New benzopyran-based openers of the mitochondrial ATP-sensitive potassium channel with potent anti-ischemic properties

Article abstract of DOI:10.1021/jm061228l

This study was aimed at evaluating, on a limited number of benzopyran compounds, whether the insertion of an electron-rich spirocyclic substituent at the C4 carbon of the benzopyran molecular nucleus may improve the cardioprotective properties against isc

Full text of DOI:10.1021/jm061228l

7600
J. Med. Chem. 2006, 49, 7600-7602
New Benzopyran-Based Openers of the
Mitochondrial ATP-Sensitive Potassium Channel
with Potent Anti-Ischemic Properties
Maria C. Breschi,‡ Vincenzo Calderone,*,‡ Alma Martelli,‡
Filippo Minutolo,# Simona Rapposelli,*,# Lara Testai,‡
Federica Tonelli,# and Aldo Balsamo#
Dipartimento di Scienze Farmaceutiche and Dipartimento di
Psichiatria, Neurobiologia, Farmacologia e Biotecnologie,
UniVersita` di Pisa, Via Bonanno, 6, I-56126, Pisa, Italy
ReceiVed October 20, 2006
Figure 1. Left: graphical representation of SAR data for anti-ischemic
properties of benzopyran derivatives, adapted with some modifications
from Mannhold.8 Right: general structure of the synthesized spiro-
morpholone- (1a,b and 2b) and spiromorpholine KCOs (3a,b and 4b).
Abstract: This study was aimed at evaluating, on a limited number
of benzopyran compounds, whether the insertion of an electron-rich
spirocyclic substituent at the C4 carbon of the benzopyran molecular
nucleus may improve the cardioprotective properties against ischemia.
Some of the new compounds (1b, 2b, and 4b) exhibited interesting
anti-ischemic properties without affecting significantly the blood
pressure parameters.
its involvement in cardioprotection is almost entirely of a
pharmacological nature based on the relative selectivity for mito-
KATP of the channel opener diazoxide and the selective inhibitor
5-HD. The possibility of triggering the above-mentioned physi-
ological step involved in the IPC process through exogenous
activators of the mito-KATP channel represents a rational basis
for the development of highly interesting, innovative anti-
ischemic drugs.6,7
KATP channel openers (KCOs) belong to a heterogeneous
group of compounds of different chemical classes, including
cyanoguanidines, thioformamides, and benzopyran derivatives.
Some first-generation KCOs (cromakalim, nicorandil, pinacidil)
showed cardioprotective effects, but their clinical use was
limited because of many side effects, like those linked to their
hypotensive properties.
In recent years many structural changes have been made,
mainly on the nucleus of benzopyran KCOs,8 in order to find
new compounds with a higher selectivity toward specific targets,
such as the KATP channel of pancreatic â-cells9 or the cardiac
mitochondrial KATP channel, such as the benzopyranylcyan-
oguanidine derivatives BMS-180448 and BMS-191095.10,11
Until now, most chemical modifications on benzopyran KCOs
have been studied at the C4 position (Figure 1), but a few cases12
have regarded the restriction of the conformation of this
substituent through its insertion into a spiro-like structure.
The absence of a detailed study of this kind of modification
led us to explore it in depth, since the constrained structure due
to the spirocyclic ring might give new compounds a higher
selectivity toward a specific type of KATP channel and give
useful information about steric requirements for their active
interaction. In this work, some new spiromorpholone or spiro-
morpholine C4-substituted benzopyran derivatives were prepared
in order to determine the effects of these structural modifications
on the cardioprotective properties and on the activity of these
new compounds on vascular smooth muscle and blood pressure.
The spiromorpholones 1a,b and 2b were synthesized follow-
ing the synthetic procedure illustrated in Scheme 1. Chro-
manones 5a,b, obtained from the appropriate 2-hydroxyace-
tophenone,13 were subjected to nucleophilic addition with
trimethylsilylcyanide (TMSCN) in the presence of ZnI2 as the
Lewis acid to afford the corresponding trimethylsilyl cyanohy-
drins (6a,b). Compounds 6a,b were directly reduced to ami-
noalcohols 7a,b with LiAlH4 in accordance with the procedure
of Amundsen and Nelson.14 The subsequent reaction with
chloroacetyl chloride of 7a,b in a heterogeneous phase yielded
the corresponding chloroacetamides 8a,b. Base-catalyzed (t-
BuOK) cyclization of 8a,b gave the spiromorpholones 9a,b,
Potassium channels are membrane proteins that selectively
allow potassium ions to flow across the cell membrane. Among
the different types of potassium channels, some of them, defined
as ATP-sensitive (KATP), are mainly regulated by changes in
the intracellular [ATP]/[ADP] ratio, thus associating the mem-
brane potential with the metabolic state of the cell.
KATP channels are located in the sarcolemmal and inner
mitochondrial membrane of the cells of many tissues, including
heart, skeletal and smooth muscle, pancreatic â-cells, and
neurons and are thus involved in many physiological processes,
including hormone secretion, smooth muscle activity,1 and
neurotransmitter release.2 In the heart, sarcolemmal and mito-
chondrial channels are involved in modulating myocardial
functions, especially in ischemia or hypoxia conditions in which
the activation of physiological cellular preservation systems may
be important.
Various mechanisms of cardioprotection are known. One of
them is the phenomenon of ischemic preconditioning (IPC), an
endogenous mechanism whereby brief periods of ischemia (3-5
min) have been shown to increase the resistance of cardiomyo-
cytes to injury induced by a subsequent prolonged period of
ischemia, reducing the extent of necrosis in the damaged region.
This phenomenon, which has not yet been completely clarified,
is triggered by several processes, both receptor-mediated (with
the activation of bradykinin, adenosine, and opioid systems)3
and receptor-independent (due to the increase in NO and reactive
oxygen species levels).4
To date, a key role in IPC seems to be played by mitochon-
drial KATP (mito-KATP) channels; mito-KATP channel openers,
such as diazoxide, at concentrations that do not activate
sarcolemmal KATP channel5 have been widely shown to mimic
IPC, producing pronounced cardioprotective effects. In addition,
these pharmacological responses are blocked, or reduced, by
the use of a selective mito-KATP channel blocking agent such
as 5-hydroxydecanoic acid (5-HD).5 However, the molecular
structure of mito-KATP is still unknown, and the evidence for
* To whom correspondence should be addressed. For V.C.: phone, +39-
050-2219589; fax, +39-050-2219609; e-mail, calderone@farm.unipi.it. For
S.R.: phone, +39-050-2219582; fax, +39-050-2219577; e-mail, rappsi@
farm.unipi.it.
‡ Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotec-
nologie.
# Dipartimento di Scienze Farmaceutiche.
10.1021/jm061228l CCC: $33.50 © 2006 American Chemical Society
Published on Web 11/29/2006

Letters
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 26 7601
Scheme 1a
Figure 2. Changes in blood systolic pressure (expressed as a % of
the basal level) following the intraperitoneal administration of the
cardioprotective compounds synthesized (40 mg kg-1), diazoxide (40
mg kg-1), and cromakalim (1 mg kg-1).
a (a) acetone, pyrrolidine; (b) TMSCN, ZnI2; (c) LiAlH4, THF; (d)
chloroacetyl chloride, NaOH; (e) t-BuOK, toluene; (f) 4-substituted benzyl
bromide, NaH, DMF; (g) NH2NH2‚H2O, FeCl3, MeOH; (h) Ac2O, acetone;
(i) MeSO2Cl, pyridine, dioxane.
Scheme 2a
Figure 3. Representative pictures of left ventricle slices submitted to
staining procedure with TTC. The pale areas represent the ischemia-
injured tissue: (A) heart isolated from a rat pretreated with the vehicle;
(B) heart isolated from a rat pretreated with compound 2b; (C) heart
isolated from a rat pretreated with 5-hydroxydecanoic acid and
compound 2b.
a (a) LiAlH4, THF; (b) BH3‚SMe2, microwave; (c) Ac2O, acetone; (d)
MeSO2Cl, pyridine, dioxane.
which reacted with p-nitrobenzyl bromide and NaH in DMF to
give the N-benzyl-substituted compounds 10a,b.15 The nitro
derivatives were then reduced to the corresponding amines 11a,b
with hydrazine hydrate in the presence of a catalytic amount of
ferric chloride and activated carbon. Treatment of 11a,b with
acetic anhydride afforded the corresponding acetamides 1a,b,
while methanesulfonylation of 11b gave compound 2b. Com-
pounds 3a,b and 4b were obtained by reduction starting from
spiromorpholones 11a,b to the corresponding spiromorpholine
derivatives 12a,b with LiAlH4 for 11a or with a borane-methyl
sulfide complex for 11b (Scheme 2). The subsequent reaction
of 12a,b with acetic anhydride and methanesulfonyl chloride
afforded the acetamido- (3a,b) and methanesulfonamido- (4b)
compounds, respectively.
All the compounds synthesized (1-4) were tested as racemic
mixtures at a dose of 40 mg kg-1 ip on Langendorff perfused
rat hearts subjected to ischemia-reperfusion cycles (30 and 120
min). Furthermore, two well-known KATP channel openers,
diazoxide and cromakalim, were also tested as reference drugs
at doses of 40 or 1 mg kg-1, respectively. Diazoxide is a
benzothiadiazine derivative widely considered to possess a
satisfactory degree of selectivity.5,16 In contrast, cromakalim is
a potent benzopyrane-based KATP channel opener (and is thus,
from a structural point of view, closer to the compounds
synthesized) that exhibits cardioprotective effects and marked
hypotensive properties. For each compound, the resulting
ischemic injury was quantified by evaluating functional, bio-
chemical, and morphological parameters. In particular, the
functional parameter at the 30th [RPP-(30)] or the 120th min
of reperfusion [RPP-(120)] has been expressed as a percentage
of the rate-pressure product (RPP) recorded during reperfusion,
with respect to the percentage of the RPP value recorded at the
last minute of the preischemic period. These parameters were
taken as indicators of the functional recovery of inotropism in
the early and final stages of reperfusion, respectively. The
biochemical marker of ischemic injury was represented by the
amount of lactate dehydrogenase (LDH) released by the heart
in the perfusion fluid during the entire reperfusion time. At the
end of reperfusion, the treatment of the heart with triphenyltet-
razolium chloride (TTC) made it possible to carry out a
Table 1. Functional (RPP-30′ and RPP-120′), Biochemical (LDH), and
Morphological (% Ischemic Area vs Total Area) Parameters Recorded in
Hearts Isolated from Rats Pretreated with the Vehicle, with the
Synthesized Compounds or with the Reference Drugs, in the Absence or
in the Presence of the Selective mito-KATP Blocker 5-Hydroxydecanoic
Acid
RPP-30′ RPP-120′ LDH U/g, % ischemic area
compd
%
%
120 min
vs total area
vehicle
(()-1a
(()-1b
(()-2b
(()-3a
(()-3b
(()-4b
(()-1a + 5-HD
(()-2b + 5-HD
(()-4b + 5-HD
(()-cromakalim
diazoxide
23 ( 12
79 ( 14
2 ( 1
23 ( 6
62 ( 20
3 ( 1
57 ( 19
17 ( 5
26 ( 4
77 ( 19
27 ( 11 13.5 ( 4.2
39 ( 19 11.7 ( 2.0
33 ( 16 13.4 ( 1.3
10.6 ( 1.0
6.0 ( 1.3
12.4 ( 0.9
3.0 ( 1.7
16.0 ( 1.7
11.3 ( 1.2
6.9 ( 3.8
35 ( 7
20 ( 4
58 ( 4
13 ( 3
50 ( 2
61 ( 2
14 ( 2
49 ( 5
47 ( 5
40 ( 1
25 ( 1
22 ( 6
52 ( 5
38 ( 3
75 ( 24
12 ( 3
15 ( 4
89 ( 11
16 ( 7
52 ( 16
17 ( 2
99 ( 1
80 ( 20
13 ( 7
84 ( 16
47 ( 3
6 ( 3
5.5 ( 0.7
5.7 ( 3.6
14.5 ( 5.4
8.6 ( 3.0
diazoxide + HD
(()cromakalim + HD 11 ( 2
18 ( 1
morphological comparison of the necrotic and healthy areas of
the left ventricular tissue, colored white (or pale pink) and red,
respectively (some representative examples are shown in Figure
3), and then to calculate the ischemia-injured area as a
percentage of the total area.
As indicated by the data shown in Table 1, global ischemia
induced severe damage in control hearts, which exhibited a low
level (23%) of postischemic functional recovery, high levels of
LDH released (>10 U g-1), and the clear presence of necrotic
areas (35%). As expected, the two reference drugs diazoxide
and cromakalim, administered at a dose of 40 and 1 mg/kg,
respectively, led to a significant improvement of these param-
eters, as shown by an increase in the postischemic functional
recovery and a clear reduction in LDH release and in the necrotic
areas. Among the compounds synthesized, 1a, 2b, and 4b
(administered at the same dose as diazoxide) exhibited a
cardioprotective behavior that was qualitatively and quantita-
tively similar to (and in some cases more satisfactory than) that

7602 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 26
Letters
Table 2. Parameters of Vasorelaxing Potency and Efficacy of the
Synthesized Compounds and the Reference KATP Openers Recorded in
Isolated Rat Aortic Rings
of substitution may effectively lead to promising compounds
possessing anti-ischemic properties at doses devoid of significant
hypotensive effects, with a pharmacological profile qualitatively
similar to those exhibited by BMS-180448 and BMS-191095.11
Further development of new structural analogues of the most
effective compounds reported in this study and their optical
resolution will make it possible to gain a better understanding
of the structure-activity relationships of this new class of
cardioprotective benzopyran-based KATP openers.
compd
pIC50
Emax, %
(()-1a
4.60 ( 0.03
4.82 ( 0.07
5.14 ( 0.03
4.88 ( 0.03
5.62 ( 0.03
5.22 ( 0.02
7.01 ( 0.09
4.72 ( 0.04
57 ( 3
70 ( 11
87 ( 3
77 ( 2
99 ( 1
98 ( 2
98 ( 1
97 ( 2
(()-1b
(()-2b
(()-3a
(()-3b
(()-4b
(()-cromakalim
diazoxide
Supporting Information Available: Chemical and pharmaco-
logical experimental procedures, 1H NMR, 13C NMR, and MS data,
histograms concerning the pharmacological data shown in Table
1, and results from elemental analysis. This material is available
free of charge via the Internet at http://pubs.acs.org.
exhibited by the reference drugs. In contrast, 1b, 3a, and 3b
did not show any cardioprotective effects.
In order to investigate the potential role of the mito-KATP
channel in cardioprotective mechanisms, the effective com-
pounds were tested in the presence of 5-hydroxydecanoic acid
(5-HD), a selective blocker of this channel type. The effects of
1a and 4b and those of the reference drugs were almost
completely abolished by 5-HD, suggesting that their anti-
ischemic properties may be due to the activation of the mito-
KATP channels. For the parameters recorded in hearts treated
with 2b in the presence of 5-HD, although the parameter of
inotropic recovery was not significantly influenced by the
antagonist, the levels of LDH and the percentage of necrotic
areas proved to be significantly enhanced, indicating that 5-HD
played at least a partial role of antagonism toward the cardio-
protective effects of 2b.
As reported above, one of the main problems in the use of
KCOs for the pharmacological prevention of ischemic injury
is linked to the lack of selectivity toward the mitochondrial
channel with respect to those expressed in the plasmalemma of
myocardiocytes and of vascular smooth muscle cells. Conse-
quently, in this work, 1-4 and the reference drugs diazoxide
and cromakalim were tested in vitro for their vasorelaxing effects
on rat aorta preparations contracted with KCl solution. Results
showed that all the compounds synthesized exhibited vasodilator
effects with full (1b, 3b, 4b) or partial (1a, 2b, 3a) efficacy
(Emax) and with modest levels of potency, with pIC50 ranging
from 4.60 and 5.62, thus showing a pharmacodynamic profile
similar to that of diazoxide (pIC50 ) 4.72). As expected,
cromakalim showed full vasorelaxing efficacy, with high levels
of potency (pIC50 ) 7.01) (Table 2).
In order to perform a more direct evaluation of the effects
produced by the new compounds showing cardioprotective
properties (1a, 2b, and 4b) and by the reference drugs diazoxide
and cromakalim on the haemodynamic parameters in vivo, these
derivatives were administered to male normotensive rats and
the systolic blood pressure was recorded for 60 min. As shown
in Figure 2, 1a, 2b, 4b, and diazoxide (administered at doses
that produced cardioprotective effects) did not show any
significant effects on the systolic blood pressure. In contrast,
cromakalim (administered at a dose that produced cardiopro-
tective effects) caused a rapid, dramatic, and long-lasting fall
of the blood pressure.
This study was designed to evaluate, on a limited number of
benzopyran compounds, whether the insertion of an electron-
rich spirocyclic substituent at the C4 carbon of the molecular
nucleus present in some potent but scarcely selective KCOs may
improve the pharmacological properties of compounds of this
type, in particular as far as selectivity is concerned. Although
the overall data obtained with the new compounds synthesized
do not allow us to advance any hypothesis about the role of the
substituents on the 4-spiroheterocyclic benzopyran derivatives
in their pharmacological activity, because of the limited number
of compounds studied, they indicate that in some cases this type
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