recep tor . The affinities of the two azido
compounds 14 and 7 were evaluated for their ability to
compete for the binding of [125I]Sar1-AII to purified rat
liver membranes which contain only the AT1 receptor
subtype,29 predominantly the AT1A isoform.30 These
experiments were carried out in the absence of BSA,
which can trap nonpeptide antagonists,32 and in the
presence of 0.1 mg/mL bacitracin which ensures satis-
factory preservation of the tracer peptide against pro-
teolysis.19 Under these conditions, the Ki values for 14
and 7 were 22 nM and 1.5 nM, respectively (Table 1);
they did not markedly differ from those obtained for the
parent compounds 2 and 5 (12.1 nM and 2.5 nM,
respectively). The direct binding properties of the tri-
tiated azido compound 28, which will be detailed in a
next paragraph, confirm the competition binding assays.
Taken together these data constitute a validation of the
selected synthetic strategies.
F igu r e 2. Binding properties of the tritiated nonpeptide
compound 21 to the AT1 receptor from membrane prepara-
tions. The binding of the tritiated nonpeptide compound 21 to
membrane preparations from rat liver or CHO cells stably
transfected with the AT1A receptor cDNA was carried out as
described in the Experimental Section. The nonspecific binding
was evaluated using an excess of either Sar1-AII or unlabeled
nonpeptide derivative 5. (A) Rat liver membranes: the binding
parameters, determined through Scatchard analysis of the
represented typical experiment (representative of three sepa-
rate experiments), were Kd ) 2.8 nM, Bmax ) 1.2 pmol/mg (9)
and Kd ) 3.0 nM, Bmax ) 1.3 pmol/mg (4) when nonspecific
binding was determined using an excess of either Sar1-AII or
5, respectively. The nonspecific binding represented only 6%
and 17% of total binding for radioligand concentrations 2 nM
and 16 nM, respectively. (B) Membranes from transfected CHO
cells: the binding parameters, determined through Scatchard
analysis of the represented typical experiment (representative
of four separate experiments), were Kd ) 1.7 nM, Bmax ) 2.8
pmol/mg (b) and Kd ) 1.8 nM, Bmax ) 3.1 pmol/mg (4) when
nonspecific binding was determined using an excess of either
Sar1-AII or 5, respectively. The nonspecific binding represented
only 6% and 16% of total binding for radioligand concentra-
tions of 1.9 nM and 18 nM, respectively. The Bmax values were
similar to those obtained for the binding of [125I]Sar1-AII (Kd
) 0.6 nM, Bmax ) 2.6 pmol/mg).
We established similar properties for the binding of
21 to membranes from CHO cells stably expressing the
AT1A receptor: the mean Kd values for four separate
experiments were 2.85 ( 0.7 nM and 2.0 ( 0.3 nM when
nonspecific binding was evaluated with excess Sar1-AII
or 5, respectively, with close values for both conditions
within the same experiment (typical experiment repre-
sented in Figure 2B).
3. Ir r ever sible Bin d in g of Azid o P r obes 7 a n d 14
to th e AT1A Recep tor . CHO cells stably expressing the
AT1A receptor at high densities (1.2 pmol/106 cells) were
chosen as a system for future analysis of covalent
nonpeptide antagonist-receptor complexes. Preliminary
evaluation of the properties of our azido compounds
were carried out as followed: intact cell binding sites
were saturated at 4 °C with each of the two probes and
the irreversible labeling upon UV irradiation was as-
The compared bindings of 21 and 22 to intact CHO
cells are represented in Figure 3. They confirm the
better receptor affinity of 21 as compared to 22, with

4576 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22
Nouet et al.
Ta ble 1. Structure and Pharmacological Properties of Parent Nonpeptide Ligands, Their Radioactive and Photoactivatable Derived
Probes
a
compd
R1
R2
R3
R4
IC50 (nM)
Kib (nM)
Kdc (nM)
2
14
5
H
H
H
H
H
H
H
H
H
N3
H
H
H
CO2H
CO2H
CONHSO2(C6H4)-2-Cl
CONHSO2(C6H4)-2-N3
CO2H
3.6 ( 0.4
6.3( 1.3
0.8 ( 0.2
3.5 ( 0.6
12.1 ( 2.9
22.0 ( 3.4
2.5 ( 0.8
1.5 ( 0.4
7
22
28
21
[3H]
[3H]
H
H
3.5 ( 0.5
11.8 ( 1.1
1.3 ( 0.2
[3H]
H
N3
H
CO2H
CONHSO2(C6H4)-2-Cl,4-[3H]
a
b
Inhibition of AII-induced contraction in rabbit aorta strips. Inhibition of [125I]Sar1-AII binding to purified rat liver membranes.
c Direct binding of tritiated compounds to the recombinant AT1A receptor stably expressed in CHO cells.
Ta ble 2. Irreversible Binding of Photoactivatable Nonpeptidic
or Peptidic Probes
[
125I]-Sar1-AII specific bindinga
(% of control)
without UV
with UV
ligand
irradiation
irradiation
none
14
100 ( 8
91 ( 1
50 ( 1
75 ( 4
84 ( 2
64 ( 3
55 ( 1
50 ( 1
7
[Sar1, Val5, Phe(4N3)8]AII
a
CHO cells expressing the AT1A receptor were incubated in the
absence or presence of the various photoactivatable compounds,
then irradiated under conditions indicated in the Experimental
Section. The remaining binding sites were titrated by incubation
of the cells in the presence of [125I]-Sar1-AII. The figures represent
the mean of triplicate assays carried out in a typical experiment.
Similar results were obtained in three separate experiments.
F igu r e 3. Binding properties of the tritiated nonpeptide
derivatives 22 and 21 to intact CHO cells expressing the
recombinant receptor. The binding of the two nonpeptide
radioligands was carried out on CHO cells expressing the AT1A
receptor as described in the Experimental Section: b, O;
binding of 22, nonspecific binding measured in the presence
of an excess of Sar1-AII or unlabeled nonpeptide ligand 2,
respectively; 2, 4: binding of 21, nonspecific binding measured
in the presence of an excess of Sar1-AII or unlabeled nonpep-
tide ligand 5, respectively. The nonspecific binding never
exceeded 5% of total 22 binding (concentration range 0.42-
46 nM) and 4% of total 21 binding (concentration range 0.18-
19 nM). The binding parameters deduced from Scatchard
analysis of this typical experiment (representative of three
separate experiments) are Kd ) 3.9 nM, Bmax ) 1.25 pmol/106
cells for 22, Kd ) 1.3 nM, Bmax ) 1.2 pmol/106 cells for 21.
affinity, we decided to use 14 for photolabeling experi-
ments and prepared its tritiated form 28.
Bin d in g P r op er ties of th e Tr itia ted Azid o Non -
p ep t id ic P r ob e 28. P h ot ola b elin g of t h e AT1A
Recep tor . 1. Bin d in g P r op er ties. The binding prop-
erties of 28 have been evaluated on CHO cells overex-
pressing the AT1A receptor. This overexpression suitable
for future mapping approaches was fortuitously ob-
tained by replacement of Cys121 (located in TM III) by
an alanine (2.4-5.4 pmol/106 cells for the C121A recep-
tor, as compared to 1.2 pmol/106 cells for the wild type
receptor); it is reminiscent of the overexpression ob-
tained upon replacement of Cys116 located in TM III of
the â2-adrenergic receptor.33 This mutant receptor was
pharmacologically identical to the wild type receptor (J .
Marie, unpublished results). Compound 28 bound to the
C121A receptor in a saturable and reversible manner
(Figure 4) and with a high affinity (Kd ) 11.8 ( 1.1 nM,
three experiments). The binding parameters were in-
dependent of the ligand (Sar1-AII or unlabeled nonpep-
tide ligand 14) added in excess to evaluate nonspecific
binding which never exceeded 3% of total binding. The
binding capacity (Bmax ) 2.4-5.4 pmol/106 cells) was
identical to that found for [125I]Sar1-AII in the same
experiments.
sessed by measuring [125I]Sar1-AII binding, after dis-
sociation of noncovalently bound ligand. The UV irra-
diation by itself (5 min, 254 nm) only moderately altered
the AII binding sites (Table 2). Cells photolyzed in the
presence of the azido compound 14 displayed a greatly
decreased binding capacity for [125I]Sar1-AII, in favor
of receptor covalent labeling with a predictive fairly good
yield. The results obtained for control nonphotolyzed
cells are indicative of a good dissociation of the ligand
under the experimental conditions used. Similar results
were obtained with the peptide azido derivative [Sar1,
Val5, Phe (4N3)8]AII, a classical photoaffinity probe,15
with the expected noncomplete dissociation of this high
affinity peptide ligand in the absence of UV irradiation.
On the contrary, the analysis of experiments carried out
on cells saturated with the azido compound 7 did not
allow one to conclude that receptor photolabeling, if any,
had occurred at a high yield. Therefore, despite its lower
2. Recep tor P h otola belin g. The photoaffinity label-
ing of the AT1A receptor with 28 was carried out as
follows: after equilibrium binding of the tritiated non-
peptide photoactivatable probe, washed cells were ir-
radiated for 5 min at 254 nm (this photolysis time was

Nonpeptide Photoprobes of the Angiotensin II AT1 Receptor
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22 4577
F igu r e 4. Binding properties of the radioactive photoactivat-
able probe 28. The binding properties of 28 have been studied
on CHO cells overexpressing the C121A AT1A mutant recep-
tor: binding of 28, nonspecific binding measured in the
presence of an excess of either Sar1-AII (9) or unlabeled 14
(4). The nonspecific binding never exceeded 3% of total binding
(concentration range 0.6-70 nM). The binding parameters
deduced from Scatchard analysis of the presented experiments
are Kd ) 11.1 nM, Bmax ) 2.4 pmol/106 cells. The mean values
( SD obtained for three separate experiments were Kd ) 11.8
( 1.1 nM, Bmax ranging from 2.4 to 5.4 pmol for 106 cells.
found to give an optimal photolabeling yield in pilot
experiments). Photolyzed cells were then efficiently
solubilized (yield ) 95-97%, five experiments) with
Triton × 100. The omission of binding medium during
irradiation together with the absence of washing be-
tween photolysis and solubilization steps allowed rigor-
ous evaluation of initially occupied binding sites and
subsequent photolabeling yield. As for the receptor
covalently labeled with peptidic probes, the receptor
photolabeled with the nonpeptide derivative was ef-
ficiently adsorbed to hydroxylapatite gels. This chro-
matography step allowed elimination of dissociated
noncovalently bound ligand. Covalent labeling yields
were estimated from covalent complexes eluted with
high ionic strength; they varied from 8% to 12% over
five experiments and were systematically identical to
those obtained for peptide-receptor covalent complexes
obtained in the same experiments. Besides a partial
purification, the hydroxylapatite step provided a way
of concentrating the tritiated complexes before SDS-
PAGE analysis.16 Autoradiographic analysis of electro-
phoretic gels revealed nonpeptide labeling of a 78 kDa
apparent molecular weight entity (Figure 5a, lane B).
The autoradiographic pattern was similar to that ob-
tained for purified receptor previously photolabeled with
the peptidic probe biotin-NH-(CH2)2-S-S-(CH2)2-CO-
[Ala1, Phe(4N3)8]AII16,17 (Figure 5A, lane A), as assessed
by the perfect superimposition of tritium and iodine
radioactivities in sliced gels (Figure 5B). This labeling
was specific, as it was suppressed when initial incuba-
tion was carried out in the presence of an excess of
either unlabeled Sar1-AII (Figure 5A, lane C) or azido
nonpeptide derivative (Figure 5B, lane D). No labeling
was observed when UV irradiation was omitted (Figure
5A, lane E).
F igu r e 5. Photoaffinity labeling of the AT1A receptor by the
nonpeptide photoactivatable probe 28. Intact CHO cells over-
expressing the mutant C121A AT1A receptor were photola-
beled, and cells were solubilized using Triton × 100 as
detergent. The samples containing covalent probe-receptor
complexes were partially purified and concentrated through
hydroxylapatite chromatography before electrophoresis (12.5%
acrylamide gels) and autoradiography. (A) SDS-PAGE and
autoradiographic analysis of photolabeled receptors. Lane A:
control purified receptor previously photolabeled with a peptide
biotinylated probe16 under identical experimental conditions
(3 fmol). Lane B: receptor photolabeled with the nonpeptide
tritiated probe (5 pmol). Lanes C and D: nonspecific photo-
labeling obtained in the presence of excess Sar1-AII or 14,
respectively, during initial receptor saturation. Lane E: control
nonphotolyzed cells. Similar results were obtained in four
separate experiments. (B) Compared electrophoretic patterns
of receptor photolabeled by peptidic and nonpeptidic probes.
Samples of receptor photolabeled by 28 and purified receptor
previously photolabeled by a radioiodinated peptide probe16
were analyzed in the same electrophoresis run. The wet
unfixed gel was cut into 5 mm slices for tritium (0) or iodine
(9) determinations. Molecular weights of precolored markers
are indicated on the abscissa in kilodaltons.
nonpeptidic compounds are distinct. However it is not
always possible to draw clear-cut conclusions from the
pharmacological properties of mutated or chimeric
receptors.42 Receptor covalent labeling using affinity or
photoaffinity probes,6-17 although it is often a difficult
task, can provide valuable information about ligand-
receptor chemical interactions as previously shown for
the AII-AT1 receptor system.15-17 Reported data rela-
tive to the biochemical mapping of peptide hormone
receptors have been obtained using peptidic probes,
while a limited number of studies have been undertaken
using nonpeptide photoactivatable probes.43,44
Discu ssion
Extensive mutagenesis experiments have been used
to study the interaction of nonpeptide antagonists with
peptide hormone receptors belonging to the GPCR
family,34-38 including the AT1 receptor.39-41 They led to
the agreement that the binding sites for peptidic and

4578 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22
Nouet et al.
The purpose of the present work was to design a
nonpeptide derivative suitable for the covalent labeling
of the AT1 angiotensin II receptor. The first step of the
strategy consisted of the synthesis of two tritiated
nonpeptide antagonists, 2219 and 21, giving negligible
nonspecific binding. On the basis of the previous devel-
opment of appropriate synthetic schemes,18-22 we have
prepared two photoactivatable compounds bearing an
azido group in two positions of the molecule potentially
interacting with different portions of the receptor.
Irreversible binding could be evidenced for one of them,
14, thus justifying the synthesis of the corresponding
tritiated compound. The tritiated azido probe 28 dis-
played a satisfactory affinity for the AT1A recombinant
receptor expressed in CHO cells (Kd ) 11.8 nM). We
demonstrated its ability to specifically and efficiently
photolabel this receptor. The apparent molecular weight
of the labeled entity (78 kDa) and the yield of covalent
labeling (about 10%) were identical to those found for
the receptor photolabeled with a peptidic probe bearing
an azido group on the C-terminal phenylalanine.15-16
be used as frameworks in order to refine the positioning
of transmembrane helices.
A previous work has demonstrated the possibility to
covalently label the AT1 receptor with agonist AII-
derived probes possessing an azido phenylalanine at
their C-terminus;15 it was further applied to receptor
purification using azido biotinylated compounds.16 We
recently reassessed the preliminary characterization of
small photolabeled tryptic fragments17 to unambigu-
ously demonstrate that the C-terminal phenylalanine
of AII interacts with a receptor zone comprising trans-
membrane domain VI and part of the third extracellular
domain.25
The photolabeling experiments carried out with pep-
tidic agonists and nonpeptide antagonists (or inverse
agonists) should thus be helpful for the building of
reliable models of ligand-receptor interactions which
integrate the molecular mechanisms underlying transi-
tions from inactive to active receptor conformations and
their differential recognition or induction50,51 by the
various classes of pharmacological agents.
If required, another probe expectedly displaying a
higher affinity might be synthesized through the intro-
duction of an azido group into the anthranilate moiety
of compound 5.
Exp er im en ta l Section
Ch em istr y. Melting points were determined either on a hot-
stage Kofler or on a Bu¨chi melting point apparatus and are
uncorrected. IR spectra were measured on a Perkin-Elmer 782
spectrophotometer. 1H and 13C NMR spectra were obtained
on a Bruker AC 300 spectrometer using tetramethylsilane as
internal reference. Structural assignments for all new com-
pounds were consistent with their spectra. Elemental analyses
were performed on a Perkin-Elmer 240 C apparatus. Molecular
formula followed by the symbols C, H, N indicate that
elemental analyses were found to be within (0.4% of the
theoretical values for C, H, and N. Sar1-AII was purchased
from Bachem (Bubendorf, Switzerland). It was radioiodinated
as previously described16 [Sar1, Val5, Phe(4N3)8]-AII was kindly
supplied by E. Escher (Sherbrooke, Canada).
Meth yl 2-[[[2-Bu tyl-1-[[4-(m eth oxyca r bon yl)p h en yl]-
m et h yl]-1H -im id a zol-5-yl]m et h yl]a m in o]b en zoa t e (1).
Methyl 4-[(2-butyl-5-chloromethyl-1H-imidazol-1-yl)methyl]-
benzoate hydrochloride22 (8 g, 22.3 mmol) was suspended in
80 mL of anhydrous toluene. Next, 10.15 g (67.1 mmol) of
methyl 2-aminobenzoate and 4.79 g (44.7 mmol) of 2,6-
dimethyl pyridine were added. The reaction mixture was
refluxed for 8 h and then poured into cold water. The product
was extracted and purified by chromatography with toluene-
2-PrOH (9:1) to afford 9 g (92%) of 1 as an orange oil: 1H NMR
(CDCl3) δ 7.91 (d, J ) 8.3 Hz, 2H), 7.82 (dd, J ) 1.57 Hz, J )
7.98 Hz, 1H), 7.67 (t, J ) 4.80 Hz, 1H), 7.28 (m, 1H), 7.04 (s,
1H), 6.92 (d, J ) 8.3 Hz, 2H), 6.63 (m, 2H), 5.18 (s, 2H), 4.20
(d, J ) 4.80 Hz, 2H), 3.90 (s, 3H), 3.77 (s, 3H), 2.56 (t, J ) 8
Hz, 2H), 1.70 (m, 2H), 1.30 (m, 2H), 0.87 (t, J ) 7.3 Hz, 3H).
2-[[[2-Bu tyl-1[(4-ca r boxyp h en yl)m eth yl]-1H-im id a zol-
5-yl]m eth yl]a m in o]ben zoic Acid (2). Compound 1 (5.1 g,
11.7 mmol) was dissolved in 50 mL of methanol. Next, 17.6
mL (35.2 mmol) of 2 N NaOH solution was added, and the
mixture was stirred under reflux for 4 h. Methanol was
evaporated, and the residue was dissolved in cold water. The
diacid was precipitated by addition of 1 N HCl to pH 4. The
solid was filtered off, washed with water, and dried over P2O5.
The crude product was recrystallized from methanol to afford
3.5 g (73.5%) of 2 as a pale yellow solid: mp 234 °C; 1H NMR
(DMSO-d6) δ 13.0 (bs, 2H), 8.0 (bs, 1H), 7.75 (d, J ) 8.26 Hz,
2H), 7.70 (d, J ) 7.95 Hz, 1H), 7.30 (m, 1H), 7.0 (d, J ) 8.13
Hz, 2H), 6.93 (s, 1H), 6.75 (d, J ) 8.42 Hz, 1H), 6.55 (m, 1H),
5.32 (s, 2H), 4.31 (s, 2H), 2.50 (t, 2H), 1.45 (m, 2H), 1.21 (m,
2H), 0.77 (t, J ) 7.35 Hz, 3H).
The elucidation of the mechanism of GPCR activation
must take into account the existence of inactive and
active receptor conformations and their preferential
recognition and (or) induction by various ligands ac-
cording to their pharmacological behavior as agonists,
inverse agonists, or pure antagonists.2,3 Recent data
suggest that transmembrane helix movements, espe-
cially movements of helices III and VI,45-48 are associ-
ated with the transition from an inactive to an active
receptor conformations, thus resulting in marked struc-
tural changes. As a consequence, binding site studies
can often be confusing when they are based on the
evaluation of mutant receptor properties through het-
erologous competition binding experiments,4,5 a situa-
tion which can be circumvented when radioactive non-
peptide ligands, such as those described in the present
paper, are available. Moreover, hypotheses about the
superimposition of peptide agonists and nonpeptide
antagonists can appear still more speculative.1,21,22
Therefore the need for the obtention of direct biochemi-
cal evidences about receptor-ligand interaction is re-
inforced. In this respect the original tritiated nonpeptide
photoactivatable probe reported in the present work
constitutes a valuable tool for mapping studies. The
absence of nonspecific binding of both radioligands 21
and 22, extended to receptors transiently expressed in
COS-7 cells, allowed accurate evaluation of mutant
receptors having lost much affinity for nonpeptidic
derivatives (i.e. AT1 N111A and S105A mutants41,49),
as well as verification of the expression of mutant
having completely lost their AII binding properties
(S105A mutant41). It is noticeable that the new non-
peptide derivatives behaved as inverse agonists when
tested in experimental situations where the AT1 recep-
tor basal activity was enhanced.49 As a consequence,
biochemical mapping using the nonpeptidic azido probe
should help to define the inactive receptor conformation.
As nonpeptidic ligands display a reduced conformational
flexibility as compared to AII-derived peptides, they can
Meth yl 2-[[[2-Bu tyl-1-[(4-ca r boxyp h en yl)-m eth yl]-1H-
im id a zol-5-yl]m eth yl]a m in o]ben zoa te (3). Sodium hydrox-
ide (0.68 g, 20 mmol) and 10 mL of water were added to a
solution of 8.3 g (19.1 mmol) of 1 in 80 mL of methanol. The

Nonpeptide Photoprobes of the Angiotensin II AT1 Receptor
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22 4579
mixture was heated at 50 °C for 3.5 h and concentrated, and
the residue was diluted with 150 mL of water. The aqueous
layer was washed with EtOAc and acidified to pH 5 with 1 N
HCl. The product was extracted with EtOAc. The organic
layers were washed with water, dried (MgSO4), and concen-
trated. The crude product was purified by chromatography on
silica with CH2Cl2-MeOH (95:5) to give 4.8 g of a solid which
was recrystallized from EtOAc, yielding 4.41 g (55%) of 3 as a
and 2.2 Hz, 1H), 8.16 (d, J ) 8.6 Hz, 1H), 3.89 (s, 3H). Anal.
(C10H7F3N2O5) C, H, N.
Met h yl (4-Am in o-2-t r iflu or om et h ylca r b on yla m in o)-
ben zoa te (10). A mixture of 9 (7 g, 24 mmol) in 70 mL of
anhydrous DMF and 0.72 g of 10% palladium on charcoal was
stirred under l atm H2 pressure for 12 h. The catalyst was
filtered off, and the DMF solution was diluted with water,
extracted with toluene, and dried (MgSO4). Evaporation gave
10 as an ochreous solid (80%): mp 161-162 °C; 1H NMR
(DMSO-d6) δ 12.44 (s, 1H), 7.73 (d, J ) 8.8 Hz, 1H), 7.62 (d, J
) 2.2 Hz, 1H), 6.48 (s, 2H), 6.43 (dd, J ) 8.8 and 2.2 Hz, 1H),
3.80 (s, 3H). Anal. (C10H9F3N2O3) C, H, N.
1
white solid: mp 160 °C; H NMR (CDCl3) δ 7.92 (d, J ) 6.64
Hz, 2H), 7.80 (d, J ) 8.61 Hz, 1H), 7.68 (bt, 1H), 7.30 (t, J )
9 Hz, 1H), 7.19 (s, 1H), 6.92 (d, J ) 8.3 Hz, 2H), 6.63 (m, 2H),
5.3 (bs, 1H), 5.22 (s, 2H), 4.20 (d, J ) 4.9 Hz, 2H), 3.79 (s,
3H), 2.88 (t, J ) 7.6 Hz, 2H), 1.88 (m, 2H), 1.33 (m, 2H), 0.88
(t, J ) 7.3 Hz, 3H).
Met h yl (4-Azid o-2-t r iflu or om et h ylca r b on yla m in o)-
ben zoa te (11). To a cold suspension (-3 °C) of 10 (4.8 g, 18
mmol) in 30 mL of aqueous 2 N HCl was added dropwise a
solution of 1.85 g (27 mmol) of NaNO2 in 12 mL of water. The
mixture was stirred for 5 h vigorously at - 3 °C. Then a
solution of 1.75 g (27 mmol) of NaN3 in 11 mL of water was
added dropwise. After the mixture was stirred for 2.5 h at -3
°C, the precipitate formed was filtered, washed with water,
and dried in a desiccator (P2O5) to give 11 as a white-cream
solid (95%) which was protected from light and kept at 4 °C:
Meth yl 2-[[[2-Bu tyl-1-[(4-((((2-a zid op h en yl)su lfon yl)-
am in o)car bon yl)ph en yl)m eth yl]-1H-im idazol-5-yl]m eth yl]-
a m in o]ben zoa te (6). To a suspension of 2.22 g (5.3 mmol) of
3 in 55 mL of CH2Cl2 were added 1.15 g (5.8 mmol) of 2-azido-
benzenesulfonamide, 0.71 g (5.8 mmol) of DMAP, and 1.11 g
(5.8 mmol) of EDCI. The mixture protected from light was
stirred during 3 days at room temperature and concentrated
in vacuo to afford a residue which was purified by flash
chromatography on silica gel using toluene-2-PrOH (6:4) as
eluent to afford 6 (2.23 g, 70%) as a yellow solid which was
1
mp 83 °C; H NMR (DMSO-d6) δ 11.99 (s, 1H), 8.03 (d, J )
8.6 Hz, 1H), 7.84 (d, J ) 2.2 Hz, 1H), 7.17 (dd, J ) 8.6 and 2.3
1
Hz, 1H), 3.87 (s, 3H). Anal. (C10H7F3N4O3) C, H, N.
kept away from light: mp 162 °C, H NMR (DMSO-d6) δ 7.89
Meth yl 2-Am in o-4-a zid oben zoa te (12). Compound 11
(1.03, 3.6 mmol) was dissolved in 50 mL of a solution of
methanol-water (5:2) containing 7% of K2CO3. The mixture
protected from light was stirred for 18 h at room temperature,
diluted with water, and extracted with EtOAc. The organic
layers were washed with water, dried, and concentrated to a
residue which was washed with Mill Q water, taken up in
ether, dried, and evaporated to yield 12 as a yellow solid (89%)
which was kept away from light: mp 69 °C; 1H NMR (DMSO-
d6) δ 7.72 (d, J ) 8.6 Hz, 1H), 6.82 (1, 2H), 6.50 (d, J ) 2.3 Hz,
1H), 6.27 (dd, J ) 8.7 and 2.3 Hz, 1H), 3.77 (s, 3H). Anal.
(C8H8N4O2) C, H, N.
(dd, J ) 7.7 Hz and J ) 1.5 Hz, 1H), 7.77 (m, 3H), 7.70 (t, J
) 5.7 Hz, 1H), 7.44 (dt, J ) 7.1 Hz and J ) 1.5 Hz, 1H), 7.33
(dt, J ) 7.1 Hz and J ) 1.5 Hz, 1H), 7.21 (m, 3H), 6.82 (m,
4H), 6.58 (t, J ) 7.8 Hz, 1H), 5.23 (s, 2H), 4.32 (d, J ) 4.6 Hz,
2H), 3.66 (s, 3H), 2.47 (t, J ) 7.8 Hz, 2H), 1.47 (m, 2H), 1.23
(m, 2H), 0.78 (t, J ) 7.3 Hz, 3H).
Meth yl 4-[2-Bu tyl-5-[[2-[(2-ch lor op h en yl)su lfon yla m i-
n oca r b on yl]p h e n yl]a m in om e t h yl]-1H -im id a zol-1-yl-
m eth yl]ben zoa te (4). Compound 4 was synthesized from 3
and 2-chloro-benzenesulfonamide by the same procedure as
decribed above for the synthesis of 6: mp 125 °C; yield 78%;
1H NMR (CDCl3) δ 8.17 (d, 1H), 7.87 (d, 2H), 7.78 (d,1H), 7.65
(s, 1H), 7.20 (m, 5H), 6.70 (d, 2H), 6.52 (m, 2H), 5.12 (s, 2H),
4.07 (s, 2H), 3.67 (s, 3H), 2.50 (m, 2H), 1.40 (m, 2H), 1.21 (m,
2H), 0.72 (t, 3H).
Meth yl 2-[[[2-Bu tyl-1-[[4-(m eth oxyca r bon yl)p h en yl]-
m e t h yl]-1H -im id a zol-5-yl]m e t h yl]a m in o]-4-a zid ob e n -
zoa te (13). Methyl 4-[(2-butyl-5-chloromethyl-1H-imidazol-1-
yl)methyl] benzoate, hydrochloride22 (1.69 g, 4.7 mmol) was
suspended in 50 mL of anhydrous toluene. Next, 2.72 g (14
mmol) of methyl 2-amino-4-azido-benzoate 12 and 1.1 mL (9.4
mmol) of 2,6-dimethylpyridine were added. The reaction
mixture was heated at 120 °C for 5.5 h and then poured into
a saturated NaHCO3 solution. The product was extracted with
EtOAc, dried, evaporated, and purified by flash chromatog-
raphy with toluene-2-PrOH (9:1). The solid obtained was
dissolved into freshly distillated acetone. Evaporation of this
solution at 60 °C under vacuum gave 13 as a beige solid (45%)
2-[[[2-Bu t yl-1-[(4-((((2-a zid op h en yl)su lfon yl)a m in o)-
ca r bon yl)p h en yl)m eth yl]-1H-im id a zol-5-yl]m eth yl]a m i-
n o]ben zoic Acid (7). To a solution of 0.83 g (1.4 mmol) of 6
in 20 mL of methanol was added 0.42 g (10.5 mmol) of NaOH
in 2 mL of water. The mixture was heated under reflux during
4 h. Evaporation of the methanol afforded a residue which was
taken up in water. The pH of the resulting solution was
adjusted to 5 with an aqueous 1 N HCl solution from which
the desired product 7 precipitated as a white solid which was
dried in vacuo in the presence of P2O5 (0.64 g, 79%) and which
1
which was kept away from light: mp 130-132 °C; H NMR
1
was kept away from light: mp 180 °C dec; H NMR (DMSO-
(CDCl3) δ 7.91 (d, J ) 8.2 Hz, 2H), 7.84 (t, J ) 5.0 Hz, 1H),
7.80 (d, J ) 8.6 Hz, 2H), 7.05 (s, 1H), 6.92 (d, J ) 8.1 Hz, 2H),
6.29 (dd, J ) 8.6 Hz, J ) 2.0 Hz, 1H), 6.21 (d, J ) 2.0 Hz, 1H),
5.17 (s, 2H), 4.16 (d, J ) 5.1 Hz, 2H), 3.91 (s, 3H), 3.76 (s,
3H), 2.57 (t, J ) 7.5 Hz, 2H), 1.70 (m, 2H), 1.36 (m, 2H), 0.88
(t, J ) 7.3 Hz, 3H). Anal. (C25H28N6O4) C, H, N.
d6) δ 8.10 (bs, 1H), 7.86 (m, 4H), 7.52 (dt, J ) 7.8 Hz and J )
1.5 Hz, 1H), 7.28 (m, 4H), 7.06 (d, J ) 8.3 Hz, 2H), 6.69 (d, J
) 8.4 Hz, 1H), 6.60 (t, J ) 7.7 Hz, 1H), 5.46 (s, 2H), 4.42 (s,
2H), 2.72 (t, J ) 7.5 Hz, 2H), 1.43 (m, 2H), 1.21 (m, 2H), 0.78
(t, J ) 7.2 Hz, 3H). Anal. (C29H29N7O5, 0.57 H2O) C, H, N.
2-[[[2-Bu tyl-1-[(4-((((2-ch lor op h en yl)su lfon yl)a m in o)-
ca r bon yl)p h en yl)m eth yl]-1H-im id a zol-5-yl]m eth yl]a m i-
n o]ben zoic Acid (5). This product was synthesized from 4
by the same procedure as decribed above for the synthesis of
7: mp 198 °C; yield 88%; 1H NMR (DMSO-d6) δ 13.20 (bs, 1H),
8.16 (m, 1H), 8.0 (d, 1H), 7.86 (d, 2H), 7.80 (d, 1H), 7.46 (m,
4H), 7.34 (t, 1H), 7.04 (d, 2H), 6.65 (m, 2H), 5.49 (s, 2H), 4.46
(s, 2H), 2.80 (t, 2H), 1.42 (m, 2H), 1.21 (m, 2H), 0.76 (t, 3H).
Anal. (C29H29ClN4O5S) C, H, N.
2-[[[2-Bu tyl-1-[(4-ca r boxyp h en yl)m eth yl]-1H-im id a zol-
5-yl]m eth yl]a m in o]-4-a zid oben zoic Acid (14). A suspen-
sion of 0.17 g (0.4 mmol) of 13 in 2 mL of distilled methanol
in 0.16 mL of water and 90 mg (1.6 mmol) of KOH was heated
under reflux for 1 h. Evaporation of the methanol afforded a
residue which was taken up in water. The pH of the resulting
solution was adjusted to 5 with an aqueous 1 N HCl solution
from which the desired product 14 precipitated as a white solid
which was dried in vacuo in the presence of P2O5 (0.13 g,
81%): mp 215 °C dec; 1H NMR (DMSO-d6) δ 8.08 (s, 1H), 7.81
(d, J ) 8.1 Hz, 2H), 7.75 (d, J ) 8.5 Hz, 1H), 6.98 (d, J ) 8.1
Hz, 2H), 6.87 (s, 1H), 6.38 (d, J ) 1.9 Hz, 2H), 6.32 (dd, J )
8.5 Hz and J ) 2.0 Hz, 1H), 5.31 (s, 2H), 4.34 (s, 2H), 2.47 (t,
J ) 7.8 Hz, 2H), 1.49 (m, 2H), 1.23 (m, 2H), 0.77 (t, J ) 7.2
Hz, 3H). Anal. (C23H24N6O4, 0.74 H2O) C, H, N.
Meth yl (4-Nitr o-2-tr iflu or om eth ylca r bon yla m in o)ben -
zoa te (9). To a solution of 8 (7 g, 36 mmol) in anhydrous
toluene was added 30 mL (214 mmol) of trifluoroacetic
anhydride. The mixture was heated under reflux for 24 h, then
washed with water until neutrality, dried (MgSO4), and
evaporated. Recrystallization from diisopropyloxide gave 9 as
ochreous needles (70%): mp 138-140 °C; 1H NMR (DMSO-
d6) δ 11.91 (s, 1H), 8.70 (d, J ) 2.2 Hz, 1H), 8.23 (dd, J ) 8.7
(1,1-Dim eth yleth yl) 4-[[2-Bu tyl-5-(h yd r oxym eth yl)-1H-
im id a zol-1-yl]m eth yl]ben zoa te (16). To a solution of 4.6 g

4580 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22
Nouet et al.
(13.4 mmol) of 1520 in 60 mL of methanol cooled to 0 °C was
added 0.61 g (16 mmol) of sodium borohydride. After 0.25 h,
the reaction mixture was concentrated, and the residue was
dissolved in water and extracted with EtOAc. The organic
layer was washed with brine, dried (MgSO4), and concentrated
to afford 16 as a crude solid which was directly used in the
stirred under 1 atm [3H]2 pressure for 6 h at room temperature.
The catalyst was then filtered off, and the solvent evaporated.
The residue is solubilized in MeOH (5 mL) and purified on
preparative TLC (silica gel 60F 254) with CHCl3-MeOH (7:
3). After elution the desired compound is obtained after
desorption by MeOH. The HPLC analysis (Lichrospher RP C8;
eluent: KH2PO4 5 mM-MeOH (53:47)) revealed that the
tritiated compound is identical to the unlabeled compound 5.
Meth yl 4-[[2-Bu tyl-5-for m yl-4-t-im id a zol-1-yl]m eth yl]-
ben zoa te (24). A mixture of 0.2 g (0.6 mmol) of methyl 4-[[2-
butyl-4-chloro-5-formyl-imidazol-1-yl]methyl]benzoate 2322 in
10 mL of methyl acetate, 0.1 mL (0.72 mmol) of triethylamine,
and 20 mg of 10% palladium on charcoal was stirred under l
atm of [3H]2 for 2 h. The catalyst was filtered off, the solvent
was evaporated, and the resulting residue was partitioned
between EtOAc and water. The organic layer was then washed
with water, dried over MgSO4, and concentrated. The crude
product was purified by preparative TLC on silica gel using
toluene-2-PrOH (9:1) as eluent to afford 0.11 g (61%) of 24
which coeluted with an authentic sample of the corresponding
nontritiated compound.
Meth yl 4-[[2-Bu tyl-5-h yd r oxym eth yl-t-4-t-im id a zol-1-
yl]m eth yl]ben zoa te (25). To a solution of 0.11 g (0.37 mmol)
of 24 in 5 mL of MeOH cooled to 0 °C were added 18.7 mg
(0.44 mmol) of NaB[3H]4. The mixture was stirred for 20 min
and the methanol was then evaporated to afford a residue
which was taken up in water. The pH of the resulting solution
was adjusted to 6 with an aqueous 1 N HCl solution from
which the desired product 25 was extracted with EtOAc. The
organic layer was washed with water, dried over MgSO4, and
concentrated to afford a crude product which was purified by
preparative TLC on silica gel using CH2Cl2/MeOH (95/5) as
eluent to afford 0.10 g (89%) of 25 which coeluted with an
authentic sample of the corresponding nontritiated compound.
Met h yl 4-[(2-Bu t yl-5-ch lor om et h yl-t-4-t-1H -im id a zol-
1-yl)-m eth yl]ben zoa te, Hyd r och lor id e (26). To a solution
of 0.1 g (0.33 mmol) of 25 in 5 mL of chloroform cooled to 0 °C
was slowly added 0.11 g (0.196 mmol) of thionyl chloride. The
resulting mixture was stirred for 1.5 h at 0 °C and then
concentrated. The residue was taken up in a few milliliters of
toluene and concentrated. This operation was reproduced three
times in order to eliminate all traces of thionyl chloride and
afforded the desired 26 which was directly used in the next
step to prepare 27.
Meth yl 2-[[[2-Bu tyl-1-[[4-(m eth oxyca r bon yl)p h en yl]-
m et h yl-4-t-]-1H -im id a zol-5-yl]m et h yl-t]a m in o]-4-a zid o-
ben zoa te (27). This product was prepared following the same
procedure described for the preparation of 13 starting from
26. Its purification has been carried out by preparative TLC
on silica gel using toluene/EtOAc (3/2) as eluent to afford 27
which coeluted with 13.
2-[[[2-Bu tyl-1-[(4-ca r boxyp h en yl)m eth yl]-4-t-1H-im id a -
zol-5-yl]m eth yl-t]a m in o]-4-a zid oben zoic Acid (28). This
product was prepared following the same procedure described
for the preparation of 14 starting from 27 instead of 13. Its
purification has been carried out by preparative TLC on silica
gel using EtOAc/HCO2H/H2O (60/5/35) as eluent to afford 28
which coeluted with 14. The chemical purity of 28 has been
measured to >99.2% by HPLC using the following conditions:
Lichrospher RP C8, 5 mm, 1 mL/min, eluent: KH2PO4 5 mM,
pH 3/H3PO4/acetonitrile (60/40), detection UV 247 nm and beta
counter. The product has been obtained with a specific activity
of 22.6 Ci/mmol.
Biology. Biologica l Assa y. The ability of the compounds
to inhibit AII-induced contractile response was examined in
the isolated rabbit aorta as previously described.19,52 A segment
of thoracic aorta was isolated, and the endothelium was
mechanically rubbed off. Rings of 4 mm in length were
prepared and suspended on triangular stainless steel wires
in 20 mL jacketed organ baths maintained at 37 °C. One hook
was suspended from a Gould-Statham UC2 or UTC1 trans-
ducer, and the other was fixed to a plastic support leg. Changes
in isometric tension were continuously recorded. Rings were
left unstretched for 30 min and were then stretched under a
1
next step: mp 163 °C; H NMR (CDCl3) δ 7.9 (d, J ) 8.3 Hz,
2H), 7.0 (d, J ) 8.36 Hz, 2H), 6.93 (s, 1H), 5.26 (s, 2H), 4.46
(s, 2H), 2.53 (t, J ) 7.8 Hz, 2H), 2.05 (bs, 1H), 1.63 (m, 2H),
1.57 (s, 9H), 1.32 (m, 2H), 0.87 (t, J ) 7.3 Hz, 3H).
(1,1-Dim eth yleth yl) 4-[[2-Bu tyl-5-(ch lor om eth yl)-1H-
im id a zol-1-yl]m eth yl]ben zoa te (17). To a solution of 4.5 g
(13 mmol) of 16 in 100 mL of CH2Cl2 cooled to 0 °C were added
5.3 mL (72.7 mmol) of thionyl chloride dropwise. The solution
was allowed to warm to room temperature during 5 min. The
mixture was concentrated, and the residue was taken up in
100 mL of toluene and concentrated again to afford 5.1 g (97%)
of crude 17 as a beige solid: mp 191 °C dec; 1H NMR (CDCl3)
δ 16.83 (s, 1H), 8.05 (d, J ) 8.34 Hz, 2H), 7.54 (s, 1H), 7.09 (d,
J ) 8.305 Hz, 2H), 5.45 (s, 2H), 4.42 (s, 2H), 3.08 (t, J ) 7.8
Hz, 2H), 1.80 (m, 2H), 1.59 (s, 9H), 1.41 (m, 2H), 0.90 (t, J )
7.3 Hz, 3H). This compound was used in the next step without
further purification.
(1,1-Dim eth yleth yl) 4-[[2-Bu tyl-5-[[2-(p h en ylm eth oxy-
ca r bon yl)p h en yl]a m in om eth yl]-1H-im id a zol-1-yl]m eth -
yl]ben zoa te (18). A mixture of 1 g (2.5 mmol) of 17, 1.83 g
(2.5 mmol) of benzyl anthranilate, and 0.53 g (2.5 mmol) of
2,6-lutidine into 35 mL of toluene was stirred under reflux
for 1 h and at room temperature for 14 h. The mixture was
partitioned between water and EtOAc. The organic layers were
washed, dried (MgSO4), and concentrated, and the crude
residue was purified by flash chromatography with methyl-
cyclohexane-acetone (7:3) to afford 0.7 g (50%) of 18 as a
colorless oil: 1H NMR (CDCl3) δ 7.90 (m, 3H), 7.70 (bt, 1H),
7.39-7.25 (m, 6H), 7.03 (s, 1H), 6.90 (d, J ) 8.34 Hz, 2H),
6.60 (m, 2H), 5.23 (s, 2H), 5.16 (s, 2H), 4.16 (d, J ) 4.87 Hz,
2H), 2.58 (t, J ) 7.58 Hz, 2H), 1.70 (m, 2H), 1.58 (s, 9H), 1.39
(m, 2H), 0.88 (t, J ) 7.2 Hz, 3H).
4-[[2-Bu t yl-5-[[2-(p h e n ylm e t h oxyca r b on yl)p h e n yl]-
am in o-m eth yl]-1H-im idazol-1-yl]m eth yl]ben zoic Acid (19).
To 2.6 g (4.7 mmol) of 18 were added 10 mL of TFA at 0 °C,
and the obtained solution was stirred at 0 °C for 3 h. The
reaction mixture was evaporated to dryness, 50 mL of water
was added, the pH of the mixture was adjusted to 6 with
NaOH, and the organic layers were washed with water, dried
(MgSO4), and concentrated. The crude product was purified
by flash chromatography with CH2Cl2-MeOH (9:1) to afford
19 (2.2 g, 94%) as a yellowish solid: mp 90 °C; 1H NMR (CDCl3)
δ 13.0 (bs, 1H), 8.03 (d, J ) 8.26 Hz, 2H), 7.95 (d, J ) 9.5 Hz,
1H), 7.88 (bs, 1H), 7.43-7.28 (m, 7H), 7.0 (d, J ) 8.25 Hz,
2H), 6.65 (dt, J ) 7.4 Hz, 1H), 6.55 (d, J ) 8.48 Hz, 1H), 5.30
(s, 2H), 5.24 (s, 2H), 4.22 (d, J ) 3.2 Hz, 2H), 2.86 (t, J ) 7.6
Hz, 2H), 1.70 (m, 2H), 1.33 (m, 2H), 0.85 (t, J ) 7.25 Hz, 3H).
Ben zyl 2-[[[2-Bu tyl-1-[(4-((((2-ch lor o-4-iod op h en yl)su l-
fon yl)a m in o)ca r bon yl)p h en yl)m eth yl]-1H-im id a zol-5-yl]-
m eth yl]a m in o]ben zoa te (20). To a suspension of 3.85 g (7.7
mmol) of 19 in 100 mL of CH2Cl2 were added 2.7 g (8.5 mmol)
of 2-chloro-4-iodo-benzene sulfonamide, 1.04 g (8.5 mmol) of
DMAP, and 1.62 g (8.5 mmol) of 1-(3-dimethyl-amino propyl)-
3-ethylcarbodiimide hydrochloride. The mixture was stirred
at room temperature for 4 days. The organic layer was washed
with water, dried (MgSO4), and concentrated to afford a crude
residue. The desired product was purified by flash chroma-
tography with toluene-2-PrOH (1:1) to afford 4.26 g (69%) of
20 as an orange solid, mp 155 °C: 1H NMR (DMSO-d6) δ 7.77
(m, 7H), 7.38 (m, 6H), 6.82 (m, 4H), 6.59 (t, J ) 7.4 Hz, 1H),
5.22 (s, 2H), 5.20 (s, 2H), 4.31 (d, J ) 5.2 Hz, 2H), 2.48 (t, J )
7.8 Hz, 2H), 1.48 (m, 2H), 1.24 (m, 2H), 0.78 (t, J ) 7.3 Hz,
3H).
2-[[[2-Bu tyl-1-[(4-((((2-ch lor o-4-t-p h en yl)su lfon yl)a m i-
n o)ca r bon yl)p h en yl)m et h yl]-1H-im id a zol-5-yl]m et h yl]-
a m in o]ben zoic Acid (21). A mixture of 0.05 g (0.063 mmol)
of 20 and 0.0378 g (0.38 mmol) of triethylamine in 5 mL of
anhydrous DMF with 0.01 g of 10% palladium on carbon was

Nonpeptide Photoprobes of the Angiotensin II AT1 Receptor
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22 4581
passive tension of 2 g. Vessel segments were maintained in a
Krebs-Henseleit solution bubbled with O2/CO2 (95%/5%)
throughout the experiments. Three to five different concentra-
tions of the antagonist were tested against the contraction to
AII added at a concentration of 3 nM which produces the
maximal response. Only one concentration of antagonist was
tested in any one ring of artery. Subsequently, the concentra-
noncovalently bound ligands. Remaining AII binding sites
were titrated by cell incubation in the presence of [125I]Sar1-
AII (5 nM, 2 h at 4 °C).
P h otoa ffin ity La belin g, Solu biliza tion , a n d An a lysis
of Non p ep tid e An ta gon ist-Recep tor Cova len t Com -
p lexes. CHO cells expressing the C121A mutant AT1A receptor
were grown to confluence in 23 cm × 23 cm square dishes as
described in the preceding paragraph. Cells were washed twice
with binding medium (PBS, pH 7.4, 5 mM MgCl2, 0.1 mg/mL
bacitracin, 0.6% Me2SO) then incubated for 4 h at 4 °C in the
dark, under gentle agitation, in 30 mL of the same medium
containing the photoactivatable probe 28 (40-60 nM), in the
presence or absence of an excess of either unlabeled Sar1-AII
or 14 (10-5 M).
The incubation medium was discarded, and each dish was
rinsed twice with 30 mL of cold binding medium without
bacitracin. The cells were irradiated at 0 °C for 5 min at 254
nm (five TUV6 Philips lamps), without any added medium.
They were then solubilized with 10 mM phosphate, pH 6.0, 5
mM EDTA, 1 mM PMSF, 0.5 mM NEM, 1.5% Triton × 100
(33 mL/dish) for 1 h at room temperature. The detergent-
treated cells were centrifuged for 1 h at 200000g.
After dissociation of noncovalently bound ligand (1 h, 30 °C)
and EDTA complexation with the appropriate amount of MgCl2
(10 mM final concentration), the solubilized samples were
incubated under gentle agitation with hydroxylapatite-Biogel
P.30 for 90 min at room temperature (3.2 mL gel/dish previ-
ously equilibrated in 10 mM phosphate, pH 6.0, 1 mM PMSF,
0.5 mM NEM, and 0.5% Triton × 100). The gel was then
packed into a column and rinsed thoroughly. Receptor was
eluted with 0.3 M phosphate, pH 6.0, 0.2% SDS, 5 mM EDTA,
1 mM PMSF, and 0.5 mM NEM, and then concentrated and
dialyzed using Amicon Centricon P30 microconcentrators
before SDS-PAGE analysis (four cycles, dilution with 0.1%
SDS between two consecutive cycles).
tion of antagonist reducing the response to AII by 50% (IC50
was determined.
)
CHO Cells. CHO cells expressing the AT1A receptor were
kindly supplied by K. E. Bernstein (Atlanta, GA) and E.
Clauser (Paris, France).
Mem br a n e P r ep a r a tion s. Rat liver membranes were
purified as described previously16 and were stored frozen in
liquid nitrogen before use. Crude membranes from CHO cells
expressing the AT1A receptor were prepared at 4 °C as follows.
Cells were lysed in ice-cold 10 mM Tris‚HCl, pH 7.4, and then
homogenized with a Dounce homogenizer. The supernatant
of a first 300g centrifugation was centrifuged at 48000g for
20 min. The pellet was resuspended in 10 mM Tris‚HCl, pH
7.4, and aliquots of the membrane suspension were frozen in
liquid nitrogen. Protein measurements in membrane prepara-
tions were carried out according to the method of Lowry et
al.52
Bin d in g Assa ys. Liver Mem br a n es. Membranes (50-100
mg/assay, volume 180 mL) were incubated for 30 min at 30
°C in binding medium (50 mM phosphate, pH 7.4, 5 mM
MgCl2, 0.1 mg/mL bacitracin, 0.6% Me2SO) with various
amounts of the tested radioligands. Nonspecific binding was
determined by addition to the assays of an excess (10-5 M final
concentration) of either unlabeled Sar1-AII or unlabeled non-
peptide antagonist). The assays were carried out in triplicate,
in polypropylene tubes to minimize the adsorption of nonpep-
tide ligands. Bound radioactivity was estimated by filtration
through GF/C filters (presoaked in a 1% BSA solution).
Competition binding assays using [125I]Sar1-AII as tracer
ligand assays were carried out with 10 µg membrane protein/
assay in a 90 µL volume. The Ki values of the nonpeptide
antagonists in competition experiments were calculated ac-
cording to Ekins equation.53
Electr op h or esis a n d Au tor a d iogr a p h y. SDS-PAGE of
covalent nonpeptide antagonist-receptor complexes was car-
ried out in reducing conditions on 12.5% acrylamide gels. The
gels were treated with EN3HANCE from Dupont-NEN before
autoradiography using Kodak XAR-5 films.
CHO Cell Mem br a n es. The binding medium was similar
to that used for rat liver membranes (no BSA, 0.1 mg/mL
bacitracin). The membrane amounts were 10 µg/assay (90 µL
volume) and 125 µg/assay (450 µL volume) for the binding of
Ack n ow led gm en t. The authors wish to thank for
their skillfull experimental analytical and chemical
work Anne-Marie Dhennequin, Gilles Martin-Gousset,
Lydia Perrin, and Dominique Viard. The authors also
wish to thank M. Guilhem for preparing the manuscript,
K. Bernstein and E. Clauser for providing us with cells
expressing the wild type AT1A receptor, and S. J ard and
F. Bellamy for helpful discussions. This work was
supported by the Institut National de la Sante´ et de la
Recherche Me´dicale (INSERM), the Centre National de
la Recherche Scientifique (CNRS), Laboratoires Fourn-
ier (Daix, France), the Ministe`re de l′Enseignement
Supe´rieur et de la Recherche, and the Fondation pour
la Recherche Me´dicale.
[
125I]Sar1-AII and 21, respectively.
In ta ct CHO Cells. CHO cells expressing the rat AT1A
receptor (wild type or C121A mutant) were grown in F12
medium supplemented with 10% fetal calf serum (heat-
inactivated), 100 U/mL penicillin, 100 µg/mL streptomycin, and
400 µg/mL Geneticin. Cells were plated in 12-well tissue
culture clusters and grown to confluence (about 5 × 105 cells/
well). After removal of the culture medium, the cells were
washed twice with binding buffer (PBS, pH 7.4, 5 mM MgCl2,
0.1 mg/mL bacitracin, 0.6% Me2SO), then incubated for 4 h at
4 °C under gentle agitation, with 300 µL of binding buffer
containing various radioligand concentrations. The reaction
was stopped by removal of medium followed by two rapid
washings with ice-cold binding buffer.
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