Design, synthesis, and biological evaluation of AT1 angiotensin II receptor antagonists based on the pyrazolo[3,4-b]pyridine and related heteroaromatic bicyclic systems

Article abstract of DOI:10.1021/jm7011563

Novel AT₁ receptor antagonists bearing the pyrazolo[3,4-b] pyridine bicyclic heteroaromatic system (or structurally related moieties) were designed and synthesized as the final step of a large program devoted to the development of new antihypertensive agents and to the understanding of the molecular basis of their pharmacodynamic and pharmacokinetic properties. The preliminary pharmacological characterization revealed nanomolar AT₁ receptor affinity for several compounds of the series and a potent antagonistic activity in isolated rabbit aortic strip functional assay for 7c and 8a. These results stimulated the study of the biopharmaceutical properties of some selected compounds, which were found to be characterized by a permeability from medium to high. Remarkably, the least permeable 7c showed both permeability and oral bioavailability (80%) higher than losartan, but its terminal half-life was shorter. These results suggest that the permeability is not a limiting factor in the pharmacokinetics of these AT₁ receptor antagonists.

Full text of DOI:10.1021/jm7011563

J. Med. Chem. 2008, 51, 2137–2146
2137
Design, Synthesis, and Biological Evaluation of AT1 Angiotensin II Receptor Antagonists Based
on the Pyrazolo[3,4-b]pyridine and Related Heteroaromatic Bicyclic Systems
Andrea Cappelli,*,† Chiara Nannicini,† Andrea Gallelli,†,‡ Germano Giuliani,† Salvatore Valenti,† Gal.la Pericot Mohr,§,†
Maurizio Anzini,† Laura Mennuni,| Flora Ferrari,| Gianfranco Caselli,| Antonio Giordani,| Walter Peris,| Francesco Makovec,|
Gianluca Giorgi, and Salvatore Vomero†
Dipartimento Farmaco Chimico Tecnologico and European Research Centre for Drug DiscoVery and DeVelopment, UniVersità di Siena,
Via A. Moro, 53100 Siena, Italy, Rottapharm S.p.A., Via Valosa di Sopra 7, 20052 Monza, Italy, and Dipartimento di Chimica, UniVersità di
Siena, Via A. Moro, 53100 Siena, Italy
ReceiVed September 17, 2007
Novel AT1 receptor antagonists bearing the pyrazolo[3,4-b]pyridine bicyclic heteroaromatic system (or
structurally related moieties) were designed and synthesized as the final step of a large program devoted to
the development of new antihypertensive agents and to the understanding of the molecular basis of their
pharmacodynamic and pharmacokinetic properties. The preliminary pharmacological characterization revealed
nanomolar AT1 receptor affinity for several compounds of the series and a potent antagonistic activity in
isolated rabbit aortic strip functional assay for 7c and 8a. These results stimulated the study of the
biopharmaceutical properties of some selected compounds, which were found to be characterized by a
permeability from medium to high. Remarkably, the least permeable 7c showed both permeability and oral
bioavailability (80%) higher than losartan, but its terminal half-life was shorter. These results suggest that
the permeability is not a limiting factor in the pharmacokinetics of these AT1 receptor antagonists.
Introduction
of the interaction with AT1 receptor, a particularly effective
heterocyclic system.4 Indeed, 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.
Angiotensin II (Ang II)a type 1 (AT1) receptor belongs to
the G protein-coupled receptor superfamily and mediates
virtually all the known physiological actions of Ang II through
interaction with heterotrimeric G-protein and subsequent activa-
tion of several effector systems (phospholipases C, D, A2,
adenyl cyclase, etc.). AT1 receptor shows the seven hydrophobic
transmembrane domains forming R-helices in the lipid bilayer
of the cell membrane and plays a key role in the renin-
angiotensin system involved in the regulation of cardiovascular
functions and pathophysiology of hypertension.1,2
The discovery of potent and orally active nonpeptide Ang II
antagonists such as losartan and eprosartan has encouraged the
development of a large number of similar compounds.3 Among
them, irbesartan, candesartan, valsartan, telmisartan, tasosartan,
and olmesartan are on the market. Most of the developed AT1
receptor antagonists are characterized by the presence in their
structure of the biphenyl fragment bearing an acidic moiety and
differ in the nature of the pendent heterocyclic system (valsartan
lacks the heterocyclic moiety) connected to the para position
of the proximal phenyl.
On the other hand, irbesartan5 shows a spirofused bicyclic
system, the main axis of which is oriented in a slightly different
manner with respect to the corresponding one of compounds 2.
The bicyclic systems of these two compounds are profoundly
different from the steric point of view, but each of them shows
two potential hydrogen bond acceptors. These results can be
considered representative of the widespread information con-
tained in the literature concerning AT1 receptor antagonists in
suggesting that a large variety of heterocyclic moieties is
tolerated by the receptor.6
At the beginning of an investigation devoted to both the
development of new antihypertensive agents and the under-
standing of the molecular basis of their pharmacodynamic and
pharmacokinetic properties, we explored the effects of the
molecular manipulation of the distal phenyl group of compounds
2 leading to the development of compounds 4–6. Some
representative compounds of the series showed in vitro proper-
ties comparable to those shown by losartan,7 but pharmacoki-
netic studies performed with the selected candidates for further
preclinical studies revealed low oral bioavailability and a rapid
excretion as a possible result of a rapid conjugation with
glucuronic acid.8
As a further step of the investigation, the structural modifica-
tion of imidazo[4,5-b]pyridine moiety of compounds 2 led to
the design of compounds 7 and 8. In the “recently” described
potent AT1 receptor antagonist 9 the hydrogen bond acceptor
imidazole nitrogen of irbesartan is transformed into a pyrazo-
lidinedione carbonyl and the lipophilic spiro-fused cyclopentane
moiety is placed between the two carbonyl groups.9 On the other
hand, in compounds 7 and 8 the second potential hydrogen
bonding acceptor is incorporated into the lipophilic moiety. In
The ortho-fused bicyclic imidazo[4,5-b]pyridine moiety of
compound 2a (L-158,809) appears to be, from the point of view
* 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, Università di Siena.
‡
Present address: Dipartimento di Scienze Farmacobiologiche, Università
degli Studi Magna Græcia di Catanzaro, Complesso Ninì Barbieri, 88021,
Roccelletta di Borgia (CZ), Italy.
§
Present address: Medicinal Chemistry, Siena Biotech, Via Torre
Fiorentina 1, 53100 Siena, Italy.
|
Rottapharm S.p.A.
Dipartimento di Chimica, Università di Siena.
a Abbreviations: Ang II, angiotensin II; DEM, diethoxymethane; DBU,
1,8-diazabicyclo[5.4.0]undec-7-ene; DMEM, Dulbecco’s modified eagle
medium.
10.1021/jm7011563 CCC: $40.75
2008 American Chemical Society
Published on Web 03/05/2008

2138 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7
Cappelli et al.
Scheme 1a
Scheme 2a
a Reagents: (a) H2, Pd/C, Na2CO3, C2H5OH; (b) morpholine, C2H5OH.
Scheme 3a
a R ) H (7j), C(C6H5)3 (17j). Reagents: (a) CH3ONa, CH3OH; (b)
CH3NH2, C2H5OH.
The synthesis of pyrazolo[3,4-b]pyridine intermediates
16a-k,m was accomplished starting from the suitable com-
mercially available nicotinic acids 10–14, which were activated
by reaction with thionyl chloride and then made to react with
the appropriate hydrazine derivatives to obtain intermediates
15a-k,m (Scheme 1). Hydrazide derivatives 15a-k,m were
then cyclized in refluxing 1-pentanol in the presence of Na2CO3
as the base, to give compounds 16a-k,m. Moreover, pyra-
zolo[3,4-b]pyridine derivatives 16j,k were used as starting
material for the preparation of 16l,p (Scheme 2).
Pyrazolo[3,4-b]pyridine derivatives 16 are intriguing com-
pounds, which show prototropic tautomerism. For instance,
crystallographic analysis of the two homologues 16a,c show
that they crystallize in different prototropic forms characterized
by different crystal colors: red prisms in the case of 16a and
colorless needles for 16c (see Figure 1).
Alkylation of 16a-m,p with 5-[4′-(bromomethyl)biphenyl-
2-yl]-2-(triphenylmethyl)-2H-tetrazole10 in the presence of NaH
as the base gave the expected biphenyl derivatives 17a-m,p
in acceptable yields, which were deprotected with formic acid
to obtain target tetrazole derivatives 17a-m,p (Scheme 1). The
structure of tetrazole derivative 7a was confirmed by single
crystal X-ray diffraction studies (Figure 2).
The remaining target pyrazolo[3,4-b]pyridine 7n,o were
prepared by nucleophilic displacement of chlorine atom of 17j
(in the case of 7n) or 7j (in the case of 7o) with sodium
methoxide or methylamine, respectively.
Indazolone derivative 18 was prepared by means of the same
chemistry described for pyrazolo[3,4-b]pyridines 7a-m,p with
a Reagents: (a) SOCl2, DMF (cat.); (b) R1-NHNH2 ·C2O4H2, CH2Cl2,
NaOH, H2O; (c) 1-pentanol, Na2CO3; (d) NaH, DMF; (e) 5-[4′-(bromo-
methyl)biphenyl-2-yl]-2-(triphenylmethyl)-2H-tetrazole, DMF; (f) HCO2H,
CH2Cl2.
the design of these compounds the attention was focused on
the effects of the orientation of the bicyclic system in order to
obtain information useful to refine the previously published AT1
receptor model.7
In this paper the synthesis, the preliminary pharmacological
and biopharmaceutical characterization and the deduction of
structure-affinity relationships of the novel Ang II antagonists
7 and 8 are described.
Results
Chemistry. The preparation of tetrazole derivatives 7a-p
was performed by means of a multistep procedure described in
Schemes 1-3.

Synthesis of AT1 Angiotensin II Receptor Antagonists
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 2139
Figure 1. Crystal structure of pyrazolo[3,4-b]pyridine derivatives 16a·H2O (left) and 16c (right). Ellipsoids enclose 50% probability.
Scheme 4a
Figure 2. Crystal structure of target compound 7a. Ellipsoids enclose
50% probability.
a Reagents: (a) SOCl2, CH2Cl2; (b) CH3CH2CH2CH2-NHNH2 ·C2O4H2,
CH2Cl2, NaOH, H2O; (c) NaOH, Pd(dppf)2Cl2, C2H5OH; (d) NaH, DMF;
(e) 5-[4′-(bromomethyl)biphenyl-2-yl]-2-(triphenylmethyl)-2H-tetrazole, DMF;
(f) HCO2H, CH2Cl2.
the peculiarity that the cyclization of hydrazide derivative
20 was carried out in the presence of a palladium catalyst
(Scheme 4).
The target pyrrolo[3,4-b]pyridine derivative 7q (Scheme 5)
was synthesized from ethyl 2-methylnicotinate (23), which was
brominated and cyclized with propylamine to obtain compound
24, the active methylene of which was acylated with dimethyl
carbonate in the presence of sodium hydride as the base to give
ester 25.11 Compound 25 was deprotonated with sodium hydride
in THF and alkylated with 5-[4′-(bromomethyl)biphenyl-2-yl]-
2-(triphenylmethyl)-2H-tetrazole to give biphenyl derivative 26,
which was promptly deprotected with sodium hydroxide to
obtain target tetrazole derivative 7q.
Target compounds 8a-c were prepared following the mul-
tistep procedure described in Scheme 6. The introduction of
the required lipophilic side chain in position 1 of the pyra-
zolo[3,4-b]pyridine nucleus was carried out in an indirect
manner by reaction of the appropriate ethyl nicotinate derivatives
27–29 with the suitable alkylhydrazine to obtain bicyclic
compounds 30–33. Crystallographic studies of compounds 30
and 32 showed that these compounds exist in the solid state in
the OH-tautomers (Figure 3). Accordingly, alkylation of 30 and
31 with 5-[4′-(bromomethyl)biphenyl-2-yl]-2-(triphenylmethyl)-
2H-tetrazole in the presence of sodium hydride as the base led
to the O-alkylated derivatives 34a,b, which were deprotected
to afford the final tetrazole compounds 35a,b. Thus, the
preparation of compounds 8a-c required the development of a
convergent procedure similar to that described for the synthesis
of losartan (Scheme 6).12
Pyrazolo[3,4-b]pyridine derivatives 31–33 were benzylated
with 4-bromobenzyl bromide via the corresponding silyl ether
derivatives 36a-c to obtain the mixtures of N- and O-alkylated
products 37 and 38, respectively. The expected N-alkylated
derivatives 37a-c were isolated from their respective mixtures
by flash chromatography (the structure of 37b was confirmed
by crystallography, see the Supporting Information) and sub-
jected to Suzuki coupling reaction with 5-(2′-boronophenyl)-
2-(triphenylmethyl)-2H-tetrazole12 to obtain the biphenyl de-
rivatives 39a–c; deprotection with formic acid gave final
tetrazole derivatives 8a–c.
Deaza derivatives 8d–g were synthesized starting from ethyl
2-methylnicotinate (23) by means of a procedure developed by
combining the chemistry11 used for the preparation of pyr-
rolo[3,4-b]pyridine derivative 7q with the assembly of the
biphenyl moiety based on the Suzuki coupling (Scheme 7).12
The structure of intermediate compounds 41 and 44d was
confirmed by crystallographic studies (see the Supporting
Information).

2140 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7
Cappelli et al.
Scheme 5a
A significant affinity decrease is observed when the hetero-
cyclic system of 8a is bound to the methylbiphenyltetrazole
component by its oxygen atom as it occurs in compounds 35a,b.
The introduction of a carbonyl group into the pyrazolo[3,4-
b]pyridine system of 35b leading to pyrido[2,3-d]pyridazino-
8-one derivative 45 has limited effects on the receptor affinity.
The replacement of the pyridine nitrogen atom of 7c with an
aromatic CH group leading to indazolone derivative 18 produces
a considerable (33-fold) decrease in the affinity. The result
demonstrates the key role played by the pyridine nitrogen in
the interaction with the AT1 receptor.
The transformation of the pyrazolo[3,4-b]pyridine heterocy-
clic system of 7a,c into the pyrrolo[3,4-b]pyridine system of
7q has dramatic effects on the AT1 receptor affinity. This
transformation involves an important center of the molecule,
which plays the role of linker between the methylbiphenyltet-
razole component and the heterocyclic moiety and affects their
relative orientation (Figure 4).
On the other hand, the effect is more limited when the same
transformation is carried out in compound 8a to obtain 8g. In
this case, the transformation involves a linking atom bound to
a lipophilic substituent, for which the binding prerequisites are
likely to be less stringent.
a Reagents: (a) NBS, benzoyl peroxide, CCl4; (b) CH3(CH2)2NH2,
C2H5OH; (c) CH3OCOOCH3, NaH, DMF; (d) NaH, THF; (e) 5-[4′-
(bromomethyl)biphenyl-2-yl]-2-(triphenylmethyl)-2H-tetrazole, THF; (f)
NaOH, H2O, C2H5OH.
(b) Effects of the Modification of the Lipophilic Substitu-
ent on the Five-Membered Ring. The work performed on
losartan congeners has demonstrated the importance of a
suitable lipophilic substituent at 2-position of imidazole, fused
imidazole, or equivalent moieties in the interaction with the
AT1 receptor. While in the case of losartan derivatives the
n-propyl and n-butyl chains are optimal,10 in imidazo[4,5-
b]pyridine derivatives 2, ethyl and n-propyl groups were
reported to be equivalent and better than the n-butyl one.4
Interestingly, the n-butyl group appears to be the optimal
lipophilic substituent in the two short series of pyrazolo[3,4-
b]pyridine derivatives 7a-f and 7g-i and in pyrrolo[3,4-
b]pyridine derivatives 8d-g. This result seems to suggest
different binding modalities between compounds 2 and our
derivatives 7 and 8 probably due to the change of the
geometry of the hydrogen bonding acceptor (see Chart 2).
Moreover, the structure-affinity relationship analysis in the
series 7a-f shows that the linear alkyl chains interact with the
binding site more tightly than the branched ones (in agreement
with the results described for losartan derivatives;10 compare
7a with 7b and 7c with 7d) and the benzyl substituent of 7f is
not tolerated at all.
(c) Effects of the Introduction of Substituents on the
Pyridine Ring. The introduction of substituents on the pyridine
moiety of compound 7c has negative effects on the affinity for
the receptor, and the extent of the affinity decrease depends on
the features of the substituent and on the position involved. In
fact, a methyl group (7i) is better tolerated than a chlorine atom
(7j), or methoxy (7n) and methylamino (7o) groups, or again a
morpholine substituent (7p). Furthermore, the chlorine atom is
better tolerated when inserted in position 3 (7m) than position
2 (7j) of the pyridine ring. Finally, the best accepted substitution
pattern appears to be a fluorine atom in position 3 (compound
7l), while the contemporary presence of the two halogen atoms
in positions 2 and 3 of the pyridine ring (7k) produces an
important affinity decrease.
Finally, the procedure for the synthesis of pyrido[2,3-
d]pyridazin-8-one derivative 45 is reported in Scheme 8.
Condensation of anhydride 46 and butylhydrazine in ethanol
afforded compound 47, which exists in the solid state as the
OH-tautomer (see the Supporting Information). Reaction with
sodium hydride and subsequently with 5-[4′-(bromomethyl)bi-
phenyl-2-yl]-2-(triphenylmethyl)-2H-tetrazole10 gave the product
of O-alkylation 48, which was deprotected with formic acid to
obtain target compound 45.
Structure-Affinity Relationship Studies. The newly syn-
thesized bicyclic derivatives 7a-q, 8a-g, 18, 35a,b, 45
(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
compounds 2a,b, losartan, and valsartan, following well-
established protocols.13 The results of the binding studies
summarized in Table 1 show that most of the tested compounds
displayed submicromolar affinity for AT1 receptor with the most
potent compound 7c and its positional isomer 8a showing IC50
values in the nanomolar range.
(a) Effects of the Modification of the Heterocyclic
System. Among the heterocyclic moieties and their relative
orientations with respect to the biphenyltetrazole component
investigated in the present work, the pyrazolo[3,4-b]pyridine
showing a 2a-like orientation typical of compound 7c is the
most effective configuration from the point of view of the
interaction with the AT1 receptor. However, the difference in
affinity between 7c and 2a suggests that the carbonyl group of
7c does not show an optimal geometry for the interaction with
the receptor hydrogen-bonding donors (e.g., N111, N294, or
S252).7 A similar conclusion has been drawn in the case of
pyrazolidinedione derivative 9.9
The variation in orientation of the pyrazolo[3,4-b]pyridine
heterocyclic system leading to 8a has limited effects on the
receptor affinity. This result suggests a partial equivalence of
the orientations of the pyrazolo[3,4-b]pyridine system shown
by compounds 7c and 8a.
It is noteworthy that the insertion of either a methyl group
(8b) or a chlorine atom (8c) in the position 2 of the pyridine
ring of compound 8a has negative effects very similar to those
observed in compounds 7c,i,j. This result suggests a common
role for the pyridine ring of compounds 7c and 8a in spite of

Synthesis of AT1 Angiotensin II Receptor Antagonists
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 2141
Scheme 6a
a R1 ) n-C3H7 (35a), n-C4H9 (35b); R2 ) H (8a), CH3 (8b), Cl (8c). Reagents: (a) R1-NH-NH2 ·C2O4H2, TEA, C2H5OH; (b) NaH, DMF; (c) 5-[4′-
(bromomethyl)biphenyl-2-yl]-2-(triphenylmethyl)-2H-tetrazole, DMF; (d) HCO2H, CH2Cl2; (e) tert-butyldimethylsilyl trifluoromethanesulfonate, N,N-
diisopropylethylamine, THF; (f) Br-C6H4-CH2Br, (n-Bu)4NF, THF; (g) 5-(2′-boronophenyl)-2-(triphenylmethyl)-2H-tetrazole, PPh3, Pd(OAc)2, K2CO3, THF,
diethoxymethane (DEM), H2O.
Scheme 7a
Figure 3. Crystal structure of pyrazolo[3,4-b]pyridine derivatives 30
(left) and 32 (right). Ellipsoids enclose 50% probability.
their different orientation with respect to the methylbiphenyltet-
razole component.
Functional Studies. The most potent AT1 receptor–ligands
7c and 8a were selected among the newly synthesized com-
pounds and their potential antagonistic activity was investigated
in vitro, using the contractile response of isolated rabbit aortic
strips as a functional assay. Tissue preincubation with increasing
amounts of compounds 7c and 8a produced progressive parallel
rightward shifts of the endogenous agonist concentration–re-
sponse curve. Shild’s plot analysis gave derived pA2 values of
7.83 and 7.98 for compound 8a and 7c, respectively. Neverthe-
less, differently from losartan and 7c, compound 8a lowered
the upper plateau of the concentration–response curve of Ang
II in a concentration-dependent way, providing unambiguous
evidence for an insurmountable antagonism (Figure 5). This
a R1 ) H (8d), C2H5 (8e), n-C3H7 (8f), n-C4H9 (8g). Reagents: (a) NBS,
result attains a particular importance in consideration of the fact
benzoyl peroxide, CCl4; (b) 4-bromobenzylamine hydrochloride, TEA,
C2H5OH; (c) 5-(2′-boronophenyl)-2-(triphenylmethyl)-2H-tetrazole, PPh3,
that 7c and 8a are position isomers showing the same pendent
heterocyclic system in two different orientations (Chart 2) and
suggests that the (in)surmountable antagonist properties can be
modulated by very small structural changes in these AT1
receptor antagonists.
In Vitro Intestinal Permeability Experiments. The perme-
ability studies performed with Caco-2 monolayers14 show that
pyrazolo[3,4-b]pyridine derivatives 7c,m, 8a,b and pyrrolo[3,4-
b]pyridine derivative 8g are characterized by a medium to high
Pd(OAc)2, K2CO3, THF, DEM, H2O; (d) HCO2H, CH2Cl2; (e) CH3OCOOCH3,
NaH, DMF; (f) DBU, DMF, R1I; (g) NaOH, H2O, C2H5OH.
permeability in apical to basolateral (AfB) experiments show-
ing Papp values ranging from 1.78 to 10.8 × 10-6 cm/s (Table
2). For comparison, propanolol shows a Papp value of 8.0 ×
10-6 cm/s in the same test, cimetidine a Papp value of 1.1 ×
10-6 cm/s, and vinblastine (a well-known P-glycoprotein–sub-

2142 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7
Cappelli et al.
Scheme 8a
Table 1. AT1 Receptor Binding Affinities of Compounds 2, 7a-q,
8a-g, 18, 35a,b, and 45
a Reagents: (a) NH2NHC4H9 C2O4H2, TEA, C2H5OH; (b) NaH, DMF;
•
(c) 5-[4′-(bromomethyl)biphenyl-2-yl]-2-(triphenylmethyl)-2H-tetrazole, DMF;
(d) HCO2H, CH2Cl2.
strate) a Papp value of 0.1 × 10-6 cm/s. All the newly
synthesized compounds show a Papp (BfA) value similar to
the Papp (AfB) value (BfA/AfB ratio < 2, data not shown)
suggesting that 7c,m, and 8a,b,g are not substrates for efflux.
In the short series of the compounds evaluated in such an assay,
the least permeable was pyrazolo[3,4-b]pyridine derivative 7c,
which shows a Papp value slightly higher than those shown by
cimetidine and losartan and is about 1 order of magnitude more
permeable than the previously described 3-tetrazolylquinolinone
derivative 4b (CR3210, Papp ) 0.1 × 10-6 cm/s).8c The variation
in orientation of the pyrazolo[3,4-b]pyridine heterocyclic system
leading to 8a has a 2-fold enhancing effect on the permeability,
while the introduction of a chlorine atom in position 5 of the
pyrazolo[3,4-b]pyridine nucleus of 7c produces a more pro-
nounced enhancing effect, so that 7m is the most permeable
among the compounds tested. On the other hand, the introduc-
tion of a methyl group in position 6 of pyrazolo[3,4-b]pyridine
nucleus of 8a, as well as the transformation of the pyrazolo[3,4-
b]pyridine heterocyclic system of 7c into pyrrolo[3,4-b]pyridine
one of 7q slightly improves the permeability. It is noteworthy
that the compound showing the highest ClogP (compound 8g)
is not the most permeable, and more in general, the permeability
shown by these compounds is apparently poorly related to their
lipophilicity.
rabbit
aortic
strips
binding
b
compd
2a
2b
7a
7b
7c
7d
7e
7f
7g
7 h
7i
7j
7k
7l
7m
7n
7o
7p
7q
8a
8b
8c
8d
8e
8f
8g
X
R1
C2H5
R2
R3 IC50 (nM) ( SEMa pA2
0.4 ( 0.1
0.8 ( 0.01
108 ( 18
2300 ( 290
18 ( 1
124 ( 8
53 ( 4
>3000
1870 ( 555
291 ( 29
81 ( 18
168 ( 15
427 ( 134
70 ( 32
76 ( 29
258 ( 27
124 ( 16
600 ( 97
>3000
n-C3H7
n-C3H7
i-C4H9
n-C4H9
i-C5H11
n-C5H11
CH2C6H5
C2H5
n-C3H7
n-C4H9
n-C4H9
n-C4H9
n-C4H9
n-C4H9
n-C4H9
n-C4H9
n-C4H9
N
H
H
H
H
H
H
CH3
CH3
CH3
Cl
Cl
H
H
H
H
H
H
H
H
H
H
H
F
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
7.98
F
H
Cl
H
H
H
H
OCH3
NHCH3
morpholino
CH n-C3H7
N
N
N
CH
CH C2H5
CH n-C3H7
CH n-C4H9
H
H
CH3
Cl
H
H
H
H
n-C4H9
n-C4H9
n-C4H9
H
39 ( 5
90 ( 9
7.83
461 ( 66
>10000
>10000
154 ( 21
95 ( 32
598 ( 72
146 ( 33
254 ( 91
114 ( 6
0.4 ( 0.1
6.7 ( 0.5
3.4 ( 0.3
Pharmacokinetic Studies. The pharmacokinetic properties
of compound 7c were evaluated in rats by standard procedures8
in comparison with reference AT1 antagonist 1 (losartan). The
results shown in Table 2 suggest that this compound is a
chemical entity characterized by very good intestinal absorption
and rapid excretion in rats. In fact, pyrazolo[3,4-b]pyridine
derivative 7c showed a bioavailability of 80%, but its terminal
half-life ranges from 0.9 to 1.0 h.
18
35a
35b
45
Ang II
losartan
valsartan
n-C3H7
n-C4H9
8.48
a Each value is the mean ( SEM of 3 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 curve was obtained from each strip.
Discussion and Conclusions
Compounds 7 and 8 were designed as part of a large program
devoted to the development of new antihypertensive agents and
to the understanding of the molecular basis of their pharmaco-
dynamic and pharmacokinetic properties.7,8 The designed
compounds were synthesized by combining the classical losartan
chemistry (linear and convergent approaches) with the chemistry
of pyrazolo[3,4-b]pyridines and pyrrolo[3,4-b]pyridines. Among
the newly synthesized bicyclic derivatives, several compounds
displayed high affinity for AT1 receptor. The most potent
compounds 7c and 8a show IC50 values in the nanomolar range
and represent interesting candidates for further preclinical
studies. It is noteworthy that compound 7c and its positional
isomer 8a show quite similar IC50 values: 18 and 39 nM,
respectively. This small difference finds a support in the
literature when 7c and 8a are compared with AT1 receptor

Synthesis of AT1 Angiotensin II Receptor Antagonists
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 2143
was found to behave as a surmountable antagonist, while
compound 8a lowered the upper plateau of the concentration–
response curve of Ang II in a concentration-dependent way,
providing unambiguous evidence for an insurmountable an-
tagonism. In view of the fact that 7c and 8a are position isomers
bearing the same pendent bicyclic system in two different
orientations, the difference in antagonist behavior suggests that
the surmountable/insurmountable antagonist properties can be
modulated by very small structural changes.16
The results prompted the characterization of the biopharma-
ceutical properties of some selected compounds. The investiga-
tions showed that pyrazolo[3,4-b]pyridine derivatives 7c,m,
8a,b, and pyrrolo[3,4-b]pyridine derivative 8g are characterized
by a permeability from medium to high and are not substrates
for efflux. The least permeable 7c shows a Papp value (1.78 ×
10-6 cm/s) slightly higher that shown by losartan and is about
1 order of magnitude more permeable than the previously
described 3-tetrazolylquinolinone derivative 4b (Papp ) 0.1 ×
10-6 cm/s).8c Moreover, pharmacokinetic studies showed the
high oral bioavailability (80%) of 7c, which is significantly
higher than that shown by losartan and 4b.17,8c The observed
differences in bioavailability can be explained on the basis of
the different permeability of 7c and 4b and by the substantial
first-pass hepatic metabolism in the case of losartan.18,3b
However, 7c shows a terminal half-life (about 1.0 h) similar
to the one shown by 4b, but shorter than that of losartan. These
results suggest that the permeability is not a limiting factor in
the pharmacokinetics of these pyrazolo[3,4-b]pyridine deriva-
tives, and further studies are required in order to evaluate the
role of UDP-glucuronosyltransferases (EC 2.4.1.17) in the
metabolism-excretion of these tetrazole derivatives.19 In par-
ticular, compound 8a showing nanomolar AT1 receptor affinity,
high permeability, and insurmountable antagonism (which may
contribute to the duration of action20) can be considered an
interesting candidate for further preclinical characterization.
Figure 4. Superposition of 7a crystallographic structure (white) with
a low energy conformer of 7q (cyan). This result suggests that 7q
can populate the solid state conformation of 7a. Thus, the difference
in the AT1 receptor affinity between 7a and 7q is probably connected
to the relative orientation of the pendent bicyclic system determined
by the linker geometry (a sp3 carbon in the case of 7q and a partially
pyramidal nitrogen atom for 7a).
Chart 1. Structures of Compounds 1–6
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
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 were recorded on either
a Varian Saturn 3 spectrometer or a ThermoFinnigan LCQ-Deca.
X-Ray Crystallography. Single crystals of 7a, 16a,c, 30, 32,
37b, 41, 44d, and 47 were submitted to X-ray data collection. Data
for compound 41 were obtained by a Bruker-Nonius FR591 rotating
anode diffractometer with a 95 mm CCD camera at 120 K. All the
other data were obtained by a Siemens P4 four-circle diffractometer
at 293 K. Both the instruments were equipped with a graphite
monochromated Mo KR radiation (λ ) 0.71069 Å).
The structures were solved by direct methods implemented in
the SHELXS-97 program.21 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.22
antagonists 9, 49, and 50 (Chart 3).9 The attempts to optimize
the interaction with the AT1 receptor failed to give ligands more
potent than 7c and 8a, but they afforded several congeners
showing interesting affinities and providing valuable information
about the interaction with the receptor.
The potential antagonist activity of compounds 7c and 8a
was investigated in vitro, using the contractile response of
isolated rabbit aortic strips as a functional assay. In this test, 7c
Biological Methods. Angiotensin II Receptor Binding As-
13
say. Male Wistar rats (Charles River, Calco, Italy) were killed
by decapitation and their livers were rapidly removed. Ang 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 about 3 g of tissue was homogenized by
Polytron Ultra-Turrax (maximal speed for 2 × 30 s) in ice-cold 20

2144 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7
Chart 2. Structures of Compounds 2a, 7–9, and Irbesartana
Cappelli et al.
a The potential hydrogen bonding acceptors are indicated in red.
[
125I]Sar1,Ile8-Angiotensin II) was estimated for the linear portion
of the competition curves.
Angiotensin II Functional Antagonism in Rabbit Aorta
Strips. 13 New Zealand White rabbits (3–4 kg body weight, Harlan
Italy) were killed by cervical dislocation, after a slight ether
anesthesia. The descending thoracic aorta, with the endothelium
removed, was cut into helical strips 3-4 mm wide and 15-20 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 ·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 four-channel pen recorder (Basile, Italy). The tissues
were allowed to equilibrate for 1 h and were washed every 10 min.
At the beginning of the experiment, a 67 mM KCl solution was
administered to check the sensitivity of the preparations as well as
to determine their maximal contractile response. After 30 min
washout, test substances or their respective vehicles were added.
Sixty minutes later, cumulative concentration–response curves of
angiotensin II were obtained. Only one curve was obtained from
each strip, and the contractile response was expressed as percentage
(%) of the maximal contraction achieved with KCl. Curve analysis
was performed by using the Allfit23 program, which calculates lower
and upper plateau, slope and agonist EC50, and allows the
comparison of two or more curves. Antagonist potency was
evaluated by the estimation of pA2 values.
Figure 5. Concentration-contractile response curves for Ang II in
isolated rabbit aortic strips, in the presence of different concentrations
[10 nM (9), 30 nM (2), or 100 nM (×)] of compound 8a or of its
vehicle ([).
In Vitro Intestinal Permeability Experiments. The perme-
ability studies were performed with Caco-2 monolayers as described
in the literature.14 Caco-2 cells were cultured in supplemented
Dulbecco’s modified eagle medium (DMEM) with 10% fetal bovine
serum, 1% nonessential amino acids, 10 mM Hepes buffer, and a
Pen/Strept mixture (50U penicillin and 50 mg/mL streptomycin)
and split at confluence by trypsinization.
For transport studies, 200000 cells/well were seeded onto
Millicell 24-well cell culture plates, and after 24 h incubation at
37 °C with 5% CO2/95% O2, the culture medium was changed with
Enterocyte Differentiation Medium with additives (Becton Dick-
inson Bioscience), which allows Caco-2 cells to establish within 3
days a differentiated enterocyte monolayer. Transepithelial electric
resistance was measured with a Millipore Millicell-ERS instrument
and was >1000 ohms.
The transport across the Caco-2 monolayer was determined in
two ways: from apical to basolateral side (AfB) by adding a 10
µM solution of the compound in DMEM (1% final concentration
of DMSO) to the apical side and from basolateral to apical side
(BfA) by adding a 10 µM solution of the compound in DMEM
(1% final concentration of DMSO) to the basolateral side; after
2 h incubation period at 37 °C, the basolateral side solution, together
with the apical and the starting solutions, was analyzed and
quantified by LC-MS/MS (column: Waters X-terra C18, 2.5 µM,
2.5 × 50 mm, room temperature; mobile phase: acetonitrile-water,
flow 250 µL/min; interface: ESI positive mode). The experiments
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 centrifuged
at 50000g 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 the assay
buffer (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 (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 Ang II and represented 5–10%
of total binding. Binding was terminated by rapid vacuum filtration
using a Millipore 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 discs were punched out
and counted in a γ-counter with 92% efficiency. The IC50 value
(concentration for 50% displacement of the specifically bound

Synthesis of AT1 Angiotensin II Receptor Antagonists
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 2145
Table 2. Pharmacokinetic Parameters of Compounds 1, 4b, 7c,m, and 8a,b,g
c
d
e
binding
IC50 (nM)
C log
Caco-2 permeability
dose (mg/kg)
(route)
AUC
(µg·h/mL)
t1/2
Cmax
tmax
oral bioavail
(%)
compd
MW
Pa
Papp (a-b)b (×10-6 cm/s)
(h)
(µg/mL)
(h)
1 (losartan)
422.91
6.7
6.9
18
4.11f
1.15g
0.10
1.78
3 (iv)
30 (os)
3 (iv)
30 (os)
5 (iv)
12.4
74.8
0.89
4.40
1.41
3.38
4.7
3.0
0.68
1.0
1.0
0.9
27.5
0.5
0.5
0.5
60
49
80
4b
7c
460.53
425.49
4.7
4.2
2.95
6.32
4.10
15 (os)
7m
8a
8b
8g
459.93
425.49
439.51
424.50
76
39
90
95
3.70
2.57
3.07
4.49
10.8
3.6
8.7
5.6
a C log P calculated by means of CS ChemDraw Ultra 8.0 (Cambridge Soft Corporation, Cambridge, MA 02140 USA). b Apical to basolateral Caco-2
c
d
e
permeability. 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 15. g See ref 15.
Chart 3. Structures of Compounds 7c, 8a, 9, 49, and 50a
a The potential hydrogen bonding acceptors are indicated in red.
were performed with buffers at different pH (6.5 apical vs 7.4
basolateral) to better mimic the physiological conditions and
propranolol, cimetidine, and vinblastine were evaluated as controls.
Caco-2 apparent permeability values (Papp) were calculated by
means of the following equation:
dell’Università e della Ricerca) - PRIN (Programmi di ricerca
di Rilevante Interesse Nazionale).
Supporting Information Available: Full experimental details
for the synthesis and the characterization of 7 and 8 and related
compounds (chemistry, NMR, MS, crystallography). This material
is available free of charge via the Internet at http://pubs.acs.org.
∆Q 1
Papp
=
(1)
∆t AC0
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 where either orally
administered or injected through the tail vein to fasted Spra-
gue–Dawley rats weighing 250–300 g. After the administration,
blood samples were taken at selected times and the plasma content
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