Studies on agents with mixed NO-dependent and calcium channel antagonistic vasodilating activities

Article abstract of DOI:10.1023/A:1011072116210

Purpose. To obtain new cardiovascular agents with mixed Ca²⁺channel antagonistic and NO-donor properties, a series of "hybrid" 1,4-dihydropyridines (1,4-DHPs), bearing NO-donating furoxan moieties on the 3-positioned lateral ester chain were synthesized and pharmacologically characterized. Furazan analogues were also prepared and investigated for control purposes, because they are unable to release NO. Methods. Synthesis of the models was achieved by a modified Hantzsch approach. All of the final furoxan 1,4-DHPs were assessed for their ability to produce nitrite in the presence of a large excess of cysteine by the Griess reaction. Vasodilating activity was evaluated on rat aorta and expressed as EC₅₀ and EC₅₀^MB values, obtained in the absence and in the presence of methylene blue (MB) respectively, a well-known guanylate cyclase inhibitor. Affinities to 1,4-DHP receptor on Ca²⁺-channels, expressed as IC₅₀ values, were determined through displacement experiments of [³H]-nitrendipine on rat cortex homogenates. Results. Some hybrid compounds (derivatives 15a, 15b, 16a, and 16b) displayed vasodilating activity depending predominantly on their Ca²⁺-channel blocker properties. By contrast, some others (derivatives 17a, 17b, and 21) behaved as well-balanced hybrids with mixed Ca²⁺-channel blocking and NO-dependent vasodilating activities. Conclusion. This work demonstrates the possibility of obtaining well-balanced hybrids endowed with mixed NO-donor and Ca²⁺-channel blocker properties using appropriate 1,4-DHP and furoxan moieties. A procedure for the individual evaluation of the NO-dependent vasodilator component and that due to Ca²⁺-channel blocking is proposed.

Full text of DOI:10.1023/A:1011072116210

Pharmaceutical Research, Vol. 18, No. 2, 2001
Research Paper
ide-like vasodilator activities. These compounds, which are
structurally related to Nifedipine 1 and to its meta-analogue 2
(Fig. 1), are characterized by having furoxan substructures
endowed with different NO-donor properties at the ortho and
meta position of the 4-phenyl ring (Fig. 1, derivatives 3a–8a
and 3b–8b). A number of these compounds behaved as well-
balanced hybrids, because they were able to display NO-
dependent and Ca2+-antagonist dependent vasodilating prop-
erties in the same range of concentration. In particular, 1,4-
DHPs bearing appropriately substituted furoxans at ortho
position were as potent as the references 1 and 2 (5).
In this paper, we report the preparation and the phar-
macological characterization of a series of 4-(3-nitrophenyl)-
1,4-dihydropyridines analogues of 1, having furoxan moieties
on the 3-positioned lateral ester chain (Fig. 2, derivatives 15a,
15b; 16a, 16b; 17a, 17b; and 21). Furoxan substituents were
appropriately chosen, in order to modulate NO-release of the
final products. These compounds should display differences
over other known NO-donor 1,4-DHPs bearing nitrate
groups in their ester side chain (7,8). In fact the presence of
suitable substituted furoxan moieties gives rise to a series of
compounds widely modulated in their NO-release properties.
In addition, these products could lack significant tolerance
development (9,10). In this paper, related furazan derivatives
(Fig. 2, derivatives 15, 16, 17, and 22) were also considered for
control purposes, because they are unable to release NO.
Nifedipine 1 and its meta-nitro analogue 2 were used as ref-
erences.
Studies on Agents with Mixed
NO-Dependent and Calcium Channel
Antagonistic Vasodilating Activities
Clara Cena,1 Sonja Visentin,1 Antonella Di Stilo,1
Donatella Boschi,2 Roberta Fruttero,1 and
Alberto Gasco1,3
Received September 30, 2000; accepted November 8, 2000
Purpose. To obtain new cardiovascular agents with mixed Ca2+
-
channel antagonistic and NO-donor properties, a series of “hybrid”
1,4-dihydropyridines (1,4-DHPs), bearing NO-donating furoxan moi-
eties on the 3-positioned lateral ester chain were synthesized and
pharmacologically characterized. Furazan analogues were also pre-
pared and investigated for control purposes, because they are unable
to release NO.4
Methods. Synthesis of the models was achieved by a modified Hantz-
sch approach. All of the final furoxan 1,4-DHPs were assessed for
their ability to produce nitrite in the presence of a large excess of
cysteine by the Griess reaction. Vasodilating activity was evaluated
MB
on rat aorta and expressed as EC50 and EC50
values, obtained in
the absence and in the presence of methylene blue (MB) respectively,
a well-known guanylate cyclase inhibitor. Affinities to 1,4-DHP re-
ceptor on Ca2+-channels, expressed as IC50 values, were determined
through displacement experiments of [3H]-nitrendipine on rat cortex
homogenates.
Results. Some hybrid compounds (derivatives 15a, 15b, 16a, and 16b)
displayed vasodilating activity depending predominantly on their
Ca2+-channel blocker properties. By contrast, some others (deriva-
tives 17a, 17b, and 21) behaved as well-balanced hybrids with mixed
Ca2+-channel blocking and NO-dependent vasodilating activities.
Conclusion. This work demonstrates the possibility of obtaining well-
balanced hybrids endowed with mixed NO-donor and Ca2+-channel
blocker properties using appropriate 1,4-DHP and furoxan moieties.
A procedure for the individual evaluation of the NO-dependent va-
sodilator component and that due to Ca2+-channel blocking is pro-
posed.
MATERIALS AND METHODS
Chemistry
Synthesis
Melting points were measured on a Bu¨chi 530 capillary
apparatus and are uncorrected. Melting points with decom-
position were determined after introducing the sample into
the bath at a temperature 10°C lower than the melting point.
A heating rate of 3°C min−1 was used. The compounds were
routinely checked by infrared spectroscopy (Shimadzu FT-IR
8101M, Shimadzu Italia, Milan, Italy) and mass spectroscopy
(Finnigan-Mat TSQ-700 spectrometer, Finnigan, Milan,
Italy), and 1H and 13C nuclear magnetic resonance at 200 and
50 MHz, respectively (Bruker AC-200 spectrometer, Bruker,
Milan, Italy). All of the spectra were in keeping with the
proposed structures. Column chromatography was performed
on silica gel (Merck Kieselgel 60, 230–400 mesh ASTM,
Merck, Milan, Italy) with the indicated solvent system. An-
hydrous magnesium sulfate was used as the drying agent of
the organic extracts. Petroleum ether (PE) 40–60°C was used.
Solvent removal was achieved under reduced pressure at
room temperature. Elemental analyses of the new com-
pounds were performed by REDOX (Cologno Monzese,
Italy) and the results are within 0.4% of theoretical values.
Intermediates 9a (11), 9b (5), and 18 (12) were synthesized
according to procedures described in the literature. Deriva-
tives 10a and 10b were a gift from Dr. K. Scho¨nafinger,
Aventis Pharma (Frankfurt am Main, Germany). Compounds
11, 13, and 14 are commercial reagents (Aldrich Chemical
Co., Milwaukee, WI, USA).
KEY WORDS: NO donors; 1,4-dihydropyridines; Ca2+-channel
blockers; hybrid drugs; furoxans; vasodilation.
INTRODUCTION
Medicinal chemical hybridization is a well-known ap-
proach to drug design. It involves the combination of two
complementary biological activities by joining appropriate
pharmacophoric groups directly or via spacers (1,2). We have
designed a number of drugs by combining suitable pharma-
cophoric groups with different NO-donor moieties (3–6).
Among these, we also realized new 1,4-dihydropyridines
(DHPs) with mixed calcium channel antagonist and nitric ox-
1 Dipartimento di Scienza e Tecnologia del Farmaco, via P. Giuria 9,
I-10125, Torino, Italy.
2 Dipartimento di Scienze Chimiche, Alimentari, Farmaceutiche, Far-
macologiche, V.le Ferrucci 33, I-28100, Novara, Italy.
3 To whom correspondence should be addressed. (e-mail: gasco@
pharm.unito.it)
4 In this paper, the term nitric oxide (NO) is used as a family name.
When necessary in the discussion, we specify the particular redox
form to which we refer.
3-(3-(Benzenesulfonyl)furoxan-4-yloxy)propanol,
19. A 50% w/w NaOH aqueous solution (2.19 g, 27.3 mmol),
157
0724-8741/01/0200-0157$19.50/0 © 2001 Plenum Publishing Corporation

158
Cena et al.
Fig. 1. Nitrosubstituted 1,4-DHPs and related furazan and furoxan derivatives.
was slowly added at room temperature to a stirred solution of
General Procedure for the Synthesis of Acetoacetate 11a,
18 (10.9 g, 27.3 mmol) and 1,3-propandiol (20.5 ml, 273 mmol) 11b, 12a, 12b, and 20. A solution of the appropriate fu-
in tetrahydrofuran (THF) (80 ml). The reaction mixture was roxanmethanol derivatives (9a, 9b, 10a, 10b, and 19) and
stirred for 30 min and then an additional amount (1.09 g) of 2,2,6-trimethyl-4H-1,3-dioxin-4-one (11) in toluene (20 ml)
50% NaOH was added (13.7 mmol). The solution was con- was refluxed. Solvent removal gave a residue that was puri-
centrated under vacuum at room temperature. The residue fied by flash chromatography. Reagent ratio, reaction times,
was treated with EtOAc and the resulting mixture was chromatography eluents, yields, melting points, and crystalli-
washed twice with water. Aqueous phases were extracted zation solvents were as follows.
with EtOAc. The combined organic layers were dried, and
11a: Reagent ratio 1:2, reaction time 10 min, eluent PE
evaporated under vacuum at room temperature to give a resi- 7/EtOAc 3, yield 42%, oil. 1H-NMR (DMSO-d6): 2.18 (s, 3H,
due that was purified by flash chromatography (PE 6/EtOAc COCH3); 2.40 (s, 3H, CH3); 3.72 (s, 2H, COCH2CO); 5.08 (s,
4, yield 69%, mp 114°C (isopropyl ether).
2H, CH2O). 13C-NMR (DMSO-d6): 7.37 (CH3); 30.0
1H-NMR (CDCl3): 1.82 (t, 1H, CH2OH), 2.15 (qi, 2H, (COCH3); 49.1 (COCH2CO); 53.8 (CH2O); 113.3 (C3-
CH2CH2OH), 3.89 (q, 2H, CH2CH2OH), 4.59 (t, 2H, Furox.); 155.7 (C4-Furox.); 170.5 (COO); 201.3 (COCH3).
OCH2CH2), 6.1–7.6 (m, 5H, C6H5). 13C-NMR (CDCl3): 31.20 Anal. (C8H10N2O5) C, H, N.
(CH2CH2OH), 59.29 (CH2CH2OH), 69.19 (OCH2CH2),
110.35 (C3-Furox.), 158.79 (C4-Furox.) 128.43, 129.57, 135.56, 7/EtOAc 3, yield 60%, oil. 1H-NMR (DMSO-d6): 2.16 (s, 3H,
137.84 (C6H5). Anal. (C11H12N2O6S) C, H, N. COCH3); 2.20 (s, 3H, CH3); 3.76 (s, 2H, COCH2CO); 5.29 (s,
11b: Reagent ratio 1:2, reaction time 2 h, eluent PE

Agents with Mixed Vasodilating Activities
159
Fig. 2. Synthesis of furazan and furoxan 1,4-DHPs.
2H, CH2O). 13C-NMR (DMSO-d6): 7.47 (CH3); 30.1 CH2CH2CH2); 2.19 (s, 3H, CH3); 3.65 (s, 2H, COCH2CO);
(COCH3); 49.2 (COCH2CO); 59.9 (CH2O); 113.4 (C3- 4.21 (t, 2H, COOCH2); 4.48 (t, 2H, CH2O); 7.72–8.05 (m, 5H,
Furox.); 154.8 (C4-Furox.); 167.0 (COO); 201.4 (COCH3). C6H5). 13C-NMR (DMSO-d6): 27.4 (CH2CH2CH2); 30.2
Anal. (C8H10N2O5) C, H, N.
(COCH3); 49.6; (COCH2CO); 60.8 (COOCH2); 68.2 (CH2O);
12a: Reagent ratio 1:1, reaction time 4 h, yield 60%, mp 110.3 (C3-Furox.); 158.9 (C4-Furox.); 128.4, 130.1, 136.3,
73–74°C (EtOAc/PE). 1H-NMR (DMSO-d6): 2.18 (s, 3H, 137.2 (C6H5); 167.4 (COO); 201.8 (COCH3). Anal.
CH3); 3.67 (s, 2H, COCH2CO); 5.23 (s, 2H, CH2O); 8.26, 8.60 (C15H16N2O8S) C, H, N.
(2s, 2H, CONH2). 13C-NMR (DMSO-d6): 30.0 (COCH3);
General Procedure for the Synthesis of Furoxan 1,4-
49.1 (COCH2CO); 54.0 (CH2O); 111.7 (C3-Furox.); 151.6 DHPs 15a, 15b, 16a, 16b, and 21. A solution of the appro-
(C4-Furox.); 158.1 (CONH2); 166.8 (COO); 201.1 (COCH3). priate acetoacetate (11a, 11b, 12a, 12b, and 20) (1 mmol),
Anal. (C8H9N3O6) C, H, N.
3-nitrobenzaldehyde (14) (0.15 g, 1 mmol) and methyl 3-ami-
12b: Reagent ratio 1:1, reaction time 4 h, yield 68%, mp nocrotonate (13) (0.12 g, 1 mmol) in 2-propanol (20 ml) was
96–97°C (EtOAc/PE). 1H-NMR (DMSO-d6): 2.22 (s, 3H, refluxed for 8 h. Solvent removal gave a residue, which was
CH3); 3.73 (s, 2H, COCH2CO); 5.42 (s, 2H, CH2O); 7.81, 8.51 purified by flash chromatography (eluent PE 7/EtOAc 3).
(2s, 2H, CONH2). 13C-NMR (DMSO-d6): 30.1 (COCH3); Yields, melting points, crystallization solvents, 1H-NMR, and
49.2 (COCH2CO); 57.3 (CH2O); 110.3 (C3-Furox.); 154.5 chemical shifts of the products were as follows (13C-NMR
(C4-Furox.); 155.7 (CONH2); 166.6 (COO); 201.0 (COCH3). data are reported in Table I).
Anal. (C8H9N3O6) C, H, N.
15a: 66%, mp 153–154°C softens 150°C (CHCl3/PE). 1H-
20: Reagent ratio 1:1, reaction time 2 h, yield 60%, mp NMR (DMSO-d6): 2.20 (s, 3H, –CH3); 2.29/2.30 (2s, 6H, C2–,
1
87–88,5°C (EtOAc /PE). H-NMR (DMSO-d6): 2.11 (m, 2H, C6–CH3); 3.56 (s, 3H, –OCH3); 4.86–5.09 (q, 2H, –OCH2);

160
Cena et al.
Fig. 2. Continued.
4.94 (s, 1H, C4–H); 7.48–8.03 (m, 4H, Ph); 9.22 (s, 1H, NH). resulting precipitate was filtered off, washed several times
Anal. (C20H20N4O8) C, H, N.
with water and dried. Yields, melting points, crystallization
15b: 61%, mp 163–164°C (CHCl3/PE). 1H-NMR solvents and 1H -NMR chemical shifts of the products were as
(DMSO-d6): 1.92 (s, 3H, –CH3); 2.30/2.34 (2s, 6H, C2–, C6– follows (13C-NMR data are reported in Table 1):
CH3); 3.58 (s, 3H, –OCH3); 4.99 (s, 1H, C4–H); 5.06–5.31 (q,
2H, –OCH2); 7.51–8.03 (m, 4H, Ph); 9.24 (s, 1H, NH). Anal. NMR (DMSO-d6): 2.18 (s, 3H, –CH3); 2.30/2.34 (2s, 6H, C2–,
15: 70%, mp 145–146°C, softens 143°C (CHCl3/PE). 1H-
(C20H20N4O8) C, H, N.
C6–CH3); 3.56 (s, 3H, –OCH3); 4.98 (s, 1H, C4–H); 5.17–5.36
16a: 55%, mp 191–192°C (CHCl3/PE). 1H-NMR (q, 2H, –OCH2); 7.47–8.05 (m, 4H, Ph); 9.23 (s, 1H, NH).
(DMSO-d6): 2.28/2.29 (2s, 6H, C2–, C6–CH3); 3.57 (s, 3H, Anal. (C20H20N4O7) C, H, N.
–OCH3); 4.89 (s, 1H, C4–H); 5.11–5.33 (q, 2H, –OCH2); 7.49–
8.01 (m, 4H, Ph); 8.22/8.57 (2s, 2H, CONH2); 9.19 (s, 1H, d6): 2.32 (s, 6H, C2–, C6–CH3); 3.56 (s, 3H, –OCH3); 5.01 (s,
16: 90%, mp 182–184°C (CHCl3/PE). 1H-NMR (DMSO-
NH). Anal. (C20H19N5O9 и 0.5 H2O) C, H, N.
1H, C4-H); 5.42–5.43 (q, 2H, –OCH2); 7.52–8.02 (m, 4H, Ph);
16b: 74%, mp 223–224°C, softens 220°C (CHCl3/PE). 1H- 8.20, 8.58 (2s, 2H, CONH2); 9.22 (s, 1H, NH). Anal.
NMR (DMSO-d6): 2.32/2.33 (2s, 6H, C2–, C6–CH3); 3.56 (s, (C20H19N5O8) C, H, N.
1
3H, –OCH3); 5.01 (s, 1H, C4–H); 5.24-5.41 (q, 2H, –OCH2);
22: 50% , mp 144–145°C, softens at 140°C (EtOH). H-
7.52–7.98 (m, 4H, Ph); 7.77/8.46 (2s, 2H, CONH2); 9.21 (s, 1H, NMR (DMSO-d6): 2.0–2.06 (m, 2H, CH2CH2CH2); 2.29/2.31
NH). Anal. (C20H19N5O9) C, H, N. (2s, 6H, C2–, C6–CH3); 3.57 (s, 3H, –OCH3); 3.98–4.08 (m,
1
21: 50%, mp 166–167°C (EtOH). H-NMR (DMSO-d6): 2H, COOCH2); 4.25 (t, 2H, CH2O ); 4.96 (s, 1H, C4-H); 7.47-
2.01–2.1 (m, 2H, CH2CH2CH2); 2.29/2.33 (2s, 6H, C2–, C6– 8.08 (m, 9H, C6H5/C6H4–DHP); 9.11 (s, 1H, NH). Anal.
CH3); 3.56 (s, 3H, –OCH3); 4.03–4.19 (m, 2H, COOCH2); (C27H26N4O10S и 0.25 H2O) C, H, N.
4.28 (t, 2H, CH2O ); 4.98 (s, 1H, C4–H); 7.46–8.02 (m, 9H,
C6H5/C6H4–DHP); 9.11 (s, 1H, NH). Anal. (C27H26N4O11S) roxan 1,4-DHPs 17a, 17b, 17. Trifluoroacetic anhydride (3
C, H, N. mmol) was added dropwise to a stirred and ice-salt cooled
General Procedure for the Synthesis of Furazan and Fu-
General Procedure for the Synthesis of Furazan 1,4-DHPs solution of the appropriate DHPs 16a, 16b, and 16 (1 mmol)
15, 16, 22. A solution of the appropriate furoxan 1,4-DHPs in dry pyridine (5 ml). The cooling bath was removed, and
(15a, 16a, and 21) (2 mmol) in trimethyl phosphite (15 ml) strirring was continued for 30 min at room temperature. The
was refluxed for 9 h. Then the reaction mixture, cooled at reaction mixture was poured into water and the solution,
room temperature, was poured into 2 N HCl (150 ml). The acidified with 2 N HCl, was extracted with EtOAc. The com-

Agents with Mixed Vasodilating Activities
161
Fig. 2. Continued.
bined organic layers were washed with 2 N HCl, dried and phate buffer (pH 7.4) at 37°C according to the procedure
evaporated to afford a residue that was purified by flash chro- previously described (13).
matography (PE 7/EtOAc 3). Yields, melting points, crystal-
1
lization solvents, and H -NMR chemical shifts of the prod-
ucts were as follows (13C-NMR data are reported in Table I).
17: 60%, mp 145–146°C (CHCl3/PE). 1H NMR (DMSO-
d6): 2.31/2.35 (2s, 6H, C2–, C6–CH3); 3.56 (s, 3H, –OCH3);
5.02 (s, 1H, C4–H); 5.37–5.53 (q, 2H, –OCH2); 7.48–8.03 (m,
4H, Ph); 9.29 (s, 1H, NH). Anal. (C20H17N5O7) C, H, N.
17a: 57%, mp 161–162°C (CHCl3/PE). 1H-NMR
(DMSO-d6): 2.30/2.32 (2s, 6H, C2–, C6–CH3); 3.56 (s, 3H,
–OCH3); 4.98 (s, 1H, C4–H); 5.05–5.16 (q, 2H, –OCH2);
7.57–8.05 (m, 4H, Ph); 9.27 (s, 1H, NH). Anal. (C20H17N5O8)
C, H, N.
Pharmacology
All experiments were done in accordance with the Prin-
ciples of Laboratory Animal Care (National Institutes of
Health Publication No. 85-23, revised 1985).
Vasoactivity Determination
Thoracic aortae were isolated from male Wistar rats
weighing 180–200 g. The vessels were helically cut, the endo-
thelium removed, 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 of the
following composition (mM): NaCl 111.2, KCl 5.0, CaCl2 2.5,
MgSO4 1.2, KH2PO4 1.0, NaHCO3 12.0, glucose 11.1, main-
tained at 37°C and gassed with 95% O2–5% CO2 (pH 7.4).
The aortic strips were allowed to equilibrate for 1 h and
were then depolarized by addition of a solution of KCl to a
final K+ concentration 50 mM. The preparations were then
17b: 67%, mp 107–108°C (CHCl3/PE). 1H-NMR
(DMSO-d6): 2.37/2.39 (2s, 6H, C2–, C6–CH3); 3.60 (s, 3H,
–OCH3); 5.17 (s, 1H, C4–H); 5.39 (s, 2H, –OCH2); 7.5–8.0 (m,
4H, Ph); 8.36 (s, 1H, NH). Anal. (C20H17N5O8) C, H, N.
Detection of Nitrite
Nitrite production by furoxan DHPs was assessed in the extensively washed with Krebs-bicarbonate buffer and a sec-
presence of a large excess of cysteine (1:50) in 50 mM phos- ond contraction was evoked by K+-depolarization (50 mM).

162
Cena et al.

Agents with Mixed Vasodilating Activities
163
When the contraction plateaued, cumulative concentrations counting, using a Beckman liquid scintillation spectropho-
of the vasodilator agents were added every 45 min, according tometer.
to Christiaans (2). Development of antagonism occurred so
Saturation experiments were performed by incubating
slowly that increasing doses had to be given at established increasing concentrations of [3H]-nitrendipine from 0.03 nM
times, without waiting for a complete equilibrium to be up to 2 nM with 50 ␮l of rat cortical membranes (170 mg
reached.
protein ml−1) and with Tris-HCl buffer 50 mM (pH 7.4) at
The effect of methylene blue (MB) 10 ␮M on relaxation 37°C for a total volume of 0.250 ml. Nonspecific binding was
was evaluated in a separate series of experiments by adding determined in the presence of 1 ␮M nifedipine. Equilibrium
this compound 5 min before K+-depolarization. All experi- dissociation constant (KD) of 0.83 0.16 nM and the maximal
ments were performed avoiding exposure to light, because of binding (Bmax) of 245
70 fmol mg−1 protein of [3H]-
the photolability of the 1,4-DHPs. The experimental room nitrendipine, and IC50 values (the concentration of each com-
was darkened and the organ bath system was shielded from pound able to display 50% of [3H]-nitrendipine binding) of all
light.
the compounds tested were calculated with the nonlinear fit-
ting program INPLOT 4.0.
Receptor Binding Assay
RESULTS AND DISCUSSION
Cerebral cortices, isolated from male Wistar rats (180–
200 g), were homogenized in 20 volumes (w/v) of 50 mM
Synthesis of furoxan 1,4-DHPs containing methyl (Fig.
Tris-HCl buffer (pH 7.4 at 4°C) in an Ultra Turrax Homog- 2A, derivatives 15a and 15b), carbamoyl (Fig. 2A, derivatives
enizer. The homogenate was centrifuged at 43,000 g for 10 16a and 16b), and phenylsulfonyl (Fig.2C, derivative 21) moi-
min and the pellet was resuspended in the buffer. This process eties required preliminary preparation of a series of acetoace-
was repeated an additional two times. The final pellet was tic esters. Intermediates 11a, 11b, 12a, and 12b were synthe-
stored at −80°C until required. As in the vasoactivity study, sized by treating the appropriate furoxanmethanol derivatives
the binding experiments were performed in a darkened room 9a, 9b, 10a, and 10b with 11, a synthon of acetylketene, in
and both incubation tubes and filtration device were shielded refluxing toluene. Acetoacetate 20 (Fig. 2C) was prepared by
from light.
the same method, starting from intermediate 19. During the
All binding assays were carried out according to Chris- reactions, no furoxan thermal isomerisation was observed.
tiaans (2), by adding 200 ␮l of Tris-buffer 50 mM (pH 7.4), Reaction of acetoacetates with ortho-nitrobenzaldehyde (14)
100 ␮l of rat brain membrane suspension (170 ␮g protein and ␤-aminocrotonate (13) in boiling isopropanol afforded
1
ml− ), 100 ␮l of [3H]-nitrendipine solution (1 nM), and 100 ␮l the expected 1,4-DHPs. Again, no furoxan isomerization was
of the drug concentration to each incubation tube for a final observed. Furazan DHPs 15, 16, and 22 were easily obtained
volume of 0.5 ml. Triplicate tubes were used for each condi- by reduction of the furoxan analogues 15a, 16a (Fig. 2B), and
tion. Specific binding was defined as the difference between 21 (Fig. 2C) in boiling trimethylphosphite. Finally, the cyano-
total binding (measured in the absence of any added ligand) substituted 1,4-DHPs 17, 17a, and 17b were prepared by de-
and nonspecific binding (determined in the presence of 1 ␮M hydration of the amide analogues dissolved in dry pyridine in
nifedipine). Reaction tubes were incubated for 60 min at the presence of trifluoroacetic anhydride (Fig. 2B). All of the
37°C, then diluted with 4 ml of ice-cold Tris-buffer (50 mM, final furoxan DHPs were assessed for their ability to produce
pH 7.4) and filtered under reduced pressure through What- nitrite (NO2−), in the presence of a large excess of cysteine
man GF/C glass fiber filters, treated with a 0.1% polyethyl- (Table II). Nitric oxide center dot (NOи), or nitroxyl anion
enimine solution. Tubes and filters were washed two addi- (NO−), or both, may be involved in thiol-induced NO-
tional times with ice-cold buffer. The amount of radioactivity donation by furoxans (14,15). These two redox species are
retained on the filters was quantified by liquid scintillation both able to activate soluble guanylate cyclase (sGC) (16).
Table II. Vasodilating Potencies (EC50, EC50MB), Radioligand Binding Affinities (IC50),
Interpolated Values from Regression Eq. (2) (EC50calcd), and NO Releasing Properties
(%NO−2) of 1,4-DPH Derivatives
MB
EC50 SE EC50
SE IC50 SE EC50calcd(CL 95%) %NO−2 SE
Compound
(nM)
(nM)
(nM)
(nM)
mol/mol
1
2
15
3.2 0.4
3.4 0.6
3.5 0.6
6.6 0.8
6.7 0.6
2.7 0.4
2.6 0.7
2.7 0.8
6.4 1.1
8.3 1.3
49 15
15a
15b
16
16a
16b
17
17a
17b
22
5.7 0.8
7.0 0.6
5.8 (4.6–7.2)
7.4 (5.9–9.3)
2.0 0.1
<0.5
50
82 12
64
8
83
64
6
2
81
1
68 (55–83)
59 (48–72)
17.4 0.1
12.0 0.2
4
71 19
2.2 0.5
28 10
1.4 0.3
6.7 0.8
4.8 0.5
0.48 0.07
8.6 1.0
21
22
3
3
24 (19–29)
26 (21–32)
40.6 0.5
35.8 0.5
30
0.65 0.27
17
9
21
16
2
6
15 (12–18)
26.8 1.3

164
Cena et al.
Under physiological conditions, the principal oxidation prod-
uct of NOи is NO2−. The reaction evolves through oxidating
and nitrosating NOx intermediates not fully characterized. By
contrast, the fate of nitroxyl anion NO− is more complex. It
can be converted to N2O, but its rapid reaction with O2 com-
petitively generates peroxy nitrite (−OONסO). In turn, this
last product partially decomposes to nitrite. Also oxidation of
NO− to NOи has been claimed. Another source of nitrite is the
slower reaction of NO− with NOи. In our experimental con-
ditions the whole picture is complicated by the presence of
thiols, which in turn can interact with the two NO-redox spe-
cies and their transient oxidation products. This complex pic-
ture is discussed in reference 14. In summary, nitrite detection
serves for inferring the previous presence of NO only in first
approximation.
Vasodilator activity of all 1,4-DHPs was evaluated on
K+-depolarized rat thoracic aorta strips. EC50 values (Table
2) were calculated from concentration-response curves. The
experiments for the NO-donor furoxan DHPs were repeated
in the presence of MB, a well-known inhibitor of soluble
guanylate cyclase (sGC). For the compounds 17a, 17b, and 21,
which are good producers of nitrite, a rightward shift of the
concentration response curves was observed. In Fig. 3, the
concentration response curves for compounds 17a and 21 are
reported. The potencies obtained in this new set of experi-
ments, expressed as EC50MB, are shown in Table 2. All DHP
derivatives were subjected to a competition study of inhibi-
tion of 3[H]-nitrendipine binding to cerebral cortices (see Ma-
terials and Methods). IC50 values (Table 2) were determined
from the competition curves. Analysis of the data reported in
the table shows that the introduction of the furazan moieties
into the 3-ester lateral chain of reference 2 gives rise to potent
Ca2+-channel antagonist vasodilators. In fact, all of these
compounds are as active as or more active than 2 and Nifed-
ipine 1. Only the carbamoyl analogue 16 is less potent and this
could be principally dependent on the low lipophilicity of the
CONH2 moiety. Furoxan analogues are less active vasodila-
tors than the corresponding furazans, but their potencies are
still comparable to the reference compounds. A problem that
arises with these compounds is individual evaluation of the
Fig. 3. Concentration-response curves for vasodilating activity of
compounds 17a and 21 in the presence and absence of MB. All points
are mean values SE from independent experiments.
residuals (observed values minus estimated values) lower
than 1.5 standard deviation(s). Slopes and intercepts of these
two linear correlations are the same, if the SEs are consid-
ered. Equation (2) indicates a very good relationship between
DHP receptor occupancy and functional effect. The value of
the slope is 1 and the intercept is 0. From Eq. (2) and the
tabulated IC50 values, it is possible to calculate EC50calcd val-
ues (Table 2) for all of the furoxan DHPs of the present work.
Analysis of the data shows a very good correlation between
NO-dependent vasodilator component and that due to Ca2+
-
channel block. To solve this problem we used the same ap-
proach discussed in a previous paper (5). In that work, by
plotting log 1/EC50 values versus log 1/IC50 values of the DHP
compounds not containing NO-donor moieties (derivatives 1,
2, and 3–8), we obtained Eq. (1):
Log 1րEC50 = 0.947 0.042͒ log 1րIC50 + 0.443 0.299͒
(1)
͑
͑
MB
calcd
MB
EC50
and EC50
values. Consequently, EC50
values
where N ס 8, r2 ס 0.988, and s ס 0.14 (figures in brackets are
SE).
Using a set of 12 compounds, namely the eight structures
described in the previous work and the four furazan DHPs
described in this paper (derivatives 15, 16, 17, 22), Eq. (1) is
changed to Eq. (2):
are a measure of the vasodilator potencies of these com-
pounds, largely dependent on their Ca2+-blocking properties.
Data in the table show that EC50MB/EC50 ratio is 1 for the
compounds 15a, 15b, 16a, and 16b. Therefore, they are not
well-balanced hybrids, because their vasodilating action, in
the tested concentration range, is due to their Ca2+-channel
antagonist activities. By contrast, DHPs 17a, 17b, and 21,
which are more potent NO-donors, show EC50MB/EC50 ratio
between 2 and 5 and behave as well-balanced hybrids. This
means that, for a large part of the tested concentration range,
they display vasodilating activity dependent both on their
Log 1րEC50 = 0.971 0.032͒log 1րIC50 + 0.286 0.246͒
(2)
͑
͑
where N ס 12, r2 ס 0.989, and s ס 0.14.
No outlier is present and all of the compounds show NO-donor and on their Ca2+-blocker properties (Fig. 3). Fi-

Agents with Mixed Vasodilating Activities
165
MB
H2-antagonists containing NO-donor moieties endowed with
mixed antisecretory and gastroselective activities. Helv. Chim.
Acta 83:287–299 (2000).
7. Y. Kawashima, T. Kato, A. Nakazato, K. Tsuchida, K. Hatayama,
S. Hirono, and I. Moriguchi. Structure-activity study of antihy-
pertensive 1,4-dihydropyridine derivatives having nitrooxyalkyl
moieties at the 3 and 5 positions. Chem. Pharm. Bull. 41:1060–
1065 (1993) and references therein.
8. N. Iqbal and E. E. Knaus. Synthesis and smooth muscle calcium
channel antagonist effects of dialkyl 1,4-dihydro-2,6-dimethyl-4-
aryl-3,5-pyridinedicarboxylates containing a nitrooxy or nitro-
phenyl moiety in the 3-alkyl ester substituent. Arch. Pharm. Med.
Chem. 329:23–26 (1996) and references therein.
9. H. Bohn, J. Brendel, P. A. Martorana, and K. Scho¨nafinger. Car-
diovascular actions of the furoxan CAS 1609, a novel nitric oxide
donor. Br. J. Pharmacol. 114:1605–1612 (1995).
nally, analysis of EC50
values for furoxan DHPs indicates that
substitution of furazan moieties with the corresponding furoxans pro-
duces a decreased affinity for DHP receptors.
In conclusion, the manipulation of the 3-ester lateral
chain in 2 gave rise to well-balanced hybrids as potent as
those obtained by manipulation of the ortho-position of the
4-phenyl ring.
ACKNOWLEDGMENTS
This work was supported by a grant from MURST Studi
e Ricerche Finalizzate 40%, Roma. The authors would like to
thank Dr. Karl Scho¨nafinger, Aventis Pharma, for providing
intermediates 10a and 10b.
10. M. Hecker, W. Vorhoff, A. T. Bara, P.I. Mordvintcev, and R.
Busse. Characterization of furoxans as a new class of tolerance-
resistant nitrovasodilators. Naunyn-Schmiedeberg’s Arch. Phar-
macol. 351:426–432 (1995).
REFERENCES
1. S. S. Baldwin. Drug design. In M. Williams and S. B. Malick
(eds.), Drug Discovery Development, Humana Press, Clifton, NJ,
1987.
2. J. A. M. Christiaans and H. Timmerman. Cardiovascular hybrid
drugs: Combination of more than one pharmacological property
in one single molecule. Eur. J. Pharm. Sci. 4:1–22 (1996) and
references therein.
3. R. Fruttero, D. Boschi, A. Di Stilo, and A. Gasco. The furoxan
system as a useful tool for balancing “hybrids” with mixed ␣1-
antagonist and NO-like vasodilator activities. J. Med. Chem. 38:
4944–4949 (1995).
4. D. Boschi, A. Di Stilo, C. Cena, M. Lolli, R. Fruttero, and A.
Gasco. Studies on agents with mixed NO-dependent vasodilating
and ␤-blocking activities. Pharm. Res. 14:1750–1757 (1997).
5. A. Di Stilo, S. Visentin, C. Cena, A. M. Gasco, G. Ermondi, and
A. Gasco. New 1,4-dihydropyridines conjugated to furoxanyl
moieties, endowed with both nitric oxide-like and calcium chan-
11. A. M. Gasco, D. Boschi, and A. Gasco. Unsymmetrically substi-
tuted furoxans. Part 15. Bromination of dimethylfuroxan related
compounds with NBS. J. Heterocyclic Chem. 32:811–813 (1995).
12. G. Sorba, G. Ermondi, R. Fruttero, U. Galli, and A. Gasco. Un-
symmetrically substituted furoxans. Part 16. Reaction of ben-
zenesulfonyl substituted furoxans with ethanol and ethanethiol in
basic medium. J. Heterocyclic Chem. 33:327–334 (1996).
13. G. Sorba, C. Medana, R. Fruttero, C. Cena, A. Di Stilo, U. Galli,
and A. Gasco. Water soluble furoxan derivatives as NO prodrugs.
J. Med. Chem. 40:2288, 463–469 (1997).
14. M. Feelisch. In M. Feelisch and J. S. Stamler (eds.), Methods in
Nitric Oxide Research, John Wiley, Chichester, 1996.
15. C. Medana, G. Ermondi, R. Fruttero, A. Di Stilo, C. Ferretti, and
A. Gasco. Furoxans as nitric oxide donors. 4-Phenyl-3-
furoxancarbonitrile: Thiol-mediated NO release and biological
evaluation. J. Med. Chem. 37:4412–4416 (1994).
nel antagonist vasodilator activities. J. Med. Chem. 41:5393–5401 16. J. M. Fukuto, K. Chiang, R. Hszieh, P. Wong, and G. Chadhuri.
(1998).
The pharmacological activity of nitroxyl: A potent vasodilator
with activity similar to nitric oxide and/or endothelium-derived
factor. J. Pharmacol. Exp. Ther. 263:546–551 (1992).
6. M. Bertinaria, G. Sorba, C. Medana, C. Cena, M. Adami, and G.
Morini. Synthesis and pharmacological characterisation of new