Syntheses, Calcium Channel Agonist-Antagonist Modulation Activities, and Nitric Oxide Release Studies of Nitrooxyalkyl 1,4-Dihydro-2,6-dimethyl-3-nitro-4-(2,1,3-benzoxadiazol-4-yl) pyridine-5-carboxylate Racemates, Enantiomers, and Diastereomers

Article abstract of DOI:10.1021/jm030333h

A novel group of hybrid calcium channel (CC) modulators was prepared where the isopropyl ester moiety of isopropyl 1,4-dlhydro-2,6-dimethyl-3-nitro-4-(2,1,3-benzoxadiazol-4-yl) pyridine-5-carboxylate (PN 202-791) was replaced by a variety of nitric oxide

Full text of DOI:10.1021/jm030333h

254
J . Med. Chem. 2004, 47, 254-261
Syn th eses, Ca lciu m Ch a n n el Agon ist-An ta gon ist Mod u la tion Activities, a n d
Nitr ic Oxid e Relea se Stu d ies of Nitr ooxya lk yl
1,4-Dih yd r o-2,6-d im eth yl-3-n itr o-4-(2,1,3-ben zoxa d ia zol-4-yl)p yr id in e-5-ca r boxyla te
Ra cem a tes, En a n tiom er s, a n d Dia ster eom er s
Rudong Shan, Carlos Velazquez, and Edward E. Knaus*
Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, T6G 2N8 Canada
Received J uly 10, 2003
A novel group of hybrid calcium channel (CC) modulators was prepared where the isopropyl
ester moiety of isopropyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-(2,1,3-benzoxadiazol-4-yl)pyridine-
5-carboxylate (PN 202-791) was replaced by a variety of nitric oxide (•NO) donor nitrooxyalkyl
ester substituents. Enantiomers, or diastereomers, having the (R)-configuration at the C-4
position of the 1,4-dihydropyridine ring (1,4-DHP) exhibited more potent in vitro CC antagonist
activity on guinea pig ileum longitudinal smooth muscle (GPILSM) than compounds having
the (4S)-configuration. None of the nitrooxyalkyl compounds exhibited a contraindicated CC
agonist effect on GPILSM that would cause smooth muscle contraction. Structure-activity
studies showed the enantiomers having the (S)-configuration at the C-4 position of the 1,4-
DHP ring or diastereomers having the a (4S)-configuration at the C-4 position of the 1,4-DHP
ring in conjunction with a (1R-)-1-methyl-2-nitrooxyethyl ester substituent exhibited the most
potent cardiac CC agonist (positive inotropic) activity on guinea pig left atrium (GPLA). This
class of compounds releases •NO in vitro that is enhanced by the presence of a thiol such as
N-acetylcysteamine. The novel •NO donor (-)-(S,R)-1-methyl-2-nitrooxyethyl 1,4-dihydro-2,6-
dimethyl-3-nitro-4-(2,1,3-benzoxadiazol-4-yl)pyridine-5-carboxylate [(-)-(S,R)-38], which acts
as a dual cardioselective calcium channel agonist (GPLA)/ smooth muscle selective calcium
channel antagonist (GPILSM), is a useful lead compound for drug discovery targeted to the
treatment of congestive heart failure, and it provides a useful research probe to study the
structure-function relationship of calcium channels.
In tr od u ction
The design of tissue selective 1,4-dihydropyridine (1,4-
DHP) calcium channel (CC) agonists to treat congestive
heart failure (CHF) necessitates removal of their con-
traindicated smooth muscle vasoconstrictor effect while
the target cardiac positive inotropic action is main-
tained.1 In this regard, racemic methyl 1,4-dihydro-2,6-
dimethyl-3-nitro-4-(2-trifluoromethylphenyl)pyridine-5-
carboxylate (Bay K8644) produces a CC agonist effect
on both smooth and cardiac muscle since the agonist
F igu r e 1. Structures for the (-)-(S)-1 and (+)-(R)-3 enantio-
mers of Bay K8644, the (-)-(S)-2 and (+)-(R)-4 enantiomers
(-)-(S)-enantiomer (1) is about 10-fold more potent
relative to the antagonist (+)-(R)-enantiomer (3).2 In a
previous study, hybrid 1,4-DHP CC modulators were
prepared where the methyl ester substituent of Bay
K8644 was replaced by a 2-nitrooxyethyl ester moiety,
which also have the potential to simultaneously release
nitric oxide (•NO).3 This hybrid design concept was
based on observations that •NO is an endogenous
activator of guanylate cyclase that is responsible for
vascular relaxation and that organic nitrovasodilators
elicit their effect in vivo by bypassing the •NO-produc-
tion system in the endothelium to deliver •NO directly
to muscle cells in the artery. Accordingly, the (-)-(S)-2
2-nitrooxyethyl enantiomer, like (-)-(S)-Bay K8644 (1),
acts as a cardiac CC agonist, but unlike (-)-(S)-Bay
K8644, (-)-(S)-2 is a smooth muscle CC antagonist that
also acts as a •NO donor in vitro (see structures in
of 2-nitrooxyethyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-triflu-
oromethylphenyl)pyridine-5-carboxylate, and PN 202-791 (5).
Figure 1). In contrast, the 2-nitrooxyethyl (+)-(R)-
enantiomer (4) exhibited a CC antagonist effect on both
cardiac and smooth muscle. The dual cardioselective CC
agonist/ smooth muscle selective CC antagonist actions
of the 2-nitrooxyethyl (-)-(S)-2 enantiomer fulfill the
clinical requirements for the treatment of CHF. The two
enantiomers of PN 202-791 (5) also show differences in
CC modulation effects where the (-)-(R)-enantiomer is
100-fold more potent as an antagonist compared to the
agonist (+)-(S)-enantiomer.4 In our ongoing program to
acquire CC modulation structure-activity relationships
and to design compounds to study structure-function
relationships with respect to CC modulation, we now
report the syntheses of nitrooxyalkyl 1,4-dihydro-2,6-
dimethyl-3-nitro-4-(2,1,3-benzoxadiazol-4-yl)pyridine-5-
carboxylate racemates, enantiomers, and/or diastereo-
* To whom correspondence should be addressed. Phone: 780-492-
5993. Fax: 780-492-1217. E-mail: eknaus@pharmacy.ualberta.ca.
10.1021/jm030333h CCC: $27.50 © 2004 American Chemical Society
Published on Web 12/09/2003

Novel Hybrid Calcium Channel Modulators
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1 255
Sch em e 1a
Sch em e 3a
a
Reagents and conditions: (a) 70% HNO3, 95% H2SO4, 25 °C,
1 h; (b) AgNO3, MeCN, 80 °C, 3 h.
Sch em e 2a
a
Reagents and conditions: (a) EtOH, 80 °C, 17 h; (b) 1,8-
diazabicyclo[5.4.0]undec-7-ene (DBU), MeOH, 25 °C, 4 h; (c)
BrCHMe2 for (+)-(S)-5 and (-)-(R)-5, or BrCH2CH2ONO2 (14) for
(+)-(S)-35 and (-)-(R)-35, DMF, K2CO3, 25 °C, 36 h for (+)-(S)-5
and (-)-(R)-5 and 24 h for (+)-(S)-35 and (-)-(R)-35.
34, by fractional crystallization of the (-)-cinchonidine
or (+)-cinchonine salts using a procedure similar to that
reported by Shibanuma et al.5 for the synthesis of
nicardipine enantiomers was not successful, since the
racemic acid 22 was extremely insoluble in solvents such
as EtOAc, EtOH, or MeOH. An alternate synthetic
strategy was therefore investigated, since it was ex-
pected that use of a chiral esterification group such as
D-threonine, which could be efficiently removed by a
â-elimination mechanism using a nonnucleophilic base
such as 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), would
be a desirable derivative for separation of the diaster-
eomeric esters (32, 33) that could be converted to the
respective target chiral acids (+)-(S)-34 and (-)-(R)-34.6
Accordingly, the modified Hantzsch condensation of
nitroacetone (29), 2,1,3-benzoxadiazol-4-carboxaldehyde
(30), and (1S,2R)-2-(3,5-dinitrophenylcarbonylamino)-
2-methoxycarbonyl-1-methylethyl 3-aminocrotonate (31)
afforded a mixture of the two diastereomers 32 and 33
that differ in configuration (S or R) at the C-4 position
of the 1,4-DHP ring (see Scheme 3). Silica gel column
separation of diastereomers 32 and 33 and then cleav-
age of the individual esters using DBU at 25 °C yielded
the individual (+)-(S)-34 and (-)-(R)-34 carboxylate
enantiomers. The absolute configuration for (+)-(S)-34
and (-)-(R)-34 was determined by their conversion to
the respective (+)-(S)-5 and (-)-(R)-5 isopropyl esters
for which the absolute configuration and optical rotation
are known.7 Reaction of (+)-(S)-34 or (-)-(R)-34 with
BrCH2CH2ONO2 in the presence of K2CO3 in DMF
afforded the respective (+)-(S)-35 or (-)-(R)-35 enan-
tiomer of 2-nitrooxyethyl 1,4-dihydro-2,6-dimethyl-3-
a
Reagents and conditions: (a) O2NO-X-Br (14, 15, 16, 18),
DMF, K2CO3, 25 °C, 24 h; (b) O2NO-X-Br (17), DMF, K2CO3,
25 °C, 7 days; (c) SOCl2, DMF-CH2Cl2, HOCH(CH2ONO2) (21),
0 °C, 6 h.
mers (23-28, 35-39), their in vitro CC modulating
effects on smooth and cardiac muscle, and •NO release
data.
Ch em istr y
Nitration of the bromoalkanols (6-12) using a mix-
ture of 70% HNO3 and 95% H2SO4 afforded the respec-
tive nitrooxyalkyl bromides (14-20) in 83-99% yield.
In contrast, 1,3-dinitrooxy-2-propanol (21) was prepared
in 96% yield by reaction of 1,3-dibromo-2-propanol (13)
with AgNO3 in MeCN at 80 °C, as illustrated in Scheme
1.
Condensation of the nitrooxyalkyl bromides (14-18)
with racemic 1,4-dihydro-2,6-dimethyl-3-nitro-4-(2,1,3-
benzoxadiazol-4-yl)pyridine-5-carboxylate (22) in the
presence of K2CO3 afforded the respective nitrooxyalkyl
ester product as a racemate (23-25) or as a mixture of
diastereomers (26, 27). The racemic 1,3-dinitrooxy-2-
propyl ester (28) was synthesized by conversion of the
racemic acid (22) to the acid chloride using SOCl2 and
then condensation with 1,3-dinitrooxy-2-propanol (21)
as illustrated in Scheme 2.
Attempts to resolve the racemic acid 22, to acquire
the individual acid enantiomers (+)-(S)-34 and (-)-(R)-

256 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1
Shan et al.
Sch em e 4a
moieties in place of the isopropyl ester substituent
present in isopropyl 1,4-dihydro-2,6-dimethyl-3-nitro-
4-(2,1,3-benzoxadiazol-4-yl)pyridine-5-carboxylate (5, PN
202-791) were synthesized. Hybrid agents of this type
that release the smooth muscle vasorelaxant •NO
constitute a useful drug design strategy to eliminate the
contraindicated smooth muscle vasoconstrictor effect
exhibited by the (+)-(S)-enantiomer of PN 202-791 while
its useful cardiac positive inotropic (calcium agonist)
effect is maintained. Additional support for this concept
is based on reports that •NO modulates the activity of
Ca2+ release channels by preventing oxidation of regu-
latory sulfhydryls,8 and replacement of the methyl group
of the ester substituent of (-)-(S)-Bay K8644 by a
2-nitrooxyethyl •NO donor substituent abolished the
undesirable calcium channel agonist effect of (-)-(S)-
Bay K8644 on vascular smooth muscle.3
The in vitro calcium channel modulating activities of
the nitrooxyalkyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-
(2,1,3-benzoxadiazol-4-yl)pyridine-5-carboxylates (23-
28, 35-39) were determined using guinea pig ileum
longitudinal smooth muscle (GPILSM) and guinea pig
left atrium (GPLA) assays.6 The molar concentration of
the antagonist compound required to produce 50%
inhibition of GPILSM Ca2+-dependent contractility
(IC50) induced by the muscarinc agonist carbachol
(1.6 × 10-7 M) are presented in Table 1. Structure-
activity data for CC antagonist activity showed that the
racemic (23-25, 28) and diastereomeric (26, 27) ni-
trooxyalkyl derivatives were weaker CC antagonists
(IC50 values in the 6.38 × 10-6 to 2.41 × 10-7 M range)
than the reference drugs nifedipine (IC50 ) 1.40 × 10-8
M) or racemic PN 202-791 (IC50 ) 4.0 × 10-8 M).
Nitrooxyalkyl substituent chain length [(CH2)nONO2]
had a variable effect on CC antagonist activity, where
the relative potency order was 24 (n ) 3) > 25 (n ) 4)
> 23 (n ) 2). The number of nitrooxy moieties present
in the ester substituent was not a determinant of
activity, since 23 [(CH2)2ONO2] and 28 [CH(CH2ONO2)2]
were equipotent CC antagonists. The point of attach-
ment of a methyl group in a nitrooxyethyl ester sub-
stituent had a modest effect, where the relative potency
order was CH2CH(Me)ONO2 (27) > CH(Me)CH2ONO2
(26). The configuration at the C-4 position of the 1,4-
DHP ring was an important determinant of CC antago-
nist activity, since compounds having the (4R)-config-
uration were always more potent (63-194-fold) than the
corresponding (4S)-enantiomer [(-)-(R)-35 > (+)-(S)-35]
or diastereomer [(-)-(R,R)-37 > (+)-(S,S)-36; (+)-(R,S)-
39 > (-)-(S,R)-38]. It is also highly relevant that none
of these nitrooxyalkyl compounds exhibited a contra-
indicated CC agonist effect on GPILSM that would
produce smooth muscle vasoconstriction.
a
Reagents and conditions: (a) dry DMF, K2CO3, 25 °C, 7 days.
nitro-4-(2,1,3-benzoxadiazol-4-yl)pyridine-5-carboxyl-
ate. Each enantiomer [(+)-(S)-5, (-)-(R)-5, (+)-(S)-35,
(-)-(R)-35] exhibited a single resonance for the dihy-
dropyridine C-6 methyl resonance upon addition of the
1H NMR chiral shift reagent (+)-Eu(hfc)3, indicating a
very high optical purity (g96% ee).
1-Methyl-2-nitrooxyethyl 1,4-dihydro-2,6-dimethyl-3-
nitro-4-(2,1,3-benzoxadiazol-4-yl)pyridine-5-carboxyl-
ate (26), prepared by reaction of racemic 22 with racemic
2-bromo-1-nitrooxypropane (17) as illustrated in Scheme
2, possesses two asymmetric centers, which resulted in
the formation of four diastereomeric products (SS, RR,
SR, RS). The four diastereomeric compounds (+)-(S,S)-
36, (-)-(R,R)-37, (-)-(S,R)-38, and (+)-(R,S)-39 have
been prepared by reaction of the optically pure acid (+)-
(S)-34 or (-)-(R)-34 with a chiral 2-bromo-1-nitrooxy-
propane [(-)-(R)-19 or (+)-(S)-20] in DMF at 25 °C as
illustrated in Scheme 4. This SN2 reaction, which occurs
with inversion of configuration at the asymmetric center
in the 2-bromo-1-nitrooxypropane [(-)-(R)-19 or (+)-(S)-
20], is favored when a relatively unhindered alkyl
halide, strong nucleophile, polar solvent, and high
concentration of nucleophile are employed. The 1H NMR
spectra of diastereomers 36-39 exhibited a single set
of resonances [diastereomer excess (de) >96%] relative
to the diastereomeric mixture (26), which exhibited dual
resonances.
The structure-activity data acquired (see Table 1) for
cardiac CC agonist (positive inotropic) activity on GPLA
showed that some members of this group of nitrooxy-
alkyl compounds, with the exception of the inactive
agents (-)-(R)-35 and (+)-(R,S)-39, exhibit appreciable
cardiac CC agonist activity (EC50 values in the 9.15 ×
10-5 to 1.56 × 10-7 M range) relative to the reference
drug racemic PN 202-791 (EC50 ) 9.4 × 10-6 M). The
relative cardiac CC agonist potency order with respect
to nitrooxyalkyl substituent chain length [(CH2)nONO2]
was n ) 4 (25) > n ) 3 (24) and n ) 2 (23). The point
Resu lts a n d Discu ssion
A group of Hantzsch 1,4-dihydropyridines having a
variety of nitrooxyalkyl ester nitric oxide (•NO) donor

Novel Hybrid Calcium Channel Modulators
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1 257
Ta ble 1. In Vitro Calcium Channel Modulation Activities for Nitrooxyalkyl
1,4-Dihydro-2,6-dimethyl-3-nitro-4-(2,1,3-benzoxadiazol-4-yl)pyridine-5-carboxylates (23-28, 35-39)
calcium channel
calcium channel agonist
activity (cardiac inotropy)
GPLA: EC50 (M)b
antagonist activity
compound
rac-23
R
GPILSM: IC50 (M)a
(CH2)2ONO2
(CH2)3ONO2
(CH2)4ONO2
CH(Me)CH2ONO2
CH2CH(Me)ONO2
CH(CH2ONO2)2
(CH2)2ONO2
3.89 ( 0.25 × 10-6
2.41 ( 0.30 × 10-7
8.78 ( 1.02 × 10-7
6.38 ( 0.52 × 10-6
1.70 ( 2.30 × 10-6
3.01 ( 0.51 × 10-6
2.24 ( 0.17 × 10-5
1.15 ( 0.09 × 10-7
3.65 ( 0.05 × 10-5
5.77 ( 0.82 × 10-7
9.43 ( 0.80 × 10-6
5.96 ( 0.50 × 10-8
1.40 ( 0.19 × 10-8
4.0 ( 0.07 × 10-8
3.11 ( 2.31 × 10-5
5.23 ( 4.92 × 10-5
4.03 ( 0.55 × 10-6
1.86 ( 0.59 × 10-6
9.72 ( 4.19 × 10-7
9.15 ( 9.07 × 10-5
3.13 ( 2.30 × 10-6
inactive
rac-24
rac-25
26c
27c
rac-28
(+)-(S)-35
(-)-(R)-35
(+)-(S,S)-36
(-)-(R,R)-37
(-)-(S,R)-38
(+)-(R,S)-39
nifedipine
rac-PN 202-791
(CH2)2ONO2
(S)-CH(Me)CH2ONO2
(R)-CH(Me)CH2ONO2
(R)-CH(Me)CH2ONO2
(S)-CH(Me)CH2ONO2
1.92 ( 0.29 × 10-6
3.37 ( 3.37 × 10-5
1.56 ( 0.19 × 10-7
inactive
-
CH(Me)2
9.4 ( 2.6 × 10-6
a
The molar concentration of the test compound causing a 50% decrease in the slow component or tonic contractile response (IC50
(
SEM, n ) 3) in guinea pig ileum longitudinal smooth muscle (GPILSM) induced by the muscarinic agonist carbachol (1.6 × 10-7 M) was
b
determined graphically from the dose-response curves. The molar concentration of the test compound causing a 50% increase in the
cardiac contractile force (EC50 ( SEM, n ) 3) in guinea pig left atrium (GPLA) was determined graphically from the dose-response
curves. c Mixture of diastereomers.
Ta ble 2. Nitric Oxide Release Data for Nitrooxyalkyl
1,4-Dihydro-2,6-dimethyl-3-nitro-4-(2,1,3-benzoxadiazol-4-yl)-
pyridine-5-carboxylates (23-28)
of attachment of a Me group to the nitrooxyethyl chain
in diastereomers 26 [R ) CH(Me)CH2ONO2] and 27
[R ) CH2CH(Me)ONO2] was not a determinant of
% nitric oxide releaseda
activity, since their <2-fold difference in cardiac CC
absence of
presence of
agonist potency is small. All four possible diastereomers
36-39 (SS, RR, SR, RS) of the compound having a
1-methyl-2-nitrooxyethyl substituent were prepared to
determine the effect of configuration at the C-4 position
of the 1,4-DHP ring and the C-1 position of the 1-meth-
yl-2-nitrooxyethyl moiety on cardiac CC agonist activity.
The configuration at the C-4 position of the 1,4-DHP is
a major determinant of activity, since compounds having
the (4S)-configuration are much more potent than the
corresponding (4R)-enantiomer [(+)-(S)-35 . inactive
(-)-(R)-35] or diastereomer [(+)-(S,S)-36 . inactive (+)-
(R,S)-39; (-)-(S,R)-38 > (-)-(R,R)-37]. In contrast, di-
astereomers having a (1R)-1-methyl-2-nitrooxyethyl
configuration were more potent than the corresponding
diastereomers having a (1S)-1-methyl-2-nitrooxyethyl
configuration [(-)-(S,R)-38 > (+)-(S,S)-36; (-)-(R,R)-37
. inactive (+)-(R,S)-39]. These latter structure-activity
correlations are consistent with the observation that (-)-
(S,R)-1-methyl-2-nitrooxyethyl 1,4-dihydro-2,6-dimethyl-
3-nitro-4-(2,1,3-benzoxadiazol-4-yl)pyridine-5-carboxyl-
ate [(-)-(S,R)-38] is the most potent cardiac CC agonist,
being about 60-fold more active than racemic PN 202-
791 (EC50 ) 9.4 × 10-6 M).
N-acetyl-
N-acetyl-
compound
23
24
25
26
27
28
R
cysteamineb cysteaminec
(CH2)2ONO2
(CH2)3ONO2
(CH2)4ONO2
CH(Me)CH2ONO2
H2CH(Me)ONO2
CH(CH2ONO2)2
1.08
0.00
0.00
1.84
0.58
0.19
0.18
2.71
1.31
1.10
1.53
1.74
1.02
5.30
glycerol
trinitrated
a
Percent nitric oxide released (mean value, n ) 3) from one
nitrooxy (ONO2) group present in the test compound. Variation
from the mean % value was e0.02%. Incubated in the absence
of N-acetylcysteamine in phosphate buffer (pH 7.4) at 37 °C for 1
h. c Incubated in the presence of N-acetylcyteamine (1 equiv per
ONO2 moiety) in phosphate buffer (pH 7.4) at 37 °C for 1 h.
b
d
Glycerol trinitrate ) O2NOCH2CH(ONO2)CH2ONO2.
effect on GPILSM is attributed to their ability to release
•NO. In contrast, (+)-(S)-PN 202-791 acts as a CC
agonist on vascular smooth muscle.4
Con clu sion s
The nitrooxyalkyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-
(2,1,3-benzoxadiazol-4-yl)pyridine-5-carboxylate race-
mates, enantiomers, and diastereomers invesitigated
constitute a novel group of nitric oxide donor compounds
with desirable calcium channel modulation activities.
In particular, the 2-nitrooxyethyl ester enantiomer (+)-
(S)-35, having the (S)-configuration at the C-4 position
of the 1,4-dihydropyridine ring, or the (-)-(S,R)-38
diastereomer, also having the (S)-configuration at the
1,4-DHP C-4 position in conjunction with a 1-methyl-
In vitro •NO release was determined by quantitation
of nitrite using the Griess reaction,9 and the results are
listed in Table 2. The percentage •NO released was
generally greater in the presence of N-acetylcysteamine
than in the absence of N-acetylcysteamine. This •NO
release data is consistent with reports that •NO release
from organic nitrates is facilitated by thiols.10 A plau-
sible explanation for the observation that the nitrooxy-
alkyl compounds described do not induce a CC agonist

258 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1
Shan et al.
(+)-(S)-2-Br om o-1-n itr ooxyp r op a n e (20): 83% yield; oil;
D
2-nitrooxyethyl ester substituent having the (R)-config-
uration at its C-1 position, exhibit distinctive profiles
in vitro that encompass a dual cardioselective agonist/
smooth muscle selective antagonist activity that could
provide a potentially new approach to drug design
directed toward the treatment of congestive heart
failure. The (+)-(S)-35 enantiomer and the (-)-(S,R)-
38 diastereomer are potentially valuable probes to
investigate the structure-function relationship of cal-
cium channel modulation.
1
[R]23 ) +17.5° (c 0.4 in CH2Cl2); H NMR (CDCl3) δ 1.76 (d,
J ) 6.7 Hz, 3H, CH3), 4.23 (ddq, J ) 6.4, 6.7, 6.7 Hz, 1H,
BrCH), 4.54 (dd, J ) 11.9, 6.7, 1H, CHH′ONO2), 4.70 (dd, J )
11.9, 6.4, 1H, CHH′ONO2). Compound 20 was used im-
mediately for the syntheses of compounds (-)-(R,R)-37 and (-)-
(S,R)-38.
1,3-Din itr ooxy-2-p r op a n ol (21). A solution of 1,3-dibromo-
2-propanol (13, 9.13 g, 41.89 mmol), AgNO3 (25.48 g, 150
mmol), and acetonitrile (150 mL) was heated at 80 °C for 3 h
with stirring. The solution was cooled to 25 °C, the solvent
was removed in vacuo, and water (200 mL) was added. The
precipitated AgBr was removed by filtration, the filtrate was
extracted with ether (3 × 500 mL), the extract was dried
(NaSO4), and the solvent was removed in vacuo to give 21 (7.32
g, 96% yield) as an oil: 1H NMR (CDCl3) δ 2.78 (br s 1H, OH),
4.31 (m, 1H, H-2), 4.54 (dd, J ) 6.1, 11.7 Hz, 2H, CH2ONO2),
4.61 (dd, J ) 4.5, 11.7 Hz, 2H, CH2ONO2). Compound 21 was
used immediately for the syntheses of compound 28.
Gen er a l Meth od for th e P r ep a r a tion of Nitr ooxya lk yl
1,4-Dih yd r o-2,6-d im eth yl-3-n itr o-4-(2,1,3-ben zoxa d ia zol-
4-yl)p yr id in e-5-ca r boxyla tes (23-27). A solution of the
respective nitrooxyalkyl bromide (14-18, 0.22 mmol), 22 (0.20
mmol), and K2CO3 (0.24 mmol) in dry DMF (10 mL) was stirred
at 25 °C for 24 h (7 days for preparation of 27). Water (40 mL)
was added, the mixture was extracted with EtOAc (3 × 300
mL), the extract was washed with water (2 × 40 mL) and then
brine (40 mL), the organic phase was dried (Na2SO4), and the
solvent was removed in vacuo. The residue was purified by
silica gel column chromatography using EtOAc-hexane (1:1,
v/v) as eluent. Physical and spectral data for 23-27 are listed
below.
Exp er im en ta l Section
Gen er a l. Melting points were recorded with a Thomas-
Hoover capillary apparatus and are uncorrected. 1H NMR
spectra were recorded on a Bruker AM-300 spectrometer (300
MHz). The assignment of exchangeable protons (NH, NH2, OH)
was confirmed by the addition of D2O. Optical rotations were
measured on a Perkin-Elmer 241 polarimeter. Infrared spectra
were acquired using a Nicolet IR-500 Series II spectrometer.
Silica gel column chromatography was carried out using Merck
7734 (60-200 mesh) silica gel. Microanalyses were within
(0.4% of theoretical values for all elements listed, unless
otherwise stated. 2-Bromoethanol, 3-bromo-1-propanol, iso-
propyl bromide, 1,3-dibromo-2-propanol, 1-bromo-2-propanol,
N-acetylcysteamine, and tris[3-(heptafluoropropylhydroxy-
methylene)-(+)-camphorato]europium(III) [Eu(hfc)3] were pur-
chased from the Aldrich Chemical Co. Nitroacetone,11 2-amino-
1-nitroprop-1-ene,12 nitroglycerin (glycerol trinitrate),13 2,1,3-
benzoxadiazol-4-carboxaldehyde,14 4-bromo-1-butanol,15 and
(S)- and (R)-2-bromo-1-propanol16 were prepared according to
the reported procedures.
2-Nit r ooxyet h yl 1,4-d ih yd r o-2,6-d im et h yl-3-n it r o-4-
(2,1,3-ben zoxadiazol-4-yl)pyr idin e-5-car boxylate (23): 91%
yield; yellow oil; IR (CHCl3) 1313, 1379, and 1488 (NO2), 1637
Gen er a l Meth od for th e P r ep a r a tion of Nitr ooxya lk yl
Br om id es (14-20). A bromo alcohol (6-12, 10 mmol) was
added dropwise to a solution of 70% HNO3 (1.1 mL) and 95%
H2SO4 (2.4 mL) at 0 °C, and the reaction was allowed to
proceed at the same temperature for 1 h with stirring. The
resulting suspension was poured into water (50 mL), extracted
with CH2Cl2 (3 × 200 mL), and dried (MgSO4), and the solvent
was removed to give the respective nitrooxyalkyl bromide (14-
1
(CdN), 1710 (CO2), 3324 (NH) cm-1; H NMR (CDCl3) δ 2.37
(s, 3H, C-6 Me), 2.56 (s, 3H, C-2 Me), 4.40-4.25 (m, 2H,
CO2CH2), 4.61 (t, J ) 4.5 Hz, 2H, CH2ONO2), 5.79 (s, 1H, H-4),
6.47 (br s, 1H, NH), 7.36 (dd, J ) 9.0, 6.5 Hz, 1H, 4-benzo-
furazanyl H-6), 7.46 (d, J ) 6.5 Hz, 1H, 4-benzofurazanyl H-5),
7.69 (d, J ) 9.0 Hz, 1H, 4-benzofurazanyl H-7). Anal.
(C16H15N5O8) C, H, N.
1
20). Some physical and H NMR spectral data for compounds
14-20, which were used immediately for the subsequent
syntheses of the nitrooxyalkyl 1,4-dihydro-2,6-dimethyl-3-
nitro-4-(2,1,3-benzoxadiazol-4-yl)pyridine-5-carboxylates (23-
27, 35-39), are listed below.
3-Nitr ooxyp r op yl 1,4-d ih yd r o-2,6-d im eth yl-3-n itr o-4-
(2,1,3-ben zoxadiazol-4-yl)pyr idin e-5-car boxylate (24): 93%
yield; yellow oil; IR (CHCl3) 1313, 1382, and 1489 (NO2), 1631
1
(CdN), 1706 (CO2), 3320 (NH) cm-1; H NMR (CDCl3) δ 2.02
2-Nitr ooxyeth yl br om id e (14): 94% yield; oil; 1H NMR
(CDCl3) δ 3.56 (t, J ) 6.4 Hz, 2H, H-1), 4.76 (t, J ) 6.4, 2H,
H-2). Compound 14 was used immediately for the syntheses
of compounds 23, (+)-(S)-35, and (-)-(R)-35.
(p, J ) 6.1 Hz, 2H, CH2CH2ONO2), 2.38 (s, 3H, C-6 Me), 2.56
(s, 3H, C-2 Me), 4.16 (t, J ) 6.1 Hz, 2H, CO2CH2), 4.40 (t, J )
6.1 Hz, 2H, CH2ONO2), 5.81 (s, 1H, H-4), 6.54 (br s, 1H, NH),
7.35 (dd, J ) 9.0, 6.5 Hz, 1H, 4-benzofurazanyl H-6), 7.44 (d,
J ) 6.5 Hz, 1H, 4-benzofurazanyl H-5), 7.70 (d, J ) 9.0 Hz,
1H, 4-benzofurazanyl H-7). Anal. (C17H17N5O8) C, H, N.
1
3-Nitr ooxyp r op yl br om id e (15): 99% yield; oil; H NMR
(CDCl3) δ 2.28 (p, J ) 6.1 Hz, 2H, H-2), 3.49 (t, J ) 6.1, 2H,
BrCH2), 4.62 (t, J ) 6.1, 2H, CH2ONO2). Compound 15 was
used immediately for the syntheses of compound 24.
4-Nit r ooxyb u t yl 1,4-d ih yd r o-2,6-d im et h yl-3-n it r o-4-
(2,1,3-ben zoxadiazol-4-yl)pyr idin e-5-car boxylate (25): 82%
yield; yellow oil; IR (CHCl3) 1312, 1380, and 1489 (NO2), 1628
4-Nitr ooxybu tyl br om id e (16): 84% yield; oil; 1H NMR
(CDCl3) δ 1.87-2.04 (m, 4H, BrCH2CH2CH2), 3.45 (t, J ) 6.0,
2H, BrCH2), 4.50 (t, J ) 6.0, 2H, CH2ONO2). Compound 16
was used immediately for the syntheses of compound 25.
2-Br om o-1-n itr ooxyp r op a n e (17): 96% yield; oil; 1H NMR
(CDCl3) δ 1.76 (d, J ) 6.7 Hz, 3H, CH3), 4.23 (ddq, J ) 6.4,
6.7, 6.7 Hz, 1H, BrCH), 4.54 (dd, J ) 11.9, 6.7, 1H, CHH′ONO2),
4.70 (dd, J ) 11.9, 6.4, 1H, CHH′ONO2). Compound 17 was
used immediately for the syntheses of compound 26.
1
(CdN), 1702 (CO2), 3318 (NH) cm-1; H NMR (CDCl3) δ 1.69
(m, 4H, CH2CH2CH2ONO2), 2.37 (s, 3H, C-6 Me), 2.55 (s, 3H,
C-2 Me), 4.08 (m, 2H, CO2CH2), 4.42 (t, J ) 5.50 Hz, 2H, CH2-
ONO2), 5.80 (s, 1H, H-4), 6.39 (br s, 1H, NH), 7.35 (dd, J )
8.8, 6.1 Hz, 1H, 4-benzofurazanyl H-6), 7.43 (d, J ) 6.1 Hz,
1H, 4-benzofurazanyl H-5), 7.70 (d, J ) 8.8 Hz, 1H, 4-benzo-
furazanyl H-7). Anal. (C18H19N5O8) C, H, N.
1-Meth yl-2-n itr ooxyeth yl 1,4-d ih yd r o-2,6-d im eth yl-3-
n it r o-4-(2,1,3-b en zoxa d ia zol-4-yl)p yr id in e-5-ca r b oxyl-
a te (26): 45% yield; yellow oil (mixture of diastereomers); IR
(CHCl3) 1312, 1381, and 1489 (NO2), 1637 (CdN), 1708 (CO2),
3321 (NH) cm-1; 1H NMR (CDCl3) δ 1.10 and 1.33 (two d, J )
6.7 Hz, 3H total, CO2CHCH3), 2.36 and 2.38 (two s, 3H total,
C-6 Me), 2.55 and 2.56 (two s, 3H total, C-2 Me), 4.21-4.62
(m, 2H, CH2ONO2), 5.13-5.22 (m, 1H, CO2CH), 5.78 and 5.80
(two s, 1H, H-4), 6.53 and 6.54 (two br s, 1H total, NH), 7.32-
7.38 (m, 1H, 4-benzofurazanyl H-6), 7.44 (d, J ) 6.4 Hz, 1H,
4-benzofurazanyl H-5), 7.69 and 7.70 (two d, J ) 8.8 Hz, 1H
total, 4-benzofurazanyl H-7). Anal. (C17H17N5O8) C, H, N.
1-Br om o-2-n itr ooxyp r op a n e (18): 98% yield; oil; 1H NMR
(CDCl3) δ 1.48 (d, J ) 6.0 Hz, 3H, CH3), 3.49 (d, J ) 6.0 Hz,
2H, BrCH2), 5.25 (m, 1H, CH3CH). Compound 18 was used
immediately for the syntheses of compound 27.
(-)-(R)-2-Br om o-1-n itr ooxyp r op a n e (19): 83% yield; oil;
1
[R]23 ) -17.5° (c 0.4 in CH2Cl2); H NMR (CDCl3) δ 1.76 (d,
D
J ) 6.7 Hz, 3H, CH3), 4.23 (ddq, J ) 6.4, 6.7, 6.7 Hz, 1H,
BrCH), 4.54 (dd, J ) 11.9, 6.7, 1H, CHH′ONO2), 4.70 (dd, J )
11.9, 6.4, 1H, CHH′ONO2). Compound 19 was used im-
mediately for the syntheses of compounds (+)-(S,S)-36 and (+)-
(R,S)-39.

Novel Hybrid Calcium Channel Modulators
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1 259
2-Meth yl-2-n itr ooxyeth yl 1,4-d ih yd r o-2,6-d im eth yl-3-
n it r o-4-(2,1,3-b en zoxa d ia zol-4-yl)p yr id in e-5-ca r b oxyl-
a te (27): 65% yield; yellow oil (mixture of diastereomers); IR
(CHCl3) 1219, 1279, and 1535 (NO2), 1642 (CdN), 1736 (Cd
O), 3314 (NH) cm-1; 1H NMR (CDCl3) δ 1.27 and 1.37 (two d,
J ) 6.4 Hz, 3H total, MeCHONO2), 2.38 (s, 3H, C-6 Me), 2.56
(s, 3H, C-2 Me), 4.05-4.34 (m, 2H, CO2CH2), 5.19-5.31 (m,
1H, MeCHONO2), 5.77 (s, 1H, H-4), 6.46 (br s, 1H, NH), 7.34
(two overlapping dd, J ) 8.8, 8.2 Hz, 1H total, 4-benzofura-
zanyl H-6), 7.45 and 7.47 (two d, J ) 8.2 Hz, 1H total,
4-benzofurazanyl H-5), 7.70 (d, J ) 8.8 Hz, 1H, 4-benzofura-
zanyl H-7). Anal. (C17H17N5O8) C, H, N.
furazanyl H-5), 7.50 (dd, J ) 8.8, 6.7 Hz, 1H, 4-benzofurazanyl
H-6), 7.83 (d, J ) 8.8 Hz, 1H, 4-benzofurazanyl H-7), 9.00 (dd,
J ) 2.1, 2.1 Hz, 1H, dinitrophenyl H-4), 9.07 (d, J ) 2.1 Hz,
2H, dinitrophenyl H-2 and H-6), 9.66 (d, J ) 7.9 Hz, 1H,
CHNH), 9.86 (br s, 1H, dihydropyridyl NH). Anal. (C26H23N7O12)
C, H, N.
1,4-Dih yd r o-2,6-d im eth yl-3-n itr o-4-(2,1,3-ben zoxa d ia -
zol-4-yl)p yr id in e-5-ca r boxylic Acid En a n tiom er s [(+)-(S)-
34 a n d (-)-(R)-34]. A solution of either 32 or 33 (5.00 g, 8.0
mmol) and DBU (3.65 g, 24.0 mmol) in MeOH (200 mL) was
stirred at 25 °C for 24 h. The solvent was removed in vacuo
and water (250 mL) was added. The water layer was washed
with ether (3 × 500 mL), acidified to pH 2 with 1 N HCl, and
extracted with EtOAc (3 × 500 mL). The combined extracts
were dried Na2SO4), and the solvent was removed in vacuo to
give the crude product as a brownish oil which was purified
by silica gel column chromatography using EtOAc-hexane (2:
1, v/v) as eluent. Recrystallization from EtOH-ether afforded
the respective product [(+)-(S)-34 or (-)-(R)-34] as yellow
crystals.
1,3-Din itr ooxy-2-p r op yl 1,4-d ih yd r o-2,6-d im eth yl-3-n i-
tr o-4-(2,1,3-ben zoxadiazol-4-yl)pyr idin e-5-car boxylate (28):
A mixture of 22 (94.8 mg, 0.30 mmol), SOCl2 (89.2 mg, 0.75
mmol), DMF (1 mL), and CH2Cl2 (5 mL) was stirred at 0 °C
for 2 h, and to this suspension a mixture of 21 (60.1 mg, 0.33
mmol) in CH2Cl2 (1 mL) was added dropwise with stirring at
the same temperature. After stirring at 0 °C for 4 h, the
reaction mixture was poured into water (10 mL), basified with
1 N NaOH to pH ∼8, extracted with EtOAc (3 × 50 mL),
washed with water (50 mL), and dried (Na2SO4), and the
solvent was removed in vacuo. The residue was purified by
silica gel column chromatography using EtOAc-hexane (1:1,
v/v) as eluent to afford 28 as a yellow oil (80.7 mg, 56.0%): IR
(CHCl3) 1314, 1381 and 1487 (NO2), 1645 (CdN), 1714 (CO2),
3325 (NH) cm-1; 1H NMR (CDCl3) δ 2.39 (s, 3H, C-6 Me), 2.56
(s, 3H, C-2 Me), 4.41 (dd, J ) 12.3, 5.8 Hz, 1H, CHH′ONO2),
4.52 (dd, J ) 12.3, 4.0 Hz, 1H, CHH′ONO2), 4.61 (dd, J ) 12.3,
5.8 Hz, 1H, CHH′ONO2), 4.74 (dd, J ) 12.3, 4.0 Hz, 1H,
CHH′ONO2), 5.36 (m, 1H, CO2CH), 5.76 (s, 1H, H-4), 6.37 (br
s, 1H, NH), 7.35 (dd, J ) 8.8, 6.1 Hz, 1H, 4-benzofurazanyl
H-6), 7.43 (d, J ) 6.1 Hz, 1H, 4-benzofurazanyl H-5), 7.71 (d,
E n a n t iom er (+)-(S)-34: 50% yield; 1.27 g; mp 196 °C;
[R]23 ) + 114.0° (c 0.4 in MeOH); IR (KBr): 1219 and 1494
D
(NO2), 1649 (CdO), 3308 (NH) cm-1
;
1H NMR (DMSO-d6) δ
2.27 (s, 3H, C-6 Me), 2.50 (s, 3H, C-2 Me), 5.70 (s, 1H, H-4),
7.40 (d, J ) 6.7 Hz, 1H, 4-benzofurazanyl H-5), 7.55 (dd, J )
9.2, 6.7 Hz, 1H, 4-benzofurazanyl H-6), 7.88 (d, J ) 9.2 Hz,
1H, 4-benzofurazanyl H-7), 9.82 (s, 1H, NH), 12.29 (br s, 1H,
COOH). Anal. (C14H12N4O5) C, H, N.
En a n tiom er (-)-(R)-34: 50% yield; 1.27 g; mp 212 °C (dec);
[R]23 ) - 114.0° (c 0.4 in MeOH); IR (KBr) and 1H NMR
D
(DMSO-d6) spectra for (-)-(R)-34 were the same as those for
(+)-(S)-34. Anal. (C14H12N4O5) C, H, N.
Gen er a l Meth od for th e P r ep a r a tion of Op tica lly P u r e
Isop r op yl a n d Nitr ooxya lk yl 1,4-Dih yd r o-2,6-d im eth yl-
3-n itr o-4-(2,1,3-ben zoxa d ia zol-4-yl)p yr id in e-5-ca r boxyl-
a tes [(+)-(S)-5, (-)-(R)-5, (+)-(S)-35, (-)-(R)-35, (+)-(S,S)-
36, (-)-(R,R)-37, (-)-(S,R)-38, a n d (+)-(R,S)-39]. A solution
of either (+)-(S)-34 or (-)-(R)-34 (126.4 mg, 0.40 mmol), K2-
CO3 (66.3 mg, 0.48 mmol), and either isopropyl bromide or
nitrooxyalkyl bromide [0.44 mmol, 14, (-)-(R)-19, or (+)-(S)-
20] in dry DMF (12 mL) was stirred at 25 °C for 24 h (7 days
for syntheses of compounds 36-39). Water (40 mL) was added,
and the mixture was extracted with EtOAc (3 × 200 mL) and
washed with water (2 × 40 mL) and then brine (40 mL). The
organic phase was dried (Na2SO4), and the solvent was
removed in vacuo. The residue was purified by silica gel
column chromatography using EtOAc-hexane (1:1, v/v) as
eluent to yield the respective optically pure enantiomer [(+)-
(S)-5, (-)-(R)-5, (+)-(S)-35, or (-)-(R)-35] or diastereomer [(+)-
(S,S)-36, (-)-(R,R)-37, (-)-(S,R)-38, or (+)-(R,S)-39] product.
The physical and spectral data for these compounds are listed
below.
J ) 8.8 Hz, 1H, 4-benzofurazanyl H-7). Anal. (C17H16N6O11
C, H, N.
)
(1S,2R)-2-(3,5-Din it r op h en ylca r b a m oyl)-2-(m et h oxy-
ca r bon yl)eth yl 1,4-Dih yd r o-2,6-d im eth yl-3-n itr o-4-(2,1,3-
b en zoxa d ia zol-4-yl)p yr id in e-5-ca r b oxyla t e Dia st er eo-
m er s [(1S,2R)-32 a n d (1S,2R)-33. A solution of (1S,2R)-31
(21.00 g, 51.2 mmol), 2,1,3-benzoxadiazol-4-carboxaldehyde
(30) (7.58 g, 51.2 mmol), and nitroacetone (29) (7.35 g, 71.3
mmol) in EtOH (300 mL) was stirred at 25 °C for 1 h prior to
heating at 80 °C for 17 h. Removal of the solvent in vacuo gave
a foamlike solid which was purified by silica gel column
chromatography using EtOAc-hexane (1:1, v/v) as eluent.
Initial elution gave (1S,2R)-32. Further elution provided a
mixture of the two diastereomers (1S,2R)-32 and (1S,2R)-33,
followed by fractions containing pure (1S,2R)-33. The fractions
containing the mixture of (1S,2R)-32 and (1S,2R)-33 were
rechromatographed using EtOAc-CH2Cl2 (1:6, v/v) as eluent.
In this way, after five column purifications, similar fractions
were combined, and the solvent was removed in vacuo to afford
(1S,2R)-32 and (1S,2R)-33 as yellow crystals after recrystal-
lization from EtOAc/hexane, respectively.
(+)-(S)-Isop r op yl 1,4-d ih yd r o-2,6-d im eth yl-3-n itr o-4-
(2,1,3-ben zoxa d ia zol-4-yl)p yr id in e-5-ca r boxyla te en a n -
tiom er [(+)-(S)-5]: 88.8% yield; [R]23 ) + 63.2° (c 0.5 in
Dia ster eom er (1S,2R)-32: 20% yield; 6.49 g; mp 244-245
D
CHCl3); 1H NMR (CDCl3) δ 1.07 (d, J ) 6.1 Hz, 3H, MeCHMe′),
1.25 (d, J ) 6.4 Hz, 3H, MeCHMe′), 2.36 (s, 3H, C-6 Me), 2.55
(s, 3H, C-2 Me), 4.95 (dq, J ) 6.4, 6.1 Hz, 1H, MeCHMe), 5.80
(s, 1H, H-4), 6.30 (br s, 1H, NH), 7.34 (dd, J ) 8.8, 6.1 Hz, 1H,
4-benzofurazanyl H-6), 7.45 (d, J ) 6.1 Hz, 1H, 4-benzofura-
zanyl H-5), 7.68 (d, J ) 8.8 Hz, 1H, 4-benzofurazanyl H-7).
(-)-(R)-Isop r op yl 1,4-d ih yd r o-2,6-d im eth yl-3-n itr o-4-
(2,1,3-ben zoxa d ia zol-4-yl)p yr id in e-5-ca r boxyla te en a n -
tiom er [(-)-(R)-5]: 89% yield; [R]23D ) -63.2° (c 0.5 in CHCl3);
1H NMR (CDCl3) spectrum for (-)-(R)-5 was the same as that
for (+)-(S)-5.
°C; [R]23 ) -129.25° (c 0.4 in CH2Cl2); IR (CHCl3): 1353 and
D
1541 (NO2), 1703 (CdO), 3314 (NH) cm-1; H NMR (DMSO-
1
d6) δ 1.26 (d, J Me,CH ) 6.1 Hz, 3H, CHMe), 2.33 (s, 3H, C-6
Me), 2.44 (s, 3H, C-2 Me), 3.60 (s, 3H, CO2Me), 4.61 (m, 1H,
CHCHNH), 5.22 (p, J CH,CH ) J CH,Me ) 6.1 Hz, 1H, OCHMe),
5.67 (s, 1H, H-4), 7.29 (dd, J ) 8.8, 6.1 Hz, 1H, 4-benzofura-
zanyl H-6), 7.35 (d, J ) 6.1 Hz, 1H, 4-benzofurazanyl H-5),
7.58 (d, J ) 8.8 Hz, 1H, 4-benzofurazanyl H-7), 8.85 (d, J )
2.1 Hz, 2H, dinitrophenyl H-2 and H-6), 8.97 (dd, J ) 2.1, 2.1
Hz, 1H, dinitrophenyl H-4), 9.26 (d, J ) 7.3 Hz, 1H, CH-NH),
9.91 (br s, 1H, dihydropyridyl NH). Anal. (C26H23N7O12) C,
H, N.
(+)-(S)-2-Nitr ooxyeth yl 1,4-d ih yd r o-2,6-d im eth yl-3-n i-
t r o-4-(2,1,3-b en zoxa d ia zol-4-yl)p yr id in e-5-ca r b oxyla t e
en a n tiom er [(+)-(S)-35]: 91% yield; [R]23D ) +60.6° (c 0.5 in
CHCl3); IR (CHCl3): 1313, 1379, and 1488 (NO2), 1636 (Cd
Dia ster eom er (1S,2R)-33. 27% yield; 8.60 g; mp 222-223
°C; [R]23 ) + 53.75° (c 0.4 in CH2Cl2); IR (CHCl3): 1347 and
D
1541 (NO2), 1709 and 1743 (CdO), 3314 (NH) cm-1; 1H NMR
(DMSO-d6) δ 1.00 (d, J Me,CH ) 6.4 Hz, 3H, CHMe), 2.25 (s, 3H,
C-6 Me), 2.45 (s, 3H, C-2 Me), 3.65 (s, 3H, CO2Me), 4.85 (m,
1H, CHCHNH), 5.28 (p, J CH,CH ) J CH,Me ) 6.4 Hz, 1H,
OCHMe), 5.72 (s, 1H, H-4), 7.42 (d, J ) 6.7 Hz, 1H, 4-benzo-
1
N), 1710 (CO2), 3321 (NH) cm-1; H NMR (CDCl3) δ 2.37 (s,
3H, C-6 Me), 2.56 (s, 3H, C-2 Me), 4.40-4.25 (m, 2H, CO2CH2),
4.61 (t, J ) 4.5 Hz, 2H, CH2ONO2), 5.79 (s, 1H, H-4), 6.47 (br
s, 1H, NH), 7.36 (dd, J ) 9.0, 6.5 Hz, 1H, 4-benzofurazanyl

260 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1
Shan et al.
H-6), 7.46 (d, J ) 6.5 Hz, 1H, 4-benzofurazanyl H-5), 7.69 (d,
J ) 9.0 Hz, 1H, 4-benzofurazanyl H-7). Anal. (C16H15N5O8) C,
H, N.
tion of a single resonance for the C-6 methyl resonance at δ
2.36 or δ 2.38, for each diastereomer, compared to two
resonance at both δ 2.36 and δ 2.38, for a mixture of the
diastereomers (26), showed that these diastereomers were
optically pure (g96% de). In addition, the opposite optical
rotation for the diastereomers (+)-(S,S)-36 and (-)-(R,R)-37
and for the diastereomers (-)-(S,R)-38 and (+)-(R,S)-39 sup-
ports this assumption.
(-)-(R)-2-Nitr ooxyeth yl 1,4-d ih yd r o-2,6-d im eth yl-3-n i-
t r o-4-(2,1,3-b en zoxa d ia zol-4-yl)p yr id in e-5-ca r b oxyla t e
en a n tiom er [(-)-(R)-35]: 91% yield; [R]23 ) -61.0° (c 0.5
D
in CHCl3); IR (CHCl3) and 1H NMR (CDCl3) spectra for (-)-
(R)-35 were the same as those for (+)-(S)-35. Anal. (C16H15N5O8)
C, H, N.
In Vitr o Ca lciu m Ch a n n el An ta gon ist a n d Agon ist
Assa ys. Calcium channel antagonist activities were deter-
mined as the molar concentration of the test compound
required to produce 50% inhibition of the muscarinic receptor-
mediated (carbachol, 1.6 × 10-7 M) Ca2+-dependent contraction
(tonic response) of guinea pig ileum longitudinal smooth
muscle (GPILSM) using the procedure reported previously.6
The IC50 value ((SEM, n )3) was determined graphically from
the dose-response curve.
Calcium channel modulation activity (positive inotropic
effect) was calculated as the molar concentration of the test
compound required to produce a 50% increase (positive ino-
tropic, or CC agonist, effect) in contractile force of isolated
guinea pig left atrium (GPLA) relative to its basal contractile
force in the absence of test compound.6
(+)-(S,S)-1-Meth yl-2-n itr ooxyeth yl 1,4-d ih yd r o-2,6-d i-
m et h yl-3-n it r o-4-(2,1,3-b en zoxa d ia zol-4-yl)p yr id in e-5-
ca r b oxyla t e d ia st er eom er [(+)-(S,S)-36]: 25% yield;
[R]23 ) +59.0° (c 0.4 in CH2Cl2); IR (CHCl3) 1312, 1381, and
D
1489 (NO2), 1637 (CdN), 1708 (CO2), 3321 (NH) cm-1; 1H NMR
(CDCl3) δ 1.33 (d, J ) 6.7 Hz, 3H, CO2CHCH3), 2.38 (s, 3H,
C-6 Me), 2.56 (s, 3H, C-2 Me), 4.25 (dd, J ) 12.0, 6.7 Hz, 1H,
CHH′ONO2), 4.44 (dd, J ) 12.0, 3.0 Hz, 1H, CHH′ONO2), 5.16
(dp, J ) 6.7, 3.0 Hz, 1H, CO2CH), 5.80 (s, 1H, H-4), 6.43 (br s,
1H, NH), 7.34 (dd, J ) 8.8, 6.4 Hz, 1H, 4-benzofurazanyl H-6),
7.44 (d, J ) 6.4 Hz, 1H, 4-benzofurazanyl H-5), 7.69 (d, J )
8.8 Hz, 1H, 4-benzofurazanyl H-7). Anal. (C17H17N5O8) C, H,
N.
(-)-(R,R)-1-Met h yl-2-n it r ooxyet h yl 1,4-d ih yd r o-2,6-
d im eth yl-3-n itr o-4-(2,1,3-ben zoxa d ia zol-4-yl)p yr id in e-5-
ca r b oxyla t e d ia st er eom er [(-)-(R,R)-37]: 30% yield;
Nitr ic Oxid e Relea se Assa y. In vitro nitric oxide release
was assayed using a modification of the previously reported
procedure.9 The test compound (0.0075 mmol) was added to a
thoroughly mixed solution of 0.1 M phosphate buffer (pH 7.40)
and acetonitrile (1:1, v/v) (1.5 mL) containing N-acetylcys-
teamine (1 equiv per -ONO2 moiety) or, in the absence of
N-acetylcysteamine, with stirring under argon at 37 °C for
1 h. After exposure to air for 10 min, the reaction mixture
(0.2 mL) was diluted with water (0.6 mL), and this solution
was treated with 0.2 mL of Griess reagent [sulfanilamide
(4 g), N-naphthylenediamine dihydrochloride (0.2 g), and 85%
phosphoric acid (10 mL) in distilled water (final volume: 100
mL)] for 10 min at room temperature. After dilution of this
solution with water to a volume of 5 mL, the absorbance was
measured at 540 nm. Solutions of 3-24 µM sodium nitrite
were used to prepare a nitrite versus concentration curve. A
control experiment (absence of test compound) was performed
for each measurement (n ) 3). The percent nitric oxide release
(quantitated as nitrite ion) was calculated from the nitrite
versus concentration curve.
[R]23 ) -59.0° (c 0.4 in CH2Cl2); IR (CHCl3) and 1H NMR
D
(CDCl3) spectra for (-)-(R,R)-37 were the same as those for
(+)-(S,S)-36. Anal. (C17H17N5O8) C, H, N.
(-)-(S,R)-1-Meth yl-2-n itr ooxyeth yl 1,4-d ih yd r o-2,6-d i-
m et h yl-3-n it r o-4-(2,1,3-b en zoxa d ia zol-4-yl)p yr id in e-5-
ca r b oxyla t e d ia st er eom er [(-)-(S,R)-38]: 30% yield;
[R]23 ) -30.5° (c 0.4 in CH2Cl2); IR (CHCl3) and 1H NMR
D
(CDCl3) spectra for (-)-(S,R)-38 were the same as those for
(+)-(R,S)-39 listed below. Anal. (C17H17N5O8) C, H, N.
(+)-(R,S)-1-Meth yl-2-n itr ooxyeth yl 1,4-d ih yd r o-2,6-d i-
m et h yl-3-n it r o-4-(2,1,3-b en zoxa d ia zol-4-yl)p yr id in e-5-
ca r b oxyla t e d ia st er eom er [(+)-(R,S)-39]: 30% yield;
[R]23D ) +30.8° (c 0.4 in CH2Cl2); IR (CHCl3): 1312, 1381, and
1489 (NO2), 1637 (CdN), 1708 (CO2), 3321 (NH) cm-1; 1H NMR
(CDCl3) δ 1.10 (d, J ) 6.7 Hz, 3H, CO2CHCH3), 2.36 (s, 3H,
C-6 Me), 2.56 (s, 3H, C-2 Me), 4.43 (dd, J ) 12.2, 6.7 Hz, 1H,
CHH′ONO2), 4.59 (dd, J ) 12.2, 3.4 Hz, 1H, CHH′ONO2), 5.19
(dp, J ) 6.7, 3.4 Hz, 1H, CO2CH), 5.78 (s, 1H, H-4), 6.41 (br s,
1H, NH), 7.35 (dd, J ) 8.8, 6.1 Hz, 1H, 4-benzofurazanyl H-6),
7.44 (d, J ) 6.1 Hz, 1H, 4-benzofurazanyl H-5), 7.70 (d, J )
8.8 Hz, 1H, 4-benzofurazanyl H-7). Anal. (C17H17N5O8) C,
H, N.
Ack n ow led gm en t. We are grateful to the Canadian
Institutes of Health Research (Grant Number MT-8892)
for financial support of this research and for a fellowship
to R.S. and to the National Council of Science and
Technology (CONACYT, Mexico) for a graduate scholar-
ship to C.V.
Op t ica l P u r it y of E n a n t iom er s a n d Dia st er eom er s.
The optical purity of (+)-(S)-5, (-)-(R)-5, (+)-(S)-35, and (-)-
(R)-35 was determined by 1H NMR spectrometry. When 20
µL of a solution (100 mg in 1 mL CDCl3) of the chiral shift
reagent (+)-Eu(hfc)3 was added to a solution of racemic
2-nitrooxyethyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-(2,1,3-ben-
zoxadiazol-4-yl)-5-pyridinecarboxylate (23) (5 mg in 0.5 mL
CDCl3), the 1,4-dihydropyridyl C-6 methyl resonance at δ 2.37
was separated into two resonances that appeared at δ 2.54
and 2.56. Addition of (+)-Eu(hfc)3 (60 µL) to (+)-(S)-35 or (-)-
(R)-35, as described above, resulted in retention of single
resonance for the C-6 methyl resonance (g96% ee). Similarly,
addition of (+)-Eu(hfc)3 (10 µL) to racemic isopropyl 1,4-di-
hydro-2,6-dimethyl-3-nitro-4-(2,1,3-benzoxadiazol-4-yl)-5-pyr-
idinecarboxylate (5) (5 mg in 0.5 mL CDCl3) resulted in the
separation of the 1,4-dihydropyridyl C-6 methyl resonance at
δ 2.36 into two resonances, which appeared at δ 2.37 and 2.38.
A similar addition of (+)-Eu(hfc)3 (40 µL) to (+)-(S)-5 or (-)-
(R)-5 resulted in retention of a single resonance for the C-6
methyl resonance (g96% ee). The optical purity of diastereo-
mers (+)-(S,S)-36, (-)-(R,R)-37, (-)-(S,R)-38, and (+)-(R,S)-
39 was determined by 1H NMR spectrometry. Similar to
previous studies, the configuration at the 1,4-dihydropyridine
C-4 position was retained during the esterification reaction,
whereas the SN2 reaction with the nitrooxyalkyl bromide
proceeded with inversion of configuration. Accordingly, reten-
Refer en ces
(1) For an excellent review, see the following. Rampe, D.; Kano, J .
M. Activators of voltage-dependent L-type calcium channels.
Drug Dev. Res. 1994, 33, 344-363.
(2) Franckowiak, G.; Bechem, M.; Schramm, M.; Thomas, G. The
optical isomers of the 1,4-dihydropyridine Bay K 8644 show
opposite effects on Ca channels. Eur. J . Pharmacol. 1985, 114,
223-226.
(3) Shan, R.; Howlett, S. E.; Knaus, E. E. Syntheses, calcium
channel agonist-antagonist modulation activities, nitric oxide
release, and voltage-clamp studies of 2-nitrooxyethyl 1,4-dihydro-
2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)pyridine-5-car-
boxylate enantiomers. J . Med. Chem. 2002, 45, 955-961.
(4) Wei, X. Y.; Luckowski, E. M.; Rutledge, A.; Su, C. M.; Triggle,
D. J . Pharmacologic and radioligand binding analysis of the
action of 1,4-dihydropyridine activator-antagonist pairs in smooth
muscle. J . Pharmacol. Exp. Ther. 1986, 239, 144-153.
(5) Shibanuma, T.; Iwanani, M.; Okuda, K.; Takenaka, T.; Mu-
rakami, M. Synthesis of optically active 2-(N-benzyl-N-methyl-
amino)ethyl methyl 2,6-dimethyl-4-(m-nitrophenyl)-1,4-dihydro-
pyridine-3,5-dicaboxylate (Nicardipine). Chem. Pharm. Bull.
1980, 28, 2809-2812.
(6) Vo, D.; Matowe, W. C.; Ramesh, M.; Iqbal, N.; Wolowyk, M. W.;
Howlett, S. E.; Knaus, E. E. Synthesis, calcium channel agonist-

Novel Hybrid Calcium Channel Modulators
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1 261
antagonist modulation activities, and voltage-clamp studies of
isopropyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-pyridinylpyridine-
5-carboxylate racemates and enantiomers. J . Med. Chem. 1995,
38, 2851-2859.
(12) Miri, R.; McEwen, C.-A.; Knaus, E. E. Synthesis and calcium
channel modulating effects of modified Hantzsch nitrooxyalkyl
1,4-dihydro-2,6-dimethyl-3-nitro-4-(pyridinyl or 2-trifluorometh-
ylphenyl)-5-pyridine-carboxylates. Drug Dev. Res. 2000, 51, 225-
232.
(7) Hof, R. P.; Ru¨egg, U. T.; Hof, A.; Vogel, A. Stereoselectivity at
the calcium channel: Opposite action of the enantiomers of a
1,4-dihydropyridine. J . Cardiovasc. Pharmacol. 1985, 7, 689-
693.
(8) Aghdasi, B.; Reid, M. B.; Hamilton, S. L. Nitric oxide protects
the skeletal muscle Ca2+ release channel from oxidation induced
activation. J . Biol. Chem. 1997, 272, 25462-26467.
(9) Sako, M.; Oda, S.; Ohara, S.; Hirota, K.; Maki, Y. Facile synthesis
and NO-generating property of 4H-[1,2,5]oxadiazolo[3,4-d]py-
rimidine-5,7-dione 1-oxides. J . Org. Chem. 1998, 63, 6947-6951.
(10) Wang, P. G.; Xian M.; Tang, X.; Wu, X.; Wen, Z.; Cai, T.; J anczuk,
A. J . Nitric oxide donors: Chemical activities and biological
applications. Chem. Rev. 2002, 102, 1091-1134; and references
therein.
(13) Marken, C. D.; Kristofferson, C. E.; Roland, M. M.; Manzara, A.
P.; Barnes, M. W. A low hazard procedure for the laboratory
preparation of polynitrate esters. Synthesis 1977, 484-485.
(14) Heitzmann, M. Preparation of metalated 2,1,3-benzoxa- or
thiadiazole and their use in synthesis of dihydropyridine deriva-
tives. Patentschrift, Switz. CH 661,728, 14 August 1987.
(15) Nguyen, J .-T.; McEwen, C.-A.; Knaus, E. E. Hantzsch 1,4-
dihydropyridines containing a nitrooxyalkyl ester moiety to
study calcium channel structure-activity relationships and
nitric oxide release. Drug Dev. Res. 2000, 51, 233-243.
(16) Pirrung, M. C.; McGeehan, G. M. Ethylene biosynthesis. 6.
Synthesis and evaluation of methylaminocyclopropanocarboxylic
acid. J . Org. Chem. 1986, 51, 2103-2106.
(11) Hurd, C. D.; Nilson, M. E. Aliphatic Nitro Ketones. J . Org. Chem.
1955, 20, 927-936.
J M030333H