Design, Synthesis, Structural Studies, Biological Evaluation, and Computational Simulations of Novel Potent AT1 Angiotensin II Receptor Antagonists Based on the 4-Phenylquinoline Structure

Article abstract of DOI:10.1021/jm031100t

Novel AT₁ receptor antagonists bearing substituted 4-phenylquinoline moieties instead of the classical biphenyl fragment were designed and synthesized as the first step of an investigation devoted to the development of new antihypertensive agen

Full text of DOI:10.1021/jm031100t

2574
J . Med. Chem. 2004, 47, 2574-2586
Design , Syn th esis, Str u ctu r a l Stu d ies, Biologica l Eva lu a tion , a n d
Com p u ta tion a l Sim u la tion s of Novel P oten t AT1 An gioten sin II Recep tor
An ta gon ists Ba sed on th e 4-P h en ylqu in olin e Str u ctu r e
Andrea Cappelli,*,‡ Gal.la Pericot Mohr,‡ Andrea Gallelli,‡ Milena Rizzo,∇ Maurizio Anzini,‡ Salvatore Vomero,‡
Laura Mennuni,§ Flora Ferrari,§ Francesco Makovec,§ M. Cristina Menziani,† Pier G. De Benedetti,† and
Gianluca Giorgi|
Dipartimento Farmaco Chimico Tecnologico and European Research Centre for Drug Discovery and Development,
Universita` di Siena, Via A. Moro, 53100 Siena, Italy, Dipartimento di Scienze Farmacobiologiche,
Universita` “Magna Græcia” di Catanzaro, Complesso “Ninı` Barbieri”, 88021 Roccelletta di Borgia, Catanzaro, Italy,
Rotta Research Laboratorium S.p.A., Via Valosa di Sopra 7, 20052 Monza, Italy, Dipartimento di Chimica,
Universita` degli Studi di Modena e Reggio Emilia, Via Campi 183, 41100 Modena, Italy, and
Centro Interdipartimentale di Analisi e Determinazioni Strutturali, Universita` di Siena, Via A. Moro, 53100 Siena, Italy
Received November 6, 2003
Novel AT1 receptor antagonists bearing substituted 4-phenylquinoline moieties instead of the
classical biphenyl fragment were designed and synthesized as the first step of an investigation
devoted to the development of new antihypertensive agents and to the understanding of the
molecular basis of their pharmacodynamic and pharmacokinetic properties. The newly
synthesized compounds were tested for their potential ability to displace [125I]Sar1,Ile8-Ang II
specifically bound to AT1 receptor in rat hepatic membranes. These AT1 receptor binding studies
revealed nanomolar affinity in several of the compounds under study. The most potent ligands
4b,t were found to be equipotent with losartan and possessed either a 3-tetrazolylquinoline or
a 2-amino-3-quinolinecarboxylic moiety, respectively. Moreover, some selected compounds were
evaluated for antagonism of Ang II-induced contraction in rabbit aortic strips, and the most
potent compounds in the binding test 4b,t were slightly more potent than losartan in inhibiting
Ang II-induced contraction. Finally, the most relevant structure-affinity relationship data were
rationalized by means of computational studies performed on the isolated ligands as well as
by computational simulations on the ligands complexed with a theoretical AT1 receptor model.
In tr od u ction
proach to modulate the RAS. In humans, two main Ang
II receptor subtypes have been characterized and called
AT1 and AT2.1,3
Angiotensin II (Ang II) is an octapeptide produced by
the renin-angiotensin system (RAS), which plays a key
role in the pathophysiology of hypertension. This vasoac-
tive hormone regulates the cardiovascular homeostasis
by acting on both the vascular resistance and the blood
volume. Ang II is produced in vivo from angiotensin I
by the angiotensin converting enzyme (ACE).1 ACE in-
hibitors such as Enalapril and Captopril are largely
used in clinic in the treatment of hypertension, and the
commercial success of these drugs demonstrates the
modulation of RAS to represent a very interesting ap-
proach to the control of hypertension. However, ACE is
not a very selective enzyme, as it possess among its sub-
strates important peptides such as bradykinin and sub-
stance P. In fact, the pharmacological properties of ACE
inhibitors, but also some of the major side effects associ-
ated with their clinical use (such as dry cough, angio-
edema, and rashes), may be related to bradykinin poten-
tiation.2 Thus, the specific block of Ang II actions at the
receptor level represents a potentially convenient ap-
The AT1 receptor subtype mediated virtually all the
known physiological actions of Ang II in cardiovascular,
neuronal, endocrine, hepatic, and in other cells. This
receptor belongs to the G protein-coupled receptor
(GPCR) superfamily and shows the seven hydrophobic
transmembrane domains forming R-helices in the lipid
bilayer of the cell membrane. The interaction of Ang II
with AT1 receptor induces a conformational change,
which promotes the coupling with the G protein(s) and
leads to the signal transduction via several effector
systems (phospholipases C, D, A2, adenyl cyclase, etc.).1,4
The parallel discovery of losartan and eprosartan,
potent and orally active nonpeptide Ang II antagonists,
has stimulated the design of a large number of conge-
ners.5 Among them, irbesartan, candesartan, valsartan,
telmisartan, and olmesartan are on the market and
about 20 other compounds are being developed. Most
of these compounds have the biphenyl fragment bearing
an acidic moiety (tetrazole ring, COOH, SO2NHCO) in
common and differ in the nature of the pendent hetero-
cyclic system (valsartan lacks the heterocyclic moiety)
connected to the para position of the distal phenyl by
means of a methylene group. In fact, in the design of
new nonpeptide Ang II antagonists, the strategy fol-
lowed by most medicinal chemists concerned the mo-
lecular modification of the imidazole moiety of losartan
(1) (Chart 1). Among the large variety of the heterocyclic
* To whom correspondence should be addressed. Tel: +39 577
234320. Fax: +39 577 234333. E-mail: cappelli@unisi.it.
‡ Dipartimento Farmaco Chimico Tecnologico European Research
Centre for Drug Discovery and Development, Universita` degli Studi
di Siena.
∇ Dipartimento di Scienze Farmacobiologiche, Universita` “Magna
Græcia” di Catanzaro.
§ Rotta Research Laboratorium S.p.A.
† Dipartimento di Chimica, Universita` degli Studi di Modena e
Reggio Emilia.
| Centro Interdipartimentale di Analisi e Determinazioni Strutturali,
Universita` di Siena.
10.1021/jm031100t CCC: $27.50 © 2004 American Chemical Society
Published on Web 04/10/2004

AT1 Angiotensin II Receptor Antagonists
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10 2575
Ch a r t 1
Ch a r t 3
Sch em e 1a
Ch a r t 2
a
Reagents: (a) NBS, dibenzoyl peroxide, CCl4; (b) 2-substituted-
5,7-dimethyl-3H-imidazo[4,5-b]pyridine, NaH, DMF; (c) HCOOH
(for tetrazole derivatives 4b ,l,o) or NaOH (for carboxylates
4a ,c,d ,k ,m ,p ,q,v).
systems developed, an outstanding position is occupied
by the imidazo[4,5-b]pyridine moiety of compound 2a
(L-158,809).6 This congener of losartan has been re-
ported to show a subnanomolar AT1 receptor affinity
about 2 orders of magnitude higher than that of losartan
and represents one of the most potent nonpeptide Ang
II antagonists developed. This suggests that the stereo-
electronic characteristics of the imidazo[4,5-b]pyridine
moiety can be considered optimal for the interaction
with the receptor. On the other hand, relatively little
information is available on the effects of the molecular
modification of the phenyl group bearing the acidic
moiety (distal phenyl ring). While the design was being
performed, our attention was captured by the report
that the introduction of a nitrogen atom in the distal
phenyl of compound 3a (SC-50560) had detrimental
effects on the AT1 receptor affinity independent of the
position occupied by the nitrogen atom (see Chart 2),
while negligible effects were seen when this kind of
modification involved the proximal phenyl group.7 On
the other hand, similar molecular modifications per-
formed on imidazo[4,5-b]pyridine derivative 2c
(L-158,338) gave slightly different results.8 For example,
the introduction of the nitrogen atom in para-position
(with respect to the proximal phenyl ring) of 3a distal
phenyl led to a 45-fold affinity decrease, while the same
modification performed on imidazo[4,5-b]pyridine de-
rivative 2c had less dramatic effects (only a 4-fold loss
in AT1 binding affinity). Moreover, 4-phenyl-3-tetra-
zolylpyridyl derivative 2f was reported to be an orally
active Ang II antagonist showing a somewhat poorer
oral bioavailability than 2c. The authors suggested that
the decrease in the lipophilicity had a negative effect
on the oral potency of compound 2f with respect to 2c.8
Taken together, these observations revealed the com-
plex nature of the ligand-receptor interaction stimulat-
ing the design of some molecular modifications involving
distal phenyl group of compounds 2 which led to the
development of compounds 4 (Chart 3). In this paper
we describe the synthesis, the structural and the
pharmacological characterization, the structure-affinity
relationships (SAFIR), and the ligand-receptor interac-
tion simulations of the novel Ang II antagonists 4 as
the first step of an investigation devoted to both the
development of new antihypertensive agents and the
understanding of the molecular basis of their pharma-
codynamic and pharmacokinetic9 properties.
Ch em istr y
The preparations of the target compounds 4a -d ,
k -m ,o-q,v were carried out in three steps starting
from the suitable toluene derivatives by benzylic bro-
mination, coupling reaction with substituted imidazo-
[4,5-b]pyridines,6,10 and unmasking of the acidic (car-
boxylic or tetrazole) moiety (Scheme 1).

2576 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10
Cappelli et al.
Sch em e 2a
Sch em e 5a
a
Reagents: (a)CNCH2CH(OCH3)2,PTSA,toluene;(b)(CH3)3SnN3,
xylene, (c) Ph3CCl, NaOH, THF, CH2Cl2.
Sch em e 6a
a
Reagents: (a) NaOH, dioxane; (b) (CH3)4Sn, Pd(PPh3)2Cl2,
DMF; (c) NaOH, C2H5OH; (d) NaN3, DMF; (e) (4e,t,w , R2
NH2): C2H5OCOCH2NH2HCl, C5H5N, then NaOH, C2H5OH; (4f,h ,
)
R2 ) NHR): RNH2, C2H5OH, then NaOH, C2H5OH; (4g,i, R2
)
NRR): RRNH, C2H5OH (or DMF), then NaOH, HOCH2CH2OH.
Sch em e 3a
a
Reagents: (a) EDCI, DMAP, p-NO2C6H4SO2NH2, CH2Cl2.
Sch em e 4a
a
Reagents: (a) C2H5OCOCH2COCl, CH2Cl2; (b) NaH, C2H5OH;
(c) POCl3; (d) CH3ONa, CH3OH; (e) H2, Pd/C, (C2H5)3N, C2H5OH;
(f) CH3I, KOH, (C4H9)4NBr, THF, DMF; (g) NaOH, C2H5OH, (h)
SOCl2; (i) NH3, CH2Cl2; (l) POCl3, C6H6; (m) (CH3)3SnN3, xylene;
(n) Ph3CCl, NaOH, THF, CH2Cl2.
a
Reagents: (a) p-CH3C6H4B(OH)2, Na2CO3, LiCl, Pd/C, DMF.
Other target 3-quinolinecarboxylic acids showing dif-
ferent substituents in position 2 of the quinoline nucleus
(compounds 4e-i,r -t,w ) were synthesized by means of
the suitable elaboration of the intermediate iminochlo-
rides 6q,x,y. The chlorine atom in position 2 of the
quinoline nucleus of these compounds was easily dis-
placed by a variety of nucleophilic reagents (Scheme 2)
such as NaOH, amines, and sodium azide. In particular,
the reaction of 6q with NaOH in dioxane led to the
contemporary hydrolysis of both the ester and the
iminochloride function (compound 4s), while the reac-
tion of 6x with sodium azide gave, after hydrolysis, the
tetrazoloquinoline derivative 4n . It is noteworthy that
the reaction of iminochlorides 6q,x,y with glycine ethyl
ester hydrochloride in pyridine gave the corresponding
2-aminoquinoline derivatives, which were hydrolyzed
into the amino acid derivatives 4e,t,w . Stille cross-
coupling11 of 6q with tetramethylstannane gave, after
hydrolysis, the expected 2-methylquinoline derivative
4r . The acylsulfonamide 4u was synthesized by reaction
of the carboxylic acid 4p with p-nitrobenzensulfonamide

AT1 Angiotensin II Receptor Antagonists
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10 2577
Sch em e 7a
Sch em e 8a
a
a
Reagents: (a) C2H5OCOCH2CH2COCl, CH2Cl2; (b) NaH,
Reagents: (a) p-CH3C6H4B(OH)2, K2CO3, Pd(PPh3)2Cl2, DMF;
C2H5OH, (c) C2H5OH, POCl3; (d) POCl3; (e) NBS, dibenzoyl
peroxide, CCl4; (f) 5,7-dimethyl-2-ethyl-3H-imidazo[4,5-b]pyridine,
NaH, DMF; (h) H2, Pd/C, (C2H5)3N, C2H5OH, (i) NaOH, C2H5OH;
(g) NaOH, THF.
(b) (CH3)3SnN3, xylene; (c) Ph3CCl, NaOH, THF, CH2Cl2; (d) NBS,
dibenzoyl peroxide, CCl4; (e) 5,7-dimethyl-2-ethyl-3H-imidazo[4,5-
b]pyridine, NaH, DMF; (f) HCOOH; (g) NaOH, HOCH2CH2OH.
role in lowering the basicity of the quinoline nitrogen
atom of 5k and prevented the self-condensation of the
corresponding bromide.
Finally, reference compounds 2a ,b,h -j were synthe-
sized in our laboratories following (or modifying, see
Scheme 8) the procedures described by Mantlo and co-
workers.6,8,10,16
in the presence of EDCI and DMAP (Scheme 3). The
intermediates 5a -d ,k -m ,q required for the synthesis
shown in Scheme 1 were prepared by means of either
the classical biaryl chemistry or the 4-phenylquinoline
chemistry. For example, naphthalene derivative 5a was
prepared by aryl coupling with heterogeneous palladium
catalyst12 as depicted in Scheme 4, while 3-tetra-
zolylquinoline intermediate 5b was obtained by means
of a three-step procedure involving the acid-catalyzed
Friedla¨nder condensation of the aminobenzophenone 813
with 3,3-dimethoxypropionitrile14 followed by the clas-
sical tetrazole chemistry (Scheme 5), and quinoline
intermediates 5c,d ,l,m ,q were synthesized by means of
the standard procedures shown in Scheme 6.15
3-Quinolineacetic acid derivatives 4j,k were obtained
(Scheme 7) from the common intermediate 6k possess-
ing a chlorine atom in position 2 of the quinoline
nucleus. This chlorine was demonstrated to be neces-
sary, from the point of view of the synthesis, because
the attempts at applying the bromination-coupling
procedure (successfully employed in the case of 3-quino-
linecarboxylate 5c) to the higher homologue ethyl 4-(4-
methylphenyl)-3-quinolineacetate were unsuccessful.
The difference between these two compounds consisted
of the attachment of the carboxylic group to the quino-
line nucleus and, consequently, the presumably higher
electron-withdrawing effect of the carboxylic ester con-
jugated to the heteroaromatic ring. Probably, the chlo-
rine in position 2 of the quinoline nucleus played a key
Str u ctu r a l Stu d ies
X-ray crystallographic studies were carried out on
compounds 4d , 6n , and 6q (Table 1, Figures 1-3). While
the asymmetric units of 6n and 6q are formed by one
molecule, in the case of 4d they consist of two molecules
showing different conformational parameters. In all the
compounds, the heterocyclic systems are planar. The
largest deviation from planarity is shown by the tetra-
zoloquinoline system in 6n where C(6) and C(9) show
the largest deviations (0.077(5) and 0.075(5)Å, respec-
tively, from two opposite sides) from the least squares
plane defined by the 13 atoms. The CdO group at
position 3 of the quinoline nucleus of 6q shows a
perpendicular orientation with respect to the plane of
the quinoline, as in the case of the two independent
molecules of 4d , while in compound 6n it is almost
coplanar with the quinoline (Table 2). To minimize steric
hindrance, in each compound the three rings are nearly
perpendicular to one another (Table 2). The phenyl ring
bridging the two heterocyclic systems forms dihedral
angles with the quinoline moiety in the range
65.93(12)° (6n ) ÷ 84.85(7)° (4d ).

2578 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10
Cappelli et al.
Ta ble 1. Crystal Data for Compounds 4d and 6n ,q
4d
6n
6q
formula
C
28H26N4O3‚0.5H2O‚
C29H27N7O2
C30H29ClN4O2
0.5C2H5OH
498.57
triclinic
P-1 (no. 2)
13.641(4)
14.136(1)
14.302(1)
72.61(1)
83.37(1)
88.93(1)
2613.8(8)
293(2)
MW
505.58
triclinic
P-1 (no. 2)
8.515(3)
9.849(2)
15.826(4)
95.45(1)
99.24(2)
104.56(3)
1255.2(6)
293(2)
2
513.02
triclinic
P-1 (no. 2)
9.311(1)
9.917(1)
15.653(1)
104.30(1)
105.96(1)
97.04(1)
1318.1(2)
293(2)
2
crystal system
space group
a/Å
b/Å
c/Å
R/°
â/°
γ/°
U/Å3
temperature/K
Z
4
F(000)
1056
532
540
Dc/g cm-3
µ(Mo-KR)/mm-1
scan mode
scan range/°
scan width/°
scan speed/
deg min-1
independent
reflections
obsd reflections
(I > 2σ(I))
no. parameters
refined
1.267
0.086
ω/2θ
1 e θ e 25
1.26
1.338
0.088
ω/2θ
1 e θ e 25
1.00
1.293
0.180
ω/2θ
1 e θ e 25
0.92
3
3
3
6757
5255
712
4409
1943
373
4569
3715
355
R1 (I > 2σ(I))
wR2 (I > 2σ(I))
0.046
0.112
0.070
0.133
0.044
0.113
F igu r e 1. Crystal structure of 4d . Ellipsoids enclose 50% of
probability. The OH group of the ethanol having site occupa-
tion factor of 0.62(1) is reported.
F igu r e 2. Crystal structure of 6n . Ellipsoids enclose 50% of
probability. The atom O(26) and the OEt group having site
occupation factor of 0.60(1) are reported.
Interestingly, the analysis of the crystal packing of
the carboxylic acid derivative 4d shows the presence of
a complex hydrogen bonding network. This is due to the
simultaneous presence of heterocyclic nitrogen atoms
and of one COOH group in the structure of 4d , and of
one molecule of water and one of ethanol in the
asymmetric unit. In particular, the cocrystallized water
molecule [OW1 (x, y, z)] establishes hydrogen bonds with
both the lutidine nitrogen atom of the same asymmetric
unit [N(19A) (x, y, z)] and the carbonyl oxygen atom of
4d belonging to another asymmetric unit [O(26) (1 - x,
-y, 1 - z)]. Another hydrogen bond interaction is
present between the COOH group [O(27) (x, y, z)] and
the imidazole nitrogen atom N(14A) (x, -1 + y, z), while
O(27A) interacts with (N14). The OH group [OEt (x, y,
z)] of the ethanol forms a hydrogen bond with quinoline
nitrogen N(1A) (x, y, -1 + z). The distances (H)O‚‚‚N
are in the range 2.59 ÷ 3.16Å (Figure 1). Thus, these
results show that all the heterocyclic (imidazole, luti-
dine, and quinoline) nitrogen atoms possessing one
unshared lone pair behave as hydrogen bonding accep-
tors in the crystal.
Str u ctu r e-Affin ity Rela tion sh ip Stu d ies
The newly synthesized bicyclic derivatives 4 (showing
a suitable degree of purity as confirmed by 1H NMR and
combustion analyses) were tested for their potential
ability to displace [125I]Sar1,Ile8-Ang II specifically
bound to AT1 receptor in rat hepatic membranes, in
comparison with reference monocyclic compounds
2a ,b ,h -j, losartan, and valsartan, following well-
established protocols.17 The results of the binding stud-

AT1 Angiotensin II Receptor Antagonists
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10 2579
in the receptor affinity of about 1 order of magnitude
(this effect was comparable with that reported in the
literature for losartan19), in the series of bicyclic deriva-
tives 4 the carboxyl and tetrazole groups appeared to
be completely bioisosteric acidic moieties.
The transformation of the carboxyl group of 4p into
an acylsulfonamide group of compound 4u led to an
affinity decrease of 1 order of magnitude, in disagree-
ment with that described in the literature concerning
some monocyclic derivatives.19 Similarly, the insertion
of a methylene spacer between the acidic moiety and
the quinoline nucleus, as in quinoline-3-acetic acid
derivative 4j, decreased the affinity by more than 1
order of magnitude.
(c) Effects of th e In tr od u ction of a Su bstitu en t
in th e Or th o-P osition w ith Resp ect to th e Acid ic
Moiety. The introduction of a substituent in the ortho-
position with respect to the acidic moiety produced
different effects which appeared to be dependent on the
stereoelectronic characteristics of the substituent (and
on the potential interaction with the acidic moiety).
Replacement of the hydrogen atom with NH2 group
appeared to be well tolerated by the receptor (compare
4c,p ,v with 4e,t,w , respectively), while the increase in
steric bulk in the series of alkyl-substituted amino
derivatives 4e-i led to a progressive decrease in the
receptor affinity. Other small substituents possessing
different electronic characteristics such as a methyl
group and a chlorine atom appeared to have only slight
effects (compare 4p with 4r ,q), while the introduction
of a hydroxy group dramatically decreased the receptor
affinity (compare 4p vs 4s). The low affinity shown by
hydroxy derivative 4s is not surprising provided its
probable prototropic side chain tautomerism to the oxo-
form20 and the similarly low affinity shown by 1-methyl-
2-quinolinone derivative 4m are taken into account
together. Interestingly, the transformation of the quino-
line nucleus of our compounds into 1-methyl-2-quinoli-
none is more negative in the case of tetrazole derivative
4l (compare 4l vs 4b) than in the case of carboxylic acid
derivative 4m (compare 4m vs 4c). Finally, the intro-
duction of either a methoxy group (compound 4d ) or of
a condensed tetrazole ring (4n ) led to a slight (but
significant) decrease in the receptor affinity.
F igu r e 3. Crystal structure of 6q. Ellipsoids enclose 50% of
probability.
ies summarized in Table 3 show that most of the tested
compounds displayed high affinity for AT1 receptor. The
most potent compounds 4b,q,r ,t can be considered
equipotent with losartan.
(a ) Effects of th e Mod ifica tion of th e Dista l
P h en yl Rin g. The introduction of an additional ben-
zene ring in the structure of carboxylic acid derivative
2i leading to naphthalene derivative 4a 18 appeared to
be tolerated by the receptor. Moreover, the transforma-
tion of the naphthalene moiety of carboxylic derivative
4a into the quinoline one of 4c seemed to have slightly
positive effects, as the AT1 receptor affinity of 4c was
twice as higher as that of its carbaisostere 4a .
The latter result appeared to be in disagreement with
those described by Mantlo et al.8 for compounds 2 and
with those obtained by Reitz7 and co-workers with
compounds 3. These authors showed that the replace-
ment of CH to N in the distal phenyl of compounds 2c
or 3a produced more or less pronounced decreases in
the AT1 receptor affinity (see Chart 2). It is noteworthy
that the comparison of the affinities of the monocyclic
derivatives 2a ,h -j, which we used as reference com-
pounds, confirmed the trend described in the litera-
ture.7,8
(d ) Effects of th e Mod ifica tion of th e Lip op h ilic
Su bstitu en t in P osition 2 of th e Im id a zo[4,5-b]-
p yr id in e Nu cleu s. The work performed on losartan
congeners has shown the importance of a short alkyl
chain at position 2 of imidazole, fused imidazole, or
(b ) E ffect s of t h e Mod ifica t ion of t h e Acid ic
Moiety. While in the series of the reference monocyclic
compounds 2a ,h -j the replacement of the tetrazole
moiety of compounds 2a and 2h with a carboxyl group
(leading to 2i and 2j, respectively) produced a decrease
Ta ble 2. Conformational Parameters for 6n ,q and 4d
4d
torsion angle (deg)
Mol 1
Mol 2
6q
6n
C(2)-C(3)-CdO
92.2(3)
-110.0(3)
80.0(3)
96.4(3)
-82.6(3)
-67.1(3)
-179.0(2)
84.4(3)
-77.6(2)
-60.0(2)
-175.9(2)
147(1)
-66.9(6)
178.7(4)
179.0(4)
C(3)-C(4)-C(1′)-C(2′)
C(3′)-C(4′)-C(11)-N(12)
N(12)-C(13)-CH2-C
-178.5(3)
4d
dihedral angle between
planes (deg)
Mol 1
Mol 2
6q
6n
1, 2
1, 3
2, 3
69.42(7)
85.79(4)
63.17(7)
84.85(7)
75.31(5)
74.59(6)
76.92(5)
75.88(4)
80.57(4)
65.93(12)
66.37(9)
82.64(11)
a
Least squares planes defined as 1: N1-C10 (10 atoms); 2: C1′-C6′ (six atoms); 3: N12-C20 (nine atoms).

2580 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10
Cappelli et al.
Ta ble 3. AT1 Receptor Binding Affinities and Inhibition of Ang II-Induced Contraction in Rabbit Aortic Strips of Compounds 2 and 4
rabbit aortic strips IC50 (nM)
compd
X
R1
R2
A
binding IC50 (nM) ( SEMa
60 min
120 minb
2a
2b
2h
2i
2j
4a
CH
CH
N
CH
N
CH
N
N
N
N
N
C2H5
n-C3H7
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
CN4H
CN4H
CN4H
COOH
COOH
COOH
CN4H
COOH
COOH
COOH
COOH
COOH
COOH
0.4 ( 0.1
0.8 ( 0.01
18 ( 2.9
10 ( 2.2
819 ( 151
33 ( 4.7
6.9 ( 1.3
17 ( 1.8
74 ( 6.4
13 ( 1.2
13 ( 1.9
29 ( 4.4
66 ( 4.1
492 ( 85
576 ( 104
215 ( 40
> 300
326 ( 36
52 ( 6.4
12 ( 2.1
11 ( 1.8
7.7 ( 1.0
9.0 ( 1.6
261 ( 65
4.2 ( 0.34
106 ( 19
41 ( 7.1
13 ( 1.5
0.4 ( 0.1
6.7 ( 0.5
1.9 ( 0.2
H
H
H
OCH3
NH2
NHCH3
N(CH3)2
NHn-C3H7
4b (CR3210)
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4.0
2.0
N
N
N
N
N(n-C3H7)2 COOH
H
Cl
dO
dO
CH2COOH
CH2COOH
CN4H
N
NCH3 C2H5
NCH3 C2H5
650
170
COOH
N
N
N
N
N
N
N
N
N
n-C3H7
n-C3H7
n-C3H7 Cl
n-C3H7 CH3
n-C3H7 OH
n-C3H7 NH2
n-C3H7
n-C4H9
H
H
CN4H
COOH
COOH
COOH
COOH
COOH
CONHSO2C6H4(p)NO2
COOH
COOH
4r
4s
4t
4u
13
3.3
H
H
4v
4w
Ang II
losartan
valsartan
n-C4H9 NH2
9.9
a
Each value is the mean ( SEM of three determinations and represents the concentration giving half the maximum inhibition of
b
[
125I]Sar1,Ile8-Ang II specific binding to rat hepatic membranes. The antagonism of Ang II-contracted rabbit aorta rings was assayed by
using different time of contact of the tested compound.
equivalent moieties for the binding efficiency.21 In the
class of imidazopyridine derivatives 2, ethyl and n-
propyl groups have been described to be fully equivalent
from the point of view of the receptor affinity.6a Accord-
ingly, ethyl derivative 2a showed in our test system a
2-fold higher affinity with respect to propyl derivative
2b. A similar SAFIR trend was found for the couple of
bicyclic tetrazole derivatives 4b and 4o, while a small
deviation from this trend was observed in the case of
the short series of carboxylic derivatives 4c,p ,v and
amino derivatives 4e,t,w . Indeed, in both series the
optimal lipophilic substituent appeared to be the n-
propyl group.
establish charge-reinforced hydrogen bond interactions
with a basic (protonated) residue of the receptor and
dispersive interactions with suitable receptor aromatic
residues, respectively. The use of this descriptor fur-
nished a semiquantitative rationalization of the struc-
ture-affinity relationship data. In fact, both the re-
placement of the tetrazole moiety with the carboxyl
group and the introduction of a nitrogen atom in the
monocyclic ligands 2a ,h -j (Figure 4, top) produce a
significant reduction of the superdelocalizability char-
acterizing the acidic moiety. This effect is less marked
for the ligands carrying the quinoline framework.
No significant variations were observed for the elec-
trophilic superdelocalizability calculated on the aromatic/
heteroaromatic rings (moiety b and c, in Figure 4). In
fact, the localization of the electronic cloud on the
highest occupied molecular orbital (HOMO) of 2h ,j is
very similar to that of their analogues 2a and 2i,
respectively, and the great difference in the value of this
index is shown by compounds 2h and 2j. It is worth
Com p u ta tion a l Stu d ies on th e Isola ted Liga n d s
The results of the computational analysis carried out
on selected isolated anionic ligands are summarized in
Figure 4. The electrophilic superdelocalizability22 on the
acidic moieties and on the adjacent aromatic rings has
been chosen as a descriptor of the ligand propensity to

AT1 Angiotensin II Receptor Antagonists
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10 2581
F igu r e 4. Electrophilic Superdelocalizability on the acidic moiety (a : SH(a)), on the adjacent aromatic/heteroaromatic rings (b:
SH(b), and c: SH(c)), and on the overall antagonist molecule where the highest occupied molecular orbital (HOMO) is localized
(SH(t)). Binding affinities data values (IC50) are reported in green. The value of the dihedral angle (φ) which regulates the mutual
position of the acidic moiety and the aromatic/heteroaromatic ring is reported, and the most significant energetic conformers of
the ligands are shown.
noting that this molecular index is relatively indepen-
dent of molecular conformation, at least in the confor-
mational range considered to be accessible by the
ligands when interacting with the receptor. Indeed, the
lowest energy conformation of compounds 2i and 4c
shows the carboxylic anionic moiety perpendicular to
the aromatic/heteroaromatic rings (φ ) 90°). Cheap
energetic costs have to be spent by these ligands (2.9
and 0.67 kcal/mol, respectively) to achieve a local
minimum in which the carboxylic moiety tends to be
more coplanar with the aromatic/heteroaromatic rings
and to show higher similarity with the tetrazole con-
formational preference (see Figure 4). A different be-
havior is observed when the energy necessary to force
the acidic moiety in a planar conformation is higher, as
shown at the bottom of Figure 4 for the φ ) 0° conformer
of ligand 4c.
An additional important observation suggested from
the analysis of the electrophilic superdelocalizability is
that the frontier orbital electronic cloud of the neutral
compound shifts from the carboxylic moiety to the
imidazo[4,5-b]pyridine nucleus, as shown at the bottom
of Figure 5. This supports the hypothesis of a two-step
binding mechanism: the first step is controlled by a
long-range electrostatic interaction for both agonists and
antagonists; in the second step short-range intermo-
lecular interactions established by the imidazo[4,5-b]-
pyridine moiety dictated the functional profile of the
ligands, as suggested by single point mutagenesis
studies on the receptor.23
Com p u ta tion a l Stu d ies on th e Liga n d -Recep tor
Com p lexes
The three-dimensional receptor model constructed at
the beginning of this study was based on both the
bacteriorhodopsin structure24 and on the helical wheel
projection model proposed by Baldwin.25 Although ap-
proximate, the model was instrumental in guiding the
synthesis of new ligands designed to explore the mo-
lecular basis of receptor interaction.
The model we present here has been updated accord-
ing to the recently determined 2.8 Å X-ray structure of
rhodopsin.26 The residues considered to form the surface
of the binding site for the antagonists 2 and 4 are L70
(Helix 2), S105, V108, S109, N111, L112, Y113, S115,
V116 (Helix 3), A159, S160, A163 (Helix 4), Y170, V179,
A181 (II Extracellular loop), L195, K199, G203, F204,
F208 (Helix 5), F249, S252, W253, V254, P 255, H256,

2582 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10
Cappelli et al.
F igu r e 5. Minimized average structure of the 4c-AT1 receptor complex (top left), detailed view of the antagonist binding site
(top right), and modification of the HOMO orbital upon neutralization of the anionic 4c inhibitor (bottom).
T260 (Helix 6), A291, F 293, N294, N295, L297 (Helix
7). The residues reported in bold capitals have been
demonstrated (by means of site-directed mutagenesis
studies) to be important for binding,23 and some of these
establish common key interactions with peptide agonists
and nonpeptide antagonits.27
induced contraction in rabbit aortic strips.17,28 The
results (Table 3) show that all the tested compounds
behaved as Ang II antagonists in this functional model
showing IC50 values well related to their binding affini-
ties. The most potent compounds in the binding test 4b,t
were highly potent in inhibiting Ang II-induced contrac-
tion. Indeed, 2-amino-3-quinolinecarboxylic acid deriva-
tive 4t and tetrazole derivative 4b were slightly more
active than losartan. Moreover, the potency of compound
4b was enhanced when the contact with the antagonist
was increased from 60 to 120 min.
The essential shape and physicochemical character-
istics of the receptor cavity assumed to accommodate
ligands 2 and 4 is shown in Figure 5 (top right). The
back- and bottom-walls are constituted by hydrophobic
residues, while polar residues are situated at the top of
the cavity, where the basic K199 residue lies, and
spotted the front wall at a depth from K199 of about
8-10 Å which is perfectly congruent with the distance
between the antagonist acidic moiety and the imidazo-
[4,5-b]pyridine nucleus. Therefore, in a dynamic view,
the hydrogen bonding acceptor propensity of the an-
tagonist pendant heterocyclic moiety could be satisfied
alternatively by the N111, or N294, or S252 residues,
once the main ion-pair interaction has been achieved.
The energy minimized average structure of the 4c-AT1
receptor complex is shown in Figure 5.
Discu ssion
Among the AT1 receptor antagonists bearing a nitro-
gen atom in the biphenyl skeleton, compound 2f repre-
sented an interesting starting point for the design of
new antihypertensive agents. It was reported both to
show an affinity just four times lower than its carbai-
sostere 2c and to be an orally active Ang II antagonist
showing a somewhat poorer oral bioavailability (prob-
ably for the decreased lipophilicity) than that of 2c.8
This observation, together with the complex effects of
the introduction of a nitrogen atom in the biphenyl
moiety (see the Introduction), led to the design of
compound 4b and its derivatives with the aim of both
developing new antihypertensive agents and under-
F u n ction a l Stu d ies
Some selected compounds showing different binding
affinities were evaluated for antagonism of Ang II-

AT1 Angiotensin II Receptor Antagonists
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10 2583
standing the molecular basis of their pharmacodynamic
and pharmacokinetic properties. We believed that the
introduction of the fused benzene ring should ensure
the lipophilicity necessary for a good oral bioavailability,
and the SAFIR study involving compounds 4 should
provide information on the ligand-AT1 receptor inter-
action.
preliminary recognition step and is shared by agonists
and antagonists. The second step differentiates the
functional activity of ligands. In the case of the antago-
nists presented here, short-range intermolecular inter-
actions of the imidazopyridine moiety with residues
N111, or N294, or S252 (according to the molecular
framework of the ligand) perturb the hydrogen bonding
network which allows the communication among helices
and therefore receptor activation and signal transduc-
tion.
Very interestingly, quinoline derivative 4b (CR3210)
showed AT1 receptor affinity in the low nanomolar range
very similar both to that described for pyridine deriva-
tive 2f and to that shown by losartan. Moreover, 4b was
slightly more active than losartan in inhibiting Ang II-
induced contraction in rabbit aortic ring, and therefore
it was considered to be an interesting candidate for
further preclinical studies. Thus, the pharmacokinetic
properties of 4b and some selected members of this class
of AT1 antagonists are under evaluation in a compre-
hensive study devoted to the understanding of their
structure-pharmacokinetics relationships.
Exp er im en ta l Section
Melting points were determined in open capillaries on a
Gallenkamp apparatus and are uncorrected. Microanalyses
were carried out using a Perkin-Elmer 240C elemental ana-
lyzer. Merck silica gel 60 (70-230 or 230-400 mesh) was used
for column chromatography. Merck TLC plates, silica gel 60
1
F
254, were used for TLC. H NMR spectra were recorded with
a Bruker AC 200 spectrometer in the indicated solvents (TMS
as internal standard): the values of the chemical shifts are
expressed in ppm and the coupling constants (J ) in Hz. Mass
spectra (EI, 70 eV) were recorded either on a VG 70-250S
spectrometer (Centro di Analisi e Determinazioni Strutturali,
Universita` di Siena) or Varian Saturn 3 (Dipartimento Far-
maco Chimico Tecnologico, Universita` di Siena). NMR spectra
and elemental analyses were performed in the Dipartimento
Farmaco Chimico Tecnologico, Universita` di Siena.
P r ep a r a tion of Ta r get Tetr a zole Der iva tives 4b,l,o
(Dep r otection of th e Tr ityl-P r otected Tetr a zole Der iva -
tives). A mixture of the appropriate trityl-protected tetrazole
derivative (1.0 mmol) with formic acid (15 mL) was stirred at
room temperature under argon for a suitable time (2-44 h),
and the reaction progress was monitored by TLC. When the
trityl-protected tetrazole derivative disappeared from the
chromatogram, the reaction mixture was evaporated under
reduced pressure. Purification of the residue by washing with
diethyl ether gave the target compounds showing a suitable
degree of purity as confirmed by 1H NMR and combustion
analyses.
The SAFIR analysis performed on our compounds
revealed that the introduction of a nitrogen atom in the
naphthalene derivative 4a to give quinolinecarboxylic
acid 4c is well tolerated by the receptor. The presence
of the additional condensed benzene ring of compounds
4a -w has little consequences on the receptor affinity
(compare 4a with 2i), but it appears to produce some
peculiarities in key SAFIR trends. For example, in the
series of bicyclic derivatives 4, the carboxylic and
tetrazole groups appeared to be completely bio-equiva-
lent acidic moieties, while the monocyclic tetrazole
derivatives 2a and 2h were found more than 1 order of
magnitude more potent than corresponding monocyclic
carboxylate derivatives 2i and 2j, respectively (in agree-
ment with the results described for losartan). These
results can be rationalized by considering the electro-
philic superdelocalizability computed over the antago-
nist moiety where the HOMO orbital is localized. In fact,
the reactivity potential of the different acidic moieties
in the series of ligands 4 is almost identical, while the
carboxylic anionic head is significantly less reactive than
the tetrazole moiety in compounds 2 (Figure 4). From a
qualitative point of view, the shape of the electronic
cloud distribution on the highest occupied molecular
orbital does not show significant differences among the
compounds (Figure 4); however, the value of the reactiv-
ity index gives a rough but useful classification of the
ligands with respect to their binding activities: sub-
nanomolar ligands possess a total electrophilic super-
delocalizability (SH(t)) of about 0.43. For nanomolar
ligands, this index ranges in the interval 0.36-0.40;
finally, micromolar ligands show a SH(t) value of about
0.32.
4-[4-[(5,7-Dim eth yl-2-eth yl-3H-im id a zo[4,5-b]p yr id in -
3-yl)m et h yl]p h en yl]-3-(2H -t et r a zol-5-yl)q u in olin e (4b ).
The title compound was prepared from 6b to obtain a white
crystalline solid (0.36 g, 78%) melting at 257-259 °C. 1H NMR
(CDCl3): 1.20 (t, J ) 7.5, 3H), 2.56 (s, 3H), 2.61 (s, 3H), 2.82
(q, J ) 7.5, 2H), 5.57 (s, 2H), 6.93 (s, 1H), 7.25 (m, 4H), 7.52
(m, 2H), 7.79 (m, 1H), 8.22 (d, J ) 8.3, 1H), 9.53 (s, 1H). Anal.
(C27H24N8‚0.5H2O) C, H, N.
4-[4-[(5,7-Dim eth yl-2-eth yl-3H-im id a zo[4,5-b]p yr id in -
3-yl)m eth yl]p h en yl]-1-m eth yl-3-(2H-tetr a zol-5-yl)- 2(1H)-
qu in olin on e (4l). The title compound was prepared from 6l
to obtain a white crystalline solid (0.34 g, 69%) melting at 270-
1
272 °C. H NMR (CDCl3): 1.38 (t, J ) 7.6, 3H), 2.61 (s, 3H),
2.63 (s, 3H), 2.90 (q, J ) 7.6, 2H), 3.93 (s, 3H), 5.59 (s, 2H),
6.89 (s, 1H), 7.22 (m, 6H), 7.52 (d, J ) 8.4, 1H), 7.68 (m, 1H).
Anal. (C28H26N8O‚0.67H2O) C, H, N.
4-[4-[(5,7-Dim eth yl-2-p r op yl-3H-im id a zo[4,5-b]p yr id in -
3-yl)m et h yl]p h en yl]-3-(2H -t et r a zol-5-yl)q u in olin e (4o).
The title compound was prepared from 6o to obtain a white
crystalline solid (0.37 g, 78%, mp 236-238 °C). 1H NMR
(CDCl3): 0.89 (t, J ) 7.5, 3H), 1.65 (m, 2H), 2.57 (s, 3H), 2.61
(s, 3H), 2.80 (t, J ) 7.4, 2H), 5.57 (s, 2H), 6.93 (s, 1H), 7.28
(m, 4H), 7.52 (m, 2H), 7.78 (m, 1H), 8.22 (d, J ) 8.4, 1H), 9.51
(s, 1H). Anal. (C28H26N8) C, H, N.
P r ep a r a tion of Ta r get Ca r boxylic Acid Der iva tives
4a ,c-k ,m ,p -t,v,w (Ba sic Hyd r olysis). To a solution of the
appropriate ester (6a ,c-k ,m ,p -t,v,w ) (1.0 mmol) in the
suitable solvent (ethanol, THF, dioxan, or ethylene glycol) (40
mL) was added 1 N NaOH (10 mL), and the resulting mixture
was refluxed while the reaction progress was monitored by
TLC. When the ester derivative disappeared from the chro-
matogram, the reaction mixture was evaporated under reduced
pressure and diluted with water (20 mL), and the pH was
Further insight into the molecular basis of the
interactions can be obtained by a dynamic analysis of
the inhibitor-receptor complexes. Figure 5 shows the
minimized average structure of the 4c-AT1 receptor
complex, a detailed view of the binding site, and the
modification of the HOMO orbital upon neutralization
of the anionic antagonist 4c. The putative antagonist
binding site is essentially hydrophobic, the only basic
residue being K199, which has been identified to be the
key residue for agonist and antagonist recognition.29 The
overall information obtained justifies the hypothesis
that a long-range electrostatic interaction controls the

2584 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10
Cappelli et al.
adjusted to 5-6 by addition of 1 N HCl. The precipitate was
collected by filtration (or extracted with chloroform when
necessary), washed with water, and dried under reduced
pressure. Purification of the solid obtained by recrystallization
from the suitable solvent or by washing with ethyl acetate or
diethyl ether gave target carboxylic acid derivatives showing
a suitable degree of purity as confirmed by 1H NMR and
combustion analyses.
Purification of the residue by flash chromatography with ethyl
acetate as the eluent gave pure compounds 6a -d ,k -m ,
o-q,v,x,y.
4-[4-[(5,7-Dim eth yl-2-eth yl-3H-im id a zo[4,5-b]p yr id in -
3-yl)m eth yl]p h en yl]-3-[2-(tr ip h en ylm eth yl)-2H-tetr a zol-
5-yl]qu in olin e (6b). The title compound was prepared from
5b and 5,7-dimethyl-2-ethyl-3H-imidazo[4,5-b]pyridine to ob-
tain a white solid (0.495 g, 47%) melting at 188-189 °C. 1H
NMR (CDCl3): 1.26 (t, J ) 7.6, 3H), 2.59 (s, 3H), 2.65 (s, 3H),
2.73 (q, J ) 7.5, 2H), 5.39 (s, 2H), 6.92 (m, 7H), 7.04 (d, J )
8.3, 2H), 7.12 (d, J ) 8.0, 2H), 7.26 (m, 9H), 7.39 (m, 2H), 7.73
(m, 1H), 8.17 (d, J ) 8.5, 1H), 9.48 (s, 1H).
Eth yl 1-[4-[(5,7-Dim eth yl-2-eth yl-3H-im id a zo[4,5-b]p y-
r idin -3-yl)m eth yl]ph en yl]-2-n aph th alen ecar boxylate (6a).
The title compound was prepared from 5a and 5,7-dimethyl-
2-ethyl-3H-imidazo[4,5-b]pyridine to obtain a white solid (0.30
g, 43%), melting at 147 °C. 1H NMR (CDCl3): 0.92 (t, J ) 7.4,
3H), 1.38 (t, J ) 7.4, 3H), 2.61 (s, 3H), 2.64 (s, 3H), 2.89 (q, J
) 7.5, 2H), 4.01 (q, J ) 7.0, 2H), 5.56 (s, 2H), 6.90 (s, 1H),
7.24 (m, 4H), 7.45 (m, 3H), 7.87 (m, 3H). MS (EI): m/z 463
(M+, 100).
Eth yl 4-[4-[(5,7-Dim eth yl-2-eth yl-3H-im id a zo[4,5-b]p y-
r id in -3-yl)m et h yl]p h en yl]-3-qu in olin eca r boxyla t e (6c).
The title compound was prepared from 5c and 5,7-dimethyl-
2-ethyl-3H-imidazo[4,5-b]pyridine to obtain a white solid (0.34
g, 49%), melting at 155 °C. 1H NMR: (CDCl3): 0.97 (t, J )
7.3, 3H), 1.38 (t, J ) 7.4, 3H), 2.60 (s, 3H), 2.64 (s, 3H), 2.88
(q, J ) 7.5, 2H), 4.08 (q, J ) 7.2, 2H), 5.56 (s, 2H), 6.91 (s,
1H), 7.25 (m, 4H), 7.47 (m, 2H), 7.76 (m, 1H), 8.16 (d, J ) 8.5,
1H), 9.31 (s, 1H).
X-r a y Cr ysta llogr a p h y. Single crystals of 4d , 6n , and 6q
were submitted to X-ray data collection on a Siemens P4 four-
circle diffractometer with graphite monochromated Mo-KR
radiation (λ ) 0.71069 Å). The ω/2θ scan technique was used
for data collection.
The three structures were solved by direct methods imple-
mented in the SHELXS-97 program.30 The refinements were
carried out by full-matrix anisotropic least-squares on F2 for
all reflections for non-H atoms by means of the SHELXL-97
program.31
While for 6n and 6q the asymmetric unit contains one
molecule, in the case of 4d , two molecules are present together
with water and ethanol molecules as crystallization solvents.
Statistical disorder is present for 6n and 4d . In the first case,
disorder at the COOEt group has been treated by refining two
different positions for atoms O(26), O(27), C(28), and C(29) and
the attached hydrogen atoms. The refined site occupation
factors are 0.60(1) for one position and 0.40(1) for the other.
For 4d statistical disorder has been found for the OH group
of the cocrystallized ethanol. Two different positions have been
refined with site occupation factors of 0.62(1) for one position
and 0.38(1).
Biologica l Meth od s. An gioten sin II Recep tor Bin d in g
Assa y.17 Male Wistar rats (Charles River, Calco, Italy) were
killed by decapitation, and their livers were rapidly removed.
Angiotensin II receptors from rat liver were prepared by
differential centrifugation. The liver was dissected free of fatty
tissue and minced accurately with small scissors, and then
about 3 g of tissue was homogenized by Polytron Ultra-Turrax
(maximal speed for 2 × 30 s) in ice cold 20 vol of Tris-HCl 5
mM, sucrose 0.25 M (pH 7.4). The homogenate was centrifuged
at 750g for 10 min, and the supernatant was filtered through
cheesecloth and saved. The pellets were homogenized and
centrifuged as before. The combined supernatants were cen-
trifuged at 50 000g for 15 min. The resulting pellet was
resuspended in Tris-HCl 5 mM, sucrose 0.25 M (pH 7.4), and
centrifuged as above. The final pellets were used immediately
or stored frozen at -70 °C before use. The membrane pellets
were resuspended in Tris-HCl 50 mM, NaCl 100 mM, MgCl2
10 mM, EDTA 1 mM, bacitracin 100 µM, PMSF 100 µM, BSA
0.1% (pH 7.4) to obtain a final protein concentration of 0.25
mg/mL. Binding of [125I]Sar1,Ile8-angiotensin II (NEN Perkin-
Elmer Life Sciences, S. A. 2000 Ci/mmol) to liver membranes
was performed at 25 °C for 180 min in 96-well filtration plates
1-[4-[(5,7-Dim eth yl-2-eth yl-3H-im id a zo[4,5-b]p yr id in -
3-yl)m eth yl]p h en yl]-2-n a p h th a len eca r boxylic Acid (4a ).
The title compound was obtained from ester 6a by means of
the basic hydrolysis procedure employing ethanol as the
solvent (yield 90%, mp 245-247 °C). 1H NMR (DMSO-d6): 1.27
(t, J ) 7.3, 3H), 2.53 (s, 3H), 2.54 (s, 3H), 2.84 (q, J ) 7.3,
2H), 5.56 (s, 2H), 6.97 (s, 1H), 7.22 (m, 4H), 7.40 (m, 2H), 7.55
(m, 1H), 7.81 (d, J ) 8.6, 1H), 8.01 (d, J ) 8.4, 2H), 12.65 (br
s, 1H). MS (EI): m/z 435 (M+, 5). Anal. (C28H25N3O2‚3H2O) C,
H, N.
4-[4-[(5,7-Dim eth yl-2-eth yl-3H-im id a zo[4,5-b]p yr id in -
3-yl)m eth yl]p h en yl]-3-qu in olin eca r boxylic Acid (4c). The
title compound was obtained from ester 6c by means of the
basic hydrolysis procedure employing ethanol as the solvent
1
(yield 98%, mp 293-295 °C). H NMR: (CDCl3): 1.00 (t, J )
7.5, 3H), 2.61 (s, 6H), 2.78 (q, J ) 7.5, 2H), 5.57 (s, 2H), 6.95
(s, 1H), 7.29 (m, 4H), 7.48 (m, 2H), 7.77 (m, 1H), 8.21 (d, J )
8.3, 1H), 9.39 (s, 1H). Anal. (C27H24N4O2‚H2O) C, H, N.
4-[4-[(5,7-Dim eth yl-2-eth yl-3H-im id a zo[4,5-b]p yr id in -
3-yl)m et h yl]p h en yl]-2-m et h oxy-3-q u in olin eca r b oxylic
Acid (4d ). This compound was obtained from ester 6d by
means of the basic hydrolysis procedure employing ethanol as
the solvent and was recrystallized from ethanol to obtain X-ray
quality crystals (yield 72%, mp 210-211 °C). 1H NMR:
(CDCl3): 0.88 (t, J ) 7.5, 3H), 2.67 (m, 8H), 4.13 (s, 3H), 5.53
(s, 2H), 6.95 (s, 1H), 7.27 (m, 3H), 7.48 (m, 3H), 7.63 (m, 1H),
7.89 (d, J ) 8.1, 1H). Anal. (C28H26N4O3‚0.5H2O‚0.5C2H5OH)
C, H, N.
4-[4-[(5,7-Dim eth yl-2-eth yl-3H-im id a zo[4,5-b]p yr id in -
3-yl)m eth yl]p h en yl]-N-(4-n itr op h en ylsu lfon yl)-3-qu in o-
lin eca r boxa m id e (4u ). A mixture of 4p (0.10 g, 0.22 mmol)
in dichloromethane (20 mL) with 4-(dimethylamino)pyridine
(DMAP) (0.029 g, 0.24 mmol), 4-nitrobenzenesulfonamide
(0.048 g, 0.24 mmol), and 1-[3-(dimethylamino)propyl]-3-
ethylcarbodiimide hydrochloride (EDCI) (0.046 g, 0.24 mmol)
was stirred at room temperature for 48 h under argon. The
solvent was removed under reduced pressure, and the residue
was purified by flash chromatography with ethyl acetate-
methanol (7:3) as the eluent to give pure 4u as a white solid
(0.093 g, yield 67%, mp 285 °C dec). 1H NMR: (DMSO-d6):
0.89 (t, J ) 7.4, 3H), 1.69 (m, 2H), 2.49 (s, 6H), 2.75 (t, J )
7.5, 2H), 5.50 (s, 2H), 6.93 (s, 1H), 7.07 (m, 4H), 7.39 (m, 2H),
7.68 (m, 3H), 7.99 (d, J ) 8.2, 1H), 8.10 (d, J ) 8.6, 2H), 8.93
(s, 1H). MS (FAB): m/z 635 (M + 1). Anal. (C34H30N6O5S‚3H2O)
C, H, N.
Gen er a l P r oced u r e for th e P r ep a r a tion of Com p ou n d s
6a -d ,k -m ,o-q,v,x,y (Br om in a tion -Cou p lin g P r oced u r e).
A mixture of the toluene derivative 5a -d ,k -m ,q (1.5 mmol)
in 40 mL of CCl4 with N-bromosuccinimide (0.27 g, 1.5 mmol)
and dibenzoyl peroxide (0.03 g, 0.12 mmol) was refluxed for a
suitable time (typically 2-3 h), and the reaction progress was
monitored by TLC. The initial solvent volume was reduced by
half under reduced pressure, the insoluble succinimide was
filtered off, and the resulting mixture was evaporated under
reduced pressure. The residue was dissolved into anhydrous
DMF (10 mL) and added to a mixture (aged at 0 °C for 20
min) of the appropriate 2-alkyl-5,7-dimethyl-3H-imidazo-
[4,5-b]pyridine6,10 (1.5 mmol) in anhydrous DMF (10 mL) with
NaH (0.036 g, 1.5 mmol). The resulting mixture was stirred
at room temperature for 15-18 h under argon, and the
reaction was quenched with water (5 mL). The bulk of the
DMF was evaporated under reduced pressure, and the residue
was diluted with water (20 mL) and extracted with chloroform.
The combined organic extracts were washed with brine, dried
over sodium sulfate, and concentrated under reduced pressure.

AT1 Angiotensin II Receptor Antagonists
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10 2585
(Millipore GFB-Multiscreen). Each 250 µL incubate contained
algorithm,37 allowing an integration time step of 0.001 ps.
Moreover, weak harmonic constraints (30 kJ /mol per Å) were
applied between the backbone oxygen atoms of residue i and
backbone nitrogen atom of residue i+4 by means of the NOE
facility in the CHARMM program36 in order to maintain the
helical structure.
The structures were thermalized to 300 K with 5 °C rise
per 6000 steps by randomly assigning individual velocities
from the Gaussian distribution. After heating, the systems
were allowed to equilibrate until the potential energy versus
time was approximately stable (34 ps). Velocities were scaled
by a single factor. An additional 10 ps period of equilibration
with no external perturbation was run. Time-averaged struc-
tures were then determined over the last 200 ps of each
simulation.
Bu ild in g a n d Refin em en t of th e Liga n d -Recep tor
Com p lexes. The main criteria followed to dock the ligands
into the minimized average structure of the receptor were (a)
the formation of a charge-reinforced hydrogen bond between
the acidic (tetrazole or carboxylic) moieties of the ligands and
the K199 residue of Helix 5, (b) the achievement of a hydrogen
bond between the imidazo[4.5-b]pyridine nucleus of the ligands
and one of the following polar residues N111 (Helix 3), N294
(Helix 7), S252 (Helix 6) which protrude into the receptor pore
at a distance of about 8-9Å deeper than K199. The complexes
were refined according to the procedure described in the
previous paragraph.
the following:
[
125I]Sar1,Ile8-angiotensin II (25 pM), liver
membrane proteins (25 µg) and standard or test compounds.
Nonspecific binding was measured in the presence of 1 µM
angiotensin II and represented 5-10% of total binding. Bind-
ing was terminated by rapid vacuum filtration using a Milli-
pore Multiscreen device. Receptor-ligand complex trapped on
filters was washed twice with 200 µL of ice cold NaCl 100 mM,
MgCl2 100 mM. Dried filters disks were punched out and
counted in a gamma-counter with 92% efficiency. The IC50
value (concentration for 50% displacement of the specifically
bound [125I]Sar1,Ile8-Angiotensin II) was estimated for the
linear portion of the competition curves.
An gioten sin II Fu n ction al An tagon ism in Rabbit Aor ta
Str ip s.17 New Zealand White rabbits (3-4 kg body weight,
Harlan Italy) were killed by cervical dislocation, after a slight
ether anaesthesia. The descending thoracic aorta, with the
endothelium removed, was cut into helical strips 3 to 4 mm
wide, and 15 to 20 mm long. These strips were mounted in 20
mL tissue baths containing Krebs-Henseleit solution of the
following composition (mM): NaCl 118; KCl 4.69; KH2PO4
1.17; MgSO4‚7H2O 1.17; CaCl2‚2H2O 2.51; NaHCO3 25; glucose
11.1. The tissue baths were kept at 37 °C and aerated with
95% O2 and 5% CO2. Each strip was connected to an isometric
transducer (Basile, Italy), and a resting tension of 2 g was
applied to the tissues. Changes in isometric tension were
displayed on a four-channel pen recorder (Basile, Italy). The
tissues were allowed to equilibrate for 1 h and were washed
every 10 min. The strips were then stimulated by increasing
concentrations of angiotensin II to obtain a cumulative con-
centration-response curve. After 30 min washout, submaxi-
mal-effect (70-80%) concentration of angiotensin II was chosen
to test the inhibitory effects of the substances under study.
Various concentrations of the antagonists or vehicles were
added, and at least 20 min contact with the tissues was
allowed before adding the agonist to the bathing fluid. The
antagonist activity was expressed as percentage of inhibition
of the agonist contractions. The regression line was calculated,
and the concentration capable of inhibiting the effect of the
agonist by 50% (IC50) and its P ) 0.05 fiducial limits were
calculated from the regression line.
Ack n ow led gm en t. Thanks are due to Italian MIUR
for financial support. Prof. Stefania D’Agata D’Ottavi’s
careful reading of the manuscript is also acknowledged.
Su p p or tin g In for m a tion Ava ila ble: Full experimental
details for the synthesis and the characterization of compounds
4, 2, and their intermediates (chemistry, NMR, MS, and X-ray
crystallographic details for compounds 4d , 6n ,q). This material
is available free of charge via Internet at http://pubs.acs.org.
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J M031100T