New 1,4-Dihydropyridines Endowed with NO-Donor and Calcium Channel Agonist Properties

Article abstract of DOI:10.1021/jm031109v

A new series of calcium channel agonists structurally related to Bay K8644, containing NO donor furoxans and the related furazans unable to release NO, is described. The racemic mixtures were studied for their action on L-type Ca ²⁺ channels expressed in cultured rat insulinoma RINm5F cells. All the products proved to be potent calcium channel agonists. All the racemic mixtures, with the only exception of the carbamoyl derivatives 9, 12 endowed with scanty solubility, were separated by chiral chromatography into the corresponding enantiomers; the (+) enantiomers were found to be potent agonists while the (-) ones were feeble antagonists. The racemic mixtures were also assessed for their positive inotropic activity on electrically stimulated rat papillary muscle and for their ability to increase Ca²⁺ entry into the vascular smooth muscle of rat aorta strips. The cyanofuroxan 8 proved to be an interesting product with dual Ca²⁺-dependent positive inotropic and NO-dependent vasodilating activity.

Full text of DOI:10.1021/jm031109v

2688
J . Med. Chem. 2004, 47, 2688-2693
New 1,4-Dih yd r op yr id in es En d ow ed w ith NO-Don or a n d Ca lciu m Ch a n n el
Agon ist P r op er ties
Sonja Visentin,† Barbara Rolando,† Antonella Di Stilo,† Roberta Fruttero,† Monica Novara,‡ Emilio Carbone,‡
Christian Roussel,§ Nicolas Vanthuyne,§ and Alberto Gasco*,†
Dipartimento di Scienza e Tecnologia del Farmaco, Universita` degli Studi di Torino, via Pietro Giuria 9, I-10125 Torino, Italy,
Dipartimento di Neuroscienze, Unita` di ricerca INFM, Universita` degli Studi di Torino, Corso Raffaello 30,
I-10125 Torino, Italy, and UMR “Chirotechnologies: catalyse et biocatalyse”, Universite´ Aix-Marseille III,
Av. Escadrille Normandie Niemen, F-13397 Marseille Cedex 20, France
Received November 24, 2003
A new series of calcium channel agonists structurally related to Bay K8644, containing NO
donor furoxans and the related furazans unable to release NO, is described. The racemic
mixtures were studied for their action on L-type Ca2+ channels expressed in cultured rat
insulinoma RINm5F cells. All the products proved to be potent calcium channel agonists. All
the racemic mixtures, with the only exception of the carbamoyl derivatives 9, 12 endowed with
scanty solubility, were separated by chiral chromatography into the corresponding enantiomers;
the (+) enantiomers were found to be potent agonists while the (-) ones were feeble antagonists.
The racemic mixtures were also assessed for their positive inotropic activity on electrically
stimulated rat papillary muscle and for their ability to increase Ca2+ entry into the vascular
smooth muscle of rat aorta strips. The cyanofuroxan 8 proved to be an interesting product
with dual Ca2+-dependent positive inotropic and NO-dependent vasodilating activity.
Ch a r t 1
In tr od u ction
1,4-Dihydropyridines (DHPs) of general structure A
(Chart 1) are an important class of drugs able to block
the Ca2+ currents through voltage-dependent L-type
channels.1 It is known that when an appropriate group,
such as the nitro group, is substituted in A for one of
the two ester functions, the resulting two enantiomers
display opposite pharmacological profiles.2 Bay K 8644
is a typical example (Chart 1). The (-) S-antipode is a
potent agonist at L-type Ca2+-channels while the (+)
R-antipode is a feeble antagonist. The Ca2+-channel
agonists are potentially useful in therapy as positive
inotropic agents, in particular for the treatment of the
congestive heart failure (CHF).3-5 The practical use is
limited by their additional capacity to increase Ca2+
entry into vascular smooth muscle with the consequent
constriction. NO donors are a class of products which
display potent vasodilating properties.6 This is because
they are able to release NO in physiological conditions.
Hybrid structures in which 1,4-dihydropyridines able
to activate L-type Ca2+ channels are connected with
suitable NO-donor moieties could represent a new
interesting class of positive inotropic agents, devoid of
vasoconstrictor properties. Two examples of this ap-
proach were recently reported in the literature.7,8 On
these bases, we designed new 1,4-dihydropyridines
formally obtained by substitution of appropriately sub-
stituted NO-donor furoxan substructures for the CF3
group in Bay K 8644 (derivatives 8, 9, 10). We herein
describe the syntheses of these products and the action
of their racemic mixtures on L-type Ca2+ channels
expressed by rat insulinoma cell line (RINm5F cells).
The racemic mixtures 8, 10 were resolved by chiral
chromatography, and the abilities of the single stere-
oisomers to modulate the calcium channels were as-
sessed. In the case of these latter racemates, namely of
products that release small and large amounts of NO
respectively, their actions on rat aorta strips partially
depolarized with 10 mM K+ as well as their inotropic
actions evaluated on electrically stimulated rat papillary
muscle are also discussed. For control purposes parallel
studies were done on the furazan analogues (derivatives
11, 12, 13) devoid of the properties to release NO.
Resu lts a n d Discu ssion
Ch em istr y. The aldehydes 3, 4, 5 were used as
starting materials for the synthesis of the final products.
Aldehydes 4, 5 were described in a previous paper,9
while 3 was obtained from the related (bromomethyl)-
furoxan 2 through nucleophilic substitution of the
bromine by o-hydroxybenzaldehyde (1) in DMF, in the
presence of potassium carbonate (Scheme 1A). The
furoxan 1,4-DHPs 8, 9, 10 were prepared by a modified
Hantzsch reaction, namely by condensation of the
appropriate aldehydes with nitroacetone 6 and methyl
3-aminocrotonate 7 (Scheme 1B) in 2-propanol. For
compound 8 the reaction was conducted at 40 °C to
avoid thermal furoxan isomerization. The furazan DHPs
11, 12, 13 were obtained by reduction of the furoxan
analogues in boiling trimethyl phosphite.
* To whom correspondence should be addressed. Telephone: 0039
011 6707670. Fax: 0039 011 6707286. E-mail: alberto.gasco@unito.it.
† Dipartimento di Scienza e Tecnologia del Farmaco, Universita` degli
Studi di Torino.
The furoxan compounds were assessed for their ability
‡ Dipartimento di Neuroscienze, Universita` degli Studi di Torino.
§ Universite´ Aix-Marseille III.
to produce nitrite (NO2-) (Griess reaction) in buffer
10.1021/jm031109v CCC: $27.50 © 2004 American Chemical Society
Published on Web 04/01/2004

New 1,4-Dihydropyridines
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10 2689
Sch em e 1a
a
(A): (i) DMF, K2CO3, 40 °C. (B): (ii) for compounds 9 and 10: i-PrOH reflux, 8 h; for compound 8: i-PrOH, 40 °C, 48 h; (iii) P(OCH3)3,
reflux, 5 h.
Ta ble 1. Chromatographic Conditions of Chiral Analytical Separation of Derivatives 8, 9, 10, 11, 12, and 13
compd
stationary phase
mobile phase
tR(1) (+)a
k′(1) (+)
tR(2) (-)
k′(2) (-)
R
8
11
9
12
10
13
Whelk R,R
Whelk R,R
Chiralpak AD
Chiralcel OJ
Chiralpak AD
Chiralcel OJ
hexane/chloroform 25/75
hexane/chloroform 25/75
hexane/ethanol 50/50
hexane/ethanol 50/50
hexane/ethanol 80/20
hexane/ethanol 80/20
10.80
8.30
4.86
5.68
9.10
15.30
2.54
1.72
0.61
0.89
2.03
3.90
13.21
10.16
6.13
7.76
11.37
18.07
3.33
2.33
1.04
1.58
2.79
4.79
1.31
1.35
1.69
1.78
1.37
1.23
a
Sign of the rotatory power in the mobile phase given by on-line polarimeter.
solution (pH 7.4) at 37 °C, in the presence of an excess
of cysteine according to a procedure previously de-
action on the L-type Ca2+ channels expressed in cul-
tured rat insulinoma RINm5F cells.11,12 Cells were
bathed in 10 mM Ba2+ and kept at -60 mV holding
potential to increase the proportion of L-type currents.
The test potential was -20 mV when either testing the
agonistic and antagonistic effects of the DHP deriva-
tives, and the return potential was set at -40 mV to
better visualize the agonistic effects of the compounds
through the slowing down of tail currents. Under these
conditions, 3 µM nifedipine quickly blocked about 67%
of the total current, and 3 µM Bay K 8644 caused an
approximately 320% increase at -20 mV and a marked
slowing of tail currents on return to -40 mV (Figure
1A). Figure 1B shows the variable degrees of potentia-
tion of Ba2+ currents induced by the six racemates
tested (8 to 13). To the right, the mean percentage of
increased Ba2+ currents in reference to control currents
(agonistic potency) are also reported. As noticed, all six
racemates increased the size of Ba2+ currents at -20
mV and produced a marked prolongation of tail currents
on return to -40 mV. Racemates 8, 10, 11, and 13 were
the most effective, with no significant difference among
them (200 to 240% agonistic potency; P < 0.1, as
established with a one-way ANOVA followed by a
Student-Newman-Keuls test for multiple compari-
sons). In contrast to this, racemates 9 and 12 had only
weak agonistic effects. Because of the weak action and
due to the difficulties in separating the corresponding
scribed.10 The results expressed as percentages of NO2
-
(mol/mol) rank the order 8 (40.3 ( 0.7) > 9 (13.6 ( 0.7)
> 10 (<1). These figures can be taken as rough indexes
of the ability of the products to produce NO under the
chosen experimental conditions.
Racemic mixtures of the products were successfully
separated by HPLC on analytical scale (Table 1). In the
case of compounds 9 and 10 an amylose tris(3,5-dimeth-
ylphenylcarbamate) (Chiralpak AD) column and hexane/
ethanol mixture as mobile phase were used. Racemic
mixture of furazan derivatives 12 and 13 were resolved
under similar conditions but using a cellulose tris(4-
methylbenzoate) (Chiralcel OJ ) column. It was not possi-
ble to use these experimental conditions to separate
racemates 8 and 11 since the products, during the elu-
tion, undertook partial reaction with the alcohol present
in the mobile phase. Therefore, both the mixtures were
separated using a (3S,4R)-4-(3,5-dinitrobenzamido)-3-
[3-(dimethylsilyloxy)propyl]-1,2,3,4-tetrahydrophenan-
trene ((R,R) Whelk-01) column and hexane/chloroform
mixture as mobile phase. Chromatographic conditions
reported in Table 1 were chosen for semipreparative
separation. The amides 9 and 12 were not separated
on the semipreparative scale because of their scanty
solubility.
Volta ge Cla m p . Racemates 8, 9, 10, 11, 12, 13 and
(()-enantiomers of 8, 10, 11, 13 were studied for their

2690 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10
Visentin et al.
F igu r e 2. Agonistic and antagonistic effects of the (()-
enantiomers of compounds 8, 10, 11, and 13 (3 µM). The
voltage protocol to test the percentage of blockade and
potentiation of the 1,4-DHPs was similar to that illustrated
in Figure 1. To the right are reported the percentage of
potentiation (positive ordinates) or blockade (negative ordi-
nates) calculated at -20 mV from a number of cells varying
from 15 to 22 for each compound. The effects are referred to
the L-type currents estimated by subtracting the nifedipine-
insensitive currents remaining after adding 3 µM nifedipine
to the bath. Notice the weak degree of antagonistic effect of
the (-)-enantiomers compared to the potent agonistic effects
of the (+)-enantiomers.
F igu r e 1. Action of furoxan 1,4-DHPs (racemates 8, 9, 10)
and furazan 1,4-DHPs (racemates 11, 12, 13) on L-type Ba2+
currents of rat insulinoma RINm5F cells. The cells were
maintained at -60 mV holding potential and depolarized to
-20 mV for 100 ms. The return potential was set to -40 mV
to assay the potentiating effects of 1,4-DHP agonists. This was
evident through the time course of tail currents which became
10 times slower and approached nonzero steady-state values
at the end of the -40 mV step. To the right are reported the
mean potentiation of each racemate calculated as the percent-
age of L-type Ba2+ current increase at -20 mV from n ) 11,
5, 5, 8, 5, and 7 cells for racemates 8, 9, 10, 11, 12, and 13,
respectively. The potentiation was calculated after subtracting
from control currents the nifedipine-insensitive component
remaining after addition of 3 µM nifedipine. As shown, all
racemates at 3 µM concentration produced a variable degree
of Ba2+ current potentiation. To the top of the figure are shown
the blocking potency of 3 µM nifedipine (mean 67% n ) 14)
and the potentiating effects of 3 µM (-)-Bay K 8644 (mean
340% n ) 15) at the same testing voltage (-20 mV). Each
panel is obtained from a different RINm5F cell.
potent agonistic action of the four racemates derives
from the predominant agonistic effects of the (+)-
enantiomers. To complete this study, we also investi-
gated the action of the cyano furoxan derivative 8 and
its (()-enantiomers on the L-type Ba2+ currents at
various voltages, using ramp commands from -50 and
+60 mV (Figure 3). The agonistic effects of the racemate
and (+)-enantiomer were evident in a wide range of
potentials (from -40 to +10 mV), and the same was true
for the weak blocking effects of the (-)-enantiomer.
P h a r m a cology. Racemic mixtures 8, 10, 11, 13 were
assessed for their positive inotropic activities on rat
papillary muscle electrically stimulated by unipolar
squarewave pulses. After an appropriate equilibration
period, cumulative concentration-response curves for
each Ca2+ agonist were constructed. The curve of the
cyanofuroxan derivative 8 is reported in Figure 4A as
an example. The positive inotropic potencies of each
compound, expressed as EC50 values, are reported in
Table 2. Products 13, 10, 11, 8 were about 5, 7, 9, 30-
fold less potent than the Bay K 8644, respectively. The
furazans were more potent than the structurally related
furoxans and the methyl-substituted products more po-
tent than the analogous cyano derivatives. Since nitric
oxide may behave as an inotropic agent,13 the experi-
ments were repeated for the NO-donor furoxan deriva-
tives 8 and 10 in the presence of 1H-[1,2,4]oxadiazolo-
[4,3-a]quinoxalin-1-one (ODQ), a well-known inhibitor
of the soluble guanylate cyclase (sGC), a cytosolic en-
enantiomers (see above), the two compounds were not
further investigated.
The results of Figure 1 allow a preliminary consid-
eration about the dual nature of furoxan derivatives
being both DHP agonists and NO donors. Their agonis-
tic action on L-type Ca2+ channels apparently occurs
regardless of their ability to produce NO at various
degrees. Notice that furoxan compounds display similar
L-type channel agonists activity to their furazan ana-
logues which are unable to release NO.
Figure 2 summarizes the agonistic and antagonistic
effects of the (+/-)-enantiomers of compounds 8, 10, 11,
and 13. As shown, the four (+)-enantiomers exert a
similarly potent agonistic action on L-type channels,
which results in an increased amplitude at -20 mV (275
to 290%, with no significant difference among them; P
< 0.2 with ANOVA) and a marked prolongation of tail
currents at -40 mV. On the contrary, the (-)-enantio-
mers exerted weak blocking effects, suggesting that the

New 1,4-Dihydropyridines
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10 2691
Ta ble 2. Positive Inotropic Effect and Smooth Muscle Contraction of Derivatives 8, 10, 11, and 13
rat papillary muscle
rat aortic strips
compd
EC50 ( SE (µM)
EC50 ( SE (µM)
% max., respectivelya
BAY K 8644
8
11
10
13
0.17 ( 0.02
0.018 ( 0.002
84 ( 4
5.2 ( 0.4 (5.0 ( 0.6)b
1.5 ( 0.2
no effect (0.21 (0.07)b
0.12 ( 0.02
0 (29 ( 2)b
62 ( 5
1.2 ( 0.3 (1.3 ( 0.2)b
0.82 ( 0.07
0.10 ( 0.02 (0.11 ( 0.01)b
0.057 ( 0.006
80 ( 4 (83 ( 5)b
85 ( 3
a
b
Relative to 50 mM KCl. In the presence of 1 µM ODQ.
F igu r e 3. Current/voltage characteristics in control condi-
tions, in the presence of racemates 8 (top) and during applica-
tion of (+) and (-) enantiomers (bottom) measured with ramp
commands of 1.4 V/s slope. Cells were maintained at -60 mV
holding potential and depolarized linearly up to +80 mV as
indicated. The two panels report experiments from two dif-
ferent RINm5F cells. Notice the marked potentiating effect of
the (+)-8 enantiomer (3 µM), which is evident between -35
and 0 mV where L-type channels mainly contribute to total
Ba2+ current.
F igu r e 4. (A) Cumulative concentration-response curves of
Bay K 8644 (solid circle) and der. 8 (open square) in rat
papillary muscle. (B) Cumulative concentration-response
curves of Bay K 8644 (solid circle) and der. 8 in the absence
(open square) and in the presence (solid square) of ODQ in
partially depolarized rat aorta. The contractile response was
expressed in terms of the percentage of the 50 mM KCl-
induced contractile force in each strips.
zyme involved in the nitric oxide signal transduction
mechanism.14 No shift of the concentration-response
curve was observed. This suggests that no NO cGMP-
dependent inotropic effects are present. Consequent-
ly the positive inotropic effects detected seem to be prin-
cipally due to the Ca2+-agonist properties of the prod-
ucts.
masked by its NO-dependent vasodilating activity. In
fact this model was able to dilate rat aorta strips
depolarized with 50 mM K+ in a dose-dependent man-
ner. The potency of the product as a vasodilator (EC50
) 0.0044 ( 0.0008 µM; EC50 in the presence of 1 µM
ODQ ) 1.9 ( 0.1 µM) was higher than that of glyceryl
trinitrate (GTN, EC50 ) 0.022 ( 0.004 µM).10 In conclu-
sion, 8 is an interesting product endowed with both
Ca2+-dependent positive inotropic properties and NO-
dependent vasodilating activities.
The capacity of the racemic mixtures to increase Ca2+
entry into the vascular smooth muscle was evaluated
on rat aorta strips partially depolarized with 10 mM
K+. Furazan derivatives 13 and 11 were capable of
contracting the tissue in a dose-dependent manner.
Their potencies were less than Bay K8644’s potency by
about 3 and 7-fold, respectively. The behavior of the
methylfuroxan derivative 10, a feeble generator of
nitrite, was similar to that of the related furazan 13.
Experiments conducted in the presence of ODQ did not
induce any modification of the concentration-response
curve. This suggests that the product behaves in the
vasoconstrictor experiments as a pure Ca2+ agonist. By
contrast, analogous experiments carried out with the
cyanofuroxan 8, which is a potent producer of nitrite,
showed that the product was able to contract the tissue
only in the presence of ODQ (Figure 4B). This indicates
that the Ca2+-agonist properties of the product are
Exp er im en ta l Section
Melting points were determined on a Bu¨chi 530 apparatus
after introducing the sample into the bath at a temperature
10 °C lower than the melting point. A heating rate of 1 °C
min-1 was used, 3 °C min-1 in the case of decomposition. The
compounds were routinely checked by infrared spectropho-
tometry (Shimadzu FT-IR 8101). 1H and 13C NMR spectra were
recorded on a Bruker AC-200 spectrometer and are reported
in δ (ppm) units (* means either not assigned or tentatively
assigned). Column chromatography was performed on silica
gel (Merck Kieselgel 60, 230-400 mesh ASTM) with the
indicated solvent system. Anhydrous MgSO4 was used as a
drying agent. Solvent removal was achieved under reduced

2692 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10
Visentin et al.
pressure at room temperature. Elemental analyses of the new
compounds were performed by REDOX (Cologno Monzese),
and the results are within (0.4% of the theoretical values.
blocker propranolol (1 µM) and the R1-receptor blocker prazosin
(0.1 µM).16 The receptor blockers were introduced into the bath
solution 15 min before addition of Ca2+-channel activator. Only
one concentration-response curve to a 1,4-DHP activator could
be determined in a single tissue.17 In the case of 8 and 10 we
determined the inotropic effect either in the absence or in the
presence of 1 µM ODQ which was added to the bath at least
15 min before the addition of the first concentration of the
agonist. EC50 values are the mean of 5-7 determinations.
Aor tic Str ip s. Thoracic aortas were helically cut, the
endothelium was removed, and three strips were obtained from
each vessel. The tissues were mounted under 0.7 g tension
(isotonic transducer).
Analytical chiral HPLC experiments on Chiralpak AD and
Chiralcel OJ (250 × 4.6 mm, 10 µm) (Daicel Co., Tokyo) and
(R,R) Whelk-01 (250 × 4.6 mm, 5 µm) (E. Merck, Darmstadt)
columns were performed with a LaChrom (Merck) screening
unit equipped with a L-7100 pump, a L-7200 auto sampler, a
L-7360 oven which accommodates 12 columns alimented by a
Valco positions valve, a L-7400 UV detector, and a J asco OR-
1590 polarimeter detector. Analysis were performed on 1 mL/
min, at a controlled temperature (25 °C) with UV (λ ) 254
nm) and polarimetric detection. Semipreparative separations
were performed with a Merck-Hitachi LiChrograph Model
L-6000 HPLC pump and a Merck-Hitachi LiChrograph L-4000
UV detector (λ ) 254 nm). For semipreparative separations,
a Chiralpak AD, Chiralcel OJ (250 × 10 mm, 10 µm), and (R,R)
Whelk-01 (250 × 5 mm, 5 µm) were used. The solvents were
HPLC grade from SDS (Peypin, France), filtered on a Millipore
membrane 0.45 µm and degassed before use. The optical
rotatory powers were measured on a 241 MC Perkin-Elmer
polarimeter with a sodium lamp and a double-jacketed cell (1
dm) at 25 °C.
Cell Cu ltu r e a n d Solu tion s. RINm5F cells were kindly
provided by Dr. E. Sher (Eli Lilly and Co., Windlesham,
Surrey, U.K.) and were cultured as previously described. The
recording external solution was (mM) 125 NaCl, 10 BaCl2, 1
MgCl, 10 HEPES (pH 7.3 with NaOH), and 300 nM TTX. The
standard internal solution was (mM) 100 CsCl, 20 TEACl, 10
EGTA, 2 MgCl2, 8 glucose, 4 ATP, 0.5 GTP, 15 phosphocreat-
ine, 10 HEPES, (pH 7.3 with CsOH). The four racemates and
their enantiomers as well as Bay K 8644 (Bayer AG, Wup-
pertal, Germany) were dissolved in ethanol (95%) and stored
in the dark at 4 °C as 1 mM stock solutions. Dilution to the
final concentration was performed daily under light protection.
The external solutions were exchanged by fast superfusing
using a multibarrelled glass pipet held close to the cell.
Experiments were carried out at room temperature (22 °C).15
To evaluate vascular contraction, the aortic strips were
allowed to equilibrate for 1 h and then depolarized twice at
60 min intervals by an addition of KCl solution of to a final
concentration 50 mM. Contractile responses to Ca-channel
agonists were determined in the presence of 10 mM KCl,
because at that K+ concentration the activator produced the
maximum response.18 In the case of furoxan derivatives 8 and
10, we determined the effect either in the absence or in the
presence of 1 µM ODQ which was added to the bath at least
15 min before the addition of the first concentration of the
agonist. EC50 values are the mean of 5-7 determinations. To
evaluate vasodilating activity, we followed a method previously
reported.9 All the experiments were performed avoiding ex-
posure to light, because of the photolability of the 1,4-DHPs.
Syn th esis of 3-[(3-Cya n ofu r oxa n -4-yl)m eth oxy]ben -
za ld eh yd e (3). K2CO3 (1.4 g, 10 mmol) was added to a stirred
solution of compound 2 (1 g, 5 mmol) and 2-hydroxybenzal-
dehyde (1) (0.73 g, 6 mmol) in dry DMF (10 mL). The stirring
was continued for 30 min at room temperature, and then the
reaction mixture was poured into water. The resulting pre-
cipitate was filtered, washed with water, and purified by flash-
chromatography (PE 7/EtOAc 3); mp 122 °C (EtOAc/PE); yield
87%. 1H NMR (DMSO-d6): 5.65 (s, 2H, -CH2); 10.4 (s, 1H,
CHO); 7.18-7.78 (Ph). 13C NMR (DMSO-d6): 61.3 (OCH2);
114.1, 122.4, 124.7, 127.8, 136.5, 159.4 (Ph); 98.6 (C3-furoxan);
106.1 (CN); 154.7 (C4-furoxan); 189.1 (CHO). Anal. (C11H7N3O4)
C, H, N.
Wh ole-Cell Cu r r en t Recor d in gs. Membrane currents
were measured in the whole-cell configuration using a Axo-
patch 200 A (Axon Instruments) patch-clamp amplifier and
pipets of borosilicate glass with a resistance of 3-4 MΩ.
Stimulation, acquisition, filtering, and data analysis were
performed as described elsewhere.12 Cells were clamped at -60
mV holding potential (Vh). Step depolarization of 100 ms from
Vh were applied at intervals of 10 s to minimize Ca2+-channel
“run down”. Capacitative currents were corrected with the
clamp-amplifier settings. Leakage currents were compensated
electronically with the P/4 pulse protocol. Data are expressed
as means ( SEM for n ) number of cells.
Gen er a l Meth od of P r ep a r a tion of DHP s 9 a n d 10. A
solution of the appropriate aldehyde 4 and 5 (5 mmol) and
nitroacetone 6 (0.62 g, 6 mmol) in 2-propanol (70 mL) was
refluxed for 4 h, and then methyl-3-aminocrotonate 7 (0.58 g,
5 mmol) was added. Reflux was continued for other 4 h; solvent
removal gave a residue which was purified by flash-chroma-
tography. Chromatographic eluents, yields, melting points, and
crystallization solvents of the products were as follows.
F u n ction a l Stu d ies. Papillary muscles16 and aortas were
isolated from male Wistar rats weighing 200-250 g which had
been anaesthetized with CO2 and killed by decapitation. All
animals were treated humanely in accordance with recognized
guidelines on experimentation. As few rats as possible were
used. The purposes and the protocols of our studies have been
approved by the Ministero della Salute, Rome, Italy. The
tissues were mounted in organ baths containing 30 mL of
Krebs-bicarbonate buffer of the following composition (mM):
NaCl 111.2, KCl 5.0, CaCl2 2.5, MgSO4 1.2, KH2PO4 1.0,
NaHCO3 12, glucose 11.1. The solution was maintained at 37
°C and continuously gassed with 95% O2-5% CO2 (pH ) 7.4).
(()-Meth yl 4-[2-[(3-Ca r ba m oylfu r oxa n -4-yl]m eth oxy]-
p h en yl]-2,6-d im eth yl-5-n itr o 1,4-d ih yd r op yr id in e-3-ca r -
boxyla te (9): eluent CH2Cl2 8/EtOAc 2; yield 42%; mp 198-
199 °C dec (EtOAc/PE). 1H NMR (DMSO-d6): 2.13, 2.33 (s,
6H, 2,6-CH3); 3.52 (s, 3H, COOCH3); 5.29 (s, 2H, OCH2); 5.40
(s, 1H, 4-CH); 6.92-7.22 (m, 4H, Ph); 7.80-8.55 (2s, 2H,
CONH2); 9.38 (s br, 1H, NH). 13C NMR (DMSO-d6): 17.4 (2-
CH3); 19.9 (6-CH3); 38.3 (C4-DHP); 50.9 (OCH3); 61.1 (OCH2);
104.5 (C5-DHP); 110.5 (C3-furoxan); 124.7 (C3-DHP); 143.6,
146.1 (C2,C6-DHP); 102.7, 112.8, 128.2, 131.4, 131.7, 155.7
(Ph); 155.0 (C4-furoxan); 156.6 (CONH2); 166.8 (COOCH3).
Anal. (C19H19N5O8) C, H, N.
P a p illa r y Mu scles. Two electrodes implanted into the
ventricular basis were used to drive the tissues electrically.
Square wave pulses (2 Hz, 1 ms, 20% above threshold voltage)
were continuously delivered, and changes in the contraction
force of papillary muscles were recorded by an isometric
transducer connected with a pen recorder. After a 90 min
equilibration period, cumulative concentration-response curves
to Ca2+ agonists were constructed by increasing doses by half-
log units after each response had reached a plateau. To avoid
indirect receptor-mediated effects of possibly released endo-
genous noradrenaline by the Ca2+-channel activators, the
muscles were incubated in the presence of the â-receptor
(()-Meth yl 2,6-Dim eth yl-4-[2-[(3-m eth ylfu r oxa n -4-yl]-
m eth oxy]ph en yl]-5-n itr o-1,4-dih ydr opyr idin e-3-car boxyl-
a te (10): eluent CH2Cl2 9.5/EtOAc 0.5; yield 25%; mp 216 °C
(EtOAc/PE). 1H NMR (DMSO-d6): 2.14, 2.36 (s, 6H, 2,6-CH3);
2.47 (s, 3H, CH3); 3.45 (s, 3H, COOCH3); 5.33 (s, 2H, OCH2);
5.49 (s, 1H, 4-CH); 9.29 (s br, 1H, NH). 13C NMR (DMSO-d6):
7.4 (CH3); 17.4 (2-CH3); 19.8 (6-CH3); 37.1 (C4-DHP); 50.8
(OCH3); 60.7 (OCH2); 105.5 (C5-DHP); 113.3* (C3-furoxan);
125.3 (C3-DHP); 143.0, 147.6 (C2,C6-DHP); 112.6*, 121.3,
128.3, 130.9, 132.4, 155.6* (Ph); 155.5* (C4-furoxan); 166.7
(COOCH3). Anal. (C19H20N4O7 × 0.5H2O) C, H, N.

New 1,4-Dihydropyridines
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10 2693
Semipreparative separation afforded ca. 50 mg of each
Semipreparative separation afforded 18 mg of each enan-
enantiomer with an optical purity higher than 98%: (+)10 (c
tiomer with an optical purity higher than 98%: (+)13 (c )
) 0.1, acetone), [R]25D) +17; (-)10 (c ) 0.1, acetone), [R]25
-16.
)
0.1, acetone), [R]25 ) +15; (-)13 (c ) 0.1, acetone), [R]25
)
D
D
D
-12.
Syn th esis of (()-Meth yl 4-[2-[(3-Cya n ofu r oxa n -4-yl]-
m et h oxy]p h en yl]-2,6-d im et h yl-5-n it r o-1,4-d ih yd r op yr i-
d in e-3-ca r boxyla te (8): A mixture of compound 3 (0.73 g, 3
mmol) and nitoacetone 6 (1.08 g, 10.5 mmol) in 2-propanol (70
mL) was heated at 40 °C for 36 h. Methyl 3-aminocrotonate 7
(0.58 g, 5 mmol) was added, and heating was continued for
other 48 h; solvent removal gave a residue which was purified
by flash-chromatography (eluent PE 7/EtOAc 3). Yield 30%;
mp188-189 °C dec (PE/EtOAc). 1H NMR (DMSO-d6): 2.16,
2.35 (s, 6H, 2,6-CH3); 3.49 (s, 3H, COOCH3); 5.33 (s, 2H,
OCH2); 5.48 (s, 1H, 4-CH); 6.97-7.20 (Ph); 9.29 (s br, 1H, NH).
13C NMR (DMSO-d6): 17.5 (2-CH3); 19.8 (6-CH3); 37.7 (C4-
DHP); 50.8 (OCH3); 60.7 (OCH2); 105.5 (C5-DHP); 106.0 (CN);
113.3* (C3-furoxan); 125.3 (C3-DHP); 143.3, 147.6 (C2,C6-
DHP); 113.6*, 121.8, 128.2, 131.3, 132.6, 155.7* (Ph); 155.4*
(C4-furoxan); 166.7 (COOCH3). Anal. (C19H17N5O7 × 0.25 H2O)
C, H, N.
Ack n ow led gm en t. This work was partially sup-
ported by MIUR (Ministero dell′Istruzione dell′Univer-
sita` e della Ricerca) grant.
Refer en ces
(1) Triggle, D. J .; Langs, D. A.; J anis, R. A. Ca2+ Channels
Ligands: Structure-Function Relationships of the 1,4-Dihydro-
pyridines. Med. Res. Rev. 1989, 9, 123-180.
(2) Goldmann, S.; Stoltefuss, J . 1,4-Dihydropyridines: Effects of
Chirality and Conformation on the Calcium Antagonist and
Calcium Agonist Activities. Angew. Chem., Int. Ed. Engl. 1991,
30, 1559-1578.
(3) Takenaka, T.; Inagaki, O.; Terai, M.; Asano, M.; Kubo, K. New
1,4-Dihydropyridines with vasoconstrictor Action Through Ac-
tivation of Ca2+-channels. J . Pharmacol. Exp. Ther. 1988, 244,
699-708.
(4) Goldenberg, I. F.; Cohn, J . N. New Inotropic Drugs for Heart
Failure. J . Am. Med. Assoc. 1987, 258, 493-496.
Semipreparative separation afforded ca. 10 mg of each
enantiomer with an optical purity higher than 99%. Unfortu-
nately, the rotatory power of this compound is too small to be
determined on our polarimeter with this low quantity of
product.
(5) Erhardt, P. W. In Search for the Digitalis Replacement. J . Med.
Chem. 1987, 30, 321-237.
Gen er a l Meth od of P r ep a r a tion of DHP s 11, 12, a n d
13. The appropriate furoxan 1,4-DHP 8-10 (2 mmol) was
refluxed in trimethyl phosphite (50 mL). Then the reaction
mixture, cooled at room temperature, was poured into 2 N HCl
(150 mL) and extracted with EtOAc (3 × 50 mL); the dried
organic phases were evaporated to give the title product as
an orange oil which was purified by flash-chromatography
(eluent CH2Cl2 95/EtOAC 5). Reaction time, yields, melting
points, and crystallization solvents of the products were as
follows.
(()-Meth yl 4-[2-[(4-Cya n ofu r a za n -3-yl]m eth oxy]p h en -
yl]-2,6-d im eth yl-5-n itr o-1,4-d ih yd r op yr id in e-3-ca r boxyl-
a te (11): 1 h; yield 83%; mp 178 °C (EtOAc/PE). 1H NMR
(DMSO-d6): 2.20, 2.40 (s, 6H, 2,6-CH3); 3.53 (s, 3H, COOCH3);
5.34 (s, 2H, OCH2); 5.49 (s, 1H, 4-CH); 6.96-7.25 (Ph); 9.50 (s
br, 1H, NH). 13C NMR (DMSO-d6): 17.4 (2-CH3); 19.7 (6-CH3);
32.3 (C4-DHP); 50.9 (OCH3); 59.1 (OCH2); 105.3 (C5-DHP);
107.6 (CN); 133.5 (C3-furazan); 125.4 (C3-DHP); 143.1, 147.3
(C2,C6-DHP); 113.4, 121.8, 128.2, 131.6, 132.8, 155.3 (Ph);
153.9 (C4-furazan); 166.7 (COOCH3). Anal. (C19H17N5O6) C, H,
N.
(6) Harabie, J . A.; Keefer, L. K. Nitric Oxide Donors. In Patophysi-
ology and Clinical Applications of Nitric Oxide; Rubany, G. M.,
Ed.; Harwood Academic Publishers: Australia, 1999; pp 455-
470.
(7) Shan, R.; Howlett, S. E.; Knaus, E. E. Syntheses, Calcium
Channels Agonist-Antagonist Modulation Activities, Nitric Oxide
Release, and Voltage-Clamp Studies of 2-Nitrooxyethyl 1,4-
Dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)pyridine-
5-carboxylate Enantiomers. J . Med. Chem. 2002, 45, 955-961.
(8) Vo, D.; Nguyen, J .-T.; McEwen, C.-A.; Shan, R.; Knaus, E. E.
Syntheses, Calcium Channels Agonist-Antagonist Modulation
Effects, and Nitric Oxide Release Studies of [3-(Benzensulfonyl)-
furoxan-4-yloxy]alkyl 1,4-Dihydro-2,6-dimethyl-5-nitro-4-(2-tri-
fluoromethyphenyl, benzofurazan-4-yl, 2-,3-, or 4-pyridyl)-3-
pyridinecarboxylates. Drug Dev. Res. 2002, 56, 1-16.
(9) Di Stilo, A.; Visentin, S.; Cena, C.; Gasco, A. M.; Ermondi, G.;
Gasco, A. New 1,4-Dihydropyridines Conjugated to Furoxanyl
Moieties, Endowed with Both Nitric Oxide-like and Calcium
Channel Antagonist Vasodilator Activities. J . Med. Chem. 1998,
41, 5393-5401.
(10) Sorba, G.; Medana, C.; Fruttero, R.; Cena, C.; Di Stilo, A.; Galli,
U.; Gasco, A. Water Soluble Furoxan Derivatives as NO-
Prodrugs. J . Med. Chem. 1997, 40, 2288, 463-469.
Semipreparative separation afforded ca. 10 mg of each
enantiomer with an optical purity higher than 98%. Unfortu-
nately, the rotatory power of this compound is too small to be
determined on our polarimeter with this low quantity of
product.
(()-Meth yl 4-[2-[(4-Ca r ba m oylfu r a za n -3-yl]m eth oxy]-
p h en yl]-2,6-d im eth yl-5-n itr o-1,4-d ih yd r op yr id in e-3-ca r -
boxyla te (12): 4 h; yield 55%; mp 205 °C (EtOAc/PE). 1H NMR
(DMSO-d6): 2.19, 2.33 (s, 6H, 2,6-CH3); 3.51 (s, 3H, COOCH3);
5.29 (s, 2H, OCH2); 5.40 (s, 1H, 4-CH); 6.92-7.22 (m, 4H, Ph);
8.25-8.58 (2s, 2H, CONH2); 9.21 (s br, 1H, NH). 13C NMR
(DMSO-d6): 17.4 (2-CH3); 19.8 (6-CH3); 38.0 (C4-DHP); 50.8
(OCH3); 59.5 (OCH2); 104.7 (C5-DHP); 143.3 (C3-furazan);
124.9 (C3-DHP); 146.7, 147.8 (C2,C6-DHP); 112.9, 120.9, 128.1,
131.4, 132.0, 156.2 (Ph); 155.0 (C4-furazan); 156.6 (CONH2);
166.8 (COOCH3). Anal. (C19H19N5O7), C, H, N.
(()-Meth yl 2,6-Dim eth yl-4-[2-[(4-m eth ylfu r a za n -3-yl]-
m eth oxy]ph en yl]-5-n itr o-1,4-dih ydr opyr idin e-3-car boxyl-
a te (13): 16 h; yield 37%; mp 198 °C (EtOAc/PE). 1H NMR
(DMSO-d6): 2.16, 2.20 (s, 6H, 2,6-CH3); 2.38 (s, 3H, CH3); 3.48
(s, 3H, COOCH3); 5.21 (s, 2H, OCH2); 5.47 (s, 1H, 4-CH); 6.90-
7.25 (m, 4H, Ph); 9.40 (s br, 1H, NH). 13C NMR (DMSO-d6):
7.8 (CH3); 17.3 (2-CH3); 19.7 (6-CH3); 36.7 (C4-DHP); 50.8
(OCH3); 58.7 (OCH2); 105.7 (C5-DHP); 155.4* (C3-furazan);
125.4 (C3-DHP); 147.5, 142.8 (C2,C6-DHP); 112.4, 121.2, 128.2,
130.7, 132.5, 155.4* (Ph); 151.9 (C4-furazan); 166.8 (COOCH3).
Anal. (C19H20N4O6), C, H, N.
(11) Pollo, A.; Lovallo, M.; Biancardi, E.; Sher, E.; Socci, C.; Carbone,
E. Sensitivity to dihydropiridines, ω-conotoxin and noradrenaline
reveals multiple HVA Ca2+ channels in rat insulinoma and
human pancreatic â-cells. Pflu¨gers Archiv. 1993, 423, 462-471.
(12) Magnelli, V.; Pollo, A.; Sher, E.; Carbone, E. Block of non-L-,
non -N-type Ca2+ channels in rat insulinoma RINm5F cells by
ω-conotoxin MVIIC. Pflu¨gers Archiv. 1995, 429, 762-771.
(13) Sarkar, D.; Vallance, P.; Harding, S. E. Nitric Oxide: not just a
negative inotrope. Eur. J . Heart Failure 2001, 3, 527-534.
(14) Kerwin, J . F., J r.; Lancaster, J . R., J r.; Feldman, P. L. Nitric
Oxide: A New Paradigm for Second Messengers. J . Med. Chem.
1995, 38, 4343-4362.
(15) Carabelli, V.; Carra, I.; Carbone, E. Localized secretion of ATP
and opioids revealed through single Ca2+ channel modulation
in bovine chromaffin cells. Neuron 1998, 20 (6), 1255-1268.
(16) Skomedal, T.; Schiander, I.; Aass, H.; Osnes, J . B. Properties of
the Inotropic Response in Rat Papillary Muscles to the Dihy-
dropyridine “Ca2+-Channel Activator” BAY K 8644. Pharmacol.
Toxicol. 1988, 63, 20-25.
(17) Kwon, Y. W.; Franckowiak, G.; Langes, D. A.; Hawthorn, M.;
J oslyn, A.; Triggle, D. J . Pharmacologic and radioligand binding
analysis of the actions of 1,4-dihydropyridine activators related
to Bay K 8644 in smoth muscle, cardiac muscle and neuronel
preparations. Naunyn-Shmiedeberg’s Arch Pharmacol 1989, 339,
19-30.
(18) Wakabayashi, I.; Sakamoto, K.; Kakishita, E.; Potentialing Effect
of Daunorubicin on vasocontractile response to KCl and BAY K
8644 in rat aorta. J . Pharm. Pharmacol. 1990, 42, 716-719.
J M031109V