1,4-Dihydropyridine derivatives as calcium channel modulators: The role of 3-methoxy-flavone moiety

Article abstract of DOI:10.1016/j.bmc.2005.03.007

It was earlier recognized that calcium antagonists, and in particular 1,4-dihydropyridines, exhibited distinct cardiovascular profiles. In addition two different splice variants of the L-type calcium channel were found in vascular and cardiac tissues. In

Full text of DOI:10.1016/j.bmc.2005.03.007

Bioorganic & Medicinal Chemistry 13 (2005) 3423–3430
1,4-Dihydropyridine derivatives as calcium channel modulators:
the role of 3-methoxy-flavone moiety
Roberta Budriesi, Alessandra Bisi, Pierfranco Ioan,* Angela Rampa, Silvia Gobbi,
Federica Belluti, Lorna Piazzi, Piero Valenti and Alberto Chiarini
Department of Pharmaceutical Sciences, University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy
Received 7 January 2005; revised 24 February 2005; accepted 1 March 2005
Available online 30 March 2005
Abstract—It was earlier recognized that calcium antagonists, and in particular 1,4-dihydropyridines, exhibited distinct cardiovascu-
lar profiles. In addition two different splice variants of the L-type calcium channel were found in vascular and cardiac tissues. In this
study, novel substituted 1,4-dihydropyridines with a 3-methoxy-flavone moiety were synthesized and structural modifications of the
substituents in the dihydropyridine ring of nifedipine were carried out in order to find tissue specific compounds. The negative ino-
tropic, chronotropic and vasorelaxant effects were investigated on guinea-pig left, right atria and aortic strips, respectively. The
introduction of an heteroaromatic ring in 4-position of the 1,4-dihydropyridine nucleus led to compounds selective for cardiac tis-
sues. Moreover, different residues in the 1,4-dihydropyridine ring could modulate the chronotropic versus inotropic activity.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
Calcium entry blockers (CEBs) are structurally divided
in two large groups: the dihydropyridines (DHP), repre-
sented by Nifedipine (1 Fig. 1), and the non-dihydro-
pyridines, represented by Verapamil and Diltiazem.1
Most of them are therapeutic agents used in the treat-
ment of many cardiovascular diseases such as angina
and hypertension. They elicit the therapeutic effects
by reversibly blocking Ca2+ influx through L-type
calcium channels (LCCs Cav1) found in cardiac and
vascular smooth muscle.2 In particular, splice variants
Cav1.2a and Cav1.2b are associated with the heart and
smooth muscle, respectively.3
Cav1 channels consist of a pore forming a1-subunit and
three auxiliary subunits, namely a2-d, b and c.4,5 The a1-
subunit is composed of four homologues domains (I–
IV); each of which has six transmembrane segments
(S1–S6).6 Photoaffinity labelling provided the first evi-
Figure 1.
dence that the DHP binding site was located on the
transmembrane strands IIIS6 and IVS6 and on the S5/
S6 helices.6
For a long time we have been interested in the study of
the 1,4-dihydropyridines bearing a different heteroaro-
matic ring in 4-position.6–13 The elucidation of the struc-
ture–activity relationships of DHPs agreed that the
substitution of o-nitrophenyl ring by aromatic or
Keywords: Calcium entry blockers; 1,4-Dihydropyridine; Cardiac
selectivity; In vitro assays.
*
Corresponding author. Tel.: +39 051 2099737; fax: +39 051
2099721; e-mail: pierfranco.ioan3@unibo.it
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.03.007

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R. Budriesi et al. / Bioorg. Med. Chem. 13 (2005) 3423–3430
heteroaromatic ring is important to improve selectivity
and activity.7,8 In particular, the potent and selective
negative chronotropic activity of compounds bearing a
4-xanthone moiety (2 Fig. 1) might indeed open new
perspectives in the search for more effective drugs for
the control of cardiac arrhythmias.
The cardiovascular activities of all synthesized com-
pounds were assayed on isolated tissues namely right
and left guinea-pig atria and K+-depolarized guinea-
pig aortic strips.
2. Chemistry
Considering that the xantone6,7,9 group can be success-
fully replaced with structurally related oxygen heterocy-
cles such as fluorenone,9–11 benzophenone11,12 and
coumarin13 and taking into account a patent from
1985 reporting substituted chromone and flavone14
derivatives as hypotensive and cardiotonic drugs, we
prepared a series of 1,4-dihydropyridines bearing a 3-
methoxyflavone ring with general formula 3 (Fig. 1).
The compounds listed in Table 1 were prepared accord-
ing to Schemes 1 and 2.
In Scheme 1, the synthesis of the key intermediate 3-
methoxy-4-oxo-2-phenyl-4H-chromene-8-carbaldehyde
6 is reported. O-Cresol was refluxed with acetic anhy-
dride in the presence of AlCl3 to give 1-(2-hydroxy-3-
methylphenyl)-ethanone, which was treated with benzal-
dehyde and 50% NaOH to yield the corresponding 1-(2-
hydroxy-3-methylphenyl)-3-phenyl-propenone. Cycliza-
tion with H2O2 in NaOH gave 3-hydroxy-8-methyl-2-
phenylchromen-4-one,15 which was methylated by
means of dimethylsulfate to give compound 4. Bromin-
ation with N-bromosuccinimide followed by reaction
with hexamethylentetramine and HCl afforded 3-meth-
oxy-4-oxo-2-phenyl-4H-chromene-8-carbaldehyde 6.
The aim of this work is to evaluate the influence of the
heteroaromatic ring in combination with different sub-
stituents in the 1,4 dihydropyridines nucleus. Though
the enantioselective property of the binding site for
1,4-dihydropiridines in the a1-subunits of Cav1.2 chan-
nels is well established, the compounds 3d, 3f, 3h, 3i–
k, and 3m, were synthesized and tested as racemates
and, at this stage of the research no attempt was made
to resolve the enantiomeric mixtures. Above all the goal
of the present paper is to clarify the influence of the
methoxyflavone moiety on the 1,4-dihydropyridine
backbone.
The compounds 3a–k were prepared (Scheme 2) accord-
ing to the classical Hantzsch reaction,16 by heating the
aldehyde with the appropriate reagents in isopropyl
alcohol.
Table 1. Cardiovascular activity of compounds
Compound
Cardiovascular activity
Negative chronotropy
Ia %c ( SEM) EC30 (lM)b (95% cl)
Negative inotropy
Vasorelaxant activity
Ia %a
( SEM)
EC50 (lM)b (95% cl)
Ia %d
( SEM)
IC50 (lM)b (95% cl)
1
97
2
0.26
(0.19–0.36)
(1.52–2.05)
(0.028–0.033) 67 3.4f
59 2.1f
79 2.3
85 4.2h
93 3.7i
87 1.3h
0.025
0.105
0.0097
0.041
0.13
(0.019–0.031)
(0.09–0.126)
(0.0074–0.013) 61 2.8
82 1.3g 0.009
(0.003–0.02)
(0.85–1.28)
(0.36–0.52)
3a
3b
3c
3d
3e
3f
24 0.7e
66 4.1
1.11
0.46
69 3.5e 1.79
63 3.2f 0.031
38 1.4
(0.038–0.043)
(0.09–0.21)
(0.29–0.43)
37 1.5
42 3.4
29 1.2j
19 0.7j
47 2.1
24 1.4j
24 1.3j
24 0.1
13 0.9
38 2.1j
47 1.4
0.35
61 1.5
20 0.3e
10 0.3
48 2.7g
1.19
(0.82–1.61)
20 1.5
25 1.3e
90 1.6
20 1.4
67 1.7
74 3.7
81 1.3
82 4.7
3g
3h
3i
0.38
(0.27–0.53)
3j
63
2
0.55
(0.39–0.62)
2.25
1.29
0.46
0.81
(1.82–2.83)
(1.07–1.55)
(0.41–0.51)
(0.76–0.87)
3k
3l
24 1.3
10 0.7
3m
8
0.6
58 1.7j 0.80
(0.73–0.88)
a Decrease in developed tension in isolated guinea-pig left atrium at 10À5 M, expressed as percent changes from the control (n = 5–6). The left atria
were driven at 1 Hz. 10À5 M gave the maximum effect for most compounds.
b Calculated from log concentration–response curves (Probit analysis by Litchfield and Wilcoxon with n = 6–7).17 When the maximum effect was
<50%, the EC50 ino., EC30 chrono., IC50 values were not calculated.
c Decrease in atrial rate on guinea-pig spontaneously beating isolated right atrium at 10À5 M, expressed as percent changes from the control (n = 7–
8). Pretreatment heart rate ranged from 165 to 190 beats/min. 10À5 M gave the maximum effect for most compounds.
d Percent inhibition of calcium-induced contraction on K+-depolarized guinea-pig aortic strip at 5 · 10À5 M (n = 5–6). 5 · 10À5 M gave the maximum
effect for most compounds.
e At the 5 · 10À5 M.
f At the 5 · 10À7 M.
g At the 10À6 M.
h At the 10À7 M.
i At the 5 · 10À6 M.
j At the 10À5 M.

R. Budriesi et al. / Bioorg. Med. Chem. 13 (2005) 3423–3430
3425
acetoacetate, compound 3g was prepared by reacting 6
with acetylacetone and ammonia (method D). Com-
pounds 3h–i were synthesized by refluxing the aldehyde
and methyl 3-aminocrotonate with 2-acetonyl-5,5-di-
methyl-2-oxo-1,3,2-dioxophosphorinane and b-keto-
propionylphosphonate, respectively (methods E and
F). Compound 3j was obtained reacting 3a with pyridi-
nium bromide perbromide and heating the not isolable
bromomethyl intermediate (method G). Compounds
3k–l were prepared according to method H, by heating
the aldehyde with an equimolar amount of allyl acetoac-
etate and ethoxy carbonyl acetamidine in dry ethanol
with a catalytic amount of piperidine. Finally, com-
pound 3m, bearing the 2-aminoethoxymethyl group,
was prepared by cleavage of the 2-phtalimido derivative
(obtained starting from ethyl 4-(2-phtalimidoeth-
oxy)acetoacetate and allyl 3-aminocrotonate) with aque-
ous methylamine (method I).
Scheme 1.
Compounds 3a–c were prepared following method A,
coupling the 3-methoxy-4-oxo-2-phenyl-4H-chromene-
8-carbaldehyde 6 with the selected acetoacetic ester in
the presence of ammonia. The asymmetric 1,4-dihydro-
pyridine 3d was synthesized by refluxing the aldehyde 6
with an equimolar amount of allyl acetoacetate and
methyl 3-aminocrotonate (method B).
3. Pharmacology
The pharmacological profile of compounds was tested
on guinea-pig isolated left and right atria to evaluate
their inotropic and chronotropic effects, respectively,
Compounds 3e–f were obtained according to method C
by the reaction of 6 with aminocrotononitrile and allyl
Scheme 2.

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R. Budriesi et al. / Bioorg. Med. Chem. 13 (2005) 3423–3430
and on K+-depolarized guinea-pig aortic strips to assess
calcium antagonist activity. At first, all compounds were
checked at increasing doses to evaluate the percent de-
crease on developed tension on isolated left atrium dri-
ven at 1 Hz (negative inotropic activity), the percent
decrease in atrial rate on spontaneously beating right at-
rium (negative chronotropic activity) and the percent
inhibition of calcium-induced contraction on K+-depo-
larized aortic strips (vasorelaxant activity). Data were
analyzed by StudentÕs t-test. The potency of drugs de-
fined as EC50, EC30 and IC50 was evaluated from log
concentration–response curves (Probit analysis by
Litchfield and Wilcoxon, n = 6–8) in the appropriate
pharmacological preparations. All data are presented
as mean SEM.17–19
With the aim to evaluate the effects of c-lactones22 of 3a
we decided to synthesize compound 3j. As shown in
Table 1, this compound was devoid of vasorelaxant
activity and showed a clear decrease in the negative chro-
notropic potency (EC30 = 2.25 lM, c.l. 95% = 1.82–
2.83). In contrast, it elicited a significant negative inotro-
pic potency (EC50 = 0.546 lM, c.l. 95% = 0.392–0.617),
only two times less than the reference compound 1.
The substitution of the methyl ester in position C-3 and
C-5 of compound 3a with an ethyl ester, as in 3b, im-
proved the cardiovascular potency of the compound,
which proved to be about 11- and 2-fold more potent
than 3a as negative chronotropic and vasorelaxant agent,
respectively. This improved potency did not injure the
negative chronotropic selectivity, indeed 3b kept a good
vasorelaxant/negative chronotropic and negative inotro-
pic/chronotropic ratio (47 and 184, respectively). When
the ethyl ester residues of 3b were changed with allyl ester
ones (3c), the compound became more selective for the
cardiac tissue. If compared with 3b, the vasorelaxant
activity decreased significantly but the negative chrono-
tropic over inotropic selective activity was lost.
4. Results and discussion
The activity of compounds, expressed as efficacy and po-
tency, was determined on guinea-pig left and right atria
to test negative inotropy and chronotropy, respectively,
while vasorelaxant activity was assessed on guinea-pig
aortic strips. All data are reported in Table 1 with those
of parent compound 1, taken as reference, to allow rele-
vant reasoning on structure–activity relationships. It is
well established that nifedipine is selective for the vascu-
lar activity over the heart, this is probably due to a dif-
ferent affinity for the different isoforms of Cav1.2
channels expressed on both tissues,20 (Cav1.2a expressed
in the heart and Cav1.2b expressed in the vascular tis-
sue). The substitution of the o-nitrophenyl ring of nifedi-
pine with a 3-methoxyflavone moiety (3a) reverses the
selectivity of compounds 3a being about a 123-fold less
potent than nifedipine on vascular smooth muscles
(EC50 = 1.11 lM and 0.0097 lM, respectively). More-
over, compound 3a shows weak negative inotropic
activity, while negative chronotropic potency is about
four fold less if compared with that of the reference
compound 1 (EC30 = 0.105 lM and 0.025 lM, respec-
tively). The drastic change in the cardiovascular activity
of compound 3a with respect to that of nifedipine could
be due to the position of the carbonyl group of the 3-
methoxyflavone moiety. Indeed we have already shown
in previous works that most of the compounds bearing
a carbonyl group in the same position of 3-methoxyflav-
one moiety, as fluorenone9 or benzophenone,12 are en-
dowed with the same cardiovascular profile as of 3a.
On the contrary, in coumarin derivatives,13 where the
carbonyl group is shifted, the activity slightly changes
showing a reduced potency on heart and vascular prep-
arations in respect to that of nifedipine. The metabolic
breakdown of 1,4-dihydropyridine follows the principle
of increasing hydrophilicity.21 The first step is oxidation
to the pyridine derivative, practically devoid of vasodila-
tory activity, which is followed by saponification of one
of the two ester functions to give the pyridine-monocarb-
oxylic acid. The final products of biotransform-
ation are the hydroxymethyl-pyridine-carboxylic
acids resulting from oxidative hydroxylation of the
methyl group neighbouring the carboxylic acid
function. These give the corresponding c-lactones after
acidification.
The most active nifedipine analogues are characterized
by the non-identical carboxylate group in C-3 and C-
5. In most cases, dihydropyridines with non-identical
ester group have a higher vasodilatory activity than their
symmetrically substituted analogues.23 In compound 3d
the methyl ester and allyl ester groups were combined.
The cardiovascular profile of 3d is similar to that of
3a, although it is a weak vasorelaxant agent compared
to the latter compound. The replacement of one allyl
ester group of 3c with a different electron withdrawing
group as CN (3f) did not affect the cardiovascular pro-
file, but when both C-3 and C-5 were substituted with
a cyano group (3e), the compound became a weak but
selective negative inotropic agent (EC50 = 1.19 lM,
c.l. = 0.82–1.61). Decreasing the electron withdrawing
effect of substituents in position C-3 and C-5 (3g) the
cardiovascular activity was lost, in good agreement with
our previous investigations.8
On the basis of the good results obtained with some
phosphonate derivatives of 1,4-dihydropyridines,24 we
decided to introduce a cyclic or open phosphonate
group in C-3 (compounds 3h and 3i, respectively). Sur-
prisingly, while compound 3h showed a good negative
chronotropic potency and selectivity, the opening of
the cyclic phosphonate group (3i), caused the loss of
the cardiovascular activity.
It is well known that both the presence of small alkyl
groups, such as methyl in C-2 and C-6, and the replace-
ment of one alkyl group by amino are well tolerated
without affecting activity.25 The compounds 3k–m were
synthesized to investigate the influence of different sub-
stituents in position C-2 and C-6 of the 1,4-dihydropyri-
dine ring in combination with the methoxyflavone
moiety.
Compound 3l, the 2,6-diamino substituted of 3b,
showed selective negative chronotropic activity but its

R. Budriesi et al. / Bioorg. Med. Chem. 13 (2005) 3423–3430
3427
potency decreased to about 47-fold if compared with 3b
(EC30 = 0.46 lM and 0.0097 lM, respectively). On the
other hand, the introduction of amino groups in C-2
and C-6 of the compound 3k with nonidentical ester
groups decreased the bradycardic potency without
affecting the selectivity.
6.1.1.1. 3-Methoxy-8-methyl-2-phenyl-chromen-4-one
(4). A suspension of 5 g (0.02 mol) of 3-hydroxy-8-
methyl-2-phenyl-chromen-4-one15,
2.5 mL
of
(CH3)2SO4, 5 g of K2CO3 in 100 mL of acetone was
heated under reflux 8 h, hot filtered and evaporated to
dryness. The residue, on crystallizing from ethanol, gave
1
5 g (96%) of 4, mp 147–150 ꢁC. H NMR d 2.6 (s, 3H,
CH3), 3.9 (s, 3H, OCH3), 7.25–8.2 (m, 8H, Ar). Anal.
Calcd (C17H14O3) C, H.
Restoring the methyl group in C-6 and introducing in 2-
position a residue of amlodipine (3m), the negative chro-
notropic potency was improved with respect to 3k, and
vasorelaxant activity was restored.
6.1.1.2. 8-Bromomethyl-3-methoxy-2-phenyl-chromen-
4-one (5). A mixture of 3-methoxy-8-methyl-2-phenyl-
chromen-4-one (5.5 g, 0.02 mol), N-bromosuccinimide
(3.7 g, 0.02 mol) in the presence of a catalytic amount
of benzoyl peroxide in 150 mL of carbon tetrachloride,
was refluxed for 4 h and then hot filtered. The solution
was evaporated to dryness and the residue, on crystalliz-
ing from ethyl acetate, gave 5 g (70%) of 5, mp 156–
159 ꢁC. 1H NMR d 3.9 (s, 3H, OCH3), 4.8 (s, 2H,
CH2Br), 7.3–8.25 (m, 8H, Ar). Anal. Calcd
(C17H13BrO3) C, H.
5. Conclusions
In this work a series of 1,4-dihydropyridines bearing a 3-
methoxyflavone ring in 4-position and different groups
from C-2 to C-6 were synthesized. Once again we could
demonstrate that the introduction of 4-heteroaromatic
group in the 1,4-dihydropyridine ring of nifedipine
could modulate the cardiovascular activity of com-
pounds. In particular most of the new compounds
showed a selective bradycardic activity in the guinea-
pig isolated right atria.
6.1.1.3. 3-Methoxy-4-oxo-2-phenyl-4H-chromene-8-
carbaldehyde (6). A solution of 8-bromomethyl-3-meth-
oxy-2-phenyl-chromen-4-one (3.44 g, 0.01 mol) and
hexamethylenetetramine (2.8 g, 0.02 mol) in 32 mL of
50% acetic acid was refluxed for 5 h. Conc. HCl
(15 mL) was added and the mixture was refluxed for
1 h, poured into ice and filtered. The solid was purified
by flash-chromatography (eluent: petroleum ether/ethyl
acetate 4.5:0.5) to give 2.23 g (80%) of 6 mp 153–
154 ꢁC. 1H NMR d 3.95 (s, 3H, OCH3), 7.5–8.6 (m,
8H, Ar), 10.75 (s, 1H, CHO). Anal. Calcd (C17H11O4)
C, H.
Recently, Zamponi et al.5 employed a 3D-QSAR model,
to explain the SARs for 4-isoxazolyl-1,4-dihydropyri-
dines. Our compounds could bind the same site in the
domain region, namely the 3-methoxyflavone moiety
could interact with tyrosine and methionine aminoacid
residues.
The substitution in 2,3,5,6-position of 1,4-dihydropyri-
dine ring could modulate the chronotropic/inotropic
activity or make molecules inactive. In particular 3b is
51 and 7-fold less potent than nifedipine as vasorelaxant
and negative inotropic agent, respectively, while is 3-fold
more potent as bradycardic. Of course, further studies
are required, but this kind of compounds could be useful
in the treatment of myocardial ischemia, where negative
inotropic and hypotensive effects could be potentially
deleterious.26,27
6.1.1.4. 4-(3-Methoxy-4-oxo-2-phenyl-4H-chromen-8-
yl)-2,6-dimethyl-1,4-dihydro-pyridine-3,5-dicarboxylic acid
dimethylester (3a). Method A: A solution of 6 (0.56 g,
0.002 mol), methyl acetoacetate (0.46 g, 0.004 mol) and
ammonia (5 mL) in isopropyl alcohol (15 mL) was re-
fluxed 30 h. The separated solid, on cooling, was filtered
and crystallized from toluene to yield 0.28 g (30%) of 3a
mp 264–265 ꢁC. 1H NMR d 2.3 (s, 6H, CH3), 3.3 (s, 6H,
COOCH3), 3.9 (s, 3H, OCH3), 5.7 (broad, 1H, NH), 5.8
(s, 1H, H-4 dihydropyridine), 7.2–8.3 (m, 8H, Ar). MS:
476 (M+1). Anal. Calcd (C27H25NO7) C, H, N.
6. Experimental
6.1. Chemistry
6.1.1.5. 4-(3-Methoxy-4-oxo-2-phenyl-4H-chromen-8-yl)-
2,6-dimethyl-1,4-dihydro-pyridine-3,5-dicarboxylic acid
diethylester (3b). Using the precedent procedure and
starting from 0.56 g (0.002 mol) of 6, 0.4 g (40%) of 3b
mp 229–230 ꢁC (toluene) were obtained. 1H NMR d
0.8 (t, 6H, CH2CH3), 2.35 (s, 6H, CH3), 3.8–3.9 (m,
4H, CH2CH3), 3.95 (s, 3H, OCH3), 5.55 (broad, 1H,
NH), 5.8 (s, 1H, H-4 dihydropyridine), 7.2–8.3 (m, 8H,
Ar). MS: 504 (M+1). Anal. Calcd (C29H29NO7) C, H, N.
6.1.1. General methods. Melting points were determined
on a Buchi apparatus and are uncorrected. H NMR
1
spectra were obtained for CDCl3 solutions on a Gemini
300 spectrometer. Chemical shifts are reported in parts
per million (ppm) relative to tetramethylsilane (TMS),
and spin multiplicities are given as s (singlet), d (dou-
blet), t (triplet), q (quartet) or m (multiplet). Purification
by gravity column chromatography on Merck silica-gel
60, 70–230 mesh and by flash chromatography on
230–400 mesh were carried out using the slurry method
for column packing. Elemental analyses were within
0.4% of the theoretical values. Compounds were
named following IUPAC rules as applied by AUTONOM,
a PC software for systematic names in organic chemis-
try, Beilstein-Institute and Springer.
6.1.1.6. 4-(3-Methoxy-4-oxo-2-phenyl-4H-chromen-8-
yl)-2,6-dimethyl-1,4-dihydro-pyridine-3,5-dicarboxylic acid
diallylester (3c). Using the precedent procedure and
starting from 0.56 g (0.002 mol) of 6, 0.47 g (45%) of
1
3c mp 194–195 ꢁC (toluene) were obtained. H NMR d
2.4 (s, 6H, CH3), 3.95 (s, 3H, OCH3), 4.2–4.4 (m, 4H,

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R. Budriesi et al. / Bioorg. Med. Chem. 13 (2005) 3423–3430
CH@CH2), 4.7–4.85 (m, 4H, COOCH2), 5.3–5.5 (m, 2H,
CH@CH2), 5.85 (broad, 1H, NH), 5.95 (s, 1H, H-4 dihy-
dropyridine), 7.25–8.3 (m, 8H, Ar). MS: 528 (M+1).
Anal. Calcd (C31H29NO7) C, H, N.
0.002 mol), 2-acetonyl-5,5-dimethyl-2-oxo-1,3,2-dioxa-
phosphorinane (0.44 g, 0.002 mol) and methyl 3-amino-
crotonate (0.23 g, 0.002 mol) in isopropyl alcohol
(15 mL) was refluxed for 10 h and evaporated to dry-
ness. The residue was purified by flash-chromatography
(eluent: toluene/ethyl acetate 7:3) to yield 0.23 g (20%)
6.1.1.7. 4-(3-Methoxy-4-oxo-2-phenyl-4H-chromen-8-
yl)-2,6-dimethyl-1,4-dihydro-pyridine-3,5-dicarboxylic acid-
3-allylester-5-methylester (3d). A solution of 6 (0.56 g,
0.002 mol), allyl acetoacetate (0.28 g, 0.002 mol), methyl
3-aminocrotonate (0.23 g, 0.002 mol) in isopropyl alco-
hol (15 mL) was refluxed for 10 h. The solvent was evap-
orated to dryness and the residue was purified by
flash-chromatography (eluent: toluene/acetone 4:1) to
1
of 3h mp 260–262 ꢁC (toluene). H NMR d 0.5 (s, 3H,
PCH3), 0.75 (s, 3H, PCH3), 2.3 (s, 6H, CH3), 3.35 (m,
5H, COOCH3 and OCH2), 3.95 (m, 5H, OCH3 and
OCH2), 5.6 (d, 1H, H-4 dihydropyridine), 6.15 (broad,
1H, NH), 7.8–8.25 (m, 8H, Ar). MS: 565 (M+1). Anal.
Calcd (C30H32NO8P) C, H, N.
1
give 0.25 g (25%) of 3d mp 232–233 ꢁC (toluene). H
6.1.1.11. 5-(Dimethoxy-phosphoryl)-4-(3-methoxy-4-
oxo-2-phenyl-4H-chromen-8-yl)-2,6-dimethyl-1,4-dihy-
dro-pyridine-3-carboxylic acid methyl ester (3i). Method
F: A solution of 6 (0.56 g, 0.002 mol), dimethyl-b-keto-
propylphosphonate (0.33 g, 0.002 mol) and methyl 3-
aminocrotonate (0.23 g, 0.002 mol) in isopropyl alcohol
(15 mL) was refluxed for 10 h and evaporated to dry-
ness. The residue was purified by flash-chromatography
(eluent: toluene/ethyl acetate 7:3) to yield 0.20 g (20%)
NMR d 2.3 (d, 6H, CH3), 3.3 (s, 3H, COOCH3), 3.95
(s, 3H, OCH3), 4.2–4.4 (m, 2H, CH@CH2), 4.65–4.8
(m, 2H, COOCH2), 5.3–5.5 (m, 1H, CH@CH2), 5.65
(broad, 1H, NH), 5.8 (s, 1H, H-4 dihydropyridine),
7.2–8.3 (m, 8H, Ar). MS: 502 (M+1). Anal. Calcd
(C29H27NO7) C, H, N.
6.1.1.8. 4-(3-Methoxy-4-oxo-2-phenyl-4H-chromen-8-
yl)-2,6-dimethyl-1,4-dihydro-pyridine-3,5-dicarbonitrile (3e).
5-Cyano-4-(3-methoxy-4-oxo-2-phenyl-4H-chromen-8-yl)-
2,6-dimethyl-1,4-dihydro-pyridine-3-carboxylic acid allyl
ester (3f). Method C: A solution of 6 (0.56 g, 0.002 mol),
allyl acetoacetate (0.28 g, 0.002 mol), aminocrotonitrile
(0.23 g, 0.002 mol) in isopropyl alcohol (15 mL) was re-
fluxed for 20 h. The solvent was evaporated to dryness
and the residue was purified by flash-chromatography
(eluent: toluene/acetone 4:1) to give first 0.19 g (20%)
of 3f mp 128–129 ꢁC (toluene), then 0.4 g (50%) of 3e
mp 214–215 ꢁC (toluene).
1
of 3i as oily compound. H NMR d 2.4 (s, 6H, CH3),
2.9 (d, 3H, POCH3), 3.4 (d, 3H, POCH3), 3.45 (s, 3H,
COOCH3), 3.95 (s, 3H, OCH3), 5.6 (d, 1H, H-4 dihydro-
pyridine), 6.1 (broad, 1H, NH), 7.3–8.4 (m, 8H, Ar). MS:
526 (M+1). Anal. Calcd (C27H28NO8P) C, H, N.
6.1.1.12. 4-(3-Methoxy-4-oxo-2-phenyl-4H-chromen-
8-yl)-2-methyl-5-oxo-1,4,5,7-tetrahydro-furo[3,4-b] pyri-
dine-3-carboxylic acid methyl ester (3j). Method G: To
a cold solution of 3a (0.24 g, 0.0005 mol) in chloroform
(10 mL), pyridine (0.06 g, 0.0008 mol) and pyridinium
bromide perbromide (0.19 g, 0.0006 mol) were added.
The solution was stirred at 0 ꢁC for 20 min and then it
was refluxed for 90 min. After cooling, chloroform was
added and the solution was washed with 2 N HCl and
brine, dried and evaporated to dryness. The residue
was crystallized form ethanol to give 0.11 (50%) of 3j
mp 298–302 ꢁC. 1H NMR (DMSO) d 2.4 (s, 3H,
CH3), 3.4 (s, 3H, COOCH3), 3.95 (s, 3H, OCH3), 4.9
(s, 2H, CH2), 5.55 (s, 1H, H-4 dihydropyridine), 7.2–8.2
(m, 8H, Ar), 9.9 (s, 1H, NH).
3f: 1H NMR d 2.05 (s, 3H, CH3), 2.4 (s, 3H, CH3), 3.9 (s,
3H, OCH3), 4.25 (m, 2H, CH@CH2), 4.7 (m, 2H,
COOCH2), 5.4 (m, 1H, CH@CH2), 5.55 (s, 1H, H-4
dihydropyridine), 6.7 (broad, 1H, NH), 7.15–8.15 (m,
8H, Ar). MS: 470 (M+1). Anal. Calcd (C28H24N2O5)
C, H, N.
1
3e: H NMR d 2.1 (s, 6H, CH3), 3.9 (s, 3H, OCH3), 5.2
(s, 1H, H-4 dihydropyridine), 6.6 (broad, 1H, NH), 7.2–
8.2 (m, 8H, Ar). MS: 409 (M+1). Anal. Calcd
(C25H19N3O3) C, H, N.
MS: 460 (M+1). Anal. Calcd (C26H21NO7) C, H, N.
6.1.1.9. 8-(3,5-Diacetyl-2,6-dimethyl-1,4-dihydro-pyri-
din-4-yl)-3-methoxy-2-phenyl-chromen-4-one (3g). Meth-
od D: Acetylacetone (0.4 g, 0.004 mol) was added to a
solution of 6 (0.56 g, 0.002 mol) in isopropyl alcohol
(15 mL) and ammonia (5 mL) with stirring. The reaction
mixture was refluxed for 12 h and then evaporated to
dryness. The residue, on crystallizing from toluene, gave
0.4 g (40%) of 3g mp 216–219 ꢁC. 1H NMR d 2.2 (s, 6H,
CH3), 2.31 (s, 6H, CH3), 3.9 (s, 3H, OCH3), 5.8 (broad,
1H, NH), 5.9 (s, 1H, H-4 dihydropyridine), 7.2–8.3 (m,
8H, Ar). MS: 445 (M+1). Anal. Calcd (C27H25NO5) C,
H, N.
6.1.1.13. 2-Amino-4-(3-methoxy-4-oxo-2-phenyl-4H-
chromen-8-yl)-6-methyl-1,4-dihydro-pyridine-3,5-dicarbo-
xylic acid 5-allylester 3-ethylester (3k). 2,6-Diamino-4-(3-
methoxy-4-oxo-2-phenyl-4H-chromen-8-yl)-1,4-dihydro-
pyridine-3,5-dicarboxylic acid diethylester (3l). Method
H: A solution of 6 (0.6 g, 0.0021 mol) in 20 mL of dry eth-
anol was prepared and ethoxy carbonyl acetamidine
hydrochloride (0.273 g, 0.0021 mol), allyl acetoacetate
(0.298 g, 0.0021 mol) and piperidine (six drops) were
added with stirring. The reaction mixture was heated
under reflux for 6 h and evaporated to dryness. The resi-
due was purified by flash chromatography (eluent: tolu-
ene/acetone 4:1) to give 0.32 g (30%) of 3k mp 235–
1
6.1.1.10. 5-(5,5-Dimethyl-2-oxo-1,3,2-dioxophosphor-
inan-2-yl)-4-(3-methoxy-4-oxo-2-phenyl-4H-chromen-8-
yl)-2,6-dimethyl-1,4-dihydro-pyridine-3-carboxylic acid
methyl ester (3h). Method E: A solution of 6 (0.56 g,
238 ꢁC. H NMR (DMSO) d 0.5 (t, 3H, CH3), 2.4 (s,
3H, CH3), 3.6 (m, 2H, COOCH3), 3.9 (s, 3H, OCH3),
4.0–4.3 (m, 2H, COOCH2), 4.55–4.7 (m, 2H, CH@CH2),
5.3 (m, 1H, CH@CH2), 5.6 (s, 1H, H-4 dihydropyridine),

R. Budriesi et al. / Bioorg. Med. Chem. 13 (2005) 3423–3430
3429
6.75 (broad, 2H, NH2), 7.1–8.3 (m, 8H, Ar), 8.8 (s, 1H,
NH). MS: 517 (M+1). Anal. Calcd (C29H28N2O7) C, H, N.
Instruments, Quincy, MA) using Power Lab software
(Basile, Italy). The left atria were stimulated by rectan-
gular pulses of 0.6–0.8 ms duration and about 50%
threshold voltage through two platinum contact elec-
trodes in the lower holding clamp (Grass S88 stimula-
tor). The right atrium was in spontaneous activity.
After the tissue was beating for several minutes, a
length–tension curve was determined, and the muscle
length was maintained at which elicited 90% of maxi-
mum contractile force observed at the optimal length.
A stabilization period of 45–60 min was allowed before
the atria were used to test compounds. During the equili-
bration period, the bathing solution was changed every
15 min and the threshold voltage was ascertained for
the left atria. Atrial muscle preparations were used to
examine the inotropic and chronotropic activity of the
compounds (0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, 10,
50 and 100 lM), first dissolved in DMSO and then di-
luted with PSS. According to this procedure, the concen-
tration of DMSO in the bath solution never exceeded
0.3%, a concentration that did not produce appreciable
inotropic and chronotropic effects. During the construc-
tion of cumulative dose–response curves, the next higher
concentration of the compounds was added only after
the preparation reached a steady state.
Compound 3l (0.11 g, 10%) was obtained from the same
column washing with methanol, mp 175–179 ꢁC. 1H
NMR d 0.6 (t, 6H, CH3), 3.7 (m, 4H, COOCH2), 3.8
(s, 3H, OCH3), 5.6 (d, 1H, H-4 dihydropyridine), 6.4
(broad, 5H, NH2 and NH), 7.2–8.3 (m, 8H, Ar).
MS: 506 (M+1). Anal. Calcd (C27H27N3O7) C, H, N.
6.1.1.14. 2-[2-Aminoethoxymethyl]-4-(3-methoxy-4-oxo-
2-phenyl-4H-chromen-8-yl)-6-methyl-1,4-dihydropyr-
idine-3,5-dicarboxylic acid allyl ethyl ester (3m). Method
I: 1.8 g of 6 (0.0064 mol) were dissolved in 20 mL of iso-
propyl alcohol. Ethyl 4-(2-phtalimido)ethoxyacetoace-
tate (2.05 g, 0.0064 mol) and allyl-3-aminocrotonate
(0.913 g, 0.0064 mol) were added with stirring and the
reaction mixture was refluxed for 20 h. The solvent
was removed under reduced pressure and the residue
was purified by flash chromatography (eluent: toluene/
ethyl acetate 7:3), to yield 1.8 g (40%) g of oily com-
pound, which was used in the subsequent step without
1
further purification. H NMR d 0.7 (t, 3H, CH3), 2.3
(s, 3H, CH3), 3.8 (m, 4H, COOCH2 + OCH2), 3.9 (m,
7H, OCH3 + CH2 + CH2N), 4.2–4.4 (m, 2H, CH@CH2),
4.65–4.8 (m, 2H, COOCH2), 5.1 (s, 1H, H-4 dihydropyri-
dine), 5.3–5.5 (m, 1H, CH@CH2), 5.8 (broad, 1H, NH),
7.5–8.4 (m, 12H, Ar).
6.2.2. Guinea-pig aortic strips. The thoracic aorta was re-
moved and placed in Tyrode solution of the following
composition (mM): 118 NaCl, 4.75 KCl, 2.54 CaCl2,
1.20 MgSO4, 1.19 KH2PO4, 25 NaHCO3 and 11 glucose
equilibrated with 95% O2–5% CO2 gas at pH 7.4. The
vessel was cleaned of extraneous connective tissue. Two
helicoidal strips (10 mm · 1 mm) were cut from each aor-
ta beginning from the end most proximal to the heart.
Vascular strips were then tied with surgical thread (6-0)
and suspended in a jacketed tissue bath (15 mL) contain-
ing aerated pharmacological salt solution (PSS) at 37 ꢁC.
Strips were secured at one end to a force displacement
(FT 0.3, Grass) transducer for monitoring changes in iso-
metric contraction. Aortic strips were subjected to a rest-
ing force of 1 g and washed every 20 min with fresh PSS
for 1 h after the equilibration period; guinea-pig aortic
strips have been contracted by washing in PSS contain-
ing 80 mM KCl (equimolar substitution of K+ for
Na+). After the contraction reached a plateau (about
45 min) the compounds (0.001, 0.005, 0.01, 0.05, 0.1,
0.5, 1, 5, 10, 50 and 100 lM) were added cumulatively
to the bath allowing for any relaxation to obtain an
equilibrated level of force. Addition of the drug vehicle
had no appreciable effect on K+-induced contraction
(DMSO for all compounds).
1.8 g of previous compound (0.026 mol) was suspended
in 50 mL of aqueous methylamine (35% w/v) and stirred
at room temperature for 72 h. After filtration, the solid
was purified by flash chromatography (eluent: metha-
1
nol), to give 0.3 g of 3m, mp 235–238 ꢁC. H NMR d
0.7 (t, 3H, CH3), 2.4 (s, 3H, CH3), 3.0 (m, 2H,
COOCH2), 3.6 (m, 2H, CH2), 3.8 (m, 2H, OCH2), 3.9
(s, 3H, OCH3), 4.1–4.4 (m, 2H, CH@CH2), 4.75–4.85
(m, 4H, COOCH2 + CH2N), 5.4 (m, 1H, H-4 dihydro-
pyridine), 5.3–5.5 (m, 1H, CH@CH2), 5.8 (broad, 1H,
NH), 7.2–8.4 (m, 10H, Ar + NH2). MS: 575 (M+1). Anal.
Calcd (C32H34N2O8) C, H, N.
6.2. Functional studies
6.2.1. Guinea-pig atrial preparations. Guinea-pigs (300–
400 g female) were sacrificed by cervical dislocation.
After thoracotomy the heart was immediately removed
and washed by perfusion through the aorta with oxy-
genated Tyrode solution of the following composition
(mM): 136.9 NaCl, 5.4 KCl, 2.5 CaCl2, 1.0 MgCl2, 0.4
NaH2PO4xH2O, 11.9 NaHCO3 and 5.5 glucose. The
physiological salt solution (PSS) was buffered at
pH 7.4 by saturation with 95% O2–5% CO2 gas, and
the temperature was maintained at 37 ꢁC. Isolated gui-
nea-pig heart preparations were used, spontaneously
beating right atria and left atria driven at 1 Hz. For each
preparation, the entire left and right atria were dissected
from the ventricles, cleaned of excess tissue, and hung
vertically in a 15 mL organ bath containing the PSS con-
tinuously bubbled with 95% O2–5% CO2 gas at 37 ꢁC,
pH 7.4. The contractile activity was recorded isometri-
cally by means of a force transducer (FT 0.3, Grass
Acknowledgements
This research was supported by grants from M.I.U.R.
(P.R.I.N. 2003) and the University of Bologna.
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