New 1,4-dihydropyridines conjugated to furoxanyl moieties, endowed with both nitric oxide-like and calcium channel antagonist vasodilator activities

Article abstract of DOI:10.1021/jm9803267

A series of 4-phenyl-1,4-dihydropyridines substituted at the ortho and meta positions of the phenyl ring with NO-donating furoxan moieties and their non-NO-releasing furazan analogues were synthesized and pharmacologically characterized. The vasodilator a

Full text of DOI:10.1021/jm9803267

J . Med. Chem. 1998, 41, 5393-5401
5393
New 1,4-Dih yd r op yr id in es Con ju ga ted to F u r oxa n yl Moieties, En d ow ed w ith
Both Nitr ic Oxid e-lik e a n d Ca lciu m Ch a n n el An ta gon ist Va sod ila tor Activities
Antonella Di Stilo, Sonja Visentin, Clara Cena, Andrea Marcello Gasco, Giuseppe Ermondi, and Alberto Gasco*
Dipartimento di Scienza e Tecnologia del Farmaco, Universita` degli Studi di Torino, Facolta` di Farmacia,
via P. Giuria 9, 10125 Torino, Italy
Received May 26, 1998
A series of 4-phenyl-1,4-dihydropyridines substituted at the ortho and meta positions of the
phenyl ring with NO-donating furoxan moieties and their non-NO-releasing furazan analogues
were synthesized and pharmacologically characterized. The vasodilator activities of these
iGC
compounds were evaluated on rat aorta and expressed as EC50 values or as EC50
values
when obtained in the presence of inhibitors of guanylate cyclase methylene blue (MB) and
1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). Affinities to 1,4-DHP receptors on Ca2+
channels, expressed as IC50 values, were determined through displacement experiments of [3H]-
nitrendipine on rat cortex homogenates. A linear correlation between IC50 and EC50 values
was found for compounds unable to release NO. EC50calcd values for derivatives containing NO-
donor moieties, expression of the Ca2+-blocking component of their vasodilator activity, were
interpolated on this linear regression. They showed a good correspondence with EC50iGC values
determined in the presence of soluble guanylate cyclase inhibitors. Analysis of EC50iGC/EC50
ratios provided a useful tool to distinguish well-balanced hybrids from derivatives biased toward
Ca2+-blocking or NO-dependent vasodilator activity. A detrimental effect on affinity to the 1,4-
DHP receptor, due to substitution at the ortho and meta positions of the 4-phenyl ring, was
observed. SAR to explain this effect is proposed.
In tr od u ction
1,4-Dihydropyridines (DHPs) are an important class
that furoxans (1,2,5-oxadiazole 2-oxides) can behave as
NO prodrugs, in the presence of thiol cofactors.8,9 The
overall reaction mechanism appears to be very complex,
and direct NO radical release or intermediate formation
of nitroxyl anion (NO-), or both, could be involved.
Using appropriate substituents at the heteroring, it is
possible to modulate the properties of the NO production
as well as the NO-dependent vasodilator activity of
these compounds over a wide range. The pharmacologi-
cal profile of furoxans as vasodilators is similar to that
of nitrates, but they could lack significant tolerance
development.10,11 This behavior renders furoxan deriva-
tives interesting tools in the design of hybrid cardio-
vascular drugs.
In previous works we reported the use of furoxan
substructures to obtain well-balanced hybrids with
mixed R1-antagonist12 or â1-antagonist13 and NO-de-
pendent vasodilator properties. In this paper we discuss
the preparation and the pharmacological characteriza-
tion of a series of 4-phenyl-1,4-dihydropyridines, having
furoxan moieties at the ortho or meta position of the
phenyl ring (Chart 1, derivatives 24-29a ,b). The furox-
an substituents were appropriately chosen in order to
modulate the NO release of the final products. The
related furazan derivatives (Chart 1, derivatives 24-
29) were also considered for control purpose, since they
are unable to release NO. Derivatives 3 and 4, in which
the nitrate NO-donor function (-O-NO2) is present,
were considered for a comparison. Nifedipine (1) and
its 3-NO2 isomer 2 were taken as references.
of drugs which are potent blockers of calcium (Ca2+
)
currents through voltage-dependent L class Ca2+ chan-
nels.1 They induce relaxation of vascular smooth muscle,
preferentially in arterial beds, and display a negative
inotropic effect on isolated cardiac muscle. In therapy,
this class of drugs is principally used in the treatment
of cardiac arrhythmias, peripheral vascular disorders,
and hypertension.2 Organic nitrates are also able to
relax vascular smooth muscle, but their effect is more
pronounced in venous beds than on arterial ones.2 This
action is a consequence of their ability to generate nitric
oxide (NO), a physiological messenger which displays a
variety of biological actions.3 In particular NO plays
important roles in maintaining microvascular and mac-
rovascular homeostasis by dilating blood vessels, inhib-
iting platelet adhesion and aggregation, and attenuating
leukocyte adhesion and activation. Combining of 1,4-
dihydropyridine with an organic nitrate could be inter-
esting for the treatment of a number of cardiovascular
diseases. Several attempts aimed at achieving these
results have been published recently.4-7 They deal with
the introduction of the nitroxy group (-O-NO2) in the
alkyl ester chain at the 3- and/or 5-position of the
4-phenyl-1,4-DHP system, as well as of nitroxyalkyloxy
moieties at the 4-phenyl ring. The limit of this approach
is that it is difficult to modulate the NO-donor properties
of the -O-NO2 substructure and consequently to bal-
ance the activity of the hybrid. This aspect is paramount
in the design of a hybrid because the combined phar-
macophoric groups must display their activity in the
same concentration range.5 There is interesting evidence
Resu lts a n d Discu ssion
Ch em istr y. Synthesis of a number of 1,4-DHPs
containing methyl- or carbamoylfuroxan moieties re-
10.1021/jm9803267 CCC: $15.00 © 1998 American Chemical Society
Published on Web 12/03/1998

5394 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 27
Di Stilo et al.
Ch a r t 1
quired preliminary preparation of a series of (halo-
methyl)furoxans (Scheme 1). Selective bromination of
the methyl group linked at the 3-position of the ester
8, obtained by the classical sequence of reactions 5 f 6
f 7 f 8, afforded 9. In this reaction N-bromosuccin-
imide (NBS) and benzoyl peroxide in boiling carbon
tetrachloride were used, according to a procedure we
previously described for synthesis of 12.22 Derivative 9
was transformed into the chlorinated analogue 10 by
the action of sodium chloride, under phase-transfer
catalysis conditions. Derivative 10 was converted to 11
in the presence of 32% ammonia. Treatment of 9 in the
same conditions gave 3-(aminomethyl)-4-furoxancar-
bonamide. Reduction of 5 with sodium borohydride
afforded 13, which was transformed into the expected
bromomethyl derivative 14 by the action of thionyl
bromide. Isomer 16 was obtained from 15 using NBS.
related (halomethyl)furoxans through nucleophilic sub-
stitution of the halogen by m-hydroxybenzaldehyde (17)
or o-hydroxybenzaldehyde (18) in DMF, in the presence
of sodium hydroxide (Scheme 2A). The final 1,4-DHPs
(24a -27a , 24b-27b) were obtained using the modified
Hantzsch approach outlined in Scheme 2A. The ap-
propriate aldehyde dissolved in absolute ethanol was
treated with methyl 3-aminocrotonate (23) in the pres-
ence of trifluoroacetic acid to give the expected products.
The furazan 1,4-DHPs 24-27 were obtained by reduc-
tion of the furoxan analogues in boiling trimethyl
phosphite (Scheme 2B). In this reaction either series a
or b furoxans can be used. Generally we started from
isomers a , owing to the better yields in their prepara-
tion. Finally the cyano-substituted 1,4-DHPs (28, 29,
28a ,b, 29a ,b) were prepared by dehydration of the
amide analogues dissolved in dry pyridine in the pres-
ence of trifluoroacetic anhydride (Scheme 2C). All of the
final furoxan DHPs were assessed for their ability to
The aldehydes 19a -22a and 19b-22b, en route to
the final 1,4-DHPs, were synthesized starting from the

1,4-Dihydropyridines Conjugated to Furoxanyl Moieties
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 27 5395
Sch em e 1a
Sch em e 2a
a
(a) KMnO4, H2SO4, acetone; (b) SOCl2, DMF, reflux; (c) MeOH,
0 °C; (d) NBS, CCl4, benzoyl peroxide, reflux; (e) NaCl, BTEAC,
CHCl3; (f) NH3 (32%), MeOH; (g) NaBH4, dioxane, 25 °C; (h)
SOBr2, DMF, CH2Cl2, 25 °C; (i) NBS, (CH3)2S, dry CH2Cl2.
generate NO in the presence of a large excess of cysteine
(1:50) in buffer solution (pH ) 7.4), at 37 °C. The extent
of NO production after 1 h was determined by detec-
tion of nitrites, which are the oxidative metabolites of
nitric oxide (Griess reaction).9 The results expressed
as percentages of NO2- (mol/mol) are reported in Table
1.
P h ar m acology an d Str u ctu r e-Activity Relation -
sh ip (SAR). Vasodilator activities of all 1,4-DHP de-
rivatives were evaluated on K+-depolarized rat thoracic
aorta strips. Concentration-response curves were de-
termined for each compound, and the potency, expressed
as EC50 value, was calculated (Table 1). The experi-
ments were repeated in the presence of methylene blue
(MB) and, in some cases, in the presence of 1H-[1,2,4]-
oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), well-known
inhibitors of the soluble guanylate cyclase (sGC). A
rightward shift of the concentration-response curves
was taken as evidence of nitric oxide involvement in the
vasodilator action. Potencies determined in these ex-
periments, expressed as EC50iGC values, are reported in
Table 1. A problem which arises in the study of hybrid
DHPs containing NO-donor moieties is individual evalu-
ation of the NO-dependent vasodilator component and
of the component due to Ca2+ channel block. We ad-
dressed this aspect by subjecting all the compounds to
a
(a) DMF, 50% NaOH, 50 °C; (b) absolute EtOH, CF3COOH, 0
°C; (c) P(OCH3)3, reflux; (d) dry Pyr, (CF3CO)2O, 0 °C.

5396 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 27
Di Stilo et al.
Ta ble 1. Vasodilator Potencies, Radioligand Binding Affinities, and NO Generation Properties of 1,4-DHP Derivatives
iGC
EC50 ( SE
(nM)
EC50
( SE
IC50 ( SE
(nM)
Ki ( SE
(nM)
%NO2- ( SE
(mol/mol)
compd
(nM)
2
4
24
24a
24b
25
25a
25b
28
3.4 ( 0.6
750 ( 90
640 ( 40
400 ( 20
500 ( 60
3500 ( 400
1200 ( 100
710 ( 70
310 ( 50
11 ( 2
3.7 ( 0.3
750 ( 100
620 ( 60
400 ( 50
540 ( 60
3300 ( 400
3700 ( 400
1500 ( 100
300 ( 30
140 ( 20
520 ( 100a
63 ( 17
2.6 ( 0.7
970 ( 350
720 ( 190
610 ( 110
820 ( 290
5100 ( 2400
3600 ( 200
2000 ( 400
590 ( 170
500 ( 60
1.2 ( 0.3
440 ( 160
320 ( 90
<0.5
280 ( 50
<0.5
<0.5
370 ( 130
2300 ( 1100
1600 ( 100
920 ( 200
270 ( 80
7.6 ( 0.5
6.0 ( 0.2
28a
220 ( 30
31.5 ( 0.3
43.8 ( 0.6
28b
3.3 ( 0.6
500 ( 220
230 ( 100
560 ( 90a
3.4 ( 0.8
54 ( 7
1
3
26
26a
26b
27
27a
27b
29
3.2 ( 0.4
56 ( 7
41 ( 2
78 ( 10
43 ( 4
970 ( 80
560 ( 40
550 ( 50
6.6 ( 0.8
9.4 ( 1.4
4.4 ( 0.7
2.7 ( 0.4
85 ( 24
59 ( 14
72 ( 2
1.2 ( 0.2
38 ( 11
27 ( 6
32 ( 1
18 ( 3
400 ( 140
350 ( 60
670 ( 220
4.7 ( 2
30 ( 10
21 ( 6
<0.5
46 ( 10
76 ( 8
1.8 ( 0.1
0.9 ( 0.2
46 ( 6
40 ( 6
910 ( 60
640 ( 40
940 ( 120
6.6 ( 0.7
50 ( 6
880 ( 300
780 ( 120
1500 ( 500
10 ( 4
12.3 ( 0.2
13.0 ( 0.1
29a
29b
67 ( 22
27 ( 14
24.0 ( 0.2
40.8 ( 0.2
22 ( 6
a
iGC
Determined in the presence of ODQ. For all the other furoxan derivatives EC50
the same as those determined in the presence of MB within the experimental error.
values determined in the presence of ODQ were
Ta ble 2. Interpolated Values (EC50calcd, M) from Regression Eq
1 and Vasodilator Potencies Obtained in the Presence of sGC
Inhibitors (EC50iGC, M), Expressed as Cologarithms, for
NO-Donor 1,4-DHPs
a competition study of inhibition of [3H]nitrendipine
binding to cerebral cortices, following the procedure
reported in the Experimental Section. The concentration
of each compound able to display 50% of [3H]nitren-
dipine binding was determined from the competition
curves (IC50 values, Table 1), and then the correspond-
ing Ki constant was calculated (Table 1). These figures
represent pure DHP receptor affinities. When log 1/EC50
values for the compounds not containing NO-donor
moieties, namely, the furazan derivatives 24-29 and
the models 1 and 2, were plotted against the corre-
sponding log 1/IC50 values, the following very satisfac-
tory correlation (eq 1) was obtained:
calcd
iGC
log 1/EC50
(95%)
( CL log 1/EC50
(95%)
( CL
compd
4
6.14 ( 0.16
6.33 ( 0.15
6.21 ( 0.15
5.61 ( 0.20
5.84 ( 0.18
6.41 ( 0.14
6.41 ( 0.14
7.24 ( 0.12
7.21 ( 0.14
7.46 ( 0.13
6.23 ( 0.15
5.97 ( 0.17
7.24 ( 0.12
7.63 ( 0.14
6.12 ( 0.15
6.40 ( 0.16
6.27 ( 0.14
5.43 ( 0.12
5.82 ( 0.08
6.85 ( 0.21
7.20 ( 0.38
7.27 ( 0.13
7.12 ( 0.12
7.34 ( 0.10
6.19 ( 0.07
6.03 ( 0.12
7.30 ( 0.12
7.66 ( 0.18
24a
24b
25a
25b
28a
28b
3
26a
26b
27a
27b
29a
29b
6.28 ( 0.34a
6.25 ( 0.26a
log 1/EC50 ) 0.947((0.042) log 1/IC50
0.443((0.299) (1)
n ) 8, r2 ) 0.988, s ) 0.14
+
a
Determined in the presence of ODQ.
The equation indicates a very good relationship
between DHP receptor occupancy and functional effect.
presence of MB. The sole exceptions are the values
relating to the cyano derivatives 28a ,b, which are potent
By using eq 1 and the tabulated log 1/IC50 values, it is
NO donors belonging to the meta series. A good cor-
calcd
iGC
possible to calculate log 1/EC50
values (Table 2) for
respondence is again restored when EC50
values for
all the DHPs containing NO-donor moieties, namely, for
the derivatives 3, 4, 24a -29a , and 24b-29b. A recent
paper reports interesting evidence that NO released by
N-acetyl-S-nitrosopenicillamine (SNAP; 100-800 µM)
could inhibit expressed cardiovascular L-type Ca2+
these compounds, determined in the presence of ODQ,
a more potent inhibitor of sGC, are taken into account.
iGC
When log 1/EC50
values (in the presence of ODQ for
calcd
derivatives 28a ,b) are plotted against log 1/EC50
values, a very satisfactory regression is obtained, in
which the slope and the intercept are 1 and 0, respec-
tively, in the range of the confidence limits (Figure 1).
The above-reported picture indicates that EC50iGC values
can be considered a measure of the vasodilator potencies
channels through a non-cGMP-dependent pathway.14
calcd
EC50
values, obtained for NO-donor models from eq
1, could then be partially influenced by this mechanism,
if it were operating. However, our findings suggest that
this mechanism is not involved, because nifedipine
of this set of structures, largely dependent on their Ca2+
-
binding is not modified by increasing amounts (10-6
-
blocker properties. In the following discussion we as-
sume that when EC50iGC/EC50 = 1, the compounds
behave as practically pure Ca2+ antagonists (e.g., 26b
in Figure 2a). This applies to all the furazan derivatives
(Chart 1, 24-29) and to the references 1 and 2, since
they are unable to release NO. Analysis of the rightward
10-9 M) of 4-phenyl-3-furoxancarbonitrile,15 a typical
NO-donor furoxan (unpublished data). Analysis of data
in Table 2 shows that there is a good correspondence
calcd
between log 1/EC50
values interpolated on the line
iGC
(eq 1) and log 1/EC50
values determined in the

1,4-Dihydropyridines Conjugated to Furoxanyl Moieties
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 27 5397
Table 1 shows that substitution of methylfurazanyl-
methyloxy and methylfuroxanylmethyloxy moieties for
the m-nitro group in 2 affords derivatives with similar
potency, about 2 logarithm units lower than that of the
reference. Since the methylfuroxan analogues 24a ,b
have EC50 = EC50iGC, we conclude that they behave as
pure Ca2+ channel blockers. Correspondingly these
-
compounds produced relatively small amounts of NO2
in the presence of cysteine. Carbamoyl congeners of this
series (25, 25a ,b) again display low potency. Furoxan
-
isomers produce similar amounts of NO2 and behave
iGC
as low-potency, well-balanced hybrids, since EC50
/
EC50 is equal to 3 and 2, respectively. Cyano-substituted
analogues 28 and 28a ,b are the most active members
of the series; in particular 28b is equipotent with the
reference. Both the furoxan isomers display vasodilator
activity that is principally dependent on their NO-donor
properties (EC50iGC/EC50 for 28a = 47, for 28b = 170).
This behavior parallels production of nitrite by the two
derivatives. Study of derivative 3, a recently described
1,4-dihydropyridine with a nitrate ester moiety,7 shows
that this compound is practically a pure Ca2+ channel
blocker (EC50iGC/EC50 = 1) in the tested concentration
range.
calcd
F igu r e 1. Correlation between log 1/EC50iGC and log 1/EC50
values for NO-donor 1,4-DHPs. Dashed lines represent 95%
confidence limits for the regression line.
Substitution of methylfurazanylmethyloxy and me-
thylfuroxanylmethyloxy moieties for the o-nitro group
in 1 affords derivatives 26 and 26a ,b, which are 1
logarithm unit less potent than the lead. In this series,
too, methylfuroxan congeners are very feeble NO donors
and display EC50 = EC50iGC. Therefore their vasodilator
activity is principally due to Ca2+ channel block. Car-
bamoyl analogues 27 and 27a ,b are the least active
members of the group. Unlike the isomer 27a , which
has EC50iGC/EC50 = 1, 27b shows EC50iGC/EC50 = 2, and
so it can be considered a well-constructed hybrid of low
potency. Also in the ortho series, cyano derivatives 29
and 29a ,b are the most active compounds. Their potency
is comparable to that of 1. Analysis of EC50iGC/EC50
ratios (29a ,b EC50iGC/EC50 = 5) indicates that both the
furoxans are potent, well-balanced hybrid structures.
Model 47 behaves as a pure Ca2+ channel antagonist in
the range of the considered concentrations.
Analysis of the Ca2+ channel-blocking activities (Table
1, EC50iGC, IC50) shows some qualitative structure-
activity relationships. The compounds belonging to the
ortho series display higher Ca2+ channel-blocker potency
than the corresponding compounds of the meta series.
In both series, NH2CO derivatives are markedly less
potent than all the other compounds. In addition, the
furoxan isomers together with the furazan analogue
display similar potency, with the exception of derivative
29. Finally, the substitution of R-furazanyl-CH2O- and
R-furoxanyl-CH2O- moieties for the NO2 group in 1 and
2, respectively, is detrimental to Ca2+-blocker activity.
The results of a QSAR study by Coburn et al.16 on a
series of 4-phenyl-1,4-dihydropyridine derivatives with
ortho and meta monosubstitutions at the phenyl ring
can help to explain these findings. In Coburn’s paper
the effect of a number of substituents on the tonic
contractile response of longitudinal muscle strips of
F igu r e 2. Concentration-response curves for vasodilator
activity of compounds 26b (a), 29b (b), and 28b (c) in the
presence and absence of MB or ODQ. All points are mean
values ( SE from independent experiments.
shift of the concentration curves was used to determine
the relative contributions of Ca2+-blocker and NO-
mediated properties whereby values for EC50iGC/EC50 of
2-5 indicate well-balanced hybrids (e.g., 29b in Figure
2b) and values greater than these indicate a predomi-
nantly NO-mediated vasodilator action (e.g., 28b in
Figure 2c).

5398 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 27
Di Stilo et al.
and 13C nuclear magnetic resonance spectra were recorded at
200 and 50 MHz, respectively, on a Brucker AC-200 spectrom-
eter and are reported in δ (ppm) units (§, tentatively assigned;
/, not 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
drying agent. Solvent removal was achieved under reduced
pressure at room temperature. Elemental analyses of the new
compounds were performed by REDOX (Cologno M.), and the
results are within 0.4% of the theoretical values. Intermediates
521 and 1222 and derivatives 2,23 3, and 47 were synthesized
according to the literature. Derivative 15 was a gift from Dr.
K. Scho¨nafinger, H.M.R., Frankfurt, and 1 was purchased from
Sigma. Nitrite production by furoxans in the presence of
L-cysteine was evaluated according to the procedure described
in ref 9.
3-Meth yl-4-fu r oxa n ca r boxylic Acid (6). KMnO4 (7.9 g,
50 mmol) was added portionwise to a stirred, iced water-cooled
mixture of 5 (7.7 g, 60 mmol) dissolved in acetone (5 mL) and
12 N H2SO4 (50 mL). The temperature was not permitted to
rise above 15 °C. Stirring was continued for 12 h at room
temperature, and the excess of KMnO4 was destroyed with
Na2SO3. The mixture was extracted with EtOAc, and the
combined organic phases were treated with 2 N NaOH (3 ×
15 mL). The aqueous phase was strongly acidified by adding
12 N H2SO4 and then extracted with EtOAc and dried. Solvent
removal afforded a residue which was filtered on a short silica
gel column (petroleum ether/EtOAc, 7:3) (4.3 g, 50%): mp 92
°C (benzene) (lit.24 mp 92 °C).
Meth yl 3-Meth yl-4-fu r oxa n ca r boxyla te (8). A solution
of 6 (18 g, 125 mmol) in thionyl chloride (60 mL) containing a
catalytic amount of dry DMF was refluxed for 1 h. Solvent
removal gave 7 which, without any further purification, was
treated under stirring and external cooling with dry methanol
(30 mL). The resulting precipitate was filtered off, dried, and
purified by flash chromatography (petroleum ether/EtOAc, 7:3)
to give the title product (11.8 g, 60%): mp 80-81 °C (EtOAc/
petroleum ether). 1H NMR (DMSO-d6): 2.36 (s, 3H, CH3); 4.03
(s, 3H, OCH3). 13C NMR (DMSO-d6): 156.3 (COO); 148.9 (C4);
112.1 (C3); 53.3 (OCH3); 8.37 (CH3). Anal. (C5H6N2O4) C, H,
N.
Meth yl 3-(Br om om eth yl)-4-fu r oxan car boxylate (9). NBS
and a catalytic amount of benzoyl peroxide were added to a
solution of 8 (4.7 g, 30 mmol) in CCl4 (70 mL). The mixture
was refluxed for 48 h under stirring. Solvent removal gave a
residue which was purified by flash chromatography (petro-
leum ether/CH2Cl2, 1:1). The title product was obtained as a
pure pale-yellow oil (4.4 g, 63%). 1H NMR (CDCl3): 4.50 (s,
2H, CH2); 4.02 (s, 3H, OCH3). 13C NMR (CDCl3): 157.0 (COO);
146.8 (C4); 112.7 (C3); 53.2 (s, 3H, OCH3); 15.8 (CH2). Anal.
(C5H5N2O4Br) C, H, N.
guinea pig ileum was correlated satisfactorily by eq 2:
log 1/IC50 ) 0.69((0.10)π + 2.32((0.49)σm -
0.49((0.10)Lm + 8.01((0.32) (2)
n ) 29, r ) 0.87, s ) 0.67, F ) 25.35
in which the appropriate σm is entered for the substitu-
ent regardless of whether it is in the ortho or meta
position. Use of eq 2 in analyzing structure-activity
relationships in the two series of derivatives described
here is questionable, because the correlation was ob-
tained using pharmacological data from a different
tissue. In addition, the substituents present at the
phenyl ring in our compounds have different shapes and
are significantly larger than any other substituent used
to derive the equation, so they could behave differently
with respect to the original group, particularly in the
ortho series. Nevertheless eq 2 seems to explain some
trends. In a recent paper we showed that methylfura-
zanyl and methylfuroxanyl substructures display σI
values close to that of the chlorine group.17 Therefore,
since σI of the -OCH2Cl moiety is +0.41,18 we expect
values near this for all our substituents. Calculation of
their L constants, using the CCPK program,19 shows
that they range between 7.21 and 7.96. Finally, π
constants were calculated by the CLogP algorithm20
(πOCH -Fx-R ) 0.045, -0.442, -1.010; πOCH -Fz-R ) 0.203,
2
2
-0.072, -1.018; R ) CN, CH3, CONH2; Fx ) furoxanyl;
Fz ) furazanyl). In this approximation, π values for
isomer furoxan substituents are identical. On the basis
of these molecular descriptors, we could infer that the
extensive length of the substituents is responsible for
the decrease in potency of the meta series with respect
to the ortho one. Since σm and L have similar values for
all the substituents, the low potency of NH2CO deriva-
tives should principally highlight the low lipophilicity
of the carbamoyl group. In each series, the similar
potency displayed by furoxan isomers and the furazan
analogue should reflect the general resemblance be-
tween their molecular descriptors. Electronic and steric
properties, combined with lipophilicity in the case of
NH2CO derivatives, should be chiefly responsible for the
decrease in the activity of all the compounds of the meta
series with respect to NO2 derivative 2 (NO2, σm ) 0.71,
π ) -0.337, L ) 3.44). Finally, electronic properties,
again combined with lipophilicity in the carbamoyl
derivatives, could be paramount in determining the
lower potency of the derivatives of the ortho series
compared to the nitro lead 1. However this analysis is
made more difficult for the possible presence of ortho
effects not scaled in eq 2.
3-(Ch lor om eth yl)-4-fu r oxa n ca r bon a m id e (11). Benzyl-
triethylammonium chloride (BTEAC) (6.6 g, 29 mmol) and
NaCl (16.9 g, 290 mmol) were added to a well-stirred solution
of 9 (7.0 g, 29 mmol) in CHCl3 (30 mL). The mixture was kept
under stirring at room temperature for 3 days. The formed
precipitate was filtered and washed with CHCl3, and the
organic phase was dried and evaporated. The residue obtained
was extracted with EtOAc. Solvent removal gave 10 which was
filtered on a short column (petroleum ether/EtOAc, 9:1). The
oil obtained after evaporation of the solvent, without any
additional purification, was dissolved in methanol (20 mL),
and 32% ammonia (15 mL) was added dropwise to the iced
water-cooled and well stirred solution. The resulting white
precipitate was filtered off, washed with water, and dried (4.1
g, 70%): mp 149 °C (CHCl3). 1H NMR (DMSO-d6): 4.84 (s,
2H, CH2); 8.32, 8.86 (2s, 2H, CONH2). 13C NMR (DMSO-d6):
158.1 (CONH2); 150.4 (C4); 113.7 (C3); 32.7 (CH2). Anal.
(C4H4N3O3Cl) C, H, N.
In conclusion this work demonstrates the possibility
of obtaining “well-balanced” hybrids with mixed Ca2+
channel-blocker and NO-dependent vasodilator activi-
ties by using appropriate furoxan substructures. In
addition, a procedure for the individual evaluation of
the two kinds of activities is proposed.
Exp er im en ta l Section
Ch em istr y. Melting points (uncorrected) were obtained 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. All of the compounds were routinely checked
3-Meth yl-4-fu r oxa n m eth a n ol (13). NaBH4 (3.0 g, 80
mmol) was added portionwise to a solution of 5 (5.1 g, 40 mmol)
in dioxane (40 mL), keeping the temperature at 20-25 °C. The
reaction mixture was stirred for 12 h at room temperature.
The residue obtained by concentration of the solution was
treated with water and extracted with EtOAc. The organic
1
by infrared spectrophotometry (Shimadzu FT-IR 8101M). H

1,4-Dihydropyridines Conjugated to Furoxanyl Moieties
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 27 5399
phase was evaporated, and the crude oil obtained was purified
by flash chromatography (petroleum ether/EtOAc, 7:1). The
title product was obtained as a pale-yellow oil (4.6 g, 50%) (lit.25
oil).
(CHO); 159.4, 136.4, 128.3, 125.0, 122.2, 114.2 (Ph); 58.7
(OCH2); 113.4 (C3); 155.8 (C4); 10.8 (CH3). Anal. (C11H10N2O4)
C, H, N.
2-[(3-Meth ylfu r oxan -4-yl)m eth oxy]ben zaldeh yde (21b):
mp 97-98 °C (CHCl3/petroleum ether, 65%). 1H NMR (DMSO-
d6): 10.4 (s, 1H, CHO); 7.1-7.8 (m, 4H, Ph); 5.49 (s, 2H, OCH2);
2.22 (s, 3H, CH3). 13C NMR (DMSO-d6): 189.2 (CHO); 159.6,
136.4, 128.3, 124.8, 121.9, 114.1 (Ph); 61.3 (OCH2); 113.4 (C3);
155.8 (C4); 7.6 (CH3). Anal. (C11H10N2O4) C, H, N.
2-[(4-Ca r ba m oylfu r oxa n -3-yl)m eth oxy]ben za ld eh yd e
(22a ): mp 149-150 °C (CHCl3/petroleum ether, 75%). 1H NMR
(DMSO-d6): 10.3 (s, 1H, CHO); 7.1-7.8 (m, 4H, Ph); 5.42 (s,
2H, OCH2); 8.28, 8.61 (2s, 2H, CONH2). 13C NMR (DMSO-d6):
189.2 (CHO); 158.2§, 136.4, 127.8, 125.1, 122.2, 114.6 (Ph); 59.1
(OCH2); 112.0 (C3); 151.4 (C4); 159.8§ (CONH2). Anal.
(C11H10N2O4) C, H, N.
2-[(3-Ca r ba m oylfu r oxa n -4-yl)m eth oxy]ben za ld eh yd e
(22b): mp 150-151 °C (EtOAc/petroleum ether, 178-179 °C).
1H NMR (DMSO-d6): 10.4 (s, 1H, CHO); 5.59 (s, 2H, OCH2);
7.13-7.76 (m, 4H, Ph); 7.85, 8.50 (2s, 2H, CONH2). 13C NMR
(DMSO-d6): 189.2 (CHO); 160.1, 136.4, 127.7, 124.7, 121.8,
114.5 (Ph); 62.1 (OCH2); 110.7 (C3); 155.8* (C4); 155.7*
(CONH2). Anal. (C11H10N2O4) C, H, N.
Gen er a l Meth od of P r ep a r a tion of 1,4-DHP s 24a -27a
a n d 24b-27b. A solution of methyl 3-aminocrotonate (23) (1.0
g, 9 mmol) in absolute ethanol (10 mL) was added dropwise
to a stirred and ice-cooled (0 °C) solution of the appropriate
benzaldehyde (3 mmol) in absolute ethanol (10 mL) and
trifluoroacetic acid (0.7 g, 6 mmol). Stirring was continued for
3 h at 0 °C. Then the resulting precipitate was filtered off,
washed with a small amount of absolute ethanol, and dried.
Dim eth yl 1,4-dih ydr o-2,6-dim eth yl-4-[3-[(4-m eth ylfu r ox-
an -3-yl)m eth oxy]ph en yl]-3,5-pyr idin edicar boxylate (24a):
mp 159-160 °C (CHCl3/petroleum ether, 60%). 1H NMR
(DMSO-d6): 8.90 (s, 1H, -NH); 3.56 (s, 3H, -OCH3); 4.88 (s,
1H, C(4)-H); 2.27 (s, 3H, 2,6-CH3); 6.7-7.2 (m, 4H, Ph); 5.04
(s, 2H, -OCH2); 2.34 (s, 3H, -CH3). 13C NMR (DMSO-d6): 18.3
(2,6-CH3); 38.6 (4-CH); 50.8 (OCH3); 101.3 (3,5-C); 146.0 (2,6-
C); 167.4 (COO); 157.0, 149.7, 129.5, 120.9, 114.0, 113.5§ (Ph);
58.0 (OCH2);112.2§ (C3);155.7 (C4);8.0 (CH3).Anal.(C21H23N3O7)
C, N, H.
Dim eth yl 1,4-dih ydr o-2,6-dim eth yl-4-[3-[(3-m eth ylfu r ox-
an -4-yl)m eth oxy]ph en yl]-3,5-pyr idin edicar boxylate (24b):
mp 163-164 °C (CHCl3/petroleum ether, 60%). 1H NMR
(DMSO-d6): 8.92 (s, 1H, -NH); 3.57 (s, 3H, -OCH3); 4.89 (s,
1H, C(4)-H); 2.28 (s, 3H, 2,6-CH3); 6.8-7.2 (m, 4H, Ph); 5.24
(s, 2H, -OCH2); 2.17 (s, 3H, -CH3). 13C NMR (DMSO-d6): 18.3
(2,6-CH3); 38.6 (4-CH); 50.8 (OCH3); 101.3 (3,5-C); 146.0 (2,6-
C); 167.4 (COO); 157.3, 149.7, 129.4, 120.9, 114.1, 113.1§ (Ph);
60.7 (OCH2); 112.2§ (C3); 155.8 (C4); 7.67 (CH3). Anal.
(C21H23N3O7) C, N, H.
Dim eth yl 1,4-d ih yd r o-2,6-d im eth yl-4-[3-[(4-ca r ba m oyl-
fu r oxa n -3-yl)m et h oxy]p h en yl]-3,5-p yr id in ed ica r b oxy-
la te (25a ): mp 215-216 °C dec (EtOAc/petroleum ether, 85%).
1H NMR (DMSO-d6): 8.87 (s, 1H, -NH); 3.57 (s, 3H, -OCH3,);
4.88 (s, 1H, C(4)-H); 2.27 (s, 3H, 2,6-CH3); 6.7-7.2 (m, 4H,
Ph); 5.13 (s, 2H, -OCH2); 8.59, 8.26 (2s, 2H, CONH2). 13C NMR
(DMSO-d6): 18.7 (2,6-CH3); 38.6 (4-CH); 50.7 (OCH3); 101.3
(3,5-C); 145.9 (2,6-C); 167.4 (COO); 157.2, 149.7, 129.2, 120.6,
113.6*, 111.7* (Ph); 57.7 (OCH2); 112.2* (C3); 151.4 (C4); 158.2
(CONH2). Anal. (C21H22N4O8) C, N, H.
Dim eth yl 1,4-d ih yd r o-2,6-d im eth yl-4-[3-[(3-ca r ba m oyl-
fu r oxa n -4-yl)m et h oxy]p h en yl]-3,5-p yr id in ed ica r b oxy-
la te (25b): mp 209-210 °C dec (EtOAc/petroleum ether, 85%).
1H NMR (DMSO-d6): 8.93 (s, 1H, -NH); 3.58 (s, 3H, -OCH3),
4.90 (s, 1H, C(4)-H); 2.28 (s, 3H, 2,6-CH3); 6.8-7.2 (m, 4H,
Ph); 5.36 (s, 2H, -OCH2); 8.52, 7.86 (2s, 2H, CONH2). 13C NMR
(DMSO-d6): 18.3 (2,6-CH3); 38.5 (4-CH); 50.8 (OCH3); 101.4
(3,5-C); 146.0 (2,6-C); 167.5 (COO); 157.5, 149.6, 129.4, 120.4,
113.8, 111.9§ (Ph); 61.0 (OCH2); 110.5§ (C3); 155.4* (C4); 155.9*
(CONH2). Anal. (C21H22N4O8) C, N, H.
4-(Br om om eth yl)-3-m eth ylfu r oxa n (14). Thionyl bro-
mide (1.55 mL, 20 mmol) was added dropwise to a stirred and
ice-salt-cooled solution (-10 °C) of 13 (1.3 g, 10 mmol) in CH2-
Cl2 (50 mL) containing a few drops of DMF. The reaction
mixture was kept under stirring at room temperature for 12
h. After this time, water (50 mL) was added, and the mixture
was extracted with CH2Cl2. By evaporation of the dried
combined organic layers, an oily residue was obtained which
was purified by flash chromatography (petroleum ether/EtOAc,
7:3) to give the title product (1.0 g, 50%) as a pale-yellow oil.
1H NMR (CDCl3): 2.20 (s, 3H, CH3); 4.38 (s, 2H, CH2). 13C
NMR (CDCl3): 154.4 (C4); 111.8 (C3); 18.5 (CH2); 7.4 (CH3).
Anal. (C4H5N2O2Br) C, H, N.
4-(Br om om eth yl)-3-fu r oxa n ca r bon a m id e (16). NBS (5.0
g, 20 mmol) and methyl sulfide (2.6 mL, 35 mmol) were mixed
under dry atmosphere in CH2Cl2 (90 mL) at 0 °C. The reaction
mixture was cooled at -15 °C, and then 15 (3.0 g, 19 mmol)
was added under stirring. The mixture was allowed to reach
room temperature and was stirred for 24 h. After this time,
equivalent amounts of NBS (3.3 g, 19 mmol) and methyl sulfide
(1.40 mL, 19 mmol) were added, and the stirring was continued
for 4 h. Then the reaction mixture was extracted with water,
and the dried organic phases were evaporated to give the title
product as a pale-yellow oil which was purified by flash
chromatography (CH2Cl2) (3.0 g, 72%): mp 91-92 °C (CHCl3,
petroleum ether). 1H NMR (CDCl3): 5.0 (s, 2H, CH2); 7.65, 8.52
(2s, 2H, CONH2). 13C NMR (CDCl3): 155.4§ (CONH2); 155.8§
(C4); 109.8 (C3); 20.4 (CH2). Anal. (C4H4N3O3Br) C, H, N.
Gen er a l Meth od of P r ep a r a tion of Su bstitu ted Ben -
za ld eh yd es 19a -22a a n d 19b-22b. NaOH (50%) (6 mmol)
was added to a stirred solution of the appropriate (halomethyl)-
furoxans (5 mmol) and hydroxybenzaldehyde 17 or 18 (6 mmol)
in DMF (20 mL) kept at 50 °C. The stirring at 50 °C was
continued for 30 min, and then the reaction mixture was
poured into water. The resulting precipitate was filtered off
and dissolved in EtOAc. The organic solution, washed with 2
N NaOH and then with water, was dried and evaporated to
give the expected compounds.
3-[(4-Meth ylfu r oxan -3-yl)m eth oxy]ben zaldeh yde (19a):
mp 81-82 °C (CHCl3/petroleum ether, 50%). 1H NMR (DMSO-
d6): 10.0 (s, 1H, CHO); 7.4-7.8 (m, 4H, Ph); 5.24 (s, 2H, OCH2);
2.42 (s, 3H, CH3). 13C NMR (DMSO-d6): 192.8 (CHO); 157.8,
137.8, 130.8, 123.9, 121.7, 114.2 (Ph); 58.2 (OCH2); 113.4 (C3);
155.8 (C4); 10.8 (CH3). Anal. (C11H10N2O4) C, H, N.
3-[(3-Meth ylfu r oxan -4-yl)m eth oxy]ben zaldeh yde (19b):
mp 103-104 °C (CHCl3/petroleum ether, 50%). 1H NMR
(DMSO-d6): 10.0 (s, 1H, CHO); 7.4-7.6 (m, 4H, Ph); 5.43 (s,
2H, OCH2); 2.27 (s, 3H, CH3). 13C NMR (DMSO-d6): 192.8
(CHO); 158.0, 137.8, 130.7, 123.8, 121.8, 114.3 (Ph); 61.0
(OCH2); 113.1 (C3); 155.4 (C4); 7.59 (CH3). Anal. (C11H10N2O4)
C, H, N.
3-[(4-Ca r ba m oylfu r oxa n -3-yl)m eth oxy]ben za ld eh yd e
(20a ): mp 155-156 °C (EtOAc/petroleum ether). 1H NMR
(DMSO-d6): 10.0 (s, 1H, CHO); 7.4-7.6 (m, 4H, Ph); 5.33 (s,
2H, OCH2); 8.29, 8.62 (2s, 2H, CONH2). 13C NMR (DMSO-d6):
197.2 (CHO); 158.0*, 137.8, 130.7, 123.5, 121.6, 114.4 (Ph); 58.3
(OCH2); 112.0 (C3); 151.4 (C4); 158.2* (CONH2). Anal.
(C11H10N2O4) C, H, N.
3-[(3-Ca r ba m oylfu r oxa n -4-yl)m eth oxy]ben za ld eh yd e
(20b): mp 150-151 °C (EtOAc/petroleum ether). 1H NMR
(DMSO-d6): 10.0 (s, 1H, CHO); 5.53 (s, 2H, OCH2); 7.4-7.6
(m, 4H, Ph); 7.86, 8.52 (2s, 2H, CONH2). 13C NMR (DMSO-
d6): 192.9 (CHO); 158.2, 137.8, 130.6, 123.7, 121.7, 114.4 (Ph);
61.5 (OCH2); 110.6 (C3); 155.8* (C4); 155.2* (CONH2). Anal.
(C11H10N2O4) C, H, N.
2-[(4-Meth ylfu r oxan -3-yl)m eth oxy]ben zaldeh yde (21a):
mp 103-104 °C (CHCl3/petroleum ether, 70%). 1H NMR
(DMSO-d6): 10.3 (s, 1H, CHO); 7.2-7.8 (m, 4H, Ph); 5.31 (s,
2H, OCH2); 2.44 (s, 3H, CH3). 13C NMR (DMSO-d6): 189.1
Dim eth yl 1,4-dih ydr o-2,6-dim eth yl-4-[2-[(4-m eth ylfu r ox-
an -3-yl)m eth oxy]ph en yl]-3,5-pyr idin edicar boxylate (26a):
mp 184-185 °C (CHCl3/petroleum ether, 70%). 1H NMR

5400 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 27
Di Stilo et al.
(DMSO-d6): 8.64 (s, 1H, -NH); 3.45 (s, 3H, -OCH3); 5.14 (s,
1H, C(4)-H); 2.16 (s, 3H, 2,6-CH3); 6.9-7.1 (m, 4H, Ph); 5.02
(s, 2H, -OCH2); 2.45 (s, 3H, -CH3). 13C NMR (DMSO-d6): 18.0
(2,6-CH3); 35.1 (4-CH); 50.4 (OCH3); 101.6 (3,5-C); 145.2 (2,6-
C); 167.7 (COO); 155.9*, 135.3, 127.5, 130.1, 112.2§ (Ph); 58.1
(OCH2); 113.7§ (C3); 154.9* (C4); 10.7 (CH3). Anal. (C21H23N3O7)
C, N, H.
Dim eth yl 1,4-d ih yd r o-2,6-d im eth yl-4-[2-[(4-ca r ba m oyl-
fu r a za n -3-yl)m et h oxy]p h en yl]-3,5-p yr id in ed ica r b oxy-
la te (27): mp 182-184 °C (EtOAc/petroleum ether, 85%). H
1
NMR (DMSO-d6): 8.60* (s, 1H, -NH); 3.45 (s, 3H, -OCH3),
5.18 (s, 1H, C(4)-H); 2.16 (s, 3H, 2,6-CH3); 6.8-7.1 (m, 4H,
Ph); 5.45 (s, 2H, -OCH2); 8.58*, 8.26 (2s, 2H, CONH2). 13C
NMR (DMSO-d6): 18.0 (2,6-CH3); 34.3 (4-CH); 50.4 (OCH3);
101.1 (3,5-C); 145.3 (2,6-C); 167.7 (COO); 155.0, 136.6, 130.1,
127.4, 121.4, 112.9 (Ph); 60.0 (OCH2); 148.8 (C3); 152.5 (C4);
158.3 (CONH2). Anal. (C21H22N4O7) C, N, H.
Gen er a l Meth od of P r ep a r a tion of 1,4-DHP s 28, 29,
28a ,b, a n d 29a ,b. Trifluoroacetic anhydride (3 mmol) was
added dropwise to a stirred and ice-salt-cooled solution of the
appropriate DHPs 25, 25a ,b, 27, and 27a ,b (1 mmol) in dry
pyridine (5 mL). The cooling bath was removed, and the
stirring was continued for 30 min at room temperature. The
reaction mixture was poured into water, and the solution,
acidified with 2 N HCl, was extracted with EtOAc. The
combined organic layers were washed with 2 N HCl, dried,
and evaporated to afford a residue which was purified by flash
chromatography (petroleum ether/EtOAc, 7:3).
Dim eth yl 1,4-d ih yd r o-2,6-d im eth yl-4-[3-[(4-cya n ofu r a -
zan -3-yl)m eth oxy]ph en yl]-3,5-pyr idin edicar boxylate (28):
157-158 °C (EtOAc/petroleum ether, 60%). 1H NMR (DMSO-
d6): 8.95 (s, 1H, -NH); 3.57 (s, 3H, -OCH3); 4.90 (s, 1H, C(4)-
H); 2.28 (s, 3H, 2,6-CH3); 6.8-7.3 (m, 4H, Ph); 5.55 (s, 2H,
-OCH2). 13C NMR (DMSO-d6): 18.2 (2,6-CH3); 38.6 (4-CH);
50.8 (OCH3); 101.3 (3,5-C); 146.0 (2,6-C); 167.4 (COO); 157.0,
149.8, 129.4, 120.9, 114.0, 111.9 (Ph); 59.4 (OCH2); 134.0 (C3);
154.6 (C4); 107.0 (CN). Anal. (C21H20N4O6) C, H, N.
Dim eth yl 1,4-dih ydr o-2,6-dim eth yl-4-[3-[(4-cyan ofu r ox-
an -3-yl)m eth oxy]ph en yl]-3,5-pyr idin edicar boxylate (28a):
145-146 °C (EtOAc/petroleum ether, 70%). 1H NMR (DMSO-
d6): 8.88 (s, 1H, -NH); 3.57 (s, 3H, -OCH3); 4.90 (s, 1H, C(4)-
H); 2.28 (s, 3H, 2,6-CH3); 6.8-7.3 (m, 4H, Ph); 5.17 (s, 2H,
-OCH2). 13C NMR (DMSO-d6): 18.1 (2,6-CH3); 38.6 (4-CH);
50.6 (OCH3); 101.2 (3,5-C); 145.9 (2,6-C); 167.3 (COO); 156.7,
149.8, 129.4, 121.2, 114.2§, 111.7 (Ph); 58. 9 (OCH2); 113.1§
(C3); 135.0 (C4); 107.8 (CN). Anal. (C21H20N4O7) C, H, N.
Dim eth yl 1,4-dih ydr o-2,6-dim eth yl-4-[3-[(3-cyan ofu r ox-
an -4-yl)m eth oxy]ph en yl]-3,5-pyr idin edicar boxylate (28b):
142-143 °C (EtOAc/petroleum ether, 80%). 1H NMR (DMSO-
d6): 8.94 (s, 1H, -NH); 3.58 (s, 3H, -OCH3), 4.91 (s, 1H, C(4)-
H); 2.28 (s, 3H, 2,6-CH3); 6.8-7.3 (m, 4H, Ph); 5.36 (s, 2H,
-OCH2). 13C NMR (DMSO-d6): 18.2 (2,6-CH3); 38.5 (4-CH);
50.8 (OCH3); 101.3 (3,5-C); 146.0 (2,6-C); 167.4 (COO); 157.0,
149.8, 129.4, 121.0, 114.3, 111.7 (Ph); 60.7 (OCH2); 98.4 (C3);
155.3 (C4); 106.4 (CN). Anal. (C21H20N4O7) C, H, N.
Dim eth yl 1,4-d ih yd r o-2,6-d im eth yl-4-[2-[(4-cya n ofu r a -
zan -3-yl)m eth oxy]ph en yl]-3,5-pyr idin edicar boxylate (29):
mp 153-156 °C (EtOAc/petroleum ether, 50%). 1H NMR
(DMSO-d6): 8.60 (s, 1H, -NH); 3.42 (s, 3H, -OCH3), 5.22 (s,
1H, C(4)-H); 2.15 (s, 3H, 2,6-CH3); 6.9-7.1 (m, 4H, Ph); 5.49
(s, 2H, -OCH2). 13C NMR (DMSO-d6): 18.1 (2,6-CH3); 34.6 (4-
CH); 50.8 (OCH3); 101.0 (3,5-C); 145.2 (2,6-C); 167.7 (COO);
155.4, 136.5, 130.1, 129.8, 121.9, 112.8 (Ph); 59.3 (OCH2);
154.2* (C3); 153.7* (C4); 107.7 (CN). Anal. (C21H20N4O6) C, H,
N.
Dim eth yl 1,4-dih ydr o-2,6-dim eth yl-4-[2-[(4-cyan ofu r ox-
an -3-yl)m eth oxy]ph en yl]-3,5-pyr idin edicar boxylate (29a):
mp 130-133 °C (EtOAc/petroleum ether, 70%). 1H NMR
(DMSO-d6): 8.65 (s, 1H, -NH); 3.46 (s, 3H, -OCH3), 5.14* (s,
1H, C(4)-H); 2.15 (s, 3H, 2,6-CH3); 6.9-7.2 (m, 4H, Ph); 5.13
(s, 2H, -OCH2). 13C NMR (DMSO-d6): 18.1 (2,6-CH3); 35.4 (4-
CH); 50.5 (OCH3); 101.7 (3,5-C); 145.3 (2,6-C); 167.6 (COO);
155.0, 136.1*, 130.4, 127.5, 122.0, 113.5* (Ph); 58. 9 (OCH2);
113.0* (C3); 135.3* (C4); 107.7 (CN). Anal. (C21H20N4O7) C, H,
N.
Dim eth yl 1,4-dih ydr o-2,6-dim eth yl-4-[2-[(3-m eth ylfu r ox-
an -4-yl)m eth oxy]ph en yl]-3,5-pyr idin edicar boxylate (26b):
mp 168-169 °C (CHCl3/petroleum ether, 60%). 1H NMR
(DMSO-d6): 8.71 (s, 1H, -NH); 3.44 (s, 3H, -OCH3), 5.18 (s,
1H, C(4)-H); 2.16 (s, 3H, 2,6-CH3); 6.9-7.2 (m, 4H, Ph); 5.21
(s, 2H, -OCH2); 2.25 (s, 3H, -CH3). 13C NMR (DMSO-d6): 18.1
(2,6-CH3); 34.5 (4-CH); 61.0 (OCH3); 100.9 (3,5-C); 145.2 (2,6-
C); 167.7 (COO); 156.0, 135.8, 129.8, 127.5, 121.5, 112.6§ (Ph);
61.0 (OCH2);113.5 (C3);154.7 (C4);7.5 (CH3). Anal. (C21H23N3O7)
C, N, H.
Dim eth yl 1,4-d ih yd r o-2,6-d im eth yl-4-[2-[(4-ca r ba m oyl-
fu r oxa n -3-yl)m et h oxy]p h en yl]-3,5-p yr id in ed ica r b oxy-
la te (27a ): mp 167-168 °C (EtOAc/petroleum ether, 85%). 1H
NMR (DMSO-d6): 8.48 (s, 1H, -NH); 3.44 (s, 3H, -OCH3),
5.04 (s, 1H, C(4)-H); 2.11 (s, 3H, 2,6-CH3); 6.9-7.1 (m, 4H,
Ph); 5.10 (s, 2H, -OCH2); 8.59, 8.30 (2s, 2H, CONH2). 13C NMR
(DMSO-d6): 18.0 (2,6-CH3); 36.0 (4-CH); 50.3 (OCH3); 101.2
(3,5-C); 145.6 (2,6-C); 167.6 (COO); 155.8, 135.1, 130.7, 127.4,
120.4, 112.4* (Ph); 57.9 (OCH2); 112.1* (C3); 151.8 (C4); 158.2
(CONH2). Anal. (C21H22N4O8) C, N, H.
Dim eth yl 1,4-d ih yd r o-2,6-d im eth yl-4-[2-[(3-ca r ba m oyl-
fu r oxa n -4-yl)m et h oxy]p h en yl]-3,5-p yr id in ed ica r b oxy-
la te (27b): mp 161-163 °C (EtOAc/petroleum ether, 85%). 1H
NMR (DMSO-d6): 8.64 (s, 1H, -NH); 3.46 (s, 3H, -OCH3),
5.15 (s, 1H, C(4)-H); 2.14 (s, 3H, 2,6-CH3); 6.8-7.1 (m, 4H,
Ph); 5.31 (s, 2H, -OCH2); 8.51, 7.82 (2s, 2H, CONH2). 13C NMR
(DMSO-d6): 18.0 (2,6-CH3); 35.1 (4-CH); 50.4 (OCH3); 101.7
(3,5-C); 145.5 (2,6-C); 167.7 (COO); 155.8*, 136.2, 130.3, 127.4,
121.2, 113.0§ (Ph); 61.7 (OCH2); 110.5§ (C3); 155.5* (C4); 155.9*
(CH3). Anal. (C21H22N4O8) C, N, H.
Gen er a l Meth od of P r ep a r a tion of 1,4-DHP s 24-27. A
solution of the appropriate furoxan 1,4-DHPs 24a -27a (2
mmol) in trimethyl phosphite (15 mL) was refluxed for 9 h.
Then the reaction mixture, cooled at room temperature, was
poured into 2 N HCl (150 mL). The resulting precipitate was
filtered off, washed several times with water, and dried.
Dim eth yl 1,4-dih ydr o-2,6-dim eth yl-4-[3-[(4-m eth ylfu r a-
zan -3-yl)m eth oxy]ph en yl]-3,5-pyr idin edicar boxylate (24):
mp 155-156 °C (CHCl3/petroleum ether, 58%). 1H NMR
(DMSO-d6): 8.93 (s, 1H, -NH); 3.56 (s, 3H, -OCH3); 4.88 (s,
1H, C(4)-H); 2.27 (s, 3H, 2,6-CH3); 6.7-7.2 (m, 4H, Ph); 5.34
(s, 2H, -OCH2); 2.42 (s, 3H, -CH3). 13C NMR (DMSO-d6): 18.3
(2,6-CH3); 38.6 (4-CH); 50.8 (OCH3); 101.3 (3,5-C); 146.0 (2,6-
C); 167.4 (COO); 157.4, 149.7, 129.4, 120.6, 114.0, 112.2 (Ph);
58. 9 (OCH2); 152.0* (C3); 151.9* (C4); 8.0 (CH3). Anal.
(C21H23N3O6) C, H, N.
Dim eth yl 1,4-d ih yd r o-2,6-d im eth yl-4-[3-[(4-ca r ba m oyl-
fu r a za n -3-yl)m et h oxy]p h en yl]-3,5-p yr id in ed ica r b oxy-
1
la te (25): mp 202-203 °C (EtOAc/petroleum ether, 90%). H
NMR (DMSO-d6): 8.91 (s, 1H, -NH); 3.57 (s, 3H, -OCH3),
4.88 (s, 1H, C(4)-H); 2.27 (s, 3H, 2,6-CH3); 6.7-7.2 (m, 4H,
Ph); 5.46 (s, 2H, -OCH2); 8.58, 8.22 (2s, 2H, CONH2). 13C NMR
(DMSO-d6): 18.4 (2,6-CH3); 38.6 (4-CH); 50.8 (OCH3); 101.4
(3,5-C); 146.1 (2,6-C); 167.5 (COO); 157.5, 149.6, 129.4, 120.4,
113.7, 112.0 (Ph); 59.5 (OCH2); 148.9 (C3); 152.5 (C4); 158.4
(CONH2). Anal. (C21H22N4O7) C, N, H.
Dim eth yl 1,4-dih ydr o-2,6-dim eth yl-4-[2-[(4-m eth ylfu r a-
zan -3-yl)m eth oxy]ph en yl]-3,5-pyr idin edicar boxylate (26):
mp 153-154 °C (CHCl3/petroleum ether, 70%). 1H NMR
(DMSO-d6): 8.64 (s, 1H, -NH); 3.40 (s, 3H, -OCH3); 5.20 (s,
1H, C(4)-H); 2.16 (s, 3H, 2,6-CH3); 6.9-7.1 (m, 4H, Ph); 5.32
(s, 2H, -OCH2); 2.51 (s, 3H, -CH3). 13C NMR (DMSO-d6): 18.0
(2,6-CH3); 34.0 (4-CH); 50.4 (OCH3); 101.1 (3,5-C); 145.1 (2,6-
C); 167.7 (COO); 154.6, 135.9, 129.6, 127.4, 121.4, 112.4 (Ph);
59. 0 (OCH2); 152.1* (C3); 155.7 (C4); 8.0 (CH3). Anal.
(C21H23N3O6) C, N, H.
Dim eth yl 1,4-dih ydr o-2,6-dim eth yl-4-[2-[(3-cyan ofu r ox-
an -4-yl)m eth oxy]ph en yl]-3,5-pyr idin edicar boxylate (29b):
mp 149-151 °C (EtOAc/petroleum ether, 50%). 1H NMR
(DMSO-d6): 8.74 (s, 1H, -NH); 3.47 (s, 3H, -OCH3), 5.18 (s,
1H, C(4)-H); 2.17 (s, 3H, 2,6-CH3); 6.9-7.2 (m, 4H, Ph); 5.31
(s, 2H, -OCH2). 13C NMR (DMSO-d6): 18.1 (2,6-CH3); 35.2 (4-

1,4-Dihydropyridines Conjugated to Furoxanyl Moieties
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 27 5401
(2) The Pharmacological Basis of Therapeutics, 9th ed.; Hardman,
J . G., Limbird, L. E., Molinoff, P. B., Ruddon, R. W., Gilman, A.
G., Eds.; McGraw-Hill: New York, 1996.
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Class of Compounds in Medicinal Chemistry. Farmaco 1996, 51,
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CH); 50.5 (OCH3); 101.8 (3,5-C); 146.3 (2,6-C); 167.6 (COO);
155.1*, 136.4, 130.4, 127.4, 122.0, 113.8 (Ph); 61.2 (OCH2); 98.4
(C3); 154.6* (C4); 106.1 (CN). Anal. (C21H20N4O7) C, H, N.
P h a r m a cology. Va soa ctivity Deter m in a tion . Thoracic
aortae were isolated from male Wistar rats weighing 180-
200 g. The vessels were helically cut, and three strips were
obtained from each aorta. The tissues were mounted under
0.7 g tension in organ baths containing 30 mL of Krebs
bicarbonate buffer (NaCl, 111.2; KCl, 5.0; CaCl2, 2.5; MgSO4,
1.2; KH2PO4, 1.0; NaHCO3, 12.0; glucose, 11.1 mM) at 37 °C
and gassed with 95% O2-5% CO2 (pH ) 7.4).
The aortic strips were allowed to equilibrate for 1 h and
then were depolarized by addition of a solution of KCl to a
final K+ concentration of 50 mM. The preparations were then
extensively washed with Krebs bicarbonate buffer, and a
second contraction was evoked by K+ depolarization (50 mM).
When the contraction plateaued, cumulative concentrations
of the vasodilator agents were added every 45 min, according
to Christiaans.26 Development of antagonism occurred so
slowly that increasing doses had to be given at established
times, without waiting for a complete equilibrium to be
reached.
(5) Christiaans, J . A. M.; Timmerman, H. Cardiovascular hybrid
drugs: combination of more than one pharmacological property
in one single molecule. Eur. J . Pharmacol. Sci. 1996, 4, 1-22.
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eration of Nitric Oxide Accounts for the Vasodilator Action of
Furoxans. Biochem. Pharmacol. 1992, 44, 1149-1157.
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drugs. J . Med. Chem. 1997, 40, 2288, 463-469 and references
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Cardiovascular actions of the furoxan CAS 1609, a novel nitric
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System as a Useful Tool for Balancing “Hybrids” with Mixed
R1-Antagonist and NO-like Vasodilator Activities. J . Med. Chem.
1995, 38, 4944-4949.
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A. Studies on Agents with Mixed NO-Dependent Vasodilator and
â-Blocking Activities. Pharm. Res. 1997, 14, 1750-1758.
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The effects of 10 µM MB and 1 µM ODQ on relaxation were
evaluated in a separate series of experiments by adding the
sGC inhibitors 5 min before K+ depolarization.
Recep tor Bin d in g Assa y. Cerebral cortices, isolated from
male Wistar rats (180-200 g), were homogenized in 20
volumes (w/v) of 50 mM Tris-HCl buffer (pH ) 7.4 at 4 °C) in
an Ultra Turrax homogenizer. The homogenate was centrifu-
gated at 43000g for 10 min, and the pellet was resuspended
in the buffer. This process was repeated a further two times.
The final pellet was stored at -80 °C until required. Binding
experiments were performed avoiding exposure to light,
because of the photolability of [3H ]nitrendipine.
All binding assays were carried out according to Chris-
tiaans26 by adding 200 µL of 50 mM Tris buffer (pH ) 7.4),
100 µL of rat brain membrane suspension (170 µg of protein/
mL), 100 µL of [3H]nitrendipine solution (1 nM), and 100 µL
of the drug concentration to each incubation tube for a final
volume of 0.5 mL. Triplicate tubes were used for each condi-
tion. Specific binding was defined as the difference between
total binding (measured in the absence of any added ligand)
and nonspecific binding (determined in the presence of 1 µM
nifedipine). Reaction tubes were incubated for 60 min at 37
°C, then diluted with 4 mL of ice-cold Tris buffer (50 mM, pH
) 7.4), and filtered under reduced pressure through Whatman
GF/C glass fiber filters, treated with a 0.1% poly(ethylenimine)
solution. Tubes and filters were washed two additional times
with ice-cold buffer. The amount of radioactivity retained on
the filters was quantitated by liquid scintillation counting,
using a Beckman liquid scintillation spectrophotometer.
Saturation experiments were performed by incubating
increasing concentrations of [3H]nitrendipine from 0.03 up to
2 nM with 50 µL of rat cortical membranes (170 mg of protein/
mL) and with 50 mM Tris-HCl buffer (pH ) 7.4) at 37 °C for
a total volume of 0.250 mL. Nonspecific binding was deter-
mined in the presence of 1 µM nifedipine. Equilibrium dis-
sociation constant (KD) of 0.83 ( 0.16 nM and the maximal
binding (Bmax) of 245 ( 70 fmol/mg of protein of [3H]nitren-
dipine and IC50 values and Ki values of all the compounds
tested were calculated with the nonlinear fitting program
INPLOT 4.0.
(19) CCPK program v.1.10; we are indebted to Dr. Ryo Shimizu for
supplying the program.
(20) CLogP for Windows, v.1.0; BioByte Corp.
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ancarbaldehydes. J . Heterocycl. Chem. 1989, 26, 1345-1347.
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Furoxans. Part 15. Bromination of Dimethylfuroxan and Related
Compounds with NBS. J . Heterocycl. Chem. 1995, 32, 811-813.
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wa, K.; Takenaka, T.; Takahashi, K.; Murakami, M. Synthesis
of New water-soluble Dihydropyridine Vasodilators. Chem.
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Synthesis and Structure of Some Asymmetrically Substituted
Furoxans. J . Heterocycl. Chem. 1972, 9, 577-580.
(25) Blinnikov, A. N.; Kulikov, A. S.; Makhova, N. N.; Khmelnitski,
L. I. R-Hydroxyalkyl(benzyl)furazans and R-Hydroxyalkyl(ben-
zyl)furoxans. Synthesis and Reactivity. Russ. Chem. Bull. 1996,
45, 1692-1698.
Ack n ow led gm en t. This work was supported by a
MURST grant.
(26) Christiaans, J . A. M.; Windhorst, A. D.; Groenenberg, P. M.; van
der Goot, H.; Timmerman, H. Synthesis and in vitro pharmacol-
ogy of new 1,4-dihydropyridines. 1. 2-(ω-Aminoalkylthiomethyl)-
1,4-dihydropyridines as potent calcium channel blockers. Eur.
J . Med. Chem. 1993, 28, 859-867.
Refer en ces
(1) Triggle, D. J .; Langs, D. A.; J anis, R. A. Ca2+ Channel Ligands:
Structure-Function Relationships of The 1,4-Dihydropyridines.
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J M9803267