NO-sartans: A new class of pharmacodynamic hybrids as cardiovascular drugs

Article abstract of DOI:10.1021/jm049681p

The aim of this work was to develop lead pharmacodynamic hybrids, NO-sartans, possessing the characteristics of a typical AT₁- antagonist and of a "slow NO donor", by adding NO-donor side chains to losartan. These new compounds, 2a and 2b, disp

Full text of DOI:10.1021/jm049681p

J. Med. Chem. 2004, 47, 5597-5600
5597
an up-regulation9 and direct activation of bradykinin
type 1 (B1) receptors by ACE inhibitors.10
NO-Sartans: A New Class of
Pharmacodynamic Hybrids as
Cardiovascular Drugs
In view of the above-mentioned reasons, ACE inhibi-
tors represent a first-choice class in hypertension therapy.
Furthermore, they also reveal an implication at the
cardiac level: angiotensin II inhibits muscular apopto-
sis, thus provoking ventricular hyperplasia, which might
aggravate a preexistent cardiac failure.
Maria C. Breschi,‡ Vincenzo Calderone,*,‡
Maria Digiacomo,§ Alma Martelli,‡
Enrica Martinotti,‡ Filippo Minutolo,§
Simona Rapposelli,§ and Aldo Balsamo§
However, ACE inhibitors also frequently present an
adverse side-effect which is invalidating in social life
and which drastically reduces the patient’s compli-
ance: this is coughing, which is the result of the ability
of ACE inhibitors to preserve bradykinin, a kinin that
stimulates the cough-center, from hydrolysis.11
Dipartimento di Psichiatria, Neurobiologia, Farmacologia e
Biotecnologie, Universita` di Pisa, Via Bonanno 6, 56126
Pisa, Italy, and Dipartimento di Scienze Farmaceutiche,
Universita` di Pisa, Via Bonanno 6, 56126 Pisa, Italy
Received April 30, 2004
In the past decade, research on drugs which can
replace ACE inhibitors in hypertension therapy, without
their collateral effects, has led to the discovery of
sartans.12 Drugs of this class are antagonists of angio-
tensin II at the AT1 receptor and block the action of
angiotensin II in a potentially more complete way than
ACE inhibitors; as is known, there are other enzymes,
besides ACE, which are able to contribute to angiotensin
II production.13 Many comparative clinical trials have
shown that sartans do not induce coughing because they
do not prevent ACE from degrading bradykinin; in the
past, they were employed when coughing was intoler-
able in a patient receiving therapy with ACE inhibitors,
but nowadays, in view of the evidence presented,
sartans can be viewed as a first-choice class of anti-
hypertensive drugs.14,15 However, sartans lack the
enhancement of the NO-mediated vasorelaxing effect
due to bradykinin preservation. The importance of NO
lies not only in its vasorelaxing action, but also in its
potent inhibition of platelet and neutrophil aggregation
in the endothelium.16,17
The so-called “nitric oxide-releasing drugs” have their
pioneers in nitrates and nitrites used in the treatment
of angina pectoris18,19 in view of their ability to release
NO. On the basis of knowledge about nitric oxide
properties, we have witnessed, in recent years, a
proliferation of hybrid drugs in which a well-known
molecule, with a certain pharmacodynamic pattern, has
been integrated with an NO-donor group, thus confer-
ring an improved pharmacological profile or a reduction
of adverse effects. For example, much interest has been
dedicated to NO-releasing nonsteroidal antiinflamma-
tory drugs (NO-NSAIDs). Among these, NO-releasing
aspirin has displayed increased antithrombotic proper-
ties in both in vitro and in vivo studies.20-22 Further-
more, NO-NSAIDs may be beneficial in the early phases
of myocardial infarction as a result of their NO-induced
myocardial energy-sparing effect and the coronary va-
sodilatation that they induce, together with the well-
known protective role played by NO during ischaemia.23
In fact, NO-NSAIDs in general protect the myocardium
during ischaemia reperfusion, by releasing NO; in
particular, NCX-4016 significantly reduced infarct size
in a rat model of myocardial infarction.24-26 As regards
other cardiovascular drugs, NO-releasing ACE inhibi-
tors, NO-releasing calcium antagonists, and NO-releas-
Abstract: The aim of this work was to develop lead pharma-
codynamic hybrids, NO-sartans, possessing the characteristics
of a typical AT1-antagonist and of a “slow NO donor”, by adding
NO-donor side chains to losartan. These new compounds, 2a
and 2b, displayed vasorelaxing effects, due to the release of
NO, and antagonized the vasocontractile effects of angiotensin
II, with potency values similar to that of losartan. In vivo, the
antihypertensive effects of 2a were similar to those of losartan
and captopril.
The pharmacotherapy of hypertension involves sev-
eral different classes of drugs; among these, the ones
that have the renin-angiotensin system (RAS) as the
target of their mechanism of action deserve special
mention. ACE inhibitors belong to this group, seeing
that they act by inhibiting the angiotensin converting
enzyme (ACE), which is mainly responsible for the
conversion of angiotensin I into angiotensin II. The
latter is one of the most hypertensive substances in our
organism: stimulation of the AT1 receptor by angio-
tensin II determines the release of aldosterone, which,
by means of a reabsorption of Na+ ions (and conse-
quently of liquids) and a loss of K+ ions, provokes
hypertension. Furthermore, angiotensin II induces a
direct vasoconstrictor action, which plays a significant
role in hypertensive effects.
ACE is also involved in the mechanism of degradation
of many other peptides, such as bradykinin. This peptide
induces the release of endothelial nitric oxide (NO), a
factor which plays a key role in the endogenous process
of vasodilatation. The fact that ACE inhibitors save
bradykinin also leads to a vasorelaxing effect, which
contributes to the antihypertensive action that is mainly
due to the lack of angiotensin II production.1,2 Recent
studies seem to indicate the possible existence of brady-
kinin-potentiating effects of ACE inhibitors, which are
independent of the reduction of bradykinin hydrolysis;
this mechanism was initially demonstrated by using
bradykinin analogues which were assumed to be ACE-
resistant.3-7 This hypothesis is based on a “cross-talk”
between ACE and bradykinin type 2 (B2) receptors8 (for
example, forming a heterodimer), which leads to an up-
regulation of the B2 receptor,5 probably accompanied by
* To whom correspondence should be addressed. Phone: +39-050-
2219589. Fax: +39-050-2219609. E-mail: calderone@farm.unipi.it.
‡ Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Bio-
tecnologie.
§ Dipartimento di Scienze Farmaceutiche.
10.1021/jm049681p CCC: $27.50 © 2004 American Chemical Society
Published on Web 10/02/2004

5598 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 23
Letters
Scheme 1a
Figure 1. Structure of losartan (1) and general structure of
the NO-releasing derivatives (2a,b) of losartan.
ing â-blocking agents have been synthesized, to improve
the antihypertensive effects of the “native” drugs.27-32
The above-mentioned biopharmacological consider-
ations, together with the appreciable results obtained
in different classes of NO-releasing drugs, prompted us
to develop a new class of drugs, by adding a NO-donor
group to a sartan molecule. This kind of chemical
manipulation was likely to give the “native” sartan an
additional NO-mediated, but bradykinin-independent,
vasorelaxing effect, thus generating the original class
of NO-sartans, pharmacodynamic hybrids possessing
the properties of a typical AT1 antagonist and of a “slow
NO donor”. The new compounds were expected to
possess a pharmacodynamic profile very similar to that
of ACE inhibitors, but without their bradykinin-medi-
ated adverse effects.33,34
An examination of the structures of commercially
available sartans showed that losartan (1) possesses
both the high activity and the molecular features, i.e.,
the presence of an easily esterifiable alcohol group,
suitable to provide a template for our purpose.
As regards the NO-donor moiety, the previously
reported35 m-nitrooxymethylbenzoate (2a, Figure 1) and
the 5-nitrooxypentanoate (2b, Figure 1) were selected,
based on the hypothesis of a different stability toward
ester bond cleavage, depending on the aliphatic or
aromatic nature of the compound.
a Key: (a) AgNO3, CH3CN, 1 h, rt; (b) losartan (1), DCC, THF,
DMAP, 2 h, rt.
to determine NO-mediated responses30 and AT1-an-
tagonist activities.36
These two compounds induced full vasorelaxing ef-
fects (efficacy ) 92 ( 6% and 95 ( 8%, for 2a and 2b,
respectively; pIC50 ) 5.55 ( 0.09 and 5.89 ( 0.14, for
2a and 2b, respectively) which were strongly inhibited
by 1 µM 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one
(ODQ), an inhibitor of guanylate cyclase, as to be
expected in the case of an NO release effect (efficacy )
34 ( 3% and 23 ( 8%, for 2a and 2b, respectively; pIC50
values not calculable).
Also the AT1-antagonist properties of nitrooxy deriva-
tives 2a,b were investigated and compared with those
of the reference AT1-antagonist losartan. The concen-
tration-contractile response curve for angiotensin II
was shifted parallel to the right by 2a and 2b, with Kb
values of 6 nM and 16 nM, respectively. Under the same
experimental conditions, losartan displayed a Kb value
of 4 nM, substantially analogous to the value recorded
for 2a and slightly lower than that of 2b.
The chemical pathway giving access to the final
products 2a and 2b is reported in Scheme 1. m-
(Chloromethyl)benzoic acid (3a) or 5-chloropentanoic
acid (3b) were converted into their nitrooxy derivatives
4a or 4b by treatment with silver nitrate in acetonitrile
at room temperature and in the dark. Condensation of
the nitro esters (4a,b) with losartan in tetrahydrofuran,
in the presence of N,N′-dicyclohexylcarbodiimide (DCC)
and a catalytic amount of N,N-(dimethylamino)pyridine
(DMAP), afforded the corresponding targets compounds
2a and 2b.
Compound 2a was selected in order to obtain further
information, in an attempt to understand the pharma-
cokinetic steps leading to the pharmacodynamic fea-
tures observed. To evaluate whether the vasorelaxing
properties of 2a are due to the release of NO from the
whole molecule or from the side chain (4a), after its
hydrolytic removal from losartan, the vasorelaxing
effects of 4a were evaluated by functional tests. Com-
pound 4a showed vasorelaxing effects lower than 2a,
both in efficacy and in potency (efficacy ) 67 ( 12%;
pIC50 ) 4.66 ( 0.13). This first finding, which seems to
indicate that the NO release from 2a is more rapid, and
thus that it precedes the possible hydrolysis of the ester
link between losartan and 4a, is confirmed by the
Pharmacological studies were carried out on the two
“pharmacodynamic hybrids” 2a and 2b, by means of
functional tests on vascular tissues, usually employed

Letters
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 23 5599
results obtained in the presence of eserin (3 µM, an
inhibitor of esterases37). As expected, the presence of
the esterase-inhibitor did not alter the vasorelaxing
effects of 4a (efficacy ) 71 ( 6%; pIC50 ) 4.64 ( 0.07);
significantly, the effects of 2a were not influenced by
eserin, either (efficacy ) 91 ( 3; pIC50 ) 5.37 ( 0.05),
confirming that the release of NO from this compound
does not require previous hydrolytic removal of the side
chain.
An analogous experimental strategy was employed to
determine whether the AT1-antagonist properties of 2a
are due to the whole molecules or to losartan (after the
hydrolytic removal of the side chains). As reported
above, the AT1-antagonist potencies of losartan and of
2a were quite indistinguishable, and in our opinion, this
experimental evidence already tended to suggest that
the antagonism was exerted by losartan itself, after the
hydrolytic removal of the side chain, as desired for our
purposes. The experimental confirmation of this hy-
pothesis was provided by the use of eserin: in the
presence of this esterase inhibitor, the antagonist
potency of 2a was dramatically lowered (Kb ) 40 nM),
while eserin did not influence the AT1-antagonist
potency of losartan (Kb ) 5 nM).
These initial functional data suggest that the main
aim of this work, i.e., the creation of the original
pharmacological class of NO-sartan AT1-antagonist/NO-
donor hybrids, has been satisfactorily achieved. Fur-
thermore, an “exploratory” in vivo protocol was carried
out on 2a, to obtain a preliminary indication about the
possible profile of the antihypertensive action of a
compound of this new pharmacological class, in com-
parison with that of an AT1-antagonist and an ACE
inhibitor. Consequently, compound 2a, losartan, and
captopril were administered orally or subcutaneously
to spontaneously hypertensive rats (SHR) for four
weeks, recording the systolic blood pressure, by the “tail
cuff” method.38 The order of magnitude of the oral doses
of captopril and losartan was selected on the basis of
similar experimental protocols, described in the litera-
ture,39 while 2a was administered at a dose equimolar
to that of losartan. For subcutaneous administration,
the doses were reduced by one-half. In both these sets
of experimental conditions, all compounds had practi-
cally equivalent effects, causing a significant reduction
in the systolic pressure, which was lowered almost to
the levels of reference normotensive animals (Figure 2).
In our opinion, this result was of fundamental impor-
tance, since the ineffectiveness or a low effectiveness of
2a was considered to be a critical factor, capable of
invalidating the rational basis of our work.
Figure 2. Histograms indicate the systolic blood pressure
values recorded in the first four weeks of oral (A) or subcuta-
neous (B) pharmacological treatment with the vehicle, losar-
tan, captopril, and compound 2a. Systolic pressure values of
reference normotensive animals are also shown. Vertical bars
indicate the standard error.
Acknowledgment. We would like to thank the
MIUR (Italian Ministry of Schooling, Higher Education
and Research) for partial support of this project through
a grant.
Supporting Information Available: Experimental pro-
1
cedures, H NMR, and MS. This material is available free of
charge via the Internet at http://pubs.acs.org.
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