Further studies on imidazo[4,5-b]pyridine AT1 angiotensin II receptor antagonists. Effects of the transformation of the 4-phenylquinoline backbone into 4-phenylisoquinolinone or 1-phenylindene scaffolds

Article abstract of DOI:10.1021/jm0603163

The 4-phenylquinoline fragment of novel AT₁ receptor antagonists 4 based on imidazo[4,5-b]pyridine moiety was replaced by 4-phenylisoquinolinone (compounds 5) or 1-phenylindene (compounds 6) scaffolds to investigate the structure-activity relat

Full text of DOI:10.1021/jm0603163

J. Med. Chem. 2006, 49, 6451-6464
6451
Further Studies on Imidazo[4,5-b]pyridine AT1 Angiotensin II Receptor Antagonists. Effects
of the Transformation of the 4-Phenylquinoline Backbone into 4-Phenylisoquinolinone or
1-Phenylindene Scaffolds
Andrea Cappelli,*,† Gal.la Pericot Mohr,†,# Germano Giuliani,† Simone Galeazzi,† Maurizio Anzini,† Laura Mennuni,‡
Flora Ferrari,‡ Francesco Makovec,‡ Eva M. Kleinrath,§ Thierry Langer,§ Massimo Valoti,| Gianluca Giorgi, and
Salvatore Vomero†
Dipartimento Farmaco Chimico Tecnologico and European Research Centre for Drug DiscoVery and DeVelopment, UniVersita` di Siena, Via A.
Moro, 53100 Siena, Italy, Rotta Research Laboratorium S.p.A., Via Valosa di Sopra 7, 20052 Monza, Italy, Department of Pharmaceutical
Chemistry, Institute of Pharmacy, UniVersity of Innsbruck, Innrain 52a, A-6020 Innsbruck, Austria, Dipartimento di Scienze Biomediche,
UniVersita` di Siena, Via A. Moro, 53100 Siena, Italy, and Dipartimento di Chimica, UniVersita` di Siena, Via A. Moro, 53100 Siena, Italy
ReceiVed March 20, 2006
The 4-phenylquinoline fragment of novel AT1 receptor antagonists 4 based on imidazo[4,5-b]pyridine moiety
was replaced by 4-phenylisoquinolinone (compounds 5) or 1-phenylindene (compounds 6) scaffolds to
investigate the structure-activity relationships. Binding studies showed that most of the synthesized
compounds display high affinity for the AT1 receptor. Because of the in vitro high potency of carboxylic
acids 5b,f, they were evaluated in permeability (in Caco-2 cells) and in pharmacokinetic studies in comparison
with quinoline derivatives 4b,i,j,k. The studies showed that these compounds are characterized by rapid
excretion, low membrane permeability, and low oral bioavailability. The structure optimization of the indene
derivatives led to compounds 6e,f possessing interesting AT1 receptor affinities. Optimization produced
polymerizing AT1 receptor ligand 6c, which forms a thermoreversible polymer (poly-6c) and is released
from the latter by a temperature-dependent kinetics. The results suggest the possibility of developing novel
polymeric prodrugs based on a new release mechanism. Finally, a set of 34 AT1 receptor antagonists was
used as a new test for the evaluation of the predictive capability of the previously published qualitative and
quantitative pharmacophore models.
Introduction
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 followed by most medicinal chemists
was concerned with the molecular modification of the imidazole
moiety of losartan (1). Among the large variety of the
heterocyclic systems developed, an outstanding position is
occupied by the imidazo[4,5-b]pyridine moiety of compound
2a (L-158,809).4 This congener of losartan has been reported
to show a subnanomolar AT1 receptor affinity about 1 order of
magnitude higher than that of losartan and represents one of
the most potent nonpeptide Ang II antagonists so far developed.
This suggests that the stereoelectronic characteristics of the
imidazo[4,5-b]pyridine moiety can be considered optimal for
the interaction with the receptor. However, relatively little
information is available on the effects of the molecular
modification of the phenyl group bearing the acidic moiety
(distal phenyl ring). Moreover, 4-phenyl-3-tetrazolylpyridyl
derivative 2d was reported to be a very potent Ang II antagonist
showing, however, a somewhat poorer oral bioavailability than
2c (L-158,338). The authors suggested elsewhere that the
decrease in lipophilicity has a negative effect on the oral potency
of compound 2d with respect to 2c.5
Angiotensin II (Ang II) is an octapeptide produced by the
renin-angiotensin system (RAS), which plays a key role in the
pathophysiology of hypertension. In humans, Ang II interacts
with two main receptor subtypes: AT1 and AT2.1 The AT1
receptor subtype mediates virtually all the known physiological
actions of Ang II in cardiovascular, neuronal, endocrine, and
hepatic cells as well as in other ones. 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 the 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.).1,2
The parallel discovery of losartan and eprosartan, potent and
orally active nonpeptide Ang II antagonists, has stimulated the
design of a large number of congeners.3 Among them, irbesartan,
candesartan, valsartan, telmisartan, and olmesartan are on the
market and some 20 other compounds are being developed. The
biphenyl fragment bearing an acidic moiety (tetrazole ring,
-COOH, -SO2-NH-CO-) is common to most of these
compounds, which differ in the nature of the pendent hetero-
cyclic system (valsartan lacks the heterocyclic moiety) connected
These observations led to some molecular modifications
involving the distal phenyl group of compounds 2 and ultimately
to the development of compounds 4.6 Some members of this
series of 4-phenylquinoline derivatives showed in vitro proper-
ties comparable to those shown by losartan. However, phar-
macokinetic studies performed with the selected candidate for
further preclinical studies (CR3210, 4b) showed a relatively low
oral bioavailability and a rapid excretion.7,8 Owing to the very
interesting in vitro properties of quinoline derivatives 4, the
program of structural modification was pursued with the
synthesis of both isoquinolinone derivatives 5 and indene
* To whom correspondence should be addressed. Tel: +39 0577 234320.
Fax: +39 0577 234333. E-mail: cappelli@unisi.it.
† Dipartimento Farmaco Chimico Tecnologico and European Research
Centre for Drug Discovery and Development, Universita` di Siena.
‡ Rotta Research Laboratorium S.p.A..
§ University of Innsbruck.
| Dipartimento di Scienze Biomediche, Universita` di Siena.
Dipartimento di Chimica, Universita` di Siena.
# Present address: Medicinal Chemistry, Siena Biotech, Via Torre
Fiorentina 1, 53100 Siena, Italy.
10.1021/jm0603163 CCC: $33.50 © 2006 American Chemical Society
Published on Web 10/06/2006

6452 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 22
Cappelli et al.
Chart 1. Structure of the AT1 Receptor Antagonists Losartan,
Valsartan, and Compounds 2,4
Scheme 1a
Chart 2. Structure and AT1 Receptor Affinity of Compounds
2c, 2d, and 4b
a Reagents: (a) Br-CH(COOC2H5)2, K2CO3, CH3COCH3; (b) HCl,
CH3COOH; (c) CH3NH2, C2H5OH; (d) H2SO4, C2H5OH; (e) SOCl2, CH2Cl2;
(f) C2H5OH, (C2H5)3N; (g) NBS, dibenzoyl peroxide, CCl4; (h) 2-substituted-
5,7-dimethyl-3H-imidazo[4,5-b]pyridine, NaH, DMF; (i) NaOH, C2H5OH.
Results
Chemistry. The preparation of isoquinoline-3-carboxylic acid
derivatives 5b,d,f was carried out starting from the commercially
available 2-(p-toluoyl)benzoic acid 7 as described in Scheme
1.
Acid 7 was transformed into isocoumarin-3-carboxylic acid
(8), which was reacted with methylamine and dehydrated to
acid 9 by modifying a procedure described in the literature.11
The corresponding ester 10 was easily obtained from 9 and
subjected to the standard bromination-coupling-deprotection
procedure (BCD: benzylic bromination, coupling reaction with
substituted imidazo[4,5-b]pyridine derivatives,4,6,12 and unmask-
ing of the acidic moiety) to obtain the target isoquinoline-3-
carboxylic acid derivatives 5b,d,f.
Chart 3. Structure of the Newly Synthesized Isoquinolinone
Derivatives 5 and Indene Derivatives 6
However, 3-tetrazolylisoquinoline derivatives 5a,c,e were
obtained by means of a multistep procedure11 involving the
formation of 3-cyanoisoquinolone derivative 12 (Scheme 2). The
application of the classical tetrazole chemistry to 12 led to
protected tetrazole intermediate 13, which was subjected to the
standard BCD procedure to give target derivatives 5a,c,e.
Indenone derivatives 6a,b were synthesized by applying the
BCD procedure to tert-butyl ester 14, which was prepared by
modifying the procedure described for the preparation of the
corresponding ethyl ester (ethyl 3-(4-methylphenyl)-1-oxo-1H-
indene-2-carboxylate).13
derivatives 6 as a further step of an investigation devoted to
both the development of new antihypertensive agents and the
understanding of the molecular basis of their pharmacodynamic
and pharmacokinetic9 properties.In this article, the synthesis,
the preliminary pharmacological characterization, and the
deduction of structure-affinity relationships (SAFIR) of the
novel Ang II antagonists 5 and 6 are described together with
an assessment study of the prediction capability of our previ-
ously published qualitative and quantitative pharmacophore
models.10
Schemes 4-7 describe the elaboration of the intermediate
indenone derivative 15b designed to explore the top of the AT1
receptor binding cavity further, where the basic amino acid

Imidazo[4,5-b]pyridine AT1
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 22 6453
Scheme 3a
Scheme 2a
a Reagents: (a) SOCl2, CH2Cl2; (b) C2H5OMgCH(COOt-Bu)2, C2H5OC2H5;
(c) K2CO3, C2H5OH; (d) NBS, dibenzoyl peroxide, CCl4; (e) 2-substituted-
5,7-dimethyl-3H-imidazo[4,5-b]pyridine, NaH, DMF; (f) HCOOH.
Scheme 4a
a Reagents: (a) SOCl2, CH2Cl2; (b) CH3NHCH2CN‚HCl, TEA, CH2Cl2;
(c) DBU, toluene; (d) (CH3)3SnN3, xylene; (e) (C6H5)3CCl, NaOH, THF,
CH2Cl2; (f) NBS, dibenzoyl peroxide, CCl4; (g) 2-substituted-5,7-dimethyl-
3H-imidazo[4,5-b]pyridine, NaH, DMF; (h) HCOOH.
Lys199 establishes the ion-pair interaction with the acidic
moiety of the antagonist.6 On the basis of the intriguing results
obtained with related 2-indenecarboxylic acid derivatives (e.g.,
BF1, Scheme 4),13,14 this synthetic work, at the same time, can
be considered an exploration of the chemical behavior of these
indene derivatives. In particular, we were interested in evaluating
(a) the stability of trans-diene 6c, (b) its propensity to spontane-
ous polymerization, and (c) the characteristics of the thermo-
induced depolymerization process. In fact, we previously found
that BF1 was stable in solution, but polymerized spontaneously
upon solvent removal to give poly-BF1, a new polymer showing
interesting properties such as thermoreversible polymerization/
depolymerization behavior, high solubility in the most common
organic solvents, a π-stacked structure, and liability to give
nanostructured aggregates.13,14 Moreover, BF1 showed the
tendency to behave as a Michael acceptor and, in the presence
of a nucleophile such as N-methylpiperazine, established an
equilibrium with its adducts.14 This chemical behavior of BF1,
taken together with the presence of the basic Lys199 on the
top of the AT1 receptor binding cavity, shows the importance
of the chemical studies and the biological characterization of
6c.
a Reagents: (a) Al(CH3)3, CH2Cl2; (b) HCOOH; (c) PTSA, CHCl3; (d)
PTSA, CDCl3.
Trans-diene 6c was obtained in trace amounts from the
deprotection reaction with formic acid of ester 16e to acid 6e
and in more substantial amounts by dehydration of 16e or acid
6e in the presence of p-toluenesulfonic acid (PTSA) in CHCl3
(or CDCl3). Interestingly, benzofulvene derivative 6c was stable
as a pure crystalline solid but polymerized spontaneously when
the mixture of the dehydration reaction of 6e was concentrated
without the elimination of PTSA. Because the ethyl ester of

6454 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 22
Cappelli et al.
acid 6c showed the same spontaneous polymerization behavior
as that of BF1 (see Supporting Information), we assume that
the stability of pure 6c is related to the possible intermolecular
interaction of its COOH with the imidazole nitrogen in the solid
state otherwise prevented by the presence of PTSA, which
protonates the heterocyclic nitrogens. This assumption is sup-
ported by the crystal structure of a previously published
quinoline derivative in which the same intermolecular interaction
stabilizes the crystal packing.6 The polymer substance obtained
1
(poly-6c‚PTSA) was characterized by H NMR spectroscopy
(e.g., thermal-induced depolymerization followed by 1H NMR
at 200 MHz, Figure 1). When a solution of poly-6c-PTSA in
DMSO-d6 was heated at different temperatures, the depoly-
merization of poly-6c occurred at different velocities as
demonstrated by the appearance of the signals typical of 6c
(Figure 1). At 55 °C, the depolymerization process was very
slow, while becoming significantly more rapid at 80 °C; at 140
°C, it is even faster because it was almost complete after 1 h of
heating. At 120 °C, the fragmentation process showed an
intermediate velocity and it could be profitably followed by 1H
NMR spectroscopy, which clearly showed that the reaction
reached equilibrium after 4 h of heating (Figure 1). For poly-
6c, these results suggest a thermoreversible polymerization/
depolymerization behavior similar to that shown by poly-BF1
but characterized by a faster kinetics.
The hydrogenation of acid 6c generated in situ from ester
16e with PTSA in toluene gave a mixture of dihydro derivatives
6d (the product of the hydrogenation in positions 1,2 of the
diene fragment) and 17 (the compound hydrogenated in posi-
tions 1,4 of the diene fragment), which were separated by flash
chromatography (Scheme 5).
Ester 15b was reacted with the anion of the di-tert-butyl
malonate to give 18, which was cleaved with formic acid to
diacid 20 (Scheme 7) or with hydrochloric acid in acetic acid
to obtain monoacid 19. The structure of the latter compound
was confirmed by single-crystal X-ray diffraction studies. In
fact, monoacid 19 crystallized from ethyl acetate to give single
crystals suitable for X-ray diffraction studies (Figure 2), whereas
diacid 20 did not. Therefore, the synthetic procedure used in
the preparation of 20 was applied to the synthesis of the
simplified analogue 21 lacking the imidazo[4,5-b]pyridine
moiety, and X-ray quality crystals were obtained from chloro-
form (Figure 3).
Finally, acylsulfonamide derivatives 6g,h were synthesized
by reaction of carboxylic acid 6b with the appropriate sulfona-
mide in the presence of EDCI and DMAP (Scheme 7).
Structure-Affinity Relationship Studies. The newly syn-
thesized bicyclic derivatives 5a-f, 6a-h, 17, 19, and 20
(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 the AT1
receptor in rat hepatic membranes in comparison with reference
compounds losartan, and valsartan, following well-established
protocols.15 The results of the binding studies are summarized
in Table 1 along with the affinities of some of the previously
published compounds 4a-n, which are included for comparative
purposes. The results show that most of the tested compounds
displayed high affinity for the AT1 receptor, although lower than
that of reference compound 2a.
Figure 1. Thermo-induced depolymerization of poly-6c, followed by
1H NMR (200 MHz). A solution of 2.5 mg of poly-6c-PTSA in 0.5
1
mL of (DMSO-d6) was heated at 120 °C, and H NMR spectra were
recorded at regular time intervals. The arrow indicates the solvent peak,
the asterisk the water peak, and the empty circles the signals attributable
to PTSA.
the naphthalene moiety of 4a into the quinoline one of 4c had
slightly positive effects. The results obtained with the newly
synthesized compounds show that the isoquinolinone moiety
of compounds 5b,d,f is better accommodated by the receptor
than benzene, naphthalene, and quinoline groups of compounds
2,4, indicating this molecular portion as an optimized group
for the interaction with the top of the AT1 receptor binding
cavity. The comparison between compounds 5b and 4f shows
the importance of the correct position/orientation of the carbonyl
dipole, which in compounds 5 occupies the position of quinoline
(a) Effects of the Modification of the Quinoline Moiety of
Compounds 4. In our previous article,6 we reported that the
introduction of an additional benzene ring into the structure of
carboxylic acid derivative 2c leading to naphthalene derivative
4a16 was tolerated by the receptor, and the transformation of

Imidazo[4,5-b]pyridine AT1
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 22 6455
Scheme 7a
Scheme 5a
a Reagents: (a) CH3-SO2-NH2, EDCI, DMAP, CH2Cl2; (b) C6H5-SO2-
NH2, EDCI, DMAP, CH2Cl2.
a Reagents: (a) H2, Pd/C, PTSA, toluene.
Scheme 6a
Figure 2. Crystal structure of 19-0.5 CH3COOC2H5. For the pendant
ethyl moiety (-C(13)H2-C(14)H3), atoms having site occupation factor
equal to 0.54(1) are reported. Ellipsoids enclose 50% probability.
a Reagents: (a) CH2[COOC(CH3)3]2, NaH, THF; (b) HCl, CH3COOH;
(c) HCOOH.
nitrogen of compounds 4. The deletion of the N-CH3 group of
compounds 5b,d leading to 6a,b produces a decrease in AT1
receptor affinity of about 1 order of magnitude, whereas the
transformation of the CdO group of the latter compounds into
the tertiary carbinol group of 6e,f restores high affinity.
Comparison of the 6e affinity (IC50 ) 11 nM) with that shown
by 6d (IC50 ) 99 nM), which lacks the hydroxyl group, shows
the importance of this OH group in the interaction with the AT1
receptor. The replacement of the carbonyl oxygen atom of 6b
with an exocyclic methylene group (e.g., compound 6c) as well
as the hybridization change of the carbon atom bearing the
phenyl spacer (compare 6c with 17) has a negligible effect on
AT1 receptor affinity. Finally, the structure-affinity relationships
Figure 3. Crystal structure of the simplified analogue 21-H2O.
Ellipsoids enclose 50% of probability.
concerning compounds 19 and 20 showed the surprising role
of a second carboxyl group in the interaction of these stand
alone indanone derivatives with the AT1 receptor. Compounds
19 and 20 can be regarded as conformationally restrained
derivatives of some diphenylpropionic acid derivatives described

6456 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 22
Cappelli et al.
Table 1. AT1 Receptor Binding Affinities of Compounds 2, 4-6, 17, 19, and 20
binding
IC50 (nM)
( SEMa
rabbit aortic
strips IC50 (nM)
( SEMb
compd
X
R1
R2
A
2a (L-158,809)
2b
2c
4a
4b (CR3210)
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
C2H5
n-C3H7
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
n-C3H7
n-C3H7
n-C3H7
n-C3H7
n-C3H7
n-C3H7
n-C4H9
n-C4H9
C2H5
CN4H
CN4H
COOH
COOH
CN4H
COOH
COOH
CN4H
COOH
CN4H
COOH
COOH
COOH
COOH
CONHSO2C6H4(p)NO2
COOH
COOH
CN4H
COOH
CN4H
COOH
CN4H
COOH
0.4 ( 0.1
0.8 ( 0.01
10 ( 2.2
33 ( 4.7
6.9 ( 1.3
17 ( 1.8
13 ( 1.2
>300
326 ( 36
12 ( 2.1
11 ( 1.8
7.7 ( 1.0
9.0 ( 1.6
4.2 ( 0.34
106 ( 19
41 ( 7.1
13 ( 1.5
>300
CH
N
N
N
N-CH3
H
H
H
NH2
dO
dO
H
H
Cl
CH3
NH2
H
H
NH2
8.9 ( 2.9 (4.0)
N-CH3
N
N
N
N
N
N
N
N
(13)
(3.3)
4n
5a
5b (CR3588)
5c
5d
5e
5f
6a
6b
6c
6d
6e
6f
6g
6h
17
19
20
C2H5
5.2 ( 0.3
>300
5.7 ( 0.9
>300
4.6 ( 0.9
n-C3H7
n-C3H7
n-C4H9
n-C4H9
C2H5
n-C3H7
n-C3H7
n-C3H7
n-C3H7
n-C3H7
n-C3H7
n-C3H7
6.2 ( 1.1
53 ( 7.9
50 ( 15
60 ( 17
99 ( 22
11 ( 1.4
15 ( 1.5
54 ( 5.1
131 ( 12
47 ( 4.4
139 ( 12
55 ( 4.9
0.4 ( 0.1
6.7 ( 0.5
1.9 ( 0.2
CdO
CdO
COOH
COOH
COOH
COOH
COOH
COOH
CONHSO2CH3
CONHSO2C6H5
CdCH2
C(H)CH3
C(OH)CH3
C(OH)C2H5
CdO
33 ( 6.4
CdO
H
COOH
Ang II
losartan
valsartan
10 ( 1.7 (9.9)
a Each value is the mean ( SEM of three determinations and represents the concentration giving half the maximum inhibition of [125I]Sar1,Ile8-Ang II
specific binding to rat hepatic membranes. b The antagonism of Ang II-contracted rabbit aorta strips was assayed by using 60 min as time of contact of the
tested compound. Only one antagonist concentration was tested on each tissue preparation. The values in parentheses are the ones described in a previous
article (see ref 6).
in the literature, in which the monoacid shows the same affinity
as that of the diacid derivative.17
(b) Effects of the Modification of the Acidic Moiety. It is
noteworthy that in the series of isoquinolinone derivatives 5,
the replacement of the carboxyl moiety of compounds 5b,d,f
with the tetrazole group (compounds 5a,c,e) produces a dramatic
decrease in receptor affinity, whereas in the series of quinoline
derivates 4b,c,g,h, the two different acidic moieties appeared
to be completely bioisoster.6 However, we previously observed
that in the series of monocyclic compounds 2, the same

Imidazo[4,5-b]pyridine AT1
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 22 6457
structural modification lead to an affinity enhancement of about
1 order of magnitude, and this effect was comparable with that
reported in the literature for losartan.18
A comparison between acid 6b and acylsulfonamide deriva-
tive 6g suggests that an extended acidic group is tolerated in
such compounds by the AT1 receptor binding site, provided it
is not excessively bulky. In fact, the replacement of the
methylsulfonamido group of 6g with the phenylsulfonamido
group of 6h produces a slight decrease in affinity as previously
observed in a series of diphenylpropionic acid derivatives
described in the literature.17
(c) Effects of the Modification of the Lipophilic Substitu-
ent in Position 2 of the Imidazo[4,5-b]pyridine Nucleus. We
previously observed that ethyl derivative 2a showed a 2-fold
higher affinity with respect to propyl derivative 2b in our test
system, and a similar SAFIR trend was found for the couple of
bicyclic tetrazole derivatives 4b and 4g.6 However, a small
deviation from this trend was observed in the case of the short
series of carboxylic derivatives 4c,h,l, where the optimal
lipophilic substituent appeared to be the n-propyl group.6 In the
present series of compounds 5 and 6, the variation of the
lipophilic substituent in position 2 of the imidazo[4,5-b]pyridine
nucleus has an even more negligible effect than that already
observed in the other series.
Figure 4. Quantitative pharmacophore model Hypo1.
Functional Studies. Three nanomolar affinity AT1 receptor
ligands (5b, 6e, and the previously described 4b as reference
standard) were selected from the three structural subclasses of
the AT1 ligands described in the article, and their potential
antagonistic activity was investigated in vitro, using the
contractile response of isolated rabbit aortic strips as a functional
assay. In control experiments, the response to Ang II (5 nM)
that induced a submaximal contraction remained stable during
the entire experimental period, and no tachyphylaxis was
observed. However, compounds 4b, 5b, and 6e were devoid of
intrinsic activity and inhibited the response to Ang II showing
IC50 values well related to their binding affinities (Table 1).
Moreover, a significant reduction in the recovery of the
contractile response was observed after the washout of antago-
nists 4b, 5b, and 6e, providing evidence for an insurmountable
antagonism.
Figure 5. Mapping of highly active compounds (2a, 2b, 4b, 4i, 5d)
to quantitative pharmacophore model Hypo1.
Molecular Modeling Studies. To assess the prediction
capability of previously generated chemical feature based
pharmacophore models of AT1 receptor antagonists,10 a com-
parative study was performed, and the following results were
obtained. All compounds with activity values in the subnano-
molar range were identified by both a quantitative and a
qualitative pharmacophore model presented earlier, Hypo1 (five
feature pharmacophore model; seven points; one hydrophobic
aromatic, one hydrophobic aliphatic, a hydrogen bond acceptor,
a negative ionizable function, and an aromatic plane function)
presented in Figure 4, and Hypo2 (seven feature pharmacophore;
11 points; two aromatic rings, two hydrogen bond acceptors, a
negative ionizable function, and two hydrophobic functions)
presented in Figure 7, respectively.10 The mapping of the highest
active compounds (compounds 2a and 2b) is shown for the two
pharmacophore hypothesis in Figures 6 and 9. In the data set
of 34 compounds reported in Table 1, 22 (65%) were retrieved
by the fast search algorithm and 34 (100%) by the best search
algorithm within the software Catalyst.19 Five of the nine
compounds with high inhibitory potency against the AT1
receptor (i.e., 9 compounds show IC50 values higher than 10
nM) mapped on the pharmacophore pattern of Hypo1 are shown
in Figure 5. The mapping of the two substances with subna-
nomolar activity (compounds 2a and 2b) against the AT1
Figure 6. Mapping of compounds 2a and 2b to quantitative pharma-
cophore model Hypo1.
receptor is presented in Figure 6. The quantitative pharmacoph-
ore model (Hypo1) is able to detect almost the entire data set
of compounds with inhibitory activity against the AT1 receptor
presented in this study. The qualitative pharmacophore model
(Hypo2) was able to retrieve 23 of the 34 active AT1 receptor
antagonists in the fast flexible search procedure (68%) and 33
of the data set (34 compounds) in the best flexible search
procedure (97%). The activity estimation of the quantitative
pharmacophore model (Hypo1, Figure 4) yielded no satisfying
results. Therefore, we tried to predict activities by the qualitative
pharmacophore modeling approach. (Hypo 2, Figure 7) For this
procedure, we submitted the compounds to a calculation of the
best fit values. The fit value represents the mapping of chemical
substructures into feature constraints, as well as the distance
deviation of chemical functions from the center of the feature.
Therefore, the geometric fit value points out exactly how the

6458 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 22
Cappelli et al.
A qualitative predictive trend was also obvious in the compari-
son of the fit values and the measured activity data when only
compounds with IC50 values of 10 nM or even higher were
considered (nine compounds). Six of these nine molecules (67%)
were retrieved by our seven chemical feature HipHop pharma-
cophore model (Hypo2), considering five a fit borderline value
(Figure 8). Hence, only molecules with a fit value higher than
five were considered in this approach. The qualitative pharma-
cophore model (Hypo2) is able to discriminate between highly
active and moderately active compounds in about two-thirds
of the experimented cases. Enrichment can accelerate the time
of detection of new active compounds in the lead identification
phase. In summary, 6 of the nine highly active compounds (i.e.,
IC50 higher than 10 nM) and 16 of the 26 moderately active
compounds (IC50 values up to 100 nM) were predicted correctly
by the pharmacophore model Hypo2, considering the borderline
of a fit value higher than five. The two compounds with the
highest activity values are mapped onto pharmacophore model
Hypo2 in Figure 9.
Figure 7. Representation of qualitative pharmacophore model Hypo2.
Intestinal Permeability Experiments in Vitro. Permeability
studies performed with Caco-2 monolayers20 show that quinoline
derivative 4b and isoquinolinones 5b,f are chemical entities
characterized by a very low permeability in apical to basolateral
experiments (Papp values ranging from 0.10 to 0.01 × 10-6 cm/
s, Table 2). For comparison, propanolol (90% absorbed in
humans) showed a Papp value of 30.5 × 10-6 cm/s in the same
test and vinblastine (a well-known P-glycoprotein substrate) a
P
app value of 0.08 × 10-6 cm/s. In the short series of compounds
evaluated in such a test system, the most permeable was
3-tetrazolylquinoline derivative 4b, which showed a Papp value
very similar to that shown by vinblastine and was about 1 order
of magnitude more permeable than isoquinolinone derivative
5f. It is noteworthy that the decreased lipophilicity of ethyl
derivative 5b with respect to butyl derivative 5f is linked to a
slightly higher permeability.
Figure 8. Representation of Hypo2 mapped to six highly active
compounds (IC50 values lower than 10 nM: compounds 2a, 2b, 4b,
4i-k).
Pharmacokinetic Studies. The pharmacokinetic properties
of compounds 4b (CR3210), 4i,j,k, and 5b (CR3588) were
evaluated in rats by standard procedures7,8 in comparison with
reference AT1 antagonist 1 (losartan). The results shown in Table
2 suggest that compounds 4b,i,j,k, and 5b are barely bioavailable
chemical entities characterized by a poor intestinal absorption
and a rapid excretion. As expected, there is a positive correlation
between the oral bioavailability and the Caco-2 permeability
values of compounds 4b and 5b.20b In fact, 3-tetrazolylquinoline
derivative 4d, which showed a Papp value of 0.10 × 10-6 cm/s
showed a bioavailability of 49%, whereas isoquinolinone
derivative 5b, which showed a Papp value of 0.03 × 10-6 cm/s
was not bioavailable at all.
Figure 9. Mapping of compounds 2a and 2b to qualitative Hypo2.
function is localized at the center of a feature sphere. This value
ranks the compounds according to their mapping goodness on
a single pharmacophore model. In qualitative pharmacophore
models, each feature makes a contribution to the fit value of
one; hence, the Hypo2 can have a maximum fit value of seven
(seven features). The qualitative pharmacophore model was able
to retrieve 20 of the 26 substances (77%) with activity values
up to 100 nM in the fast flexible search algorithm and 26 of
the 26 substances (100%) using the best flexible search
algorithm. A best fit threshold of 5 was used to distinguish
between highly active and moderately active compounds. Under
these considerations, 16 compounds with activity values up to
100 nM were found to be above this borderline. Hence, 16 of
the 26 compounds (62%) were predicted by the pharmacophore
model Hypo2, considering five a fit threshold value in the fast
flexible search algorithm within the Catalyst software package.
Discussion
The starting point of our research program, focused on the
development of new AT1 receptor antagonists as antihyperten-
sive agents, was 4-phenyl-3-tetrazolylpyridine derivative 2d,
which was described both to show an affinity four times lower
than its carbaisoster 2c and to be an orally active Ang II
antagonist showing a poorer oral bioavailability (probably for
the decreased lipophilicity) than that of 2c.5 Our working
hypothesis concerned the introduction of a fused benzene ring
into the tetrazolylpyridyl derivative 2d structure in order to
ensure the lipophilicity necessary for good oral bioavailability.
The first step of the work led to the discovery of a series of
quinoline derivatives 4 showing in vitro properties comparable
to those shown by losartan. Investigations on the biopharma-
ceutical properties of the selected candidate for further pre-

Imidazo[4,5-b]pyridine AT1
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 22 6459
Table 2. Pharmacokinetic Parameters of Compounds 1, 2a, 4b,i,j,k, and 5b,f
Caco-2 permeability
dose
(mg/kg)
(route)
oral
bioavail.
(%)
app (a-b)b
AUC
(µg‚h/mL)
t1/2
Cmax
tmax
c
d
e
P
compd
MW
C log Pa
(× 10-6 cm/s)
(h)
(µg/mL)
(h)
4b (CR3210)
460.53
4.7
0.10
3 (iv)
30 (os)
3 (iv)
3 (iv)
3 (iv)
3 (iv)
10 (os)
0.89
4.4
0.68
1.0
0.30
0.33
0.28
0.22
4.2
0.5
49
0
4i
4j
4k
484.98
464.56
465.55
466.53
5.8
5.8
5.6
4.3
0.34
0.50
0.52
0.63
0
5b (CR3588)
0.03
5f
494.58
422.91
5.3
0.01
1 (losartan)
4.1f
1.15g
3 (iv)
30 (os)
12.4
74.8
4.7
3.0
27.5
0.5
60
a C log P calculated by means of CS ChemDraw Ultra 8.0 (Cambridge Soft Corporation, Cambridge, MA 02140). b Apical to basolateral Caco-2 permeability.
d
e
c t1/2: terminal half-life. Cmax: observed maximum concentration after administration. tmax: time to reach maximum concentration. f For comparison, the
log PHPLC value described in the literature was 4.2; see ref 21. g See ref 21.
Interestingly, the SAFIR obtained with the newly synthesized
compounds showed that the isoquinolinone moiety of com-
pounds 5b,d,f is better (from the point of view of the interaction
with the AT1 receptor) than benzene, naphthalene, and quinoline
of compounds 2 and 4. The isoquinolinone moiety looks like a
molecular fragment optimized for the interaction with the top
of the AT1 receptor binding cavity, but it appears to be
incompatible with the presence of the tetrazole moiety because
the tetrazolyl derivatives 5a,c,e were found (to our surprise) to
be inactive. Owing to the in vitro potency of carboxylic acids
5b,d,f, their evaluation in pharmacokinetic studies was per-
formed showing that the bioavailability problems of compound
5b are probably related to its very low permeability (Papp (a-
b) ) 0.03 × 10-6 cm/s).20b
clinical studies (CR3210, 4b) revealed a rather complex picture
from which the low permeability, the relatively low oral
bioavailability (49%, Table 2), the rapid excretion (t1/2 ) 0.68-
1.0 h),8 and the fast conjugation with glucuronic acid were
clearly demonstrated.22 Therefore, the transformation of the
distal phenyl ring of L-158,809 (or the pyridine one of 2d) into
the quinoline nucleus of 4b was tolerated at the receptor level,
but it was deleterious for pharmacokinetic properties. In other
words, our hypothesis on the importance of lipophilicity for oral
bioavailability and, more in general, for suitability of suitable
pharmacokinetic properties was not correct in the case of this
class of AT1 receptor antagonists. Because the main difference
between 4b (CR3210) and 2a (L-158,809) appeared to be the
higher effectiveness of the conjugation of 4b with glucuronic
acid,22 the design of the new AT1 receptor antagonists should
appropriately consider the interaction of the UDP-glucurono-
syltransferases (EC 2.4.1.17) because these appear to play a key
role in the metabolism (and excretion) of tetrazole derivatives.23
Moreover, the pharmacokinetic studies performed on other
quinoline derivatives selected on the basis of their in vitro
potency (compounds 4i,j,k) showed very rapid excretion (t1/2
values in the range from 0.22 to 0.33 h). Because small structural
modifications could affect the interaction with hepatic transport-
ers and/or with UDP-glucuronosyltransferases in a significant
way, both isoquinolinone derivatives 5 and indene derivatives
6 were designed and synthesized.
As a final step of the work, we explored the chemistry and
the structure-affinity relationships of indene derivatives 6. The
results of the binding studies performed on compounds 6 were
not very exciting, because the first members of this subclass
(6a,b) proved to be 1 order of magnitude less potent than
isoquinolinones 5b,d,f, and only after meticulous optimization
work, we were able to obtain compounds 6e,f provided with
more interesting AT1 receptor affinity. The optimization also
produced interesting SAFIR data, which provided information
on the binding site topology and above all a polymerizing AT1
receptor ligand (6c). This bioactive monomer formed a ther-
moreversible polymer (poly-6c) and was released from the latter

6460 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 22
Cappelli et al.
1
for TLC. H NMR spectra were recorded with a Bruker AC 200
spectrometer in the indicated solvents (TMS as the internal
standard): the values of the chemical shifts are expressed in ppm
and the coupling constants (J) in Hz. Mass spectra were recorded
on either a Varian Saturn 3 spectrometer or a ThermoFinnigan LCQ-
Deca.
General Procedure for the Preparation of Compounds 11a-f
and 15a,b (Radical Bromination-Coupling Procedure). A mix-
ture of the toluene derivative 10, 13, and 14 in 40 mL of CCl4
with N-bromosuccinimide (1.02 equiv) and dibenzoyl peroxide (0.1
equiv) 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 succin-
imide 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]pyridine1
(1.0 equivalent) in anhydrous DMF (10 mL) with NaH (1.0
equivalent). The resulting mixture was stirred at room temperature
for 15-18 h under argon, and the reaction was quenched with ice-
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 ethyl acetate. The combined organic extracts were
washed with brine, dried over sodium sulfate and concentrated under
reduced pressure. Purification of the residue by flash chromatog-
raphy with ethyl acetate-petroleum ether (7:3) (or ethyl acetate) as
the eluent gave pure compounds 11a-f and 15a,b.
Figure 10. Competition curves of poly-6c, its monomer 6c, and PTSA
obtained in AT1 receptor binding studies. Stock solutions of 6c and
poly-6c (see Biological Methods section for details) were freshly
prepared and then stored at 37 °C for 3 or 6 days. PTSA (×) was
assayed because of its presence as an impurity of poly-6c and was
revealed to be inactive. The assays performed on freshly prepared
solutions of poly-6c ([) revealed the presence of a detectable dose-
dependent inhibition of the radioligand specific binding to AT1
receptors. The inhibition curve of poly-6c is significantly shifted to
the left because of the use of a stock solution stored in buffer at 37 °C
for the first 3 days (9), but it did not change because of being stored
for an additional 3 days (2). On the contrary, monomer 6c, tested in
parallel in each assay, was insensitive to the storage conditions; values
(O) indicated in the Figure are the mean ( standard error of three
replicates.
Ethyl 1,2-Dihydro-4-[4-[(5,7-dimethyl-2-ethyl-3H-imidazo-
[4,5-b]pyridin-3-yl)methyl]phenyl]-2-methyl-1-oxo-3-isoquino-
linecarboxylate (11b). The title compound was prepared in 36%
yield (0.33 g, mp 152-155 °C) starting from 10 (0.60 g, 1.87 mmol)
and 5,7-dimethyl-2-ethyl-3H-imidazo[4,5-b]pyridine (0.33 g, 1.9
mmol) according to the general procedure for radical bromination
1
and coupling. H NMR (CDCl3): 0.87 (t, J ) 7.1, 3H), 1.34 (t, J
with temperature-dependent kinetics until the establishment of
a temperature-dependent equilibrium as it occurs in equilibrium
polymerization processes (in these phenomena, the equilibrium
monomer concentration is temperature-dependent).24 Preliminary
binding studies performed on poly-6c suggested that a detectable
depolymerization occurred in buffer (pH 7.4) at room temper-
ature, it increased its extent after a storage of the polymer
solution at 37 °C and appeared to reach equilibrium within about
3 days at 37 °C (Figure 10). Interestingly, the apparent IC50 of
poly-6c determined at the polymerization equilibrium appeared
to be about 2 orders of magnitude higher than that shown by
monomer 6c. This result suggests that in these conditions poly-
6c released one molecule of monomer 6c each about 100
monomeric units. Further studies are in progress to define with
precision the physicochemical constants of this equilibrium
polymerization phenomenon and to explore the potentiality of
this mechanism in drug controlled release from new polymeric
prodrugs.
Finally, 34 compounds belonging to these structural sub-
classes of AT1 receptor antagonists (4,5, and 6) were used as a
new test to evaluate the predictive capability of the previously
published qualitative and quantitative pharmacophore mod-
els.10,25 The results obtained in the validation of these models
suggest that they can be used to evaluate new virtual libraries
of potential AT1 receptor antagonists in order to discover novel
leads.
) 7.6, 3H), 2.59 (s, 3H), 2.63 (s, 3H), 2.84 (q, J ) 7.6, 2H), 3.57
(s, 3H), 3.97 (q, J ) 7.1, 2H), 5.51 (s, 2H), 6.90 (s, 1H), 7.18-
7.27 (m, 5H), 7.47-7.56 (m, 2H), 8.45-8.50 (m, 1H). MS(ESI)
m/z 495 (M + H+).
Ethyl 1,2-Dihydro-4-[4-[(5,7-dimethyl-2-propyl-3H-imidazo-
[4,5-b]pyridin-3-yl)methyl]phenyl]-2-methyl-1-oxo-3-isoquino-
linecarboxylate (11d). The title compound was prepared in 32%
yield (0.10 g) starting from 10 (0.20 g, 0.62 mmol) and 5,7-
dimethyl-2-propyl-3H-imidazo[4,5-b]pyridine (0.12 g, 0.63 mmol)
according to the general procedure for radical bromination and
1
coupling. H NMR (CDCl3): 0.86 (t, J ) 7.3, 3H), 0.97 (t, J )
7.3, 3H), 1.68-1.85 (m, 2H), 2.57 (s, 3H), 2.61 (s, 3H), 2.79 (t, J
) 7.6, 2H), 3.56 (s, 3H), 3.96 (q, J ) 7.3, 2H), 5.50 (s, 2H), 6.88
(s, 1H), 7.17-7.26 (m, 5H), 7.47-7.51 (m, 2H), 8.43-8.48 (m,
1H).
Ethyl 1,2-Dihydro-4-[4-[(2-butyl-5,7-dimethyl-3H-imidazo-
[4,5-b]pyridin-3-yl)methyl]phenyl]-2-methyl-1-oxo-3-isoquino-
linecarboxylate (11f). The title compound was prepared in 34%
yield (0.14 g) starting from 10 (0.25 g, 0.78 mmol) and 2-butyl-
5,7-dimethyl-3H-imidazo[4,5-b]pyridine (0.16 g, 0.79 mmol) ac-
cording to the general procedure for radical bromination and
1
coupling. H NMR (CDCl3): 0.86-0.96 (m, 6H), 1.32-1.51 (m,
2H), 1.67-1.82 (m, 2H), 2.60 (s, 3H), 2.64 (s, 3H), 2.81 (t, J )
7.9, 2H), 3.59 (s, 3H), 3.99 (q, J ) 7.2, 2H), 5.52 (s, 2H), 6.91 (s,
1H), 6.90-7.14 (m, 1H), 7.19-7.25 (m, 4H), 7.50-7.54 (m, 2H),
8.47-8.51 (m, 1H). MS(ESI) m/z 523 (M + H+).
4-[4-[(5,7-Dimethyl-2-ethyl-3H-imidazo[4,5-b]pyridin-3-yl)-
methyl]phenyl]-2-methyl-3-[2-(triphenylmethyl)-2H-tetrazol-5-yl]-1(2H)-
isoquinolinone (11a). The title compound was prepared in 45%
yield (0.070 g, mp 174-180 °C) starting from 13 (0.12 g, 0.21
mmol) and 5,7-dimethyl-2-ethyl-3H-imidazo[4,5-b]pyridine (0.040
g, 0.23 mmol) according to the general procedure for radical
Experimental Section
Chemistry. All chemicals used were of reagent grade. Yields
refer to purified products and are not optimized. Melting points
were determined in open capillaries on a Gallenkamp apparatus
and are uncorrected. Microanalyses were carried out by means of
a Perkin-Elmer 240C or a Perkin-Elmer Series II CHNS/O Analyzer
2400. Merck silica gel 60 (230-400 mesh) was used for column
chromatography. Merck TLC plates, silica gel 60 F254 were used
1
bromination and coupling. H NMR (CDCl3): 1.25 (t, J ) 7.6,
3H), 2.58 (s, 3H), 2.66 (s, 3H), 2.71 (q, J ) 7.6, 2H), 3.30 (s, 3H),
5.42 (s, 2H), 6.88-6.93 (m, 7H), 7.04-7.15 (m, 4H), 7.21-7.34
(m, 10H), 7.51-7.56 (m, 2H), 8.49-8.58 (m, 1H). MS(ESI) m/z
733 (M + H+).

Imidazo[4,5-b]pyridine AT1
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 22 6461
4-[4-[(5,7-Dimethyl-2-propyl-3H-imidazo[4,5-b]pyridin-3-yl)-
methyl]phenyl]-2-methyl-3-[2-(triphenylmethyl)-2H-tetrazol-5-
yl]-1(2H)-isoquinolinone (11c). The title compound was prepared
in 41% yield (0.065 g, mp 170-174 °C) starting from 13 (0.12 g,
0.21 mmol) and 5,7-dimethyl-2-propyl-3H-imidazo[4,5-b]pyridine
(0.04 g, 0.21 mmol) according to the general procedure for radical
6H), 2.83 (t, J ) 7.7, 2H), 3.70 (s, 3H), 5.47 (s, 2H), 6.88 (s, 1H),
7.05 (d, J ) 7.7, 1H), 7.19 (d, J ) 8.1, 2H), 7.32-7.47 (m, 4H),
8.42-8.45 (m, 1H). MS(ESI negative ions) m/z 479 (M - H+).
Anal. (C29H28N4O3‚0.5 H2O) C,H,N.
1,2-Dihydro-4-[4-[(2-butyl-5,7-dimethyl-3H-imidazo[4,5-b]py-
ridin-3-yl)methyl]phenyl]-2-methyl-1-oxo-3-isoquinolinecarbox-
ylic Acid (5f). This compound was prepared in 79% yield (0.090
g, mp 292-295 °C) starting from ethyl ester 11f (0.12 g, 0.23
mmol) according to the general procedure for the basic hydrolysis.
1H NMR (CDCl3): 0.70 (t, J ) 7.1, 3H), 1.03-1.14 (m, 2H), 1.32-
1.40 (m, 2H), 2.58-2.69 (m, 8H), 3.74 (s, 3H), 5.53 (s, 2H), 7.00
(s, 1H), 7.08-7.12 (m, 1H), 7.22 (d, J ) 8.0, 2H), 7.41-7.55 (m,
4H), 8.48-8.52 (m, 1H). MS(ESI negative ions) m/z 493 (M -
H+). Anal. (C30H30N4O3‚0.5 H2O) C,H,N.
Preparation of Target Tetrazole Derivatives 5a,c,e (Depro-
tection of the Trityl-Protected Tetrazole Derivatives). A mixture
of the appropriate trityl-protected tetrazole derivative (0.4-0.6
mmol) with formic acid (15 mL) was stirred at room temperature
under argon for a suitable time (18-48 h), and the reaction progress
was monitored by TLC. When the trityl-protected tetrazole deriva-
tive disappeared from the chromatogram, the reaction mixture was
evaporated under reduced pressure. Purification of the residue by
washing with diethyl ether or ethyl acetate gave the pure target
compounds.
4-[4-[(5,7-Dimethyl-2-ethyl-3H-imidazo[4,5-b]pyridin-3-yl)-
methyl]phenyl]-2-methyl-3-(2H-tetrazol-5-yl)-1(2H)-isoquinolinone
(5a). This compound was prepared in 68% yield (0.020 g of white
solid melting at 194-198 °C) starting from the protected tetrazolyl
derivate 11a (0.044 g, 0.060 mmol) according to the general
procedure for acid hydrolysis. 1H NMR (CDCl3): 0.95 (br t, 3H),
2.59 (s, 3H), 2.63 (s, 3H), 2.85 (br q, 2H), 3.30 (s, 3H), 3.98 (br s,
H+ + H2O), 5.52 (s, 2H), 7.02-7.06 (m, 3H), 7.13-7.20 (m, 3H),
7.53-7.57 (m, 2H), 8.52-8.57 (m, 1H). MS(ESI) m/z 491 (M +
H+). Anal. (C28H26N8O‚2 H2O) C,H,N.
4-[4-[(5,7-Dimethyl-2-propyl-3H-imidazo[4,5-b]pyridin-3-yl)-
methyl]phenyl]-2-methyl-3-(2H-tetrazol-5-yl)-1(2H)-isoquinoli-
none (5c). This compound was prepared in 73% yield (0.022 g of
white solid melting at 174-179 °C) starting from protected
tetrazolyl derivate 11c (0.045 g, 0.060 mmol) according to the
general procedure for acid hydrolysis. 1H NMR (CDCl3): 0.77 (br
t, 3H), 1.40-1.60 (br m, 2H), 2.64 (s, 6H), 2.86 (br t, 2H), 3.29 (s,
3H), 3.50 (br s, H+ + H2O), 5.53 (s, 2H), 7.02-7.28 (m, 6H),
7.49-7.75 (m, 2H), 8.49-8.64 (m, 1H). MS(ESI) m/z 505 (M +
H+). Anal. (C29H28N8O‚H2O) C,H,N.
4-[4-[(2-Butyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-yl)-
methyl]phenyl]-2-methyl-3-(2H-tetrazol-5-yl)-1(2H)-isoquino-
linone (5e). This compound was prepared in 98% yield (0.023 g
of white solid melting at 169-173 °C) starting from protected
tetrazolyl derivate 11e (0.034 g, 0.045 mmol) according to the
general procedure for acid hydrolysis. 1H NMR (CDCl3, TEA): 0.88
(t, J ) 7.2, 3H), 1.30-1.41 (m, 2H), 1.56-1.71 (m, 2H), 2.55 (s,
3H), 2.59 (s, 3H), 2.75 (t, J ) 7.3, 2H), 3.28 (s, 3H), 4.30 (br s,
H+ + H2O), 5.33 (s, 2H), 6.86 (s, 1H), 6.96 (d, J ) 7.9, 2H), 7.08-
7.15 (m, 3H), 7.45-7.49 (m, 2H), 8.49-8.54 (m, 1H). MS(ESI)
m/z 519 (M + H+). Anal. (C30H30N8O‚0.5 H2O) C,H,N.
Preparation of Target Carboxylic Acid Derivatives 6a,b,e,f
(Acid Hydrolysis). A mixture of the suitable ester (0.1-0.39 mmol)
with formic acid (15 mL) was stirred at room temperature under
argon for a suitable time (typically 18h), and the reaction progress
was monitored by TLC. When the ester disappeared from the
chromatogram, the reaction mixture was evaporated under reduced
pressure. Purification of the residue by washing with diethyl ether
gave the pure target compounds.
3-[4-[(5,7-Dimethyl-2-ethyl-3H-imidazo[4,5-b]pyridin-3-yl)-
methyl]phenyl]-1-oxo-1H-indene-2-carboxylic Acid (6a). This
compound was prepared in 80% yield (0.035 g of yellow solid
melting at 197-198 °C) starting from the tert-butyl ester 15a (0.050
g, 0.10 mmol) according to the general procedure for acid
hydrolysis. 1H NMR (CDCl3): 1.31 (t, J ) 7.5, 3H), 2.58 (s, 3H),
2.62 (s, 3H), 2.71 (q, J ) 7.5, 2H), 5.53 (s, 2H), 6.90 (s, 1H), 7.16
1
bromination and coupling. H NMR (CDCl3): 0.90 (t, J ) 7.3,
3H), 1.57-1.73 (m, 2H), 2.58 (s, 3H), 2.64-2.71 (m, 5H), 3.29
(s, 3H), 5.42 (s, 2H), 6.88-6.91 (m, 7H), 7.04-7.14 (m, 4H), 7.21-
7.37 (m, 10H), 7.51-7.55 (m, 2H), 8.50-8.57 (m, 1H). MS(ESI)
m/z 747 (M + H+).
4-[4-[(2-Butyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-yl)-
methyl]phenyl]-2-methyl-3-[2-(triphenylmethyl)-2H-tetrazol-5-yl]-1(2H)-
isoquinolinone (11e). The title compound was prepared in 74%
yield (0.13 g, mp 160-162 °C) starting from 13 (0.13 g, 0.23 mmol)
and 5,7-dimethyl-2-butyl-3H-imidazo[4,5-b]pyridine (0.050 g, 0.25
mmol) according to the general procedure for radical bromination
and coupling. 1H NMR (CDCl3): 0.86 (t, J ) 7.1, 3H), 1.21-1.41
(m, 2H), 1.54-1.70 (m, 2H), 2.58 (s, 3H), 2.64 (s, 3H), 2.70 (t, J
) 7.6, 2H), 3.29 (s, 3H), 5.41 (s, 2H), 6.88-6.91 (m, 7H), 7.04-
7.14 (m, 4H), 7.22-7.38 (m, 10H), 7.50-7.57 (m, 2H), 8.50-
8.55 (m, 1H). MS(ESI) m/z 761 (M + H+).
tert-Butyl 3-[4-[(5,7-Dimethyl-2-ethyl-3H-imidazo[4,5-b]py-
ridin-3-yl)methyl]phenyl]-1-oxo-1H-indene-2-carboxylate (15a).
The title compound was prepared in 33% yield (0.10 g of yellow
solid melting at 119-123 °C) starting from 14 (0.20 g, 0.62 mmol)
and 5,7-dimethyl-2-ethyl-3H-imidazo[4,5-b]pyridine (0.11 g, 0.63
mmol) according to the general procedure for radical bromination
1
and coupling. H NMR (CDCl3): 1.32 (t, J ) 7.4, 3H), 1.29 (s,
9H), 2.56 (s, 3H), 2.61 (s, 3H), 2.78 (q, J ) 7.4, 2H), 5.51 (s, 2H),
6.88 (s, 1H), 7.04 (m, 1H), 7.23 (d, J ) 8.1, 2H), 7.33 (m, 2H),
7.41 (d, J ) 8.1, 2H), 7.53 (m, 1H). MS(ESI) m/z 494 (M + H+).
tert-Butyl 3-[4-[(5,7-Dimethyl-2-propyl-3H-imidazo[4,5-b]py-
ridin-3-yl)methyl]phenyl]-1-oxo-1H-indene-2-carboxylate (15b).
The title compound was prepared in 46% yield (0.80 g of yellow
glassy solid) starting from 14 (1.1 g, 3.4 mmol) and 5,7-dimethyl-
2-propyl-3H-imidazo[4,5-b]pyridine (0.67 g, 3.5 mmol) according
to the general procedure for radical bromination and coupling.1H
NMR (CDCl3): 0.97 (t, J ) 7.4, 3H), 1.31 (s, 9H), 1.69-1.88 (m,
2H), 2.58 (s, 3H), 2.62 (s, 3H), 2.77 (t, J ) 7.7, 2H), 5.53 (s, 2H),
6.90 (s, 1H), 7.06 (m, 1H), 7.24 (d, J ) 8.3, 2H), 7.34 (m, 2H),
7.42 (d, J ) 8.3, 2H), 7.53 (m, 1H). MS(ESI) m/z 508 (M + H+).
Preparation of Target Carboxylic Acid Derivatives 5b,d,f
(Basic Hydrolysis). To a solution of the appropriate ester (11b,d,f)
(0.2-0.6 mmol) in ethanol (20 mL), 2 N NaOH (2.0 mL) was
added, and the resulting mixture was refluxed while the reaction
progress was monitored by TLC. When the ester derivative
disappeared from the chromatogram, the reaction mixture was
evaporated under reduced pressure and diluted with water (20 mL),
and the pH was adjusted to 5-6 by the 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 washing with ethyl
acetate or diethyl ether gave the pure target carboxylic acid
derivatives.
1,2-Dihydro-4-[4-[(5,7-dimethyl-2-ethyl-3H-imidazo[4,5-b]py-
ridin-3-yl)methyl]phenyl]-2-methyl-1-oxo-3-isoquinolinecarbox-
ylic Acid (5b). This compound was prepared in 83% yield (0.22
g, white solid, mp >300 °C) starting from the ethyl ester 11b (0.28
g, 0.57 mmol) according to the general procedure for basic
hydrolysis. 1H NMR (CDCl3): 1.38 (t, J ) 7.6, 3H), 2.61 (s, 6H),
2.88 (q, J ) 7.6, 2H), 3.69 (s, 3H), 5.46 (s, 2H), 6.88 (s, 1H), 7.05
(d, J ) 8.1, 1H), 7.19 (d, J ) 8.1, 2H), 7.32-7.47 (m, 4H), 8.42-
8.45 (m, 1H). MS(ESI negative ions) m/z 465 (M - H+). Anal.
(C28H26N4O3‚H2O) C,H,N.
1,2-Dihydro-4-[4-[(5,7-dimethyl-2-propyl-3H-imidazo[4,5-b]-
pyridin-3-yl)methyl]phenyl]-2-methyl-1-oxo-3-isoquinolinecar-
boxylic Acid (5d). This compound was prepared in 83% yield
(0.080 g, mp >300 °C) starting from ethyl ester 11d (0.10 g, 0.20
mmol) according to the general procedure for basic hydrolysis. 1H
NMR (CDCl3): 0.91 (t, J ) 7.5, 3H), 1.74-1.89 (m, 2H), 2.59 (s,

6462 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 22
Cappelli et al.
(m, 1H), 7.26 (d, J ) 8.4, 2H), 7.44 (m, 2H), 7.60 (m, 3H). MS-
(ESI negative ions) m/z 436 (M - H+). Anal. (C27H23N3O3‚0.33
H2O) C,H,N.
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 the assay buffer (50 mM Tris-HCl,
100 mM NaCl, 10 mM MgCl2, 1 mM EDTA, 100 µM bacitracin,
100 µM PMSF, 0.1% BSA at pH 7.4) to obtain a final protein
concentration of 0.25 mg/mL. The binding of [125I]Sar1,Ile8-
Angiotensin II (Perkin-Elmer Life and Analytical Sciences, S. A.
2000 Ci/mmol) to liver membranes was performed at 25 °C for
180 min in 96-well filtration plates (Millipore GFB-Multiscreen).
Each 250 µL incubate contained 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. Binding was terminated by rapid vacuum filtration
using a Millipore Multiscreen device. The receptor-ligand complex
trapped on filters was washed twice with 200 µL of ice-cold 100
mM NaCl and 100 mM MgCl2. 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
3-[4-[(5,7-Dimethyl-2-propyl-3H-imidazo[4,5-b]pyridin-3-yl)-
methyl]phenyl]-1-oxo-1H-indene-2-carboxylic Acid (6b). This
compound was prepared in 91% yield (0.16 g of yellow solid
melting at 209-213 °C) starting from tert-butyl ester 15b (0.20 g,
0.39 mmol) according to the general procedure for acid hydrolysis.
1H NMR (CDCl3): 0.94 (t, J ) 7.3, 3H), 1.66-1.85 (m, 2H), 2.58
(s, 3H), 2.62 (s, 3H), 2.78 (t, J ) 7.7, 2H), 5.54 (s, 2H), 6.90 (s,
1H), 7.17 (m, 1H), 7.26 (d, J ) 8.4, 2H), 7.44 (m, 2H), 7.61 (m,
3H). MS(ESI negative ions) m/z 450 (M - H+). Anal. (C28H25N3O3‚
0.5 H2O) C,H,N.
3-[4-[(5,7-Dimethyl-2-propyl-3H-imidazo[4,5-b]pyridin-3-yl)-
methyl]phenyl]-1-hydroxy-1-methyl-1H-indene-2-carboxylic Acid
(6e). This compound was prepared from tert-butyl ester 16e (0.15
g, 0.29 mmol) according to the general procedure for acid hydrolysis
(reaction time 45 min) and was purified by washing with ether to
give 0.045 g (yield 33%) of 6e as a white solid (mp 246-247 °C).
1H NMR (DMSO-d6): 0.88 (t, J ) 7.3, 3H), 1.58-1.70 (m, 5H),
2.49 (s, 6H), 2.74 (t, J ) 7.5, 2H), 5.27 (br s, 1H), 5.50 (s, 2H),
6.96 (m, 2H), 7.13-7.38 (m, 6H), 7.47 (d, J ) 7.2, 1H), 12.20 (br
s, 1H). MS (ESI): m/z 468 (M + H+). Anal. (C29H29N3O3‚0.33
H2O) C,H,N.
[
125I]Sar1,Ile8-Angiotensin II) was estimated for the linear portion
of the competition curves.
To evaluate the effect of depolymerization of poly-6c, stock
solutions of 6c and poly-6c in 40% (v/v) DMSO in buffer (50 mM
Tris-HCl, 100 mM NaCl, 10 mM MgCl2, 1 mM EDTA, pH 7.4)
were freshly prepared, divided into aliquots, and stored at 37 °C
for 3 or 6 days. The day of the assay, the solutions were diluted in
assay buffer (working solutions) and tested. The final DMSO
concentrations did not significantly interfere with the [125I]Sar1,-
Ile8-Angiotensin II specific binding.
3-[4-[(5,7-Dimethyl-2-propyl-3H-imidazo[4,5-b]pyridin-3-yl)-
methyl]phenyl]-1-ethyl-1-hydroxy-1H-indene-2-carboxylic Acid
(6f). This compound was prepared from tert-butyl ester 16f (0.030
g, 0.056 mmol) according to the general procedure for acid
hydrolysis (reaction time 45 min) and was purified by washing with
1
ether to obtain 0.011 g, mp 232-234 °C (yield 41%). H NMR
Angiotensin II Functional Antagonism in Rabbit Aorta
Strips.15 New Zealand White rabbits (3-4 kg of 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‚7 H2O 1.17;
CaCl2‚2 H2O 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 4-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
concentration-response curve. After 30 min of 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 60
min contact with the tissues was allowed before adding the agonist
to the bathing fluid. Only one antagonist concentration was tested
on each tissue preparation. The antagonist activity was expressed
as percentage of inhibition of the agonist contractions. The
regression line was calculated, and the concentration effective in
inhibiting the effect of the agonist by 50% (IC50) was calculated
from the regression line.
Intestinal Permeability Experiments In Vitro. The perme-
ability studies were performed with Caco-2 monolayers as described
in the literature.20 Caco-2 cells were cultured in supplemented
Dulbecco’s modified eagle medium with 10% fetal bovine serum
and seeded onto polycarbonate membranes for test compound
transport experiments. Caco-2 cell membranes were grown by
seeding on Snapwell supports incubated at 37 °C with 5% CO2/
95% O2 and approximately 95% humidity for 15 to 21 days. Drug
permeability experiments were performed at 37 °C at a final drug
concentration of 10-100 µM in HBSS buffer (1% DMSO) in the
apical chamber (donor side in the apical to basolateral (a-b)
permeability study). These concentrations did not show any
cytotoxic effect on Caco-2 cells. The samples were obtained from
the apical side and from the basolateral chamber at regular time
intervals and snap frozen on dry ice/methanol. Drug concentrations
(DMSO-d6): 0.41 (t, J ) 7.3, 3H), 0.88 (t, J ) 7.3, 3H), 1.58-
1.77 (m, 2H), 1.98-2.07 (m, 1H), 2.28-2.38 (m, 1H), 2.49 (s,
6H), 2.74 (t, J ) 7.6, 2H), 5.50 (s, 2H), 6.94 (m, 2H), 7.15-7.43
(m, 7H), 12.10 (br s, 1H). MS (ESI negative ions): m/z 480 (M -
H+). Anal. (C30H31N3O3‚1.5 H2O) C,H,N.
X-ray Crystallography. Single crystals of 19‚0.5 CH3COOC2H5
and 21‚H2O were submitted for 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 two structures were solved by direct methods implemented
in the SHELXS-97 program.26 The refinements were carried out
by full-matrix anisotropic least-squares on F2 for all reflections for
non-H atoms by using the SHELXL-97 program.27
The structure of 19‚0.5 CH3COOC2H5 shows a statistical disorder
for the -CH2-CH3 group of the pendant propyl moiety. For these
atoms, two different positions were refined with site occupation
factors of 0.54(1) and 0.46(1), respectively. The atoms of the ethyl
acetate moiety have been treated with a common site occupation
factor that has been refined to 0.47(1).
Crystallographic data (excluding structure factors) for both the
crystal structures reported in this article have been deposited with
the Cambridge Crystallographic Data Centre as supplementary
publication numbers CCDC600440 (19‚0.5 CH3COOC2H5) and
CCDC600439 (21‚H2O). Copies of the data can be obtained, free
of charge, on application to CCDC, 12 Union Road, Cambridge
CB2 1EZ, U.K. (fax: 144-(0)1223-336033 or e-mail: deposit@
ccdc.cam.ac.uk).
Biological Methods. Angiotensin II Receptor Binding Assay.15
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 centrifuga-
tion. The liver was dissected free of fatty tissue and minced
accurately with small scissors, and about 3 g of tissue were
homogenized by Polytron Ultra-Turrax (maximal speed for 2 ×
30 s) in ice cold 20 vol of 5 mM Tris-HCl and 0.25 M sucrose (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 centrifuged at 50 000g for 15 min. The resulting
pellet was resuspended in 5 mM Tris-HCl and 0.25 M sucrose (pH

Imidazo[4,5-b]pyridine AT1
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 22 6463
were determined by HPLC-MS. [14C]Mannitol permeability was
used as a parameter for assessing monolayer integrity, and
propanolol and vinblastine were evaluated as controls. Caco-2
apparent permeability values (Papp) were calculated by means of
the following equation
(8) Rizzo, M.; Ventrice, D.; Monforte, F.; Procopio, S.; De Sarro, G.;
Anzini, M.; Cappelli, A.; Makovec, F. Sensitive SPE-HPLC Method
to Determine a Novel Angiotensin-AT1 Antagonist in Biological
Samples. J. Pharm. Biomed. Anal. 2004, 35, 321-329.
(9) As far as the pharmacokinetic properties are concerned, it should be
noted that even the marketed AT1 receptor antagonists show limited
oral bioavailability (eprosartan 13%, valsartan 25%, losartan 33%,
candesartan 42%, telmisartan 50%, and irbesartan 60-80%). ((a)
Israili, Z. H. Clinical Pharmacokinetics of Angiotensin II (AT1)
Receptor Blockers in Hypertension. J. Hum. Hypertens. 2000, 14,
S73-S86. (b) Brunner, H. R. The New Angiotensin II Receptor
Antagonist, Irbesartan. Am. J. Hypertens. 1997, 10, 311S-317S.)
Furthermore, losartan exhibiting highly variable oral bioavailability
is transported by P-glycoprotein, whereas its carboxylic acid
metabolite is not a P-glycoprotein substrate (Soldner, A.; Benet, L.
Z.; Mutschler, E.; Christians, U. Active Transport of the Angiotensin-
II Antagonist Losartan and Its Main Metabolite EXP 3174 Across
MDCK-MDR1 and Caco-2 Cell Monolayers. Br. J. Pharmacol. 2000,
129, 1235-1243).
(10) Krovat, E. M.; Langer, T. Non-Peptide Angiotensin II Receptor
Antagonists: Chemical Feature Based Pharmacophore Identification.
J. Med. Chem. 2003, 46, 716-726.
(11) Natsugari, H.; Ikeura, Y.; Kiyota, Y.; Ishichi, Y.; Ishimaru, T.; Saga,
O.; Shirafuji, H.; Tanaka, T.; Kamo, I.; Doi, T.; Otsuka, M. Novel,
Potent, and Orally Active Substance P Antagonists: Synthesis and
Antagonist Activity of N-Benzylcarboxamide Derivatives of Pyrido-
[3,4-b]pyridine. J. Med. Chem. 1995, 38, 3106-3120.
(12) (a) Senanayake, C. H.; Fredenburgh, L. E.; Reamer, R. A.; Liu, J.;
Roberts, F. E.; Humphrey, G.; Thompson, A. S.; Larsen, R. D.;
Verhoeven, T. R.; Reider, P. J.; Shinkai, I. New Approach to the
Imidazolutidine Moiety of MK-996. Heterocycles 1996, 42, 821-
830. (b) Chakravarty, P. K.; Greenlee, W. J.; Mantlo, N. B.; Patchett,
A. A.; Walsh, T. F. Substituted Imidazo-Fused 6-Membered Het-
erocycles as Angiotensin II Antagonists. U.S. Patent 5,332,744, 1994.
(13) Cappelli, A.; Pericot Mohr, G.; Anzini, M.; Vomero, S.; Donati, A.;
Casolaro, M.; Mendichi, R.; Giorgi, G.; Makovec, F. Synthesis and
Characterization of a New Benzofulvene Polymer Showing a
Thermoreversible Polymerization Behavior. J. Org. Chem. 2003, 68,
9473-9476.
(14) Cappelli, A.; Anzini, M.; Vomero, S.; Donati, A.; Zetta, L.; Mendichi,
R.; Casolaro, M.; Lupetti, P.; Salvatici, P.; Giorgi, G. New π-Stacked
Benzofulvene Polymer Showing Thermoreversible Polymerization:
Studies in Macromolecular and Aggregate Structures and Polymer-
ization Mechanism. J. Polym. Sci., Part A: Polym. Chem. 2005, 43,
3289-3304.
(15) (a) Chang, R. S. L.; Siegl, P. K. S.; Clineschmidt, B. W.; Mantlo, N.
B.; Chakravarty, P. K.; Greenlee, W. J.; Patchett, A. A.; Lotti, V. J.
In vitro Pharmacology of L-158,809, a New Highly Potent and
Selective Angiotensin II Receptor Antagonists. J. Pharmacol. Exp.
Ther. 1992, 262, 133-138. (b) Robertoson, M. J.; Cunoosamy, M.
P.; Clark, K. L. Effects of Peptidase Inhibition on Angiotensin
Receptor Agonist and Antagonist Potency in Rabbit Isolated Thoracic
Aorta. Br. J. Pharmacol. 1992, 106, 166-172.
(16) Naphthalene derivative 4a was described in a patent application and
resynthesized in our laboratory as a reference compound for our
quinoline derivatives (Suzuki, K.; Fujiwara, S.; Machii, D.; Ochifuji,
N.; Takai, H.; Oono, T.; Furuta, S.; Yamada, K. Preparation of
Imidazopyridines and Analogs as Angiotensin II Antagonists. Jpn.
Kokai Tokkyo Koho JP 06 92,939, 1994. and Fujiwara, S.; Takai,
H.; Mizukami, T.; Myaji, H.; Sato, S.; Oomori, T.; Nukui, E.
Preparation of N-[4-(4-Quinolinyl)benzyl]benzimidazole Derivatives
as Immunosuppressants. Jpn. Kokai Tokkyo Koho JP 06, 340,658,
1994.)
∆Q
∆t AC0
1
Papp
)
(1)
where ∆Q/∆t was the rate of appearance of the drug in the receiver
chamber, C0 was the initial concentration of the drug in the donor
chamber, and A was the surface area of the monolayer. All Papp
values were standardized and reported as 10-6 cm/s.
Pharmacokinetics in Rats. Compounds were either orally
administered or injected through the tail vein to fasted Sprague-
Dawley rats weighing 250-300 g. After the administration, blood
samples were taken at selected times, and the plasma content was
analyzed by HPLC, followed by t1/2 and area under curve calcula-
tions (AUC).
Computational Methods. All molecular modeling studies were
performed as previously described10 using Catalyst 4.719 installed
on a Silicon Graphic O2 desktop workstation equipped with a 200
MHz MIPS R5000 processor (128 MB RAM) running the Irix 6.5
operating system. All 2D chemical structures were produced within
the ISIS/Draw2.1 drawing program.28
Acknowledgment. Thanks are due to Italian MIUR (PRIN)
for financial support. Professor Stefania D’Agata D’Ottavi’s
careful reading of the manuscript is also acknowledged.
Supporting Information Available: Full experimental details
for the synthesis and the characterization of 5 and 6 and related
compounds (chemistry, NMR, MS). This material is available free
of charge via the Internet at http://pubs.acs.org.
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