Coumarin 1,4-dihydropyridine derivatives

Article abstract of DOI:10.1016/S0968-0896(98)00037-6

A series of 1,4-dihydropyridines bearing a coumarin moiety in 4-position was synthesized. The compounds were evaluated for inotropic, chronotropic and calcium antagonist activities. The replacement of the o-nitrophenyl moiety of nifedipine with a coumarin

Full text of DOI:10.1016/S0968-0896(98)00037-6

BIOORGANIC &
MEDICINAL
CHEMISTRY
Bioorganic & Medicinal Chemistry 6 (1998) 803±810
Coumarin 1,4-Dihydropyridine Derivatives
P. Valenti,* A. Rampa, R. Budriesi, A. Bisi and A. Chiarini
Department of Pharmaceutical Sciences, University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy
Received 22 July 1997; accepted 22 December 1997
AbstractÐA series of 1,4-dihydropyridines bearing a coumarin moiety in 4-position was synthesized. The compounds
were evaluated for inotropic, chronotropic and calcium antagonist activities. The replacement of the o-nitrophenyl
moiety of nifedipine with a coumarin or phenylcoumarin system is accompained by a decrease of the activity on
myocardial and vascular parameters, but the synthesized compounds showed selective inhibiting e€ects on cardiac
contractility and frequency. # 1998 Elsevier Science Ltd. All rights reserved.
Introduction
The rationale for these modi®cations is based on the
theory that the xanthone moiety can be successfully
replaced with structurally related oxygen heterocycles
such as chromone, ¯avone and coumarin moieties. In
fact, in a number of cases this replacement has allowed
us to obtain important results with regard to analeptics,17
adrenergic b-blocking agents,18 clo®brate-like hypo-
lipemic drugs,19 and more recently, antitumor drugs.20±22
The asymmetric compounds were tested as racemates.
The introduction of 4-aryl-1,4-dihydropyridines (DHPs)
with highly potent calcium-channel blocking activity led
to a new direction in cardiovascular therapy. Calcium-
channel modulators are now well established in the
treatment of angina pectoris, hypertension, certain car-
diac arrhythmias and peripheral vascular disorders.1±5
A
great number of 4-aryl-1,4-dihydropyridine-3,5-dicarb-
oxylates have been synthesized and evaluated biologi-
cally for cardiovascular activity allowing delineation of
well-de®ned structure±activity relationships (SAR).6±8
The 1,4-dihydropyridine-type calcium antagonists, in
spite of the extensive SAR studies, further deserve the
interest of the medicinal chemists. For a long time we
have been interested9±16 in the study of the 1,4-di-
hydropyridines, of general formula 1, bearing a xanthone
moiety in 4-position. The presence in the synthesized
derivatives of a potent and selective chronotropic nega-
tive activity might indeed open new perspectives in the
search of more e€ective drugs for the control of cardiac
arrhythmias. With this aim we have prepared a series of
coumarin 1,4-dihydropyridines of general formula 2:
Chemistry
The studied compounds, collected in Table 1, were pre-
pared as shown in Schemes 1 and 2.
In Scheme 1 is reported the synthesis of the key inter-
mediates 4a,b. o-Cresol was treated with formaldehyde
in presence of SnCl4 to give the corresponding salicyl-
aldehyde,23 which was condensed with sodium acetate
or sodium phenylacetate, and acetic anhydride to a€ord
8-methylcoumarin and 8-methyl-3-phenylcoumarin,
respectively. Bromination with N-bromosuccinimide
(compounds 3a,b), followed by reaction with hexa-
methylenetetramine a€ord the aldehydes 4a,b.
In Scheme 2 is reported the synthesis of the studied
derivatives.
The compounds, listed in Table 1, were prepared
according to the classical Hantzsch reaction,24 heating
the selected aldehyde in isopropyl alcohol with the
appropriate reagents. Compounds 2a±f were prepared
*Corresponding author.
0968-0896/98/$19.00 # 1998 Elsevier Science Ltd. All rights reserved
PII: S0968-0896(98)00037-6

804
P. Valenti et al./Bioorg. Med. Chem. 6 (1998) 803±810
Table 1. Inotropic, chronotropic and calcium antagonist activities
Compound
R
R1
R2
ED50
ino
95% ED50
Conf. lim. chrono
95%
Conf. lim
ED50
CCB
95%
Conf. lim.
2a
2b
2c
2d
2e
2f
H
H
H
COOMe
COOEt
COOMe
COOEt
COOCH2CH=CH2 COOCH2CH=CH2
7.90
0.54
1.76
0.41
0.93
0.89
6.60±9.37
0.46±0.64
1.47±2.15
0.33±0.50
0.75±1.15
0.81±1.04
2.70
0.055 0.042±0.071 0.056 0.048±0.071
2.55±2.91
1.31
1.04±1.59
0.60
2.47
0.51±0.71
2.30±2.63
0.25
0.19±0.32
Ph COOCH2CH=CH2 COOCH2CH=CH2
34‹1.5% at 50 mM
15‹ 0.4% at 50 mM
35‹1.5% at 50 mM
12‹ 0.4% at 50 mM
Ph
Ph
H
COOMe
COOEt
COOMe
COOEt
14‹0.3% at 10 mM
0.27
2.36
0.19±0.38
2.18±2.53
2g
2h
2i
COOMe
COOMe
COOMe
COOCH2CH=CH2 38‹ 1.4% at 50 mM
COOCH2CH=CH2
Ph
H
2.69
2.11±3.40
45 ‹ 1.9% at 10 mM
0.57
0.49±0.65
34 ‹ 1.4% at 50 mM
3.05
2.87±3.25
39‹2.7% at 50 mM
39‹ 1.4% at 50 mM
45‹ 2.3% at 50 mM
2j
Ph
H
COOMe
COOMe
1.25
0.96±1.60
2.95
2.77±3.15
2k
5‹ 0.3% at 50 mM
5 ‹ 0.1% at 50 mM
41 ‹ 2.3% at 1 mM 18 ‹ 0.6% at 50 mM
2.06 1.57±2.75
7‹ 0.3% at 50 mM
2l
2m
H
Ph
H
COOMe
COOMe
COOMe
NO2
NO2
8 ‹ 0.5% at 10 mM 10 ‹ 0.3% at 50 mM
2.63 2.11±3.34
24 ‹ 1.3% at 10 mM 45 ‹ 2.7% at 50 mM
2n
2o
COMe
COMe
H
*
COMe
*
10‹ 0.3% at 50 mM 19 ‹ 0.7% at 50 mM 37 ‹ 1.4% at 50 mM
19 ‹ 0.1% at 50 mM 33 ‹ 1.4% at 50 mM 14 ‹ 0.8% at 50 mM
0.26
*
2p
Nifedip.
0.2±0.3
0.039 0.031±0.048 0.009
0.003±0.02
*
ED50 values with 95% conf. lim. are calculated from log-concn in response curves (probit analysis by Litch®eld and Wilcoxon).30 Each
compound was tested at least ®ve times. `ED50 ino' shows the ED50 values for the negative inotropic potency of tested compounds on
stimulated guinea pig left atrium in mM or as a percent inhibition [mean ‹ SEM] at a particular concentration if no ED50 has been
measured. Similarly the `ED50 chrono' in spontaneously beating right atrium, and `ED50 CCB' in K+ depolarized guinea pig aortic
strips, re¯ect the negative chronotropic activity and calcium channel blocking activity.
according to method A, coupling the coumarinic alde-
hydes with the selected acetoacetic ester in presence of
ammonia.
ester and 3-aminocrotonate. According to method C com-
pounds 2i,j were prepared by reaction of the aldehydes
with 2-acetonyl-5,5-dimethyl-2-oxo-1,3,2-dioxophos-
phorinane25 and methyl 3-aminocrotonate. Compound
2k was synthesized using dimethyl b-chetopropionyl-
phosphonate26 and methyl 3-aminocrotonate (Method
D). Compounds 2l,m were obtained by reaction with
The asymmetric 1,4-dihydropyridines 2g±h were syn-
thesized according to method B, by re¯uxing the alde-
hydes with an equimolar amount of selected acetoacetic

P. Valenti et al./Bioorg. Med. Chem. 6 (1998) 803±810
805
Scheme 1.
Scheme 2.
nitroacetone27 and methyl 3-aminocrotonate (Method
E). Compounds 2n,o were prepared by reaction of cou-
marinic aldehyde with acetylacetone, methyl 3-amino-
crotonate and, respectively, ammonia (Methods F and
G). Finally, compound 2p was obtained reacting 2a with
pyridinium bromide perbromide and heating the not
isolable bromomethyl intermediate (Method H).
Pharmacology
The biological activity of all new compounds was tested
by functional studies on isolated guinea pig cardiac
preparations to evaluate the inotropic and chronotropic
e€ects in left driven atria and in spontaneously beating
right atria, respectively. The vasorelaxing activity,

806
P. Valenti et al./Bioorg. Med. Chem. 6 (1998) 803±810
expression of calcium antagonism, was assessed in
potassium depolarized guinea pig aortic strips.
appearing as nearly pure negative chronotropic agents.
Considering vasorelaxing activity, which is a measure of
calcium antagonist action on smooth muscle again
compounds 2b, 2c and 2h possessing allyl and ethyl
radicals to the ester moiety elicit signi®cant calcium
antagonist activity being from about 6±63 fold less
potent than nifedipine. For all the other derivatives,
except 2a and 2m bearing methyl ester groups, calcium
antagonism is poor or undetectable.
The potency of the compounds was expressed as ED50
and IC50 values accordingly to the di€erent functional
experiments or as percent inhibition at a particular
concentration if no ED50 or IC50 values have been
measured.
Finally, some of these new compounds showed a very
intriguing behaviour. In fact, in our experiments on left
and right atria a rapid and transient positive inotropic
e€ect appeared and was not changed both by pretreat-
ment with a non-selective b-antagonist (1 mM propra-
nolol) and by employing reserpine-treated guinea pigs
(data not shown). The mechanism involved in this phe-
nomenon is elusive and further research on this topic is
needed. In conclusion the replacement of the o-nitro-
phenyl moiety of nifedipine with a coumarin or phenyl-
coumarin system is accompanied by a decrease of the
activity on myocardial and vascular parameters, but the
synthesized compounds show selective cardiodepressant
e€ects expecially negative chronotropic action. Further-
more, the results con®rm our hypotesis that the xan-
thone moiety can be successfully replaced with a
coumarin system.
Results and discussion
Table 1 lists negative inotropic, chronotropic and
vasorelaxing activities of studied compounds in com-
parison with nifedipine used as reference standard.
A ®rst analysis of the results reveals that the replace-
ment of the o-nitrophenyl moiety of nifedipine with
coumarin or phenylcoumarin system is accompanied by
a decrease of the activity on myocardial and vascular
parameters. The derivatives possessing the coumarin
system (2a, 2b, 2c, and 2g) are more potent than the
correspondent phenylcoumarin analogues (2d, 2e, 2f,
and 2h), but less selective. In fact 2d, 2e, and 2f show
appreciable negative inotropic potency whereas 2b dis-
plays fairly good negative chronotropic ecacy and
potency.
Generally speaking, the most active compounds of the
series appear to be compounds 2b, 2d, and 2f. The ratio
between ED50 (negative inotropic activity) and ED50
(negative chronotropic activity) and the corresponding
values of nifedipine are 3.3 and 6.9 for 2f; 2 and 1.4 for
2b; 1.5 and 63 for 2d; 6.5 and 15 for 2c; 29 and 69 for 2a;
4.6 and 76 for 2j.
Experimental
Chemistry
Melting points were determined on a Buchi apparatus
and are uncorrected. 1H NMR 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 multi-
plicities are given as s (singlet), d (doublet), t (triplet), q
(quartet) or m (multiplet). Puri®cation by gravity col-
umn chromatography on Merck silica gel 60, 70±230
mesh and by ¯ash 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 chemistry, Beilstein-
Institut and Springer.
Taking a closer look at these potency ratio it emerges
that in these derivatives there is an increase of negative
inotropic activity with respect to the other e€ects except
for 2b which shows a negative chronotropic potency
comparable to nifedipine. Furthermore, the ecacy,
expressed by % decreases both in left atrium [92‹4.2%
at 50 mM] and in right atrium [69‹1.2% at 0.1 mM], is
similar in the corresponding ®gures of nifedipine
[97‹2% at 10 mM; 85‹4.2% at 0.1 mM], but this latter
compound triggers these e€ects at lower concentration.
It is to note that these results are in keeping with our
previous studies,11,12 which underlined, as far as cardiac
activity is concerned, the relevant importance of allyl
and ethyl functions to the ester moiety at 3- and 5-posi-
tions of the dihydropyridine ring.
8-Methyl-2H-chromen-2-one. A mixture of 2-hydroxy-3-
methylbenzaldehyde23 (6.8 g, 0.05 mol), anhydrous
sodium acetate (4.1 g, 0.05 mol) and acetic anhydride
(10.2 g, 0.1 mol) was heated at 180±190 ꢀC for 5 h. After
cooling the mixture was poured in 10% solution of
K2CO3. The separated solid was collected by ®ltration,
dried and crystallized from ligroin to give 5.2 g (65%) of
Another additional interesting ®nding is the clear selec-
tivity of 2i and 2b (ED50 values 3.05 mM and 0.055 mM,
respectively) for myocardial parameter chronotropism

P. Valenti et al./Bioorg. Med. Chem. 6 (1998) 803±810
807
product mp 112±115 ꢀC [lit.28 mp 109.6±109.9 ꢀC]. 1H
NMR d 2.45 (s, 3H, CH3), 6.4 (d, 1H, H-3), 7.15±7.4 (m,
3H, Ar), 7.7 (d, 1H, H-4).
Ar and H-4 coumarin). MS: m/z (relative abundance):
369 (M+, 7.19), 225 (13.96), 224 (100). Anal. calcd
(C20H19NO6) C, H, N.
8-Methyl-3-phenyl-2H-chromen-2-one. Using the pre-
cedent procedure and starting from 2-hydroxy-3-
methylbenzaldehyde (6.8 g, 0.05 mol) and anhydrous
sodium phenylacetate (7.9 g, 0.05 mol), 8.26 g (70%) of
product mp 114±115 ꢀC (ligroin) [lit.29 mp 107.5±109 ꢀC]
were obtained. 1H NMR d 2.5 (s, 3H, CH3), 7.2±7.8 (m,
9H, Ar and H-4).
Diethyl 1,4-dihydro-4-(2-oxo-2H-chromen-8-yl)-2,6-di-
methylpyridine-3,5-dicarboxylate (2b). Using the pre-
cedent procedure and starting from 1.74 g (0.01 mol) of
4a, 0.99 g (25%) of 2b mp 197±200 ꢀC (EtOH) were
obtained. 1H NMR d 1.15 (t, 6H, CH2CH3) 2.32 (s, 6H,
CH3), 4.05 (m, 4H, CH2CH3), 5.4 (s, 1H, H-4 dihydro-
pyridine), 6.1 (s, 1H, NH), 6.38 (d, 1H, H-3 coumarin),
7.1±7.7 (m, 4H, Ar and H-4 coumarin). MS: m/z
(relative abundance): 397 (M+, 39.65), 252 (100),
106 (11.95), 89 (15.74). Anal. calcd (C22H23NO6) C,
H, N.
8-Bromomethyl-2H-chromen-2-one (3a). A mixture of 8-
methyl-2H-chromen-2-one (8 g, 0.05 mol), N-bromo-
succinimide (8.9 g, 0.05 mol) in the presence of a cata-
lytic amount of benzoyl peroxide in 150 mL of carbon
tetrachloride, was re¯uxed for 4 h and then hot ®ltered.
The solution was evaporated to dryness and the residue,
on crystallizing from ligroin, gave 8 g (70%) of 3a mp
Diallyl 1,4-dihydro-4-(2-oxo-2H-chromen-8-yl)-2,6-di-
methylpyridine-3,5-dicarboxylate (2c). Using the pre-
cedent procedure and starting from 1.74 g (0.01 mol) of
4a, 0.63 g (15%) of 2c mp 157±160 ꢀC (EtOH) were
90±92 ꢀC. H NMR d 4.75 (s, 2H, CH2), 6.5 (d, 1H, H-
1
1
3), 7.2±7.75 (m, 4H, Ar and H-4). Anal. calcd (C10H7
BrO2) C, H.
obtained. H NMR d 2.32 (s, 6H, CH3), 4.45 (m, 4H,
CH=CH2), 5.1 (m, 4H, COOCH2), 5.42 (s, 1H, H-4
dihydropyridine), 5.75 (m, 2H, CH=CH2), 6.0 (broad,
1H, NH), 6.35 (d, 1H, H-3 coumarin), 7.1±7.7 (m, 4H,
Ar and H-4 coumarin). MS: m/z (relative abundance):
421 (M+, 8.73), 277 (20.16), 276 (100). Anal. calcd
(C24H23NO6) C, H, N.
8-Bromomethyl-3-phenyl-2H-chromen-2-one (3b). Using
the precedent procedure and starting from 8-methyl-3-
phenyl-2H-chromen-2-one (11.8 g, 0.05 mol), 11 g (70%)
of 3b mp 157±160 ꢀC were obtained. 1H NMR d 4.78 (s,
2H, CH2), 7.2±7.8 (m, 9H, Ar and H-4). Anal. calcd
(C16H11BrO2) C, H.
Diallyl 1,4-dihydro-4-(3-phenyl-2-oxo-2H-chromen-8-yl)-
2,6-dimethylpyridine-3,5-dicarboxylate (2d). Using the
precedent procedure and starting from 1.25 g
(0.005 mol) of 4b, 1.12 g (45%) of 2d mp 213±215 ꢀC
8-Formyl-2H-chromen-2-one (4a).
A solution of 3a
(4.78 g, 0.02 mol) and hexamethylenetetramine (5.6 g,
0.04 mol) in 40 mL of 50% acetic acid was re¯uxed for
4 h. Twenty milliliters of conc. HCl were added and the
mixture was re¯uxed for 15 min. After cooling the mix-
ture was ®ltered and crystallized from ligroin to give
1
(toluene) were obtained. H NMR d 2.35 (s, 6H, CH3),
4.5 (m, 4H, CH=CH2), 5.1 (m, 4H, COOCH2), 5.45 (s,
1H, H-4 dihydropyridine), 5.8 (m, 2H, CH=CH2), 6.2
(s, 1H, NH), 7.1±7.8 (m, 9H, Ar and H-4 coumarin).
MS: m/z (relative abundance): 497 (M+, 3.21), 276
(32.58), 91 (100). Anal. calcd (C30H28NO6) C, H, N.
2.78 g (80%) of 4a mp 114±116 ꢀC. H NMR d 6.55 (d,
1
1H, H-3), 7.4±8.15 (m, 4H, Ar and H-4), 10.75 (s, 1H,
CHO). Anal. calcd (C10H6O3) C, H.
Dimethyl 1,4-dihydro-4-(3-phenyl-2-oxo-2H-chromen-8-
yl)-2,6-dimethylpyridine-3,5-dicarboxylate (2e). Using
the precedent procedure and starting from 1.25 g
(0.005 mol) of 4b, 0.44 g (20%) of 2e mp 265±267 ꢀC
8-Formyl-3-phenyl-2H-chromen-2-one (4b). Using the
precedent procedure and starting from 3.15 g (0.01 mol),
1.86 g (75%) of 4b mp 165±168 ꢀC were obtained. 1H
NMR d 7.4±8.15 (m, 9H, Ar and H-4), 10.8 (s, 1H,
CHO). Anal. calcd (C16H10O3) C, H.
1
(toluene) were obtained. H NMR d 2.35 (s, 6H, CH3),
3.6 (s, 6H, COOCH3), 5.4 (s, 1H, H-4 dihydropyridine),
6.1 (s, 1H, NH), 7.15±8.8 (m, 9H, Ar and H-4 coumarin).
MS: m/z (relative abundance): 445 (M+, 6.46), 224
(100), 223 (21.73). Anal. calcd (C26H24NO6) C, H, N.
Dimethyl 1,4-dihydro-4-(2-oxo-2H-chromen-8-yl)-2,6-di-
methylpyridine-3,5-dicarboxylate (2a). Method A. A
solution of 4a (1.74 g, 0.01 mol), methyl acetoacetate
(2.32 g, 0.02 mol) and ammonia (10 mL) in isopropyl
alcohol (30 mL) was re¯uxed 30 h. After cooling the
reaction mixture was ®ltered and the solid obtained was
crystallized from toluene to yield 0.74 g (20%) of 2a mp
Diethyl 1,4-dihydro-4-(3-phenyl-2-oxo-2H-chromen-8-yl)-
2,6-dimethylpyridine-3,5-dicarboxylate (2f). Using the
precedent procedure and starting from 1.25 g
(0.005 mol) of 4b, 1.2 g (50%) of 2f mp 180±183 ꢀC
(toluene) were obtained. 1H NMR d 1.15 (t, 6H,
CH2CH3), 2.3 (s, 6H, CH3), 4.03 (m, 4H, CH2CH3), 5.4
(s, 1H, H-4 dihydropyridine), 6.1 (s, 1H, NH), 7.1±7.8
242±245 ꢀC. H NMR d 2.32 (s, 6H, CH3), 3.6 (s, 6H,
1
COOCH3), 5.4 (s, 1H, H-4 dihydropyridine), 6.15 (broad,
1H, NH), 6.35 (d, 1H, H-3 coumarin), 7.15±7.7 (m, 4H,

808
P. Valenti et al./Bioorg. Med. Chem. 6 (1998) 803±810
(m, 9H, Ar and H-4 coumarin). MS: m/z (relative
abundance): 473 (M+, 9.52), 252 (100), 45 (60.71). Anal.
calcd (C28H28NO6) C, H, N.
PCH3), 2.3 (s, 6H, CH3), 3.6 (s, 3H, COOCH3), 3.7 (m,
2H, OCH2), 4.2 (m, 2H, OCH2), 5.28 (d, 1H, H-4 dihy-
dropyridine), 6.15 (broad, 1H, NH), 7.1±7.8 (m, 9H, Ar
and H-4 coumarin). MS: m/z (relative abundance): 535
(M+, 13.06), 314 (100), 228 (25.62). Anal. calcd
(C29H31NO7P) C, H, N.
Methyl, allyl 1,4-dihydro-4-(2-oxo-2H-chromen-8-yl)-2,6-
dimethylpyridine-3,5-dicarboxylate (2g). Method B. A
solution of 4a (1.74 g, 0.01 mol), allyl acetoacetate
(1.42 g, 0.01 mol), methyl 3-aminocrotonate (1.15 g,
0.01 mol) in isopropyl alcohol (30 mL) was re¯uxed for
10 h. The solvent was evaporated to dryness and the
residue was puri®ed by ¯ash chromatography (eluent:
toluene:acetone, 4:1) to give 1.58 g (40%) of 2g mp 190±
Methyl 5-dimethoxyphosphinyl-2,6-dimethyl-4-(2-oxo-
2H-chromen-8-yl)-1,4-dihydropyridine-3-carboxylate (2k).
Method D. A solution of 4a (1.74 g, 0.01 mol), dimethyl
b-chetopropylphosphonate (1.64 g, 0.01 mol) and
methyl 3-aminocrotonate (1.15 g, 0.01 mol) in isopropyl
alcohol (30 mL) was re¯uxed for 10 h and evaporated to
dryness. The residue was puri®ed by ¯ash-chromato-
graphy (eluent: toluene:ethyl acetate, 7:3) to yield 0.42 g
(10%) of 2k mp 227±230 ꢀC (toluene). 1H NMR d 2.3 (s,
6H, CH3), 3.2 (d, 3H, POCH3), 3.55 (d, 3H, POCH3),
3.6 (s, 3H, COOCH3), 5.15 (d, 1H, H-4 dihydropyr-
idine), 6.15 (broad, 1H, NH), 6.35 (d, 1H, H-3 cou-
marin), 7.1±7.7 (m, 4H, Ar and H-4 coumarin). MS: m/z
(relative abundance): 419 (M+, 6.60), 274 (100), 273
(10.86). Anal. calcd (C20H22NO7P) C, H, N.
193 ꢀC (toluene). H NMR d 2.35 (s, 6H, CH3), 3.6 (s,
1
3H, COOCH3), 4.45 (m, 2H, CH=CH2), 5.1 (m, 2H,
COOCH2), 5.4 (s, 1H, H-4 dihydropyridine), 5.8 (m,
1H, CH=CH2), 6.1 (broad, 1H, NH), 6.35 (d, 1H, H-3
coumarin), 7.1±7.7 (m, 4H, Ar and H-4 coumarin). MS:
m/z (relative abundance): 395 (M+, 7.14), 250 (100),
249Â(9.33). Anal. calcd (C22H21NO6) C, H, N.
Methyl, allyl 1,4-dihydro-4-(3-phenyl-2-oxo-2H-chromen-
8-yl)-2,6-dimethylpyridine-3,5-dicarboxylate (2h). Using
the precedent procedure and starting from 1.25 g
(0.005 mol) of 4b, 0.34 g (20%) of 2h mp 200±203 ꢀC
Methyl 1,4-dihydro-2,6-dimethyl-3-nitro-(2-oxo-2H-
chromen-8-yl)-pyridine-5-carboxylate 2l. Method E.
Nitroacetone (1 g, 0.01 mol) was added to a solution of
4a (1.74 g, 0.01 mol) in isopropyl alcohol (30 mL) and
then methyl 3-aminocrotonate (1.15 g, 0.01 mol) was
added with stirring. The reaction mixture was re¯uxed
for 10 h and evaporated to dryness. The residue was
crystallized from toluene to yield 1.42 g (40%) of 2l mp
1
(toluene) were obtained. H NMR d 2.33 (s, 6H, CH3),
3.58 (s, 3H, COOCH3), 4.48 (m, 2H, CH=CH2), 5.1 (m,
2H, COOCH2), 5.4 (s, 1H, H-4 dihydropyridine), 5.8
(m, 1H, CH=CH2), 6.1 (broad, 1H, NH), 7.1±7.8 (m,
9H, Ar and H-4 coumarin). MS: m/z (relative abun-
dance): 471 (M+, 8.44), 251 (16.38), 250 (100). Anal.
calcd (C28H26NO6) C, H, N.
266±269 ꢀC. H NMR d 2.35 (s, 3H, CH3), 2.55 (s, 3H,
1
Methyl 1,4-dihydro-5-(5,5-dimethyl-2-oxo-1,3,2-dioxo-
phosphorinan-2-yl)-4-(2-oxo-2H-chromen-8-yl)-2,6-dimeth-
ylpyridine-3-carboxylate (2i). Method C. A solution of
4a (1.74 g, 0.01 mol), 2-acetonyl-5,5-dimethyl-2-oxo-
1,3,2-dioxaphosphorinane (2.2 g, 0.01 mol) and methyl
3-aminocrotonate (1.15 g, 0.01 mol) in isopropyl alcohol
(30 mL) was re¯uxed for 10 h and evaporated to dry-
ness. The residue was puri®ed by ¯ash chromatography
(eluent: toluene:ethyl acetate, 7:3) to yield 0.92 g (20%)
CH3), 3.6 (s, 3H, COOCH3), 5.65 (s, 1H, H-4 dihy-
dropyridine), 6.35 (d, 1H, H-3 coumarin), 6.95 (broad,
1H, NH), 7.1±7.75 (m, 4H, Ar and H-4 coumarin). MS:
m/z (relative abundance): 356 (M+, 14.50), 211 (100), 91
(91.36). Anal. calcd (C18H16N2O6) C, H, N.
Methyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-(3-phenyl-2-
oxo-2H-chromen-8-yl)-pyridine-5-carboxylate (2m). Using
the precedent procedure and starting from 1.25 g
(0.005 mol) of 4b, 0.65 g (30%) of 2m mp 272±275 ꢀC
of 2i mp 252±255 ꢀC (toluene). H NMR d 0.85 (s, 3H,
1
1
PCH3), 1.05 (s, 3H, PCH3), 2.25 (s, 6H, CH3), 3.6 (s,
3H, COOCH3), 3.7 (m, 2H, OCH2), 4.2 (m, 2H, OCH2),
5.28 (d, 1H, H-4 dihydropyridine), 6.15 (broad, 1H,
NH), 6.35 (d, 1H, H-3 coumarin), 7.1±7.7 (m, 4H, Ar
and H-4 coumarin). MS: m/z (relative abundance): 459
(M+, 12.82), 314 (100), 228 (19.69). Anal. calcd
(C23H26NO7P) C, H, N.
(toluene) were obtained. H NMR d 2.3 (s, 3H, CH3),
2.5 (s, 3H, CH3), 3.6 (s, 3H, COOCH3), 5.62 (s, 1H, H-4
dihydropyridine), 6.95 (broad, 1H, NH), 7.1±7.8 (m, 9H,
Ar and H-4 coumarin). MS: m/z (relative abundance):
432 (M+, 17.71), 211 (100), 165 (26.65). Anal. calcd
(C24H20NO6) C, H, N.
Methyl 1,4-dihydro-2,6-dimethyl-3-acetyl-4-(2-oxo-2H-
chromen-8-yl)-pyridine-5-carboxylate (2n). Method F.
Acetylacetone (1 g, 0.01 mol) was added to a solution of
4a (1.74 g, 0.01 mol) in isopropyl alcohol (30 mL) and
then methyl 3-aminocrotonate (1.15 g, 0.01 mol) was
added with stirring. The reaction mixture was re¯uxed
for 10 h and evaporated to dryness. The residue was
Methyl 1,4-dihydro-5-(5,5-dimethyl-2-oxo-1,3,2-dioxo-
phosphorinan-2-yl)-4-(3-phenyl-2-oxo-2H-chromen-8-yl)-
2,6-dimethylpyridine-3-carboxylate (2j). Using the pre-
cedent procedure and starting from 1.25 g (0.005 mol) of
4b, 0.27 g (10%) of 2j mp 290±292 ꢀC (EtOH) were
obtained. 1H NMR d 0.9 (s, 3H, PCH3), 1.03 (s, 3H,

P. Valenti et al./Bioorg. Med. Chem. 6 (1998) 803±810
809
puri®ed by ¯ash-chromatography (eluent: methylene
chloride:ethyl acetate, 1:1) to yield 0.88 g (25%) of 2n
mp 242±246 ꢀC (toluene). 1H NMR d 2.28 (s, 3H, CH3),
2.31 (s, 3H, CH3), 2.35 (s, 3H, CH3), 3.15 (s, 3H, CH3),
5.5 (s, 1H, H-4 dihydropyridine), 6.1 (broad, 1H, NH),
6.4 (d, 1H, H-3 coumarin), 7.15±7.7 (m, 4H, Ar and H-4
coumarin). MS: m/z (relative abundance): 353 (M+,
15.61), 208 (100), 207 (12.54). Anal. calcd (C20H19NO5)
C, H, N.
atrium were dissected from ventricles, cleaned of excess
tissue, hung vertically in a 15 mL organ bath containing
the PSS continously bubbled with 95% O2-5% CO2 gas
at 35 ꢀC, pH 7.4. The contractile activity was recorded
isometrically by means of a force transducer (FT. 0.3,
Grass Instrument, Quincy, MA, USA) connected to a
pen recorder (KV 380), Battaglia-Rangoni, Bologna
Italy). The left atria were stimulated by rectangular
pulses of 0.6±0.8 ms duration and 50% above threshold-
voltage through two platinum contact electrodes in the
lower holding clamp (Grass S88 stimulator). After the
tissue was beating for several minutes, a length-tension
curve was determined and the muscle length was main-
tained at that which elicited 90% of maximum con-
1,4-Dihydro-2,6-dimethyl-4-(2-oxo-2H-chromen-8-yl)-3,5-
diacetylpyridine (2o). Method G. Acetylacetone (2 g,
0.02 mol) was added to a solution of 4a (1.74 g,
0.01 mol) in isopropyl alcohol (30 mL) and ammonia
(10 mL) with stirring. The reaction mixture was re¯uxed
for 12 h and then evaporated to dryness. The residue, on
crystallizing from toluene, gave 0.34 g (10%) of 2o mp
tractile force observed at the optimal length.
A
stabilization period of 45±60 min was allowed before the
atria were challenged by various agents. During this
equilibration period, the beating 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 activities of
the compounds (0.01, 0.05, 0.1, 0.5, 1, 5, 10, and
50 mmol/L) ®rst dissolved in dimethylformamide
(DMF). According to this procedure the concentration
of DMF in the bath solution never exceeded 0.3%, a
concentration which did not produce appreciable ino-
tropic and/or chronotropic e€ects. During generation of
cumulative dose±response curve the next higher con-
centration of the compound was added only after the
preparation reached a steady-state.
135±138 ꢀC. H NMR d 2.28 (s, 6H, CH3), 2.31 (s, 6H,
1
CH3), 5.52 (s, 1H, H-4 dihydropyridine), 6.02 (broad,
1H, NH), 6.38 (d, 1H, H-3 coumarin), 7.15±7.7 (m, 4H,
Ar and H-4 coumarin). MS: m/z (relative abundance):
337 (M+, 25.21), 294 (12.92), 192 (100). Anal. calcd
(C20H19NO4) C, H, N.
Methyl 2-methyl-5-oxo-4-(2-oxo-2H-chromen-8-yl)-1,4,-
5,7-tetrahydrofuro-[3,4-b]-pyridine-3-carboxylate (2p).
Method H. To a cold solution of 2a (0.74 g, 0.002 mol)
in chloroform (20 mL), pyridine (0.26 g, 0.0033 mol) and
pyridinium bromide perbromide (0.74 g, 0.0023 mol)
were added. The solution was stirred at 0 ꢀC for 20 min
and then it was re¯uxed for 90 min. After cooling
chloroform was added and the solution was washed
with 2 N HCl and brine, dried, and evaporated to dry-
ness. The residue was crystallized form ethanol to give
Guinea pig aortic strip preparations. The thoracic aorta
was removed and placed in Tyrode solution of the fol-
lowing composition (mmol/L): 118 NaCl; 4.75 KCl;
2.54 CaCl2; 1.20 MgSO4; 1.19 KH2PO4; 25 NaHCO3; 11
glucose equilibrate with 95% O2 and 5% CO2 gas at pH
7.4. The vessel was cleaned of extraneous connective
tissue. Two helicoidal strips (15 mmÂ3 mm) were cut
from each aorta 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) containing aerated PSS at 35 ꢀC. Strips were
secured at one end to plexiglass hooks and connected
via the surgical thread to a force displacement (FT. 0.3,
Grass) transducer for monitoring changes in isometric
contraction. Aortic strips were subjected to a resting
force of 1 g and washed every 20 min with fresh PSS for
1 h. After the equilibration period guinea-pig aortic
strips were contracted by being washed in PSS contain-
ing 80 mmol/l KCl (equimolar substitution of K+ for
Na+).
0.07 g (10%) of 2p mp 158±162 ꢀC. H NMR d 2.4 (s,
1
3H, CH3), 3.55 (s, 3H, COOCH3), 4.75 (s, 2H, CH2),
5.25 (s, 1H, H-4 dihydropyridine), 6.4 (d, 1H, H-3 cou-
marin), 7.2±7.8 (m, 4H, Ar and H-4 coumarin). MS: m/z
(relative abundance): 353 (M+, 33.69), 208 (100), 179
(16.57). Anal. calcd (C19H13NO6) C, H, N.
Pharmacology
Isolated guinea pig left and right atrial preparations.
Guinea pig (350±400 g male and female) were sacri®ed
by cervical dislocation. After thoracotomy, the hearts
were immediately removed and washed by perfusion
through the aorta with oxygenated Tyrode solution of
the following composition (mmol/L): 136.9 NaCl; 5.4
KCl; 2.5 CaCl2; 1.0 MgCl2; 0.4 NaH2PO4ÂH2O; 11.9
NaHCO3; 5.5 glucose. The physiological salt solution
(PSS) was bu€ered to pH 7.4 by saturation with 95%
O2-5% CO2 gas and the temperature was maintained at
35 ꢀC. Isolated guinea pig heart preparations were used:
spontaneously beating right atria and left atria driven at
1 Hz. For each preparation the entire left and right
Subsequent to the contraction reaching a plateau
(approximately 30 min) the compounds (0.01, 0.05, 0.1,
0.5, 1, 5, 10, and 50 mmol/L) were added cumulatively
to the bath allowing for any relaxation to obtain an

810
P. Valenti et al./Bioorg. Med. Chem. 6 (1998) 803±810
equilibrate level of force. Addition of the drug vehicle
had no appreciable e€ect on the K+-induced level of
force (DMF for all compounds).
12. Rampa, A.; Budriesi, R.; Bisi, A.; Chiarini, A.; Valenti, P.
Arzneim. Forsch./Drug Res. 1992, 42, 1284.
13. Bisi, A.; Budriesi, R.; Chiarini, A.; Rampa, A.; Valenti, P.
Il Farmaco 1993, 48, 1491.
Statistical evaluation. Data were analyzed by Student's
t-test. The criterion for signi®cance was a P value less
than 0.05. The ED50 values were calculated from log
concentration-response curves (Probit analysis by
Litch®eld and Wilcoxon, n=6±8). All data are pre-
sented as mean ‹SEM.30
14. Rampa, A.; Budriesi, R.; Bisi, A.; Fabbri, G; Barili, P.G.;
Chiarini, A.; Valenti, P. Arzneim. Forsch/Drug Res. 1995, 45, 957.
15. Budriesi, R.; Rampa, A.; Bisi, A.; Fabbri, G.; Chiarini, A.;
Valenti, P. Arzneim. Forsch/Drug Res. 1996, 46, 374.
16. Bisi, A.; Budriesi, R.; Rampa, A.; Fabbri, G.; Chiarini, A.;
Valenti, P. Arzneim. Forsch./Drug Res. 1996, 46, 848.
17. Da Re, P.; Sagramora, L.; Mancini, V.; Valenti, P.; Cima,
L. J. Med. Chem. 1970, 13, 527.
18. Da Re, P.; Valenti, P.; Borraccini, A.; Primo®ore, G. P. J.
Med. Chem. 1972, 15, 198.
Acknowledgements
19. Gaion, R. M.; Valenti, P.; Montanari, P.; Da Re, P. Arz-
neim. Forsch. 1982, 32, 499.
This work was supported by a grant from M.U.R.S.T.
and C.N.R. Rome.
20. Valenti, P.; Recanatini, M.; Da Re, P.; Galimbeni, W.;
Avanzi, N.; Filippeschi, S. Arch. Pharm. 1985, 318, 923.
21. Valenti, P.; Da Re, P.; Rampa, A.; Montanari, P.; Carrara,
M.; Cima, L. Anti-Cancer Drug Design, 1993, 8, 349.
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