Synthesis and biological evaluation of 1,4-dihydropyridine calcium channel modulators having a diazen-1-ium-1,2-diolate nitric oxide donor moiety for the potential treatment of congestive heart failure

Article abstract of DOI:10.1016/j.bmc.2004.05.008

A group of racemic 4-aryl(heteroaryl)-1,4-dihydro-2,6-dimethyl-3- nitropyridines possessing nitric oxide donor O²-acetoxymethyl-1-(N- ethyl-N-methylamino, or 4-ethylpiperazin-1-yl)diazen-1-ium-1,2-diolate, C-5 ester substituents were synthesized by coupling the respective 4-aryl(heteroaryl)-1,4-dihydro-2,6-dimethyl-3-nitropyridine-5-carboxylic acids with either O²-acetoxymethyl-1-[N-(2-methylsulfonyloxyethyl)-N- methylamino]diazen-1-ium-1,2-diolate, or O²-acetoxymethyl-1-[4-(2- methylsulfonyloxyethyl)piperazin-1-yl]diazen-1-ium-1,2-diolate. Compounds having a C-4 2-pyridyl, 4-pyridyl, 2-trifluoromethylphenyl, or benzofurazan-4-yl substituent exhibited more potent smooth muscle calcium channel antagonist activity (IC₅₀'s in the 0.37-1.09μM range) than related analogs having a C-4 3-pyridyl substituent (IC₅₀'s=3.03-9.14μM range) relative to the reference drug nifedipine (IC₅₀=9.13nM). The point of attachment of C-4 isomeric pyridyl substituents was a determinant of smooth muscle calcium channel antagonist activity where the relative potency profile was 4-pyridyl>2-pyridyl>3-pyridyl. Replacement of the C-5 methyl ester substituent of methyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-(2- trifluoromethylphenyl)pyridine-5-carboxylate (Bay K 8644) by an O ²-acetoxymethyl-1-(N-ethyl-N-methylamino)diazen-1-ium-1,2-diolate, or O²-acetoxymethyl-1-(4-ethylpiperazin-1-yl)diazen-1-ium-1,2-diolate, C-5 ester substituent provided compounds, which exhibited a lower, yet respectable, cardiac positive inotropic effect (IC₅₀'s=4.82 and 4.05μM, respectively) relative to the reference drug Bay K 8644 (IC ₅₀=0.30μM). All compounds released nitric oxide upon incubation with either phosphate buffer at pH7, or porcine liver esterase. However, the percentage nitric oxide released was up to 3-fold higher (76%) when these O ²-acetoxymethyl-1-(alkylamino)diazen-1-ium-1,2-diolates were incubated with guinea pig serum. These results suggest that ^·NO would be released in vivo, upon cleavage by nonspecific serum esterases, preferentially in the vascular endothelium where it may enhance smooth muscle calcium channel antagonist activity.

Full text of DOI:10.1016/j.bmc.2004.05.008

Bioorganic & Medicinal Chemistry 12 (2004) 3831–3840
Synthesis and biological evaluation of 1,4-dihydropyridine
calcium channel modulators having a diazen-1-ium-1,2-diolate
nitric oxide donor moiety for the potential treatment
of congestive heart failure
Carlos Vel ꢀa zquez and Edward E. Knaus*
Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2N8
Received 26 February 2004; revised 29 April 2004; accepted 7 May 2004
Available online 4 June 2004
2
Abstract—Agroup of racemic 4-aryl(heteroaryl)-1,4-dihydro-2,6-dimethyl-3-nitropyridines possessing nitric oxide donor O -acet-
oxymethyl-1-(N-ethyl-N-methylamino, or 4-ethylpiperazin-1-yl)diazen-1-ium-1,2-diolate, C-5 ester substituents were synthesized by
2
coupling the respective 4-aryl(heteroaryl)-1,4-dihydro-2,6-dimethyl-3-nitropyridine-5-carboxylic acids with either O -acetoxy-
2
methyl-1-[N-(2-methylsulfonyloxyethyl)-N-methylamino]diazen-1-ium-1,2-diolate, or O -acetoxymethyl-1-[4-(2-methylsulfonyloxy-
ethyl)piperazin-1-yl]diazen-1-ium-1,2-diolate. Compounds having
a C-4 2-pyridyl, 4-pyridyl, 2-trifluoromethylphenyl, or
benzofurazan-4-yl substituent exhibited more potent smooth muscle calcium channel antagonist activity (IC50’s in the 0.37–1.09 lM
range) than related analogs having a C-4 3-pyridyl substituent (IC50’s ¼ 3.03–9.14 lM range) relative to the reference drug nifedipine
(
antagonist activity where the relative potency profile was 4-pyridyl>2-pyridyl>3-pyridyl. Replacement of the C-5 methyl ester
IC50 ¼ 9.13 nM). The point of attachment of C-4 isomeric pyridyl substituents was a determinant of smooth muscle calcium channel
2
substituent of methyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)pyridine-5-carboxylate (Bay K 8644) by an O -
2
acetoxymethyl-1-(N-ethyl-N-methylamino)diazen-1-ium-1,2-diolate, or O -acetoxymethyl-1-(4-ethylpiperazin-1-yl)diazen-1-ium-
1
,2-diolate, C-5 ester substituent provided compounds, which exhibited a lower, yet respectable, cardiac positive inotropic effect
(
oxide upon incubation with either phosphate buffer at pH 7, or porcine liver esterase. However, the percentage nitric oxide released
IC50’s ¼ 4.82 and 4.05 lM, respectively) relative to the reference drug Bay K 8644 (IC50 ¼ 0.30 lM). All compounds released nitric
2
was up to 3-fold higher (76%) when these O -acetoxymethyl-1-(alkylamino)diazen-1-ium-1,2-diolates were incubated with guinea
Å
pig serum. These results suggest that NO would be released in vivo, upon cleavage by nonspecific serum esterases, preferentially in
the vascular endothelium where it may enhance smooth muscle calcium channel antagonist activity.
ꢀ
2004 Elsevier Ltd. All rights reserved.
1. Introduction
tive 1,4-dihydropyridine (1,4-DHP) calcium channel
CC) agonists to treat CHF necessitates removal of their
(
Despite a steady decline in morbidity from coronary
artery disease (CAD) during the past 25 years, cardio-
vascular diseases remain the leading cause of mortality
contraindicated smooth muscle vasoconstrictor effect
while the target cardiac positive inotropic action is
2
maintained. One of the classical examples in this regard
is Bay K 8644 (1), which in spite of its significant posi-
1
in Canada accounting for 37% of total deaths. Con-
3
gestive heart failure (CHF), which is defined as the
inability of the heart to expel sufficient blood to keep
pace with the metabolic demands of the body, develops
in 50–60% of patients with CAD, valvular insufficiency,
and rheumatic heart disease. The design of tissue selec-
tive inotropic activity, also exhibits an undesirable
vascular smooth muscle vasoconstrictor side effect that
contraindicates its clinical use to treat CHF (Fig. 1).
It has been reported that increasing endothelial nitric
Å
oxide ( NO) generation may be expected to produce
beneficial effects such as lowering blood pressure, pre-
vention of atherosclerosis, and inhibition of restenosis
after angioplasty, which constitute potential medical
Keywords: Nitric oxide donor; Positive inotrope; Calcium channel
modulation.
4
*
Corresponding author. Tel.: +1-780-492-5993; fax: +1-780-492-1217;
e-mail: eknaus@pharmacy.ualberta.ca
uses for nitric oxide donors. In nanomolar concentra-
Å
5
tions, NO reversibly activates soluble guanylate cyclase
0
968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.05.008

3832
C. Vel ꢀa zquez, E. E. Knaus / Bioorg. Med. Chem. 12 (2004) 3831–3840
12
of drug upon metabolism by porcine liver esterase. In
Å
an earlier attempt to combine the NO-donor properties
of diazen-1-ium-1,2-diolates with several 1,4-DHP
structures, we observed that unlike the prodrug 4,
2
compounds containing an O -alkyl-1-(pyrrolidin-1-
yl)diazen-1-ium-1,2-diolate moiety did not release sig-
Å
nificant amounts of NO upon incubation with porcine
Å
liver esterase, and therefore were not suitable NO do-
nors, even though they showed moderate CC modula-
13
tion effects.
Figure 1. Structures of Bay K 8644 (1) and PN 202-791 (2).
It was therefore of interest to evaluate the efficacy of an
acetoxymethyl (CH CO CH –) moiety as a protective
by 400-fold, catalyzing the conversion of guanosine tri-
phosphate to cyclic guanosine monophosphate (cGMP).
Elevation of cGMP relaxes smooth muscle in blood
vessels, inhibits platelet aggregation and adhesion, and
3
2
2
2
group for the O -oxygen atom present in diazen-1-ium-
1,2-diolates coupled to a 1,4-DHP CC modulator.
Accordingly, we now report the synthesis, calcium
channel modulation effects and nitric oxide release
studies for a group of racemic 4-aryl(heteroaryl)-1,4-
6
blocks the adhesion of white cells to blood vessel walls.
We have previously reported that it is possible to design
compounds having a dual cardioselective CC agonist
effect in conjunction with a smooth muscle selective CC
antagonist vasorelaxant effect, by replacement of the
methyl or isopropyl ester groups of Bay K 8644 (1) or
dihydro-2,6-dimethyl-3-nitropyridine-5-carboxylates
2
possessing
O -acetoxymethyl-1-(N-ethyl-N-methyl-
2
amino)diazen-1-ium-1,2-diolate (18–22), and O -acet-
oxymethyl-1-(4-ethylpiperazin-1-yl)diazen-1-ium-1,2-di-
olate (23–27), C-5 ester substituents.
Å
7–9
PN 202-791 (2) by a NO donor moiety. In this regard,
we have also proposed that tissue selective 1,4-DHP CC
modulators having a nitric oxide donor group, would
satisfy the clinical requirements for the treatment of
CHF by increasing the force of the heart contractions,
while simultaneously inducing a CC vasorelaxant effect
2. Chemistry
The synthetic reactions employed for the preparation of
2
O -acetoxymethyl-1-[N-(2-methylsulfonyloxyethyl)-N-
2
methylamino]diazen-1-ium-1,2-diolate (8), and O -acet-
Å
enhanced by the release of NO directly into peripheral
Å
blood vessels. However, a compound that releases NO
spontaneously would also produce, in addition to the
desired effect at the localized site, unwanted actions at
oxymethyl-1-[4-(2-methylsulfonyloxyethyl)piperazin-1-
yl]diazen-1-ium-1,2-diolate (12), are illustrated in
Scheme 1. Thus, reaction of 2-(N-methylamino)ethanol
(5), or 1-(2-hydroxyethyl)piperazine (9), with pressur-
ized nitric oxide (at 40 psi) and sodium methoxide at
Å
10
other NO-sensitive sites throughout the organism.
2
The chemical properties of ionic (or zwitterionic) dia-
Å
room temperature, afforded the correspondent O -so-
dium 1-[N-(2-hydroxyethyl)-N-methylamino]diazen-1-
zen-1-ium-1,2-diolates (3) as NO-donor groups in
physiological media, have shown a wide range of rates
2
ium-1,2-diolate (6), or O -sodium 1-[4-(2-hydroxy-
Å
and extents of NO release, making them ideal agents for
probing many aspects of cardiovascular functions, and
ethyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (10), salt.
Reaction of these nucleophilic 1,2-diolate sodium salts
with iodomethyl acetate, previously prepared by reac-
tion of chloromethyl acetate with sodium iodide in
11
2
therefore related disorders. The recently reported O -
acetoxymethyl-1-(N,N-diethylamino)diazen-1-ium-1,2-
diolate (4) is a novel prodrug that is stable in neutral
2
acetone, afforded O -acetoxymethyl-1-[N-(2-hydroxy-
ethyl)-N-methylamino]diazen-1-ium-1,2-diolate (7), or
Å
aqueous media, but it released 1.8 equiv of NO per mol
2
O -acetoxymethyl-1-[4-(2-hydroxyethyl)piperazin-1-yl]-
diazen-1-ium-1, 2-diolate (11), respectively. The termi-
nal hydroxy groups of 7 and 11 were subsequently
transformed into a good leaving group (mesylate) by
reaction with methanesulfonyl chloride and 4-(dimethyl-
amino)pyridine in THF (Scheme 1). It is noteworthy
that compounds 7 and 11 did not react with para-
toluenesulfonyl chloride under the same reaction con-
ditions that worked successfully for methanesulfonyl
chloride.
The final coupling reactions proceeds by nucleophilic
2
displacement (S
N
reaction) of the mesyloxy group
present in 8 or 12 by the correspondent sodium 4-aryl-
,4-dihydro-2,6-dimethyl-3-nitropyridine-5-carboxylate
(13–17) in hexamethylphosphoramide (HMPA) to af-
ford the target products (18–27) in moderate yields (19–
36%). Similar reactions using dimethylformamide,
dimethylsulfoxide, tetrahydrofuran, acetonitrile or
1
Figure 2. General structures of zwitterionic diazen-1-ium-1,2-diolates
2
(
(
3); O -acetoxymethyl-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate
2
4); O -sodium 1-[N-(2-aminoethyl)-1,2-ethanediamine-N-yl]diazen-1-
ium-1,2-diolate (DETA/NO); 1-(prolin-1-yl-2-carboxyl)diazen-1-ium-
,2-diolate disodium salt (PROLI/NO).
1

C. Vel ꢀa zquez, E. E. Knaus / Bioorg. Med. Chem. 12 (2004) 3831–3840
3833
Scheme 2. Reagents and conditions: (i) Na
2
CO
3
, HMPA, 25 ꢁC, 72 h.
guinea pig ileum longitudinal smooth muscle (GPILSM),
where the relative potency profile was 4-pyridyl>2-pyr-
idyl>3-pyridyl. Compounds having a 2-pyridyl (18, 23),
4
-pyridyl (20, 25), 2-trifluoromethylphenyl (21, 26) and
benzofurazan-4-yl (22, 27) substituent at the 1,4-DHP C-
position, exhibited more potent smooth muscle calcium
4
channel antagonist activity (IC50’s in the 0.37–1.09 lM
range) than related analogs having a C-4 3-pyridyl sub-
stituent (19 and 24, IC50’s ¼ 9.14 and 3.03 lM), relative to
the reference drug nifedipine (IC50 ¼ 9.13 nM). Replace-
ment of the methyl ester moiety of Bay K 8644 by an
Scheme 1. Reagents and conditions: (i) Nitric oxide (at 40 psi),
CH ONa/CH CO CH I, CH CN,
OH, ether, 25 ꢁC, 72 h; (ii) CH
5 ꢁC, overnight; (iii) CH SO Cl, DMAP, THF, 25 ꢁC, 15 h; (iv)
CH CO CH
I, THF, )10 to 25 ꢁC, 15–18 h.
3
3
3
2
2
3
2
3
2
2
3
2
2
O -acetoxymethyl-1-(N-ethyl-N-methylamino)diazen-1-
2
ium-1,2-diolate group (21), or an O -acetoxymethyl-1-
group
different mixtures of these solvents, in place of HMPA,
gave lower product yields (Scheme 2).
(4-ethylpiperazin-1-yl)diazen-1-ium-1,2-diolate
(26), retained the desired cardiac positive inotropic effect
on guinea pig left atrium (GPLA, IC50’s ¼ 4.82 and
4.05 lM) although 21 and 26 were less potent than the
3
. Results and discussion
reference drug Bay K 8644 (IC ¼ 0.30 lM). In contrast
5
0
to Bay K 8644, which exhibited a contraindicated CC
agonist effect on GPILSM (EC50 ¼ 0.23 lM), none of the
compounds possessing a nitric oxide donor moiety (18–
27) showed a CC agonist effect on GPILSM. Unlike
analogs of PN 202-791 (2) having nitrooxyalkyl
Agroup of racemic 4-aryl(heteroaryl)-1,4-dihydro-2,6-
dimethyl-3-nitropyridine-5-carboxylates possessing
a
potential nitric oxide donor O -acetoxymethyl-1-(N-
2
ethyl-N-methylamino)diazen-1-ium-1,2-diolate (18–22),
2
and O -acetoxymethyl-1-(4-ethylpiperazin-1-yl)diazen-
1-ium-1,2-diolate (23–27), C-5 ester substituent were
synthesized.
2 2
[O NO(CH )n] C-5 ester substituents reported in a recent
14
study, replacement of the isopropyl ester group of PN
2
202-791 by an O -acetoxymethyl-1-(N-ethyl-N-methyl-
amino)diazen-1-ium-1,2-diolate group (22), or an
2
The point of attachment of C-4 isomeric pyridyl sub-
stituents was a determinant of CC antagonist activity on
O -acetoxymethyl-1-(4-ethylpiperazin-1-yl)diazen-1-ium-
1,2-diolate group (27), decreased (22) or even abolished

3834
C. Vel ꢀa zquez, E. E. Knaus / Bioorg. Med. Chem. 12 (2004) 3831–3840
Table 1. In vitro calcium channel modulation activities for 4-aryl(heteroaryl)-1,4-dihydro-2,6-dimethyl-3-nitropyridine-5-carboxylates possessing a
2
2
O -acetoxymethyl-1-(N-ethyl-N-methylamino)diazen-1-ium-1,2-diolate (18–22), or O -acetoxymethyl-1-(4-ethylpiperazin-1-yl)diazen-1-ium-1,2-dio-
late (23–27), C-5 ester substituent
a
b
c
Compound No.
R
GPILSM IC50 (lM)
GPILSM EC50 (lM)
GPLAEC 50 (lM)
d
e
1
1
2
2
2
2
2
2
2
2
8
9
0
1
2
3
4
5
6
7
2-Pyridyl
3-Pyridyl
4-Pyridyl
0.89 ± 0.01
9.14 ± 6.25
0.37 ± 0.03
0.57 ± 0.13
0.46 ± 0.07
1.09 ± 0.02
3.03 ± 0.20
0.60 ± 0.05
0.84 ± 0.01
0.97 ± 0.01
0.0091 ± 0.0026
––
No effect
No effect
No effect
No effect
d
e
d
No effect
No effect
32.2 ± 3.2
4.82 ± 1.41
22.7 ± 1.7
d
2-Trifluoromethylphenyl
Benzofurazan-4-yl
2-Pyridyl
d
No effect
No effect
d
e
No effect
d
3-Pyridyl
4-Pyridyl
No effect
No effect
8.18 ± 0.71
12.7 ± 6.7
4.05 ± 0.23
d
d
2-Trifluoromethylphenyl
Benzofurazan-4-yl
No effect
No effect
––
d
e
No effect
––
Nifedipine
BAY K 8644 (1)
PN 202-791 (2)
0.23 ± 0.01
––
0.30 ± 0.01
9.4 ± 1.2
––
––
a
The micromolar concentration of the test compound causing a 50% decrease in the slow component or tonic contractile response (IC50ÆSEM,
n ¼ 3) in guinea pig ileum longitudinal smooth muscle (GPILSM) induced by the muscarinic agonist carbachol (0.167 lM) was determined
graphically from the dose–response curve.
b
c
The micromolar concentration of the test compound causing a 50% increase in the slow component or tonic contractile response in guinea pig ileum
longitudinal smooth muscle (GPILSM), in the absence of carbachol, was determined graphically from the dose–response curves.
The micromolar concentration of the test compound causing a 50% increase in the cardiac contractile force (EC50ÆSEM, n ¼ 3) in guinea pig left
atrium (GPLA) was determined graphically from the dose–response curves.
d
e
No smooth muscle calcium channel agonist response was observed at the highest test compound concentration employed (44.66 lM).
No calcium channel agonist response (positive inotropic effect) on heart was observed at the highest test compound concentration employed
(44.66 lM).
2
(
27) the desired cardiac positive inotropic effect on
was relatively constant (6.3–9.4% range). The O -acet-
2
GPLA. A complete list of the CC modulation results are
presented in Table 1.
oxymethyl moiety of O -acetoxymethyl-1-(alkyl-
amino)diazen-1-ium-1,2-diolate moieties, attached to
4
-aryl(heteroaryl)-1,4-dihydro-2,6-dimethyl-3-nitropyr-
Å
It has been reported that the rate of NO release from
2
idine-5-carboxylic acid derivatives, upon incubation
with 10 equiv of pig liver esterase (PLE) is hydrolyzed
less extensively compared to when the O -acetoxymethyl
O -sodium N-substituted-diazen-1-ium-1,2-diolate salts
varies greatly depending on the nature of the structure
of the substrate with half-lives from 1.8 s for PROLI/NO
One type of chemical
modification used to control the rate of nitric oxide re-
lease from diazen-1-ium-1,2-diolates is the attachment
of alkyl substituents to the O -position. O -substituted
diazen-1-ium-1,2-diolates are stable compounds that
hydrolyze slowly even in acidic solution. Consistent
with these observations, when compounds 18–27 were
2
moiety is present in smaller molecules such as the pro-
drug 4. In this regard, incubation of compounds 18–27
in the presence of PLE produced a 21–34% release of
15–19
to 20 h for DETA/NO (Fig. 2).
Å
Å
NO compared with the 90% NO release from the
prodrug 4.
2
20
2
21
Å
The effect of nonspecific esterases on the NO release
properties of compounds 18–27 was determined by their
incubation in the presence of guinea pig serum for 1.5 h
at 37 ꢁC (pH 7.4). The percentage NO released was
substantially higher (60–75% range) than that observed
upon incubation with PLE (see Table 2). These data
indicate the nonspecific serum esterases present in gui-
Å
incubated in PBS at pH 7.4, the percentage of NO
Å
released varied from 6.3% to 9.4% suggesting a
slow decomposition even when the media was acidi-
fied by quenching with the Griess reagent (pH about
1
–2).
2
nea pig serum cleave these O -acetoxymethyl-1-(alkyl-
2
As mentioned previously, the prodrug O -acetoxy-
methyl-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (4)
amino)diazen-1-ium-1,2-diolates more effectively than
pig liver esterase (PLE). Compounds 18–27 are pro-
drugs, which must be cleaved by esterases before they
Å
was reported to release up to 1.8 mol of NO per mol
2
Å
hydrolysis of the O -acetoxymethyl group by porcine
liver esterase (PLE). The first enzymatic hydrolysis
are able to release NO. In contrast, the unprotected
2
diazen-1-ium-1,2-diolates 6 and 10, which lack the O -
acetoxymethyl moiety and do not require cleavage by an
2
product of 4, O -hydroxymethyl-1-(N,N-diethyl-
amino)diazen-1-ium-1,2-diolate, is reported to be
unstable in water as it spontaneously eliminates form-
aldehyde and the zwitterion 1-(N,N-diethylamino)dia-
zen-1-ium-1,2-diolate, which in turn releases nitric oxide
esterase, released about 85% (6) and 70% (10) of the
Å
theoretical amount of NO irrespective of whether the
compound was incubated with PBS (pH 7.4), PLE, or
guinea pig serum.
Å
in phosphate buffer (see the mechanism in Fig. 3). NO
release data acquired in this investigation indicated that
Å
The hybrid CC modulation/ NO donor drugs (18–27)
possess a number of potential advantages relative to
using a physical mixture of a CC modulator and nitrate
Å
the amount of NO released upon incubation of the test
compounds 18–27 in PBS at pH 7.4 for 1.5 h at 37 ꢁC

C. Vel ꢀa zquez, E. E. Knaus / Bioorg. Med. Chem. 12 (2004) 3831–3840
3835
Figure 3. Theoretical ester cleavage and nitric oxide release from prodrug (4), and 4-aryl(heteroaryl)-1,4-dihydro-2,6-dimethyl-3-nitropyridine-5-
2
2
carboxylates possessing a O -acetoxymethyl-1-(N-ethyl-N-methylamino)diazen-1-ium-1,2-diolate (18–22) or O -acetoxymethyl-1-(4-ethylpiperazin-1-
yl)diazen-1-ium-1,2-diolate (23–27) C-5 ester substituent, where R ¼ 2-, 3-, or 4-pyridyl, 2-CF –C –, benzofurazan-4-yl.
3
6 4
H
2
2
Table 2. Nitric oxide release studies for 4-aryl(heteroaryl)-1,4-dihydro-
2
cells, the vasodilating effect induced by CC antagonists
Å
2,6-dimethyl-3-nitropyridine-5-carboxylates possessing a O -acetoxy-
methyl-1-(N-ethyl-N-methylamino)diazen-1-ium-1,2-diolate (18–22), or
23;24
is increased by NO-donor drugs,
and the combined
Å
effects of basal NO release and CC antagonists produce
an inhibition greater than additive where the concen-
trations of CC antagonist drug required (IC50) is 3-fold
2
O -acetoxymethyl-1-(4-ethylpiperazin-1-yl)diazen-1-ium-1,2-diolate (23–
27), C-5 ester substituent
a
Compd
% of Nitric oxide released
Å
lower in the presence of basal NO release than in its
25
b
c
d
PBS (pH 7.4)
PLE
GP-Serum
absence. ; (ii) in contrast to organic nitrate vasodila-
tors, these hybrid compounds do not require a thiol
1
1
2
2
2
2
2
2
2
2
6
1
8
9
0
1
2
3
4
5
6
7
9.42 ± 0.01
7.71 ± 0.04
9.03 ± 0.04
7.92 ± 0.05
6.32 ± 0.09
6.92 ± 0.05
6.93 ± 0.52
7.81 ± 0.04
6.54 ± 0.06
7.84 ± 0.04
84.90 ± 0.06
70.45 ± 0.04
26.43 ± 0.06
23.48 ± 0.07
26.62 ± 0.05
23.95 ± 0.03
25.12 ± 0.06
34.90 ± 0.06
31.00 ± 0.05
23.20 ± 0.05
21.85 ± 0.05
32.08 ± 0.05
84.92 ± 0.06
70.41 ± 0.05
68.19 ± 0.04
63.37 ± 0.05
62.41 ± 0.06
75.80 ± 0.04
69.84 ± 0.05
61.20 ± 0.04
––
cofactor as
Å
L-cysteine or glutathione to enhance the
release of NO from the diazen-1-ium-1,2-diolate moi-
Å
ety, and a redox activation is not needed for NO
Å
26
release ; (iii) the rate of NO release from the diazen-1-
ium-1,2-diolate moiety can be controlled by the nature
þ
À
À
of the R-substituent in R–N (O )@N–O
com-
and compounds of general structure
60.73 ± 0.05
69.01 ± 0.06
72.53 ± 0.09
84.94 ± 0.05
70.46 ± 0.06
15;27
pounds,
1
2
þ
À
R (R )N–N (O )@N–O(CH
2
)nNH
2
with t1=2 values of
1
.3–3400 min at 22 ꢁC and pH 7.4 in phosphate buffer
28
Å
have been reported; (iv) after release of NO from the
diazen-1-ium-1,2-diolate moiety at pH 7.4 (no enzyme
required), the DHP released with a C-5 amino-alkyl
ester substituent is expected to have a long duration of
action (like amlodipine, which has an aminoethoxy-
methyl substituent) that would be amenable to once-a-day
dosing, which would circumvent the peak and trough
plasma levels observed with short acting CC antagonist
drugs such as nifedipine; and (v) hybrid compounds
0
a
b
c
Percent of nitric oxide released (±SEM, n ¼ 3) relative to a theoret-
ical maximum release of 2 mol of NO/mol of test compound.
Incubated in phosphate buffer solution (PBS) only (pH 7.4) at 37 ꢁC
for 1.5 h.
Incubated in the presence of 10 equiv of pig liver esterase (based on a
ratio of 1 mol of test compound/10 mol of esterase) in phosphate
buffer solution (pH 7.4) at 37 ꢁC for 1.5 h.
d
À4
Test compound (2.0 · 10 mmol) incubated with guinea pig
2
serum (260 lL) in phosphate buffer solution (pH 7.4) at 37 ꢁC
for 1.5 h.
having an O -acetoxymethyldiazen-1-ium-1,2-diolate
moiety may inhibit platelet aggregation like aspirin that
is used chronically in low doses for prophylaxis of stroke
and myocardial infarction, and undergo rapid cleavage
of the acetoxy group by plasma esterases prior to con-
29
Å
vasodilator as glycerol trinitrate: (i) the 1,4-DHP moiety
can act as a carrier to simultaneously deliver the 1,4-
version to the diazen-1-ium-1,2-diolate NO donor
Å
moiety, which will subsequently release NO (see Fig. 3).
Å
Å
DHP CC modulator and NO moiety to the target tissue
that could provide a synergistic effect. CC antagonists
In addition, NO also reduces blood clotting and inhibits
Å
platelet aggregation and adhesion, whereas a NO defi-
30
ciency may favor thrombosis.
Å
enhance the effect of NO in vascular smooth muscle

3836
C. Vel ꢀa zquez, E. E. Knaus / Bioorg. Med. Chem. 12 (2004) 3831–3840
4. Conclusions
5.1. Synthesis of iodomethyl acetate
The group of racemic 4-aryl(heteroary)-1,4-dihydro-2,6-
dimethyl-3-nitropyridine-5-carboxylates possessing
Chloromethyl acetate (108.5 g, 1 mol) was added slowly
to a solution of sodium iodide (180 g, 1.2 mol) in dry
acetone (600 mL) at 25 ꢁC with stirring. The reaction
was allowed to proceed for 3 h with stirring at 25 ꢁC, the
insoluble inorganic salts were removed by filtration,
the solvent was evaporated under reduced pressure, the
residue was dissolved in dichloromethane (300 mL), and
this solution was washed with 2 N sodium thiosulfate
solution (2 · 100 mL). The organic phase was dried
(Na SO ), the solvent was removed in vacuo and the
a
nitric oxide donor O -acetoxymethyl-1-(N-ethyl-N-
2
2
methylamino)diazen-1-ium-1,2-diolate (18–22) or O -
acetoxymethyl-1-(4-ethylpiperazin-1-yl)diazen-1-ium-1,2-
diolate (23–27) C-5 ester substituent, constitute a
novel group of nitric oxide donor compounds with
desirable calcium channel modulation activities. In
particular, compounds having a C-4 2-trifluoromethyl-
phenyl substituent (21 and 26) exhibit dual cardioselec-
tive agonist/smooth muscle selective antagonist activities,
in conjunction with an enhanced nitric oxide release
profile compared with other nitric oxide functional
group donors reported in the literature, such as furox-
2
4
residue was purified by fractional distillation (27 ꢁC/
3 mmHg) to afford iodomethyl acetate as a yellow liquid
(82.4 g, 41%), which was stored under nitrogen to avoid
1
decomposition prior to use. H NMR (CDCl ): d 5.75 (s,
3
3
1;32
ans.
Bay K 8644 by an O -acetoxymethyl-1-(N-ethyl-N-
methylamino)diazen-1-ium-1,2-diolate group (21), or an
The replacement of the methyl ester moiety of
2
2 3
2H, ICH O), 2.11 (s, 3H, CH ).
2
2
O -acetoxymethyl-1-(4-ethylpiperazin-1-yl)diazen-1-ium-
1
5.2. O -Sodium 1-[N-(2-hydroxyethyl)-N-methylamino]-
diazen-1-ium-1,2-diolate (6)
,2-diolate group (26), retained the desired cardiac
positive inotropic effect while abolishing the smooth
muscle agonist effect on guinea pig ileum. Remarkably,
2-(Methylamino)ethanol (6, 20 g, 0.26 mol) was added to
a solution of sodium methoxide (14.6 g, 0.26 mol, 61 mL
of a 25% w/v solution in MeOH) and diethyl ether
(300 mL) with stirring at 25 ꢁC. This mixture was flushed
with dry nitrogen for 5 min and then the reaction was
allowed to proceed under an atmosphere of nitric oxide
(40 psi internal pressure) with stirring at 25 ꢁC for 72 h.
The product, which precipitated as a fine white powder,
was isolated by filtration and then suspended in diethyl
ether (100 mL) upon stirring for 15 min. The suspension
was filtered, the solid collected was dried at 25 ꢁC under
reduced pressure until a constant weight was achieved
2
the O -acetoxymethyl-1-(N-ethyl-N-methylamino)dia-
2
zen-1-ium-1,2-diolate and O -acetoxymethyl-1-(4-ethyl-
piperazin-1-yl)diazen-1-ium-1,2-diolate
C-5
ester
substituents, released up to 3-fold higher amounts of
nitric oxide in the presence of serum esterases, compared
Å
with the NO release in the presence of a liver esterase.
Å
This selective and enhanced NO release might provide a
useful therapeutic advantage towards the treatment of
cardiovascular diseases, including congestive heart fail-
ure, because it may be possible to increase endothelial
Å
NO with only minor effects at other sensitive sites
throughout the organism.
after about 2 h to afford 6 as a fine white powder (36.1 g,
1
8
8%); mp 150–151 ꢁC; H NMR (D O) d 4.63 (t,
2
J ¼ 5:7 Hz, 2H, CH
2
CH
2
OH), 3.35 (s, 3H, NCH
CH OH). Product 6 was used
3
), 2.86
5. Experimental
(t, J ¼ 5:7 Hz, 2H, CH
2
2
immediately after drying without further purification for
the synthesis of compound 7.
Melting points were determined using a Thomas–Hoo-
ver capillary apparatus and are uncorrected. Infrared
(
IR) spectra were recorded using a Nicolet 550 Series II
1
2
Magna FT-IR spectrometer. H NMR nuclear magnetic
resonance spectra were recorded on a Bruker AM-300
spectrophotometer. The assignment of exchangeable
5.3. O -Sodium 1-[4-(2-hydroxyethyl)piperazin-1-yl]-dia-
zen-1-ium-1,2-diolate (10)
protons (NH) was confirmed by the addition of D
2
O.
Reaction of 1-(2-hydroxyethyl)piperazine (9, 20 g,
0.15 mol) under an atmosphere of nitric oxide at 40 psi,
using the procedure described for the synthesis of 6
above, afforded 10 (29.5 g, 91%) as a white solid; mp
1
Ultraviolet (UV) spectra and quantitative analyses were
measured using a Philips PU 8740 UV/vis scanning
spectrophotometer. Silica gel column chromatography
ꢂ
was performed using Silicycle (silica gel 70–230 mesh).
Elemental analyses were performed for C, H, and N
137–140 ꢁC; H NMR (D
2
O) d 3.73 (t, J ¼ 6:3 Hz, 2H,
CH OH), 3.17 (t, J ¼ 4:8 Hz, 4H, piperazin-1-yl H-
2, H-6), 2.80 (t, J ¼ 4:8 Hz, 4H, piperazin-1-yl H-3, H-
CH OH). Product 10
2
CH
2
(
Chemistry, University of Alberta). Chloromethyl ace-
microanalytical service laboratory, Department of
5), 2.61 (t, J ¼ 6:3 Hz, 2H, CH
2
2
33
tate and the 4-aryl-1,4-dihydro-2,6-dimethyl-3-nitro-
7
was used immediately after drying without further
purification for the synthesis of compound 11.
pyridine-5-carboxylic acids (13–17) were prepared
according to procedures reported in the literature. All
other reagents (including the porcine liver esterase,
2
3
.2 M ammonium sulfate suspension) were purchased
5.4. O -Acetoxymethyl-1-[N-(2-hydroxyethyl)-N-methyl-
amino]diazen-1-ium-1,2-diolate (7)
from Aldrich Chemical (Milwaukee, WI) and used
without further purification. In vitro calcium channel
antagonist and agonist activities were determined using
protocols approved by the Health Sciences Animal
Welfare Committee at the University of Alberta.
Freshly distilled iodomethyl acetate (27.8 g, 0.14 mol)
was added drop wise to a solution of 6 (20 g, 0.12 mol)
in acetonitrile (200 mL, HPLC grade) at 25 ꢁC with

C. Vel ꢀa zquez, E. E. Knaus / Bioorg. Med. Chem. 12 (2004) 3831–3840
3837
stirring. The reaction was allowed to proceed for 19 h at
5 ꢁC with stirring, insoluble inorganic salts were re-
pyridine (1.37 g, 11.3 mmol) in dry THF (30 mL) and
this solution was stirred for 10 min at 25 ꢁC. Asolution
of 11 (2.7 g, 10.3 mmol) in dry THF (30 mL) was added
dropwise, the reaction was allowed to proceed for 16 h
at 25 ꢁC with stirring, the solids were filtered off, and the
solvent was removed in vacuo. The residue obtained was
purified by silica gel column chromatography (hexane–
ethyl acetate; 1:2, v/v) to afford 12 as a pale yellow oil
2
moved by filtration, and the solvent was removed in
vacuo. Dichloromethane was added (150 mL) to the
residue, and once again insoluble inorganic salts were
removed by filtration. Removal of the solvent in vacuo
gave a residue that was purified by silica gel column
chromatography using hexane–ethyl acetate, 1:2, v/v) as
1
eluant to afford 7 (15.54 g, 62%) as a pale yellow liquid;
(2.86 g, 81%); H NMR (CDCl ) d 5.78 (s, 2H, OCH O),
3
2
1
H NMR (CDCl
3
) d 5.78 (s, 2H, OCH
CH
OH), 3.50 (t, J ¼ 5:1 Hz, 2H,
CH CH OH), 3.10 (s, 3H, NCH ), 2.12 (s, 3H, COCH ).
2
O), 3.78 (t,
4.33 (t, J ¼ 5:1 Hz, 2H, CH
2
OMs), 3.51 (t, J ¼ 4:8 Hz,
J ¼ 5:1 Hz, 2H, CH
2
2
4H, piperazin-1-yl H-2, H-6), 3.06 (s, 3H, SO
(t, J ¼ 5:1 Hz, 2H, NCH ), 2.72 (t, J ¼ 4:8 Hz, 4H,
2
CH ), 2.76
3
2
2
3
3
2
Anal. Calcd for C H N O : C, 34.78; H, 6.32; N, 20.28.
piperazin-1-yl H-3, H-5), 2.12 (s, 3H, COCH ). Com-
3
6
13
3
5
Found: C, 34.52; H, 6.68; N, 20.49.
pound 12 was used immediately after purification for the
synthesis of compounds 23–27.
2
5.5. O -Acetoxymethyl-1-[4-(2-hydroxyethyl)piperazin-1-
yl]diazen-1-ium-1,2-diolate (11)
5.8. General procedure for the synthesis of racemic 4-aryl-
(
heteroaryl)-1,4-dihydro-2,6-dimethyl-3-nitropyridine-5-
2
Freshly distilled iodomethyl acetate (20.6 g, 103.7 mmol)
was added dropwise with stirring to a solution of 10
carboxylates possessing an O -acetoxymethyl-1-(N-eth-
yl-N-methylamino)diazen-1-ium-1,2-diolate (18–22), or
O -acetoxymethyl-1-(4-ethylpiperazin-1-yl)diazen-1-ium-
(20 g, 94.3 mmol) in dry THF (200 mL) at )50 to )60 ꢁC
(acetone–dry ice bath). Once the addition was complete,
2
1
,2-diolate (23–28), C-5 ester substituent
the reaction mixture was allowed to warm to 25 ꢁC, the
reaction was allowed to proceed for 19 h, and ethyl
acetate (200 mL) was added to quench the reaction. The
organic phase was washed with a 0.2 N sodium thio-
sulfate solution until a pale yellow solution (organic
phase) was obtained. The organic phase was dried
Amixture of a 4-aryl-1,4-dihydro-2,6-dimethyl-3-nitro-
pyridine-5-carboxylic acid (13–17, 0.47 mmol), sodium
carbonate (0.56 mmol), and hexamethylphosphoramide
(HMPA, 2 mL) was stirred for 4–6 h at 25 ꢁC to form the
sodium carboxylate salt. Asolution of the mesylate
(either 8 or 12, 0.56 mmol) in HMPA(1 mL) was added,
the reaction was allowed to proceed for 72 h at 25 ꢁC
with stirring, and then a mixture of ice-water (1:1, v/v;
50 mL) was added to quench the reaction. Extraction
with EtOAc (3 · 30 mL), washing the combined EtOAc
extracts with water (5 · 30 mL), drying the EtOAc frac-
tion (Na SO ), and removal of the solvent in vacuo gave
(Na SO ), the solvent was removed in vacuo, and the
2 4
residue was purified by silica gel column chromatogra-
phy (ethyl acetate–methanol; 9:1, v/v) to yield 11 (7.34 g,
1
2
9.6%) as a pale yellow liquid; H NMR (CDCl ) d 5.71
3
(
3
1
5
s, 2H, OCH
2
O), 3.58 (t, J ¼ 5:4 Hz, 2H, CH
2 2
CH OH),
.45 (t, J ¼ 5:1 Hz, 4H, piperazin-1-yl H-2, H-6), 2.82 (s,
H, OH), 2.63 (t, J ¼ 5:1 Hz, 4H, piperazin-1-yl H-3, H-
), 2.53 (t, J ¼ 5:4 Hz, 2H, CH CH OH), 2.05 (s, 3H,
2
4
2
2
the respective product (18–27), which was purified by
silica gel column chromatography (hexane–EtOAc, 1:2,
v/v; or EtOAc) and then recrystallization from ether–
chloroform. Physical and spectroscopic data for com-
pounds 18–27 are listed below.
COCH
3
). Anal. Calcd for C
9
H
18
N
4 5
O : C, 41.22; H, 6.92;
N, 21.36. Found: C, 41.02; H, 6.79; N, 21.63.
2
5.6. O -Acetoxymethyl-1-[N-(2-methylsulfonyloxyethyl)-
N-methylamino]diazen-1-ium-1,2-diolate (8)
2
5.8.1. O -Acetoxymethyl-1-(N-methyl-N-ethylamino)dia-
zen-1-ium-1,2-diolate 4-(2-pyridyl)-1,4-dihydro-2,6-di-
methyl-3-nitropyridine-5-carboxylate (18). Yellow powder
Methanesulfonyl chloride (4.0 g, 35.0 mmol) in dry THF
(50 mL) was added to a solution of 4-dimethylamino-
pyridine (4.24 g, 35.0 mmol) in dry THF (50 mL) and this
(
32%); mp 133–135 ꢁC; IR (KBr): 3273 (NH), 1776
mixture was stirred for 10 min at 25 ꢁC. Asolution of 7
À1
(CO), 1662 (C@C), 1528 (NO ), 1212, 829 (N–O) cm ;
(
wise, and the reaction was allowed to proceed for 15 h at
6.6 g, 31.9 mmol) in dry THF (90 mL) was added drop-
2
1
H NMR (CDCl
3
) d 10.13 (s, 1H, NH), 8.46 (d,
J
J
1
5;6 ¼ 4:5 Hz, 1H, pyridyl H-6), 7.73 (dd, J3;4 ¼ 7:5,
2
5 ꢁC with stirring. The solids were filtered off, and the
4;5 ¼ 6:8 Hz, 1H, pyridyl H-4), 7.61 (d, J3;4 ¼ 7:5 Hz,
solvent was removed in vacuo to give a pale yellow oil,
which was purified by silica gel column chromatography
H, pyridyl H-3), 7.26 (dd, J ¼ 4:5, J ¼ 6:8 Hz, 1H,
5;6
4;5
pyridyl H-5), 5.76 (s, 2H, OCH
2
O), 5.57 (s, 1H, H-4),
(
hexane–ethyl acetate; 1:2, v/v) to furnish 8 as a colorless
1
2 2
4.14-4.26 (m, 2H, CO CH ), 3.56–3.61 (m, 2H,
oil (3.2 g, 35.2%); H NMR (CDCl
OCH
3
) d 5.73 (s, 2H,
OMs), 3.69 (t,
CH CH N), 3.01 (s, 3H, NCH ), 2.41 (s, 3H, C-2 CH ),
3
2
O), 4.37 (t, J ¼ 5:1 Hz, 2H, CH
2
2
2
3
2.21 (s, 3H, C-6 CH
Calcd for C19H N O : C, 49.14; H, 5.21; N, 18.10.
3
), 2.10 (s, 3H, COCH
3
). Anal.
J ¼ 5:1 Hz, 2H, NCH ), 3.11 (s, 3H, NCH ), 3.01 (s, 3H,
2
3
SO
2
CH
3
), 2.08 (s, 3H, COCH
immediately after purification for the synthesis of 18–22.
3
). Compound 8 was used
24 6 8
Found: C, 49.14; H, 5.06; N, 17.60.
2
5.7. O -Acetoxymethyl-1-[4-(2-methylsulfonyloxyethyl)-
piperazin-1-yl]diazen-1-ium-1,2-diolate (12)
2
5.8.2. O -Acetoxymethyl-1-(N-methyl-N-ethylamino)dia-
zen-1-ium-1,2-diolate 4-(3-pyridyl)-1,4-dihydro-2,6-dime-
thyl-3-nitropyridine-5-carboxylate (19). Yellow powder
(21%); mp 122–125 ꢁC; IR (KBr): 3288 (NH), 1770
Methanesulfonyl chloride (1.3 g, 11.3 mmol) in dry THF
(
30 mL) was added to a solution of 4-dimethylamino-

3838
C. Vel ꢀa zquez, E. E. Knaus / Bioorg. Med. Chem. 12 (2004) 3831–3840
À1
2
(
CO), 1649 (C@C), 1508 (NO
2
), 1313, 829 (N–O) cm ;
5.8.6. O -Acetoxymethyl-1-(4-ethylpiperazin-1-yl)diazen-
1
H NMR (CDCl
H-2), 8.42 (dd, J5;6 ¼ 4:8, J ¼ 1:5 Hz, 1H, pyridyl H-
4;6
3
) d 8.50 (d, J2;4 ¼ 1:8 Hz, 1H, pyridyl
1-ium-1,2-diolate 4-(2-pyridyl)-1,4-dihydro-2,6-dimethyl-
3-nitropyridine-5-carboxylate (23). Yellow powder
(29%); mp 170–171 ꢁC; IR (KBr): 3328 (NH), 1602
6
J
), 8.31 (s, 1H, NH), 7.77 (ddd, J4;5 ¼ 7:8, J2;4 ¼ 1:8,
À1
1
4;6 ¼ 1:5 Hz, 1H, pyridyl H-4), 7.25 (dd, J4;5 ¼ 7:8,
(C@C), 1427 (NO ), 1266, 897 (N–O) cm ; H NMR
2
J5;6 ¼ 4:8 Hz, 1H, pyridyl H-5), 5.76 (s, 2H, OCH O),
(CDCl ) d 8.74 (s, 1H, NH), 8.26 (d, J ¼ 6:3 Hz, 1H,
2
3
5;6
5
3
2
.35 (s, 1H, H-4), 4.22–4.26 (m, 2H, CO
2
CH
2
), 3.55–
pyridyl H-6), 7.37 (dd, J3;4 ¼ 7:5, J4;5 ¼ 6:3 Hz, 1H,
pyridyl H-4), 7.25 (d, J3;4 ¼ 7:5 Hz, 1H, pyridyl H-3),
.63 (m, 2H, CH
CH ), 2.35 (s, 3H, C-6 CH ), 2.11 (s, 3H, COCH ).
2 3
N), 3.01 (s, 3H, NCH ), 2.51 (s, 3H, C-
6.90 (dd, J5;6 ¼ J ¼ 6:3 Hz, 1H, pyridyl H-5), 5.61 (s,
4;5
3
3
3
Anal. Calcd for C19
1
H
24
N
6
O
8.10. Found: C, 49.00; H, 5.07; N, 17.87.
8
: C, 49.14; H, 5.21; N,
2
2H, OCH O), 5.34 (s, 1H, H-4), 3.95–4.00 (m, 2H,
CO
2
2
CH ), 3.18–3.30 (m, 4H, piperazin-1-yl H-2, H-6),
.59 (m, 2H, CH CH N), 2.44 (m, 4H, piperazin-1-yl H-
, H-5), 2.38 (s, 3H, C-2 CH ), 2.19 (s, 3H, C-6 CH ),
3
.96 (s, 3H, COCH
50.86; H, 5.63; N, 18.87. Found: C, 50.46; H, 5.39; N,
18.69.
2
3
1
2
2
3
3
). Anal. Calcd for C22
H
29
N
7
O
8
: C,
2
5.8.3. O -Acetoxymethyl-1-(N-methyl-N-ethylamino)dia-
zen-1-ium-1,2-diolate 4-(4-pyridyl)-1,4-dihydro-2,6-dime-
thyl-3-nitropyridine-5-carboxylate (20). Yellow powder
(
19%); mp 150–152 ꢁC; IR (KBr): 3180 (NH), 1763
À1
2
(
CO), 1649 (C@C), 1494 (NO
2
), 1313, 829 (N–O) cm ;
) d 8.48 (d, Jortho ¼ 5:4 Hz, 2H, pyridyl
5.8.7. O -Acetoxymethyl-1-(4-ethylpiperazin-1-yl)diazen-
1
H NMR (CDCl
3
1-ium-1,2-diolate 4-(3-pyridyl)-1,4-dihydro-2,6-dimethyl-
3-nitropyridine-5-carboxylate (24). Yellow powder
(19%); mp 75–77 ꢁC; IR (KBr): 3308 (NH), 1763 (CO),
1649 (C@C), 1474 (NO
NMR (CDCl
1H, pyridyl H-2), 8.42 (dd, J5;6 ¼ 4:8, J4;6 ¼ 1:2 Hz, 1H,
pyridyl H-6), 7.74 (ddd, J4;5 ¼ 7:5, J2;4 ¼ 2:1, J4;6
1:2 Hz, 1H, pyridyl H-4), 7.24 (dd,
H-2, H-6), 7.62 (s, 1H, NH), 7.28 (d, Jortho ¼ 5.4 Hz, 2H,
pyridyl H-3, H-5), 5.76 (s, 2H, OCH O), 5.39 (s, 1H, H-
), 4.23–4.29 (m, 2H, CO CH ), 3.57–3.67 (m, 2H,
CH N), 3.00 (s, 3H, NCH ), 2.53 (s, 3H, C-2 CH ), 2.38
2
À1
1
4
2
2
2
), 1273, 742 (N–O) cm ; H
3
) d 8.55 (s, 1H, NH), 8.52 (d, J2;4 ¼ 2:1 Hz,
2
3
3
(
s, 3H, C-6 CH ), 2.11 (s, 3H, COCH ). Anal. Calcd for
3 3
C
4
19
H
24
N
9.05; H, 5.08; N, 17.88.
6
O
8
: C, 49.14; H, 5.21; N, 18.10. Found: C,
¼
J
4;5 ¼ 7:5,
2
J
5
3
5;6 ¼ 4:8 Hz, 1H, pyridyl H-5), 5.79 (s, 2H, OCH O),
.39 (s, 1H, H-4), 4.13–4.19 (m, 2H, CO CH ), 3.40–
2
2
.49 (m, 4H, piperazin-1-yl H-2, H-6), 2.56–2.64 (m, 6H,
CH N), 2.53 (s, 3H, C-2
CH ), 2.38 (s, 3H, C-6 CH ), 2.05 (s, 3H, COCH ).
2
piperazin-1-yl H-3, H-5, CH
2
2
5
.8.4. O -Acetoxymethyl-1-(N-methyl-N-ethylamino)dia-
zen-1-ium-1,2-diolate 4-(2-trifluoromethylphenyl)-1,4-di-
hydro-2,6-dimethyl-3-nitropyridine-5-carboxylate (21).
Yellow powder (36%); mp 91–93 ꢁC; IR (KBr): 3328
3
3
3
Anal. Calcd for C22 O
8.87. Found: C, 50.79; H, 5.79; N, 18.65.
H
29
N
7
8
: C, 50.86; H, 5.63; N,
1
(
(
1
7
2
NH), 1763 (CO), 1656 (C@C), 1494 (NO ), 1313, 776
À1
1
N–O) cm ; H NMR (CDCl ) d 7.51 (d, J ¼ 7:8 Hz,
3
3;4
2
5
1
3
.8.8. O -Acetoxymethyl-1-(4-ethylpiperazin-1-yl)diazen-
-ium-1,2-diolate 4-(4-pyridyl)-1,4-dihydro-2,6-dimethyl-
-nitropyridine-5-carboxylate (25). Yellow powder
H, phenyl H-3), 7.40–7.44 (m, 2H, phenyl H-5, H-6),
.27 (dd, J4:5 ¼ 6:9, J3;4 ¼ 7.8 Hz, 1H, phenyl H-4), 7.03
(
2
s, 1H, NH), 5.90 (s, 1H, H-4), 5.75 (s, 2H, OCH O),
0
.27–4.37 (m, 1H, CO CHH ), 4.07–4.15 (m, 1H,
2
(
(
29%); mp 180–182 ꢁC; IR (KBr): 3053 (NH), 1716
4
CO
À1
2 3
CO), 1602 (C@C), 1427 (NO ) cm ; H NMR (CDCl )
1
0
0
2
CHH ), 3.65–3.75 (m, 1H, CHH N), 3.49–3.57 (m,
0
d 8.46 (d, Jortho ¼ 5:4 Hz, 2H, pyridyl H-2, H-6), 7.60 (s,
1
CH ), 2.29 (s, 3H, C-6 CH ), 2.11 (s, 3H, COCH ).
H, CHH N), 2.97 (s, 3H, NCH
3
), 2.46 (s, 3H, C-2
1
5
2
6
H, NH), 7.24 (d, Jortho ¼ 5:4 Hz, 2H, pyridyl H-3, H-5),
.79 (s, 2H, OCH O), 5.36 (s, 1H, H-4), 4.21–4.27 (m,
H, CO CH ), 3.39–3.47 (m, 4H, piperazin-1-yl H-2, H-
3
3
3
Anal. Calcd for C21
1
H
24
F
3
N
3.18. Found: C, 47.21; H, 4.58; N, 13.06.
5
O
8
: C, 47.46; H, 4.55; N,
2
2
2
), 2.52–2.61 (m, 6H, piperazin-1-yl H-3, H-5,
CH N), 2.51 (s, 3H, C-2 CH ), 2.36 (s, 3H, C-6
CH ), 2.05 (s, 3H, COCH ). Anal. Calcd for
CH
2
2
3
3
3
2
5
.8.5. O -Acetoxymethyl-1-(N-methyl-N-ethylamino)dia-
C
50.79; H, 5.61; N, 18.63.
22
H
29
N
7
O
8
: C, 50.86; H, 5.63; N, 18.87. Found: C,
zen-1-ium-1,2-diolate 4-(benzofurazan-4-yl)-1,4-dihydro-
,6-dimethyl-3-nitropyridine-5-carboxylate (22). Yellow
powder (28%); mp 120–122 ꢁC; IR (KBr): 3348 (NH),
756 (CO), 1649 (C@C), 1474 (NO ), 1273, 803 (N–O)
) d 7.60 (d, J6;7 ¼ 8:7 Hz, 1H,
benzofurazan-4-yl H-7), 7.39 (d, J5;6 ¼ 6:3 Hz, 1H,
benzofurazan-4-yl H-5), 7.30 (dd, J6;7 ¼ 8:7, J5;6
:3 Hz, 1H, benzofurazan-4-yl H-6), 6.46 (s, 1H,
NH), 5.72 (s, 1H, H-4), 5.70 (s, 2H, OCH O), 4.12–4.17
2
2
1
2
5.8.9. O -Acetoxymethyl-1-(4-ethylpiperazin-1-yl)diazen-
1-ium-1,2-diolate
À1
1
cm ; H NMR (CDCl
3
4-(2-trifluoromethylphenyl)-1,4-dihy-
dro-2,6-dimethyl-3-nitropyridine-5-carboxylate (26). Yel-
low powder (26%); mp 70–72 ꢁC; IR (KBr): 3321 (NH),
¼
6
2
1763 (CO), 1649 (C@C), 1501 (NO ), 1320, 769 (N–O)
À1
1
cm ; H NMR (CDCl ) d 7.53 (d, J ¼ 7:5 Hz, 1H,
2
3
3;4
(
m, 2H, CO
2
CH
H, NCH ), 2.48 (s, 3H, C-2 CH
2
), 3.50–3.55 (m, 2H, CH
2
N), 2.95 (s,
), 2.29 (s, 3H, C-6
phenyl H-3), 7.40–7.44 (m, 2H, phenyl H-3, H-5), 7.26–
7.28 (m, 1H, phenyl H-4), 6.45 (s, 1H, NH), 5.93 (s, 1H,
H-4), 5.78 (s, 2H, OCH O), 4.21–4.25 (m, 1H,
3
CH ), 2.05 (s, 3H, COCH ). Anal. Calcd for
3
3
3
3
2
0
0
C
4
20
H
7.13; H, 4.68; N, 19.05.
23
N
7
O
9
: C, 47.53; H, 4.59; N, 19.40. Found: C,
CO CHH ), 4.06–4.19 (m, 1H, CO
piperazin-1-yl H-2, H-6), 2.54–2.56 (m, 6H, piperazin-1-
2
2
CHH ), 3.37 (m, 4H,

C. Vel ꢀa zquez, E. E. Knaus / Bioorg. Med. Chem. 12 (2004) 3831–3840
3839
yl H-3, H-5, CH
H, C-6 CH ), 2.12 (s, 3H, COCH
C H F N O : C, 49.15; H, 4.98; N, 14.33. Found: C,
2
CH
2
N), 2.48 (s, 3H, C-2 CH
3
), 2.33 (s,
ethyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (10) using
the reported procedures.
13
3
3
3
). Anal. Calcd for
24
29
3
6
8
4
8.99; H, 4.88; N, 14.13.
Acknowledgements
2
5
.8.10. O -Acetoxymethyl-1-(4-ethylpiperazin-1-yl)dia-
We are grateful to the Canadian Institutes of Health
Research for financial support of this research, and to
the National Council of Science and Technology (CO-
NACYT, Mexico) for a graduate scholarship to C.V.
zen-1-ium-1,2-diolate 4-(benzofurazan-4-yl)-1,4-dihydro-
,6-dimethyl-3-nitropyridine-5-carboxylate (27). Yellow
2
powder (28%); mp 85–88 ꢁC; IR (KBr): 3314 (NH), 1763
À1
(
CO), 1649 (C@C), 1501 (NO
2
), 1266, 803 (N–O) cm ;
1
H NMR (CDCl
furazan-4-yl H-7), 7.45 (d, J5;6 ¼ 6:6 Hz, 1H, benzo-
3
) d 7.70 (d, J6;7 ¼ 9:0 Hz, 1H, benzo-
furazan-4-yl H-5), 7.35 (dd, J6;7 ¼ 9:0, J ¼ 6.6 Hz, 1H,
5;6
benzofurazan-4-yl H-6), 6.66 (s, 1H, NH), 5.81 (s, 1H,
O), 4.16 (t, J ¼ 6:3 Hz, 2H,
References and notes
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