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-carboxylate enantiomers

Article abstract of DOI:10.1021/jm010394k

The novel (-)-(S)-2 and (+)-(R)-3 enantiomers of 2-nitrooxyethyl 1,4-dihydro-2,6-dimethyl- 3-nitro-4-(2-trifluoromethylphenyl)pyridine-5-carboxylate were synthesized for evaluation as calcium channel modulators. Determination of their in vitro calcium-cha

Full text of DOI:10.1021/jm010394k

J . Med. Chem. 2002, 45, 955-961
955
Syn th eses, Ca lciu m Ch a n n el Agon ist-An ta gon ist Mod u la tion Activities, Nitr ic
Oxid e Relea se, a n d Volta ge-Cla m p Stu d ies of 2-Nitr ooxyeth yl 1,4-Dih yd r o-
2,6-d im eth yl-3-n itr o-4-(2-tr iflu or om eth ylp h en yl)p yr id in e-5-ca r boxyla te
En a n tiom er s
Rudong Shan,† Susan E. Howlett,‡ and Edward E. Knaus*,†
Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta T6G 2N8, Canada, and
Department of Pharmacology, Dalhousie University, Halifax, Nova Scotia B3H 4H7, Canada
Received August 20, 2001
The novel (-)-(S)-2 and (+)-(R)-3 enantiomers of 2-nitrooxyethyl 1,4-dihydro-2,6-dimethyl-3-
nitro-4-(2-trifluoromethylphenyl)pyridine-5-carboxylate were synthesized for evaluation as
calcium channel modulators. Determination of their in vitro calcium-channel-modulating
activities using guinea pig left atria (GPLA) and ileum longitudinal smooth muscle (GPILSM)
showed that the (-)-(S)-2 enantiomer acted as a dual cardioselective calcium channel agonist
(GPLA)/ smooth muscle selective calcium channel antagonist (GPILSM). In contrast, the (+)-
(R)-3 enantiomer exhibited calcium channel antagonist activity on both GPLA and GPILSM.
The 2-nitrooxyethyl racemate is a nitric oxide (•NO) donor that released 2.7% •NO, relative to
•
the reference drug glyceryl trinitrate (5.3% NO release/ONO2 moiety), in the presence of
N-acetylcysteamine. Whole-cell voltage-clamp studies using isolated guinea pig ventricular
myocytes indicated that both enantiomers inhibit calcium current but that the (-)-(S)-2
enantiomer is a weaker antagonist than the (+)-(R)-3 enantiomer. These results indicate that
replacement of the methyl ester substituent of (-)-(S)-methyl 1,4-dihydro-2,6-dimethyl-3-nitro-
•
4-(2-trifluoromethylphenyl)pyridine-5-carboxylate [(-)-(S)-1] by the 2-nitrooxyethyl ester NO
donor substituent present in (-)-(S)-2 provides a useful drug design concept to abolish the
contraindicated calcium channel agonist effect of (-)-(S)-1 on vascular smooth muscle. The
novel (-)-(S)-2 enantiomer is a useful lead compound for drug discovery targeted toward the
treatment of congestive heart failure, and it provides a useful probe to study the structure-
function relationships of calcium channels.
In tr od u ction
ylphenyl)pyridine-5-carboxylate (Bay K 8644) acts as a
CC agonist on both smooth and cardiac muscle because
the agonist (-)-(S)-enantiomer (1) is approximately 10-
fold more potent as an activator compared to the (+)-
(R)-antipode that acts as an antagonist.4 The discovery
that nitric oxide (•NO) serves as an endogenous activator
of guanylate cyclase, an enzyme responsible for vascular
muscular relaxation, and that organic nitrovasodilators
The 1,4-dihydropyridine (1,4-DHP) L-type voltage-
sensitive calcium ion channel represents an important
drug target that possesses specific binding sites for both
antagonist and agonist ligands that modulate the closed
or open conformational state of the channel. Different
states of the channel have different affinities and/or
access for drugs, and drugs may exhibit both a quanti-
tative and a qualitative structure-activity difference,
including stereoselectivity between channel states.1 In
this regard, ion channels can be considered to be
multiple-drug binding receptors typically having four
to eight discrete binding sites that may be individually
linked to each other and to the gating and permeation
action of the ion channel by complex allosteric interac-
tions.2
The development of tissue-selective 1,4-DHP calcium
channel (CC) modulators suitable for the treatment of
congestive heart failure (CHF) requires that their
adverse smooth muscle vasoconstrictor effect be elimi-
nated and/or separated from the target cardiac positive
inotropic stimulant action.3 For example, racemic meth-
yl 1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluorometh-
•
exert their effect in vivo by bypassing the NO-produc-
•
tion system in the endothelium to deliver NO directly
to muscle cells in the artery prompted a number of
studies5-8 to design hybrid Hantzsch-type 1,4-DHP CC
antagonists that also have the potential to simulta-
•
neously release NO. In a preliminary communication,9
we reported that (-)-(S)-2-nitrooxyethyl 1,4-dihydro-2,6-
dimethyl-3-nitro-4-(2-trifluoromethylphenyl)pyridine-5-
carboxylate (2), similar to (-)-(S-)-1, acts as a cardiac
CC agonist, but in contrast to (-)-(S)-1, (-)-(S)-2 is a
•
smooth muscle CC antagonist that also acts as a NO
donor in vitro (see Figure 1). This dual combination of
tissue-selective CC modulating activities satisfies the
clinical requirements for the treatment of CHF. In our
ongoing program to acquire structure-activity relation-
ships with respect to CC modulation and to design
compounds to study CC structure-function relation-
ships, we now report the chiral syntheses of (-)-(S)-2
and its (+)-(R)-enantiomer (3), their in vitro CC modu-
lating effects on smooth and cardiac muscle and modu-
* To whom correspondence should be addressed. Phone: 780-492-
5993. Fax: 780-492-1217. E-mail: eknaus@pharmacy.ualberta.ca.
† University of Alberta.
‡ Dalhousie University.
10.1021/jm010394k CCC: $22.00 © 2002 American Chemical Society
Published on Web 01/22/2002

956 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 4
Shan et al.
L-valine, to a Knoevenagel acceptor as reported earlier10
for the efficient synthesis of chiral dialkyl 1,4-dihydro-
pyridine-3,5-dicarboxylates afforded (-)-(S)-2 or (+)-
(R)-3 in unacceptably low enantiomeric excess (70% ee).
Alternatively, it was anticipated that use of a chiral
esterifying group such as D-threonine, which could be
readily removed by a â-elimination reaction using a
nonnucleophilic base such as 1,8-diazabicyclo[5.4.0]-
undec-7-ene (DBU), would be a suitable derivative for
separation of diastereomeric esters (9, 10) that could
be converted to the chiral acids (11, 12).11 Accordingly,
the reaction of methyl (2R,3S)-2-amino-3-hydroxy-
butanoate hydrochloride (5), prepared by esterification
of D-threonine with SOCl2 in MeOH, with 3,5-dini-
trobenzoyl chloride gave the N-(3,5-dinitrobenzoyl) de-
rivative 6 (see Scheme 1). Reaction of the alcohol 6 with
2,2,6-trimethyl-4H-1,3-dioxin-4-one (a diketene precur-
sor) yielded the acetoacetate ester 7. Elaboration of 7
to the â-aminocrotonate derivative 8 and its subsequent
modified Hantzsch condensation with 2-trifluorometh-
ylbenzaldehyde and nitroacetone afforded a mixture of
the 1,4-dihydropyridine diastereomers 9 and 10. These
two diastereomers, which differ in configuration (S or
R) at the C-4 position of the 1,4-dihydropyridine ring,
were separated by silica gel column chromatography.
Cleavage of the individual esters 9 and 10 using DBU
at 25 °C yielded the individual (+)-(S)-11 and (-)-(R)-
F igu r e 1. Structures of (-)-(S)-methyl 1,4-dihydro-2,6-di-
methyl-3-nitro-4-(2-trifluoromethylphenyl)pyridine-5-carbox-
ylate [(-)-(S)-1], (-)-(S)-2-nitrooxyethyl 1,4-dihydro-2,6-di-
methyl-3-nitro-4-(2-trifluoromethylphenyl)pyridine-5-carbox-
ylate (2), and the (+)-(R)-enantiomer (3).
lation of L-type voltage-sensitive CCs in isolated guinea
•
pig ventricular myocytes, and NO release data for the
racemic mixture of 2-nitrooxyethyl compounds (-)-(S)-2
and (+)-(R)-3.
Ch em istr y
The first synthetic strategy investigated to prepare
the individual enantiomers (-)-(S)-2 and (+)-(R)-3 by a
variant of the Hantzsch dihydropyridine synthesis
involving the asymmetric Michael addition of a meta-
lated chiral aminocrotonate, derived from D-valine or
Sch em e 1a
a
Reagents and conditions: (a) SOCl2, MeOH, 25 °C, 48 h; (b) K2CO3, 3,5-dinitrobenzoyl chloride, EtOAc/H2O, 25 °C, 17 h; (c) 2,2,6-
trimethyl-4H-1,3-dioxin-4-one, xylene, 150 °C, 30 min; (d) 4-Me-C6H4-SO3H, NH3, toluene, 130 °C, 4 h; (e) 2-CF3-C6H4-CHO,
MeCOCH2NO2, EtOH, reflux, 80 °C, 17 h; (f) 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), 25 °C, 4 h; (g) BrCH2CH2ONO2, DMF, K2CO3, 25
°C, 24 h; (h) CH2N2, MeOH, 25 °C, 15 min.

2-Nitrooxyethyl Enantiomers
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 4 957
F igu r e 2. ICa is inhibited by the 5-(2-nitrooxyethyl) racemate [(-)-(S)-2/(+)-(R)-3], as well as by the (-)-(S)-2 and (+)-(R)-3
enantiomers. Shown are the mean ( SEM current-voltage relationships for ICa under control conditions and in the presence of
1 µM (A) and 10 µM (B) racemate (-)-(S)-2/(+)-(R)-3. At a concentration of 1 µM, the (-)-(S)-2 enantiomer had no effect on ICa (C),
but 10 µM (-)-(S)-2 inhibited ICa (D). In contrast, the (+)-(R)-3 enantiomer markedly inhibited ICa at both 1 µM (E) and 10 µM (F).
Asterisks indicate points that are significantly different from the control (n ) 4-7 cells per group; p < 0.05).
12 carboxylate enantiomers, respectively. Reaction of
(+)-(S)-11 or (-)-(R)-12 with BrCH2CH2ONO2 in the
presence of K2CO3 in DMF afforded the respective (-)-
(S)-2 or (+)-(R)-3 enantiomers of 2-nitrooxyethyl 1,4-
dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)-
pyridine-5-carboxylate.
diac positive inotropic (calcium agonist) effect. Credence
for this concept is based on reports that the CC
antagonist nitrendipine enhances the release of NO
•
from vascular endothelium, which may increase it
vascular relaxation effect,12 that •NO modulates the
activity of the Ca2+ release channel by preventing
oxidation of regulatory sulfhydryls,13 and that organic
The absolute configuration of the (-)-(4S)-2 enanti-
omer {[R]23 -48.75°} was assigned based on the fact
nitrates and NO donors evoke a small but constant
•
D
that (+)-(S)-11 was elaborated to the known (-)-(S)-1,
positive inotropic effect in vivo that is not induced by
which shows an optical rotation {[R]23 -56.75°} identi-
coronary vasodilation.14
D
cal to the reported value {[R]23D -56.7°}.4 Therefore, the
In vitro calcium channel (CC) modulating activities
of the (-)-(S)-2 and (+)-(R)-3 enantiomers were deter-
mined using guinea pig ileum longitudinal smooth
muscle (GPILSM) and guinea pig left atrium (GPLA).
(-)-(S)-2 [GPLA ED50 ) (9.2 ( 6.8) × 10-7 M, n ) 3],
like (-)-(S)-1,4 acts as a cardiac CC agonist (positive
inotrope) but, unlike (-)-(S)-1, is a smooth muscle CC
antagonist [GPILSM IC50 ) (1.6 ( 0.1) × 10-5 M, n )
3]. The EC50 value for agonist activity (GPLA) is the
molar concentration eliciting 50% of the maximum
contractile response produced by the test drug on GPLA,
as determined graphically from the dose-response
curve. In contrast, the (+)-(R)-3 enantiomer exhibited
CC antagonist effects on both cardiac [GPLA IC50 ) (1.6
( 8.2) × 10-5 M, n ) 3] and smooth muscle [GPILSM
IC50 ) (7.9 ( 0.4) × 10-8 M, n ) 3].
dextro enantiomer (+)-(4R)-3 {[R]23 +48.75°} must
D
have the (4R)-configuration. The two enantiomers (-)-
(S)-2 and (+)-(R)-3 each exhibited a single resonance
for the dihydropyridine C-6 methyl resonance upon
addition of the 1H NMR chiral shift reagent (+)-Eu(hfc)3,
indicating a very high optical purity (g96% ee).
Resu lts a n d Discu ssion
Two hybrid Hantzsch-type 1,4-DHP enantiomers [(-)-
(S)-2 and (+)-(R)-3] having a 2-nitrooxyethyl ester nitric
oxide (•NO) donor moiety in place of the methyl ester
substituent in methyl 1,4-dihydro-2,6-dimethyl-3-nitro-
4-(2-trifluoromethylphenyl)pyridine-5-carboxylate were
synthesized. It was anticipated that the smooth muscle
•
vasorelaxant effect of released NO offered a drug design
concept5-8 to counteract the contraindicated smooth
muscle vasconstrictor effect exhibited by methyl 1,4-
dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)-
pyridine-5-carboxylate while retaining its desired car-
In vitro •NO release was determined by quantitation
of nitrite using the Griess reaction.15 The percentage of
•NO release for the 2-nitrooxyethyl racemate [(-)-(S)-
2/(+)-(R)-3] was 1.09 ( 0.02% and 2.70 ( 0.02% in the

958 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 4
absence and presence of N-acetycysteamine, respectively
Shan et al.
exhibits a distinctive profile in vitro that encompasses
a dual cardioselective agonist/ smooth muscle selective
antagonist activity that could provide a potentially new
approach to drug design targeted toward the treatment
of congestive heart failure. These (-)-(S)-2 and (+)-(R)-3
enantiomers may also be valuable probes to study the
structure-function relationship of calcium channels.
•
(n ) 3). In comparison, the percentage of NO release
for the reference drug glycerol trinitrate was 0.18 (
0.00% per ONO2 moiety and 5.30 ( 0.00% per ONO2
moiety in the absence and presence of N-acetylcyste-
•
amine, respectively (n ) 3). These NO release data are
consistent with the previous observation that (-)-(S)-2
acts as a CC antagonist on GPILSM, in contrast to (-)-
(S)-Bay K8644, which acts as a CC agonist on smooth
muscle4 and which is most likely the result of smooth
Exp er im en ta l Section
Melting points were recorded with a Thomas-Hoover capil-
lary 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. D-Threonine, 2-bromoethanol, R,R,R-trifluoro-
o-tolualdehyde, 2,2,6-trimethyl-4H-1,3-dioxin-4-one, N-acetyl-
cysteamine, and tris[3-(heptafluoropropylhydroxymethylene)-
(+)-camphorato]europium(III) [Eu(hfc)3] were purchased from
Aldrich Chemical Co. Nitroacetone,17 nitroglycerin,18 and
racemic 2-nitrooxyethyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-
trifluoromethylphenyl)pyridine-5-carboxylate19 were prepared
using literature procedures.
2-Nitr ooxyeth yl Br om id e. 2-Bromoethanol (12.5 g, 100
mmol) was added dropwise to a mixture of HNO3 (11 mL of
70% w/v) and H2SO4 (24 mL of 98% w/v) at 0 °C with stirring,
the reaction was allowed to proceed at 0 °C for 1 h, and the
resulting mixture was poured into cold water (100 mL).
Extraction with CH2Cl2 (4 × 100 mL), drying of the CH2Cl2
extract (MgSO4), and removal of the solvent in vacuo gave the
title compound (16 g, 94%) as an oil. 1H NMR (CDCl3): δ 3.56
(t, J ) 6.4 Hz, 2H, H-1), 4.76 (t, J ) 6.4, 2H, H-2). The title
compound was used immediately for the subsequent syntheses
of (-)-(S)-2 and (+)-(R)-3.
•
muscle relaxation induced by release of NO.
The modulation of L-type sensitive calcium channels
by the 2-nitrooxyethyl racemate [(-)-(S)-2/(+)-(R)-3] and
the (-)-(S)-2 and (+)-(R)-3 enantiomers in guinea pig
ventricular myocytes using the whole-cell voltage-clamp
technique was investigated. In Figure 2 are the mean
((SEM) current-voltage relations for ICa under control
conditions and in the presence of 1 and 10 µM concen-
trations of the racemate (panels A and B), (-)-(S)-2
(panels C and D), and (+)-(R)-3 (panels E and F). In all
experimental groups under control conditions, the thresh-
old for activation of ICa was between -40 and -30 mV,
and the current reached a maximum between potentials
of -10 and 0 mV (Figure 2, panels A-F). At a concen-
tration of 1 µM, the 2-nitrooxyethyl racemate had little
effect on the magnitude of ICa (panel A). However, 10
µM racemate significantly inhibited ICa, in particular
near the peak of the current-voltage curve (panel B).
(-)-(S)-2 had no effect on the magnitude of ICa at 1 µM
but exerted a weak antagonist effect at the higher
concentration (10 µM, panel D). In contrast, (+)-(R)-3
exerted significant antagonist effects at both 1 and 10
µM concentrations (panels E and F). All three com-
pounds inhibited peak inward current but did not
appear to shift the voltage dependence of ICa. These
results suggest that (-)-(S)-2 is a weaker antagonist of
calcium current than (+)-(R)-3 in guinea pig ventricular
myocytes. Furthermore, the effects of the 2-nitrooxy-
ethyl racemate appear to be between the effects of the
two enantiomers. Although 1 µM (-)-(S)-2 had little
effect on the magnitude of ICa as measured in isolated
guinea pig ventricular myocytes, similar concentrations
of (-)-(S)-2 actually enhanced contractility in guinea pig
atria. This difference in results may be due to differ-
ences in atrial and ventricular myocytes or due to
differences in the voltage dependence of calcium channel
blockade in the two preparations. Ventricular myocytes
were voltage-clamped from a membrane potential of
-40 mV, whereas atrial cells in the isolated tissue
experiments were at their resting membrane potential,
presumably more negative than -40 mV. Certain 1,4-
DHP’s act as calcium channel agonists at more negative
activating potentials and have much less effect at more
positive potentials.16 It is therefore possible that the
calcium channel agonist activity of (-)-(S)-2 also is
voltage-dependent. Additional experiments will be re-
quired to test this possibility.
Meth yl (2R,3S)-2-Am in o-3-h yd r oxybu ta n oa te Hyd r o-
ch lor id e (5). A solution of thionyl chloride (74.9 g, 0.63 mol)
in MeOH (100 mL) was cooled to 0 °C, D-threonine (25.0 g,
0.21 mol) was added in aliquots with stirring, and the reaction
mixture was allowed to proceed at 0 °C for 1 h and then at 25
°C for 48 h. Removal of the solvent in vacuo afforded 5 as a
viscous oil (35.6 g, 100%), which was used immediately for the
subsequent preparation of 6.
Meth yl (2R,3S)-2-(3,5-Din itr op h en ylca r bon yla m in o)-
3-h yd r oxybu ta n oa te (6). A mixture of 5 (35.6 g, 0.21 mol)
in EtOAc (300 mL) and K2CO3 (43.0 g, 0.31 mol) in water (150
mL) was cooled to 0 °C, and 3,5-dinitrobenzoyl chloride (48.4
g, 0.21 mol) was added in small aliquots with stirring. The
reaction was allowed to proceed at 5-10 °C for 3 h and then
at 25 °C for 17 h, the organic layer was separated and washed
with brine (5 × 40 mL), the organic fraction was dried (Na2-
SO4), and the solvent was removed in vacuo to give a solid
that was recrystallized from EtOAc/Et2O to yield 6 as a white
solid (57.8 g, 84.1%): mp 69-70 °C; [R]23 - 19.50° (c 0.4,
D
MeOH). IR (CHCl3): 1743 (CO2), 3320 (OH), 3419 (NH) cm-1
.
1H NMR (CDCl3): δ 1.32 (d, J Me,CH ) 7.0 Hz, 3H, CH-Me),
2.83 (br s, 1H, OH), 3.82 (s, 3H, CO2Me), 4.55 (qd, J CH,Me
)
7.0 Hz, J CH,CH ) 2.0 Hz, 1H, CH-Me), 4.87 (dd, J CH,NH ) 9.0
Hz, J CH,CH ) 2.0 Hz, 1H, -CHCHNH), 7.62 (d, J NH,CH ) 9.0
Hz, 1H, CHNH), 9.06 (d, J ) 2.0 Hz, 2H, phenyl H-2 and H-6),
9.17 (dd, J ) 2.0, J ) 2.0 Hz, 1H, phenyl H-4). Anal.
(C12H13N3O8) C, H, N.
(1S,2R)-2-(3,5-Din it r op h en ylca r b on yla m in o)-2-m et h -
oxyca r bon yl-1-m eth yleth yl Acetoa ceta te (7). A solution
of 6 (45.57 g, 139.37 mmol) and 2,2,6-trimethyl-4H-1,3-dioxin-
4-one (19.81 g, 139.37 mmol) in xylene (80 mL) was placed in
a 250 mL Erlenmeyer flask. The flask was immersed in an oil
bath preheated to 150 °C, and the reaction was allowed to
proceed with vigorous stirring for 30 min at which time no
further evolution of acetone occurred. The reaction mixture
Con clu sion s
The (-)-(S)-2 and (+)-(R)-3 enantiomers of 2-nitrooxy-
ethyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoro-
methylphenyl)pyridine-5-carboxylate represent a novel
type of compound with different calcium channel modu-
lation activities. In particular, the (-)-(S)-2 enantiomer

2-Nitrooxyethyl Enantiomers
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 4 959
was cooled to 25 °C, and the product was filtered and
recrystallized from EtOAc/Et2O to yield 7 as white crystals
(53.96 g, 94.2%): mp 79-80 °C; [R]23D - 105.75° (c 0.4, CHCl3).
IR (CHCl3): 1718 (CO), 1748 (CO2), 3350 (NH) cm-1. 1H NMR
(CDCl3): δ 1.42 (d, J Me,CH ) 7.0 Hz, 3H, CHMe), 2.34 (s, 3H,
COMe), 3.60 (d, J gem ) 16.0 Hz, 2H, COCH2CO), 3.81 (s, 3H,
CO2Me), 4.98 (dd, J CH,NH ) 9.0 Hz, J CH,CH ) 2.0 Hz, 1H,
CHCHNH), 5.60 (qd, J CH,Me ) 7.0 Hz, J CH,CH ) 2.0 Hz, 1H,
O-CH-Me), 8.07 (d, J NH,CH ) 9.0 Hz, 1H, CHNH), 9.21 (dd,
J ) 2.0, J ) 2.0 Hz, 1H, phenyl H-4), 9.24 (d, J ) 2.0 Hz, 2H,
phenyl H-2, and H-6). Anal. (C16H17N3O10) C, H, N.
phenyl H-2 and H-6), 9.15 (dd, J ) 2.0, J ) 2.0 Hz, 1H,
dinitrophenyl H-4). Anal. (C27H24N5O11F3) C, H, N.
1,4-Dih yd r o-2,6-d im et h yl-3-n it r o-4-(2-t r iflu or om et h -
ylp h en yl)p yr id in e-5-ca r boxylic Acid En a n tiom er s [(+)-
(S)-11 a n d (-)-(R)-12]. A solution of either 9 or 10 (0.8 g,
1.23 mmol) and DBU (0.56 g, 3.70 mmol) in MeOH (40 mL)
was stirred at 25 °C for 4 h, the solvent was removed in vacuo,
and water (40 mL) was added. The water fraction was washed
with ether (3 × 100 mL), acidified to pH 2 with 0.5 N HCl,
and extracted with EtOAc (3 × 100 mL). The combined organic
extracts were dried (Na2SO4), and the solvent was removed
in vacuo to give the crude yellow product as a tacky residue
that was recrystallized from MeOH/ether to afford (+)-(S)-11
or (-)-(R)-12 as yellow crystals, respectively.
(1S,2R)-2-(3,5-Din it r op h en ylca r b on yla m in o)-2-m et h -
oxyca r bon yl-1-m eth yleth yl 3-Am in ocr oton a te (8). Am-
monia gas was bubbled into a solution of 7 (23.2 g, 56.4 mmol)
and p-toluenesulfonic acid (150 mg, 0.87 mmol) in dry toluene
(300 mL) that was preheated to 130 °C for 20 min. The reaction
was allowed to proceed with azeotropic removal of water using
a Dean-Stark apparatus for 4 h. Removal of the solvent in
vacuo, purification of the residue by silica gel column chro-
matography (EtOAc/hexane; 1:1, v/v), and recrystallization of
the product from EtOAc/hexane afforded 8 as yellowish
En a n tiom er (+)-(S)-11: 236.7 mg, 56.3%; mp 205-206 °C
(dec); [R]23 + 48.75° (c 0.4, MeOH). IR (KBr): 1300 and 1480
D
(NO2), 1671 (CO2), 2500-3500 (CO2H) cm-1. 1H NMR (DMSO-
d6): δ 2.24 (s, 3H, C-6 Me), 2.47 (s, 3H, C-2 Me), 5.76 (s, 1H,
H-4), 7.36 (dd, J ) 8.0, J ) 7.3 Hz, 1H, phenyl H-4), 7.43 (d,
J ) 8.0 Hz, 1H, phenyl H-6), 7.51 (d, J ) 8.0 Hz, 1H, phenyl
H-3), 7.56 (dd, J ) 8.0, J ) 7.3 Hz, 1H, phenyl H-5), 9.49 (s,
1H, NH), 12.19 (br s, 1H, COOH). Anal. (C15H13N2O4F3‚1/9H2O)
C, H, N.
crystals (12.6 g, 54.7%): mp 170 °C; [R]23 -83.25° (c 0.4,
D
CHCl3). IR (CHCl3): 1680 and 1760 (CO2), 3320 and 3408
1
(NH2), 3500 (NH) cm-1. H NMR (CDCl3): δ 1.38 (d, J Me,CH
)
En a n tiom er (-)-(R)-12: 232.5 mg, 55.3%; mp 204-205 °C
(dec); [R]23 -49.75° (c 0.4, MeOH). IR (KBr) and 1H NMR
6.4 Hz, 3H, CHMe), 1.94 (s, 3H, dC-Me), 3.81 (s, 3H, CO2Me),
4.55 (s, 1H, CHdC-NH2), 4.73 (dd, J CH,NH ) 7.3 Hz, J CH,CH
D
)
(DMSO-d6) spectral data for (-)-(R)-12 were the same as those
for (+)-(S)-11. Anal. (C15H13N2O4F3‚1/9H2O) C, H, N.
6.1 Hz, 1H, CHCHNH), 4.90 (br s, 1H, dC-NHH′), 5.41 (qd,
J CH,Me ) 6.4 Hz, J CH,CH ) 6.1 Hz, 1H, OCHMe), 7.93 (d, J NH,CH
) 7.3 Hz, 1H, CHNH), 8.09 (br s, 1H, dC-NHH′), 9.02 (d, J
) 2.1 Hz, 2H, phenyl H-2, and H-6), 9.18 (dd, J ) 2.1, J ) 2.1
Hz, 1H, phenyl H-4). Anal. (C16H18N4O9‚1/2H2O) C, H, N.
2-Nitr ooxyeth yl 1,4-Dih yd r o-2,6-d im eth yl-3-n itr o-4-(2-
tr iflu or om eth ylp h en yl)p yr id in e-5-ca r boxyla te En a n ti-
om er s [(-)-(S)-2 a n d (+)-(R)-3]. A solution of either (+)-(S)-
11 or (-)-(R)-12 (136.8 mg, 0.40 mmol), 2-nitrooxyethyl
bromide (74.8 mg, 0.44 mmol), and K2CO3 (66.3 mg, 0.48 mmol)
in dry DMF (12 mL) was stirred at 25 °C for 24 h. Water (40
mL) was added, the mixture was extracted with EtOAc (3 ×
100 mL), and the organic extracts were washed with water (2
× 40 mL) and then brine (40 mL). The organic fraction was
dried (Na2SO4), the solvent was removed in vacuo, and the
residue was purified by silica gel column chromatography
using EtOAc/hexane (1:1, v/v) as eluent to yield (-)-(S)-2 or
(+)-(R)-3 as a yellow oil, respectively.
(1S,2R)-2-(3,5-Din it r op h en ylca r b on yla m in o)-2-m et h -
oxyca r bon yl-1-m eth yleth yl 1,4-Dih yd r o-2,6-d im eth yl-3-
n it r o-4-(2-t r iflu or om et h ylp h en yl)p yr id in e-5-ca r b oxy-
la te Dia ster eom er s (9 a n d 10). A solution of 8 (10.0 g, 24.4
mmol), R,R,R-trifluoro-o-tolualdehyde (4.25 g, 24.4 mmol), and
nitroacetone (3.51 g, 34.0 mmol) in EtOH (150 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 that was separated
by silica gel column chromatography using EtOAc/hexane (1:
1, v/v) as eluent. Upon elution, 9 and 6 eluted first (ratio of
1:2 according to the 1H NMR spectrum), which could not be
separated using the EtOAc/hexane system but which were
successfully separated using toluene/EtOAc (2:1, v/v) as eluent.
Further elution gave a mixture of the two diastereomers 9 and
10 followed by fractions containing pure 10. The fractions
containing the mixture of 9 and 10 were rechromatographed
using the same procedure. In this way, after five column
purifications, similar fractions were combined and the solvent
was removed in vacuo to afford 9 and 10 as yellow crystals
after recrystallization from EtOAc/hexane.
Dia ster eom er 9: 1.85 g; 11.65%; mp 231 °C; [R]23D +87.50°
(c 0.4, MeOH). IR (CHCl3): 1309 and 1474 (NO2), 1670 and
1738 (CO2) cm-1. 1H NMR (CDCl3): δ 1.39 (d, J Me,CH ) 6.4 Hz,
3H, CHMe), 2.48 (s, 3H, C-6 Me), 2.49 (s, 3H, C-2 Me), 3.73 (s,
3H, CO2Me), 4.56 (dd, J CH,NH ) 7.9 Hz, J CH,CH ) 7.0 Hz, 1H,
CHCHNH), 5.45 (qd, J CH,CH ) 7.0 Hz, J CH,Me ) 6.4 Hz, 1H,
OCHMe), 5.93 (s, 1H, H-4), 5.96 (s, 1H, dihydropyridyl NH),
7.02 (d, J NH,CH ) 7.9 Hz, 1H, CH-NH), 7.07 (dd, J ) 7.9, J )
7.3 Hz, 1H, trifluoromethylphenyl H-4), 7.15 (dd, J ) 7.9, J )
7.3 Hz, 1H, trifluoromethylphenyl H-5), 7.39 (d, J ) 7.9 Hz,
2H, trifluoromethylphenyl H-3 and H-6), 8.70 (d, J ) 2.0 Hz,
2H, dinitrophenyl H-2 and H-6), 9.18 (dd, J ) 2.0, J ) 2.0 Hz,
1H, dinitrophenyl H-4). Anal. (C27H24N5O11F3) C, H, N.
En a n tiom er (-)-(S)-2: 139.2 mg; 80.7%; [R]23 -48.75° (c
D
0.4, CHCl3). IR (CHCl3): 1271, 1308, 1492, and 1638 (ONO2
and NO2), 1704 (CO2), 3315 (NH) cm-1 1H NMR (CDCl3): δ
.
2.36 (s, 3H, C-6 Me), 2.51 (s, 3H, C-2 Me), 4.21-4.28 (m, 1H,
CHH′ONO2), 4.36-4.44 (m, 1H, CHH′ONO2), 4.56 (t, J ) 4.58
Hz, 2H, CO2CH2), 5.95 (s, 2H, H-4 and NH), 7.30-7.34 (m,
1H, phenyl H-4), 7.44 (m, 2H, phenyl H-5 and H-6), 7.55 (d, J
) 7.63 Hz, 1H, phenyl H-3). Anal. (C17H16N3O7F3) C, H, N.
En a n tiom er (+)-(R)-3: 139.6 mg; 81.0%; [R]23 +48.75° (c
D
1
0.4, CHCl3). IR (CHCl3) and H NMR (CDCl3) spectra for (+)-
(R)-3 were the same as those for (-)-(S)-2. Anal. (C17H16N3O7F3)
C, H, N.
(-)-(S)-Meth yl 1,4-Dih yd r o-2,6-d im eth yl-3-n itr o-4-(2-
tr iflu or om eth ylp h en yl)p yr id in e-5-ca r boxyla te [(-)-(S)-
1]. To a solution of (+)-(S)-11 (16 mg, 0.0468 mmol) in MeOH
(2 mL) was added a saturated solution of diazomethane in
ether with stirring for 15 min until no further gas evolution
occurred. Evaporation of solvents in vacuo gave a residue that
was purified by silica gel chromatography (EtOAc/hexane 1:1,
v/v) to yield (-)-(S)-1 as a yellow oil (12.4 mg, 74.5%), [R]23
D
-56.75° (c 0.4, dioxane) [lit.4 [R]D -56.7° (dioxane)]. H NMR
1
(CDCl3): δ 2.34 (s, 3H, C-6 Me), 2.51 (s, 3H, C-2 Me), 3.60 (s,
3H, CO2Me), 5.88 (br s, 1H, NH), 5.95 (s, 1H, H-4), 7.27-7.31
(m, 1H, phenyl H-4), 7.42 (m, 2H, phenyl H-5 and H-6), 7.54
(d, J ) 7.9 Hz, 1H, phenyl H-3).
Op tica l P u r ity of (-)-(S)-2, (+)-(R)-3, a n d (-)-(S)-1. The
optical purity of (-)-(S)-2, (+)-(R)-3, and (-)-(S)-1 were
determined by 1H NMR spectrometry. When 50 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-trifluoromethylphenyl)pyri-
dine-5-carboxylate (5 mg in 0.5 mL of CDCl3), the original 1,4-
dihydropyridyl C-6 methyl resonance at δ 2.36 was separated
into two resonances that appeared at δ 2.52 and 2.53. Addition
Dia ster eom er 10: 1.59 g; 10.0%; mp 214 °C; [R]23D +136.75°
(c 0.4, MeOH). IR (CHCl3): 1309 and 1474 (NO2), 1670 and
1738 (CO2) cm-1. 1H NMR (CDCl3): δ 1.11 (d, J Me,CH ) 6.2 Hz,
3H, CHMe), 2.34 (s, 3H, C-6 Me), 2.41 (s, 3H, C-2 Me), 3.64 (s,
3H, CO2Me), 4.82 (dd, J CH,NH ) 8.2 Hz, J CH,CH ) 3.8 Hz, 1H,
CHCHNH), 5.46 (m, 1H, OCHMe), 5.81 (s, 1H, H-4), 5.84 (s,
1H, dihydropyridyl NH), 7.15 (d, J NH,CH ) 8.2 Hz, 1H, CHNH),
7.22 (m, 1H, trifluoromethylphenyl H-4), 7.36 (m, 2H, trifluo-
romethylphenyl H-5 and H-6), 7.43 (d, J ) 7.6 Hz, 1H,
trifluoromethylphenyl H-3), 8.93 (d, J ) 2.0 Hz, 2H, dinitro-

960 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 4
Shan et al.
of (+)-Eu(hfc)3 (80 µL) to (-)-(S)-2 or (+)-(R)-3, as described
above, resulted in retention of a single resonance for the C-6
methyl resonance (g96% ee). Similarly, addition of (+)-Eu-
(hfc)3 (30 µL) to racemic Bay K 8644 (5 mg in 0.5 mL of CDCl3)
resulted in the separation of 1,4-dihydropyridyl C-6 methyl
and the ester methyl resonances at δ 2.34 and 3.60 into two
resonances that appeared at δ 2.59 and 2.61, and 3.90 and
3.91, respectively. Addition of (+)-Eu(hfc)3 (80 µL) to (-)-(S)-
1, as described above for racemic Bay K 8644, resulted in
retention of single resonances for the C-6 methyl and the ester
methyl resonances (g96% ee).
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.11
The IC50 value ((SEM, n ) 3) was determined graphically
from the dose-response curve.
the holding potential of -80 mV, followed by a return to -40
mV. The calcium current (ICa) was activated by 200 ms test
steps to potentials between -40 and +80 mV in 10 mV
increments.
3. Exp er im en ta l P r otocol. Stock solutions of drugs were
prepared by dissolving the test compound in distilled DMSO.
Because DMSO exerts a weak inhibitory effect11 on ICa, we
controlled for solvent effects by including the same amount of
DMSO in all control and drug solutions. Cells were first
exposed to control solution for 10 min, then to 1 or 10 µM
concentrations of the test compound for 10 min. Drug was
washed out for up to 15 min, although the effects of test
compound on ICa was not reversed within that time.
4. An a lysis. The peak amplitude of ICa was measured as
the difference between the peak inward current and the
current at 200 ms. In a separate series of experiments, the
validity of this measure of ICa was tested in cells exposed to
known calcium channel antagonists. Current-voltage rela-
tionships were constructed by plotting the peak amplitude of
ICa as a function of membrane potential. Data are presented
as mean ( SEM where “n” is the number of myocytes sampled.
Differences between drug treatment and control recordings
were assessed with two-way repeated measures analysis of
variance. Differences were significant when p < 0.05.
Calcium channel modulation activity (positive or negative
inotropic effect) was calculated as the molar concentration of
the test compound required to produce a 50% increase (positive
inotropic, or CC agonist, effect) or a 50% decrease (negative
inotropic, or CC antagonist, effect) in contractile force of
isolated guinea pig left atrium (GPLA) relative to its basal
contractile force in the absence of test compound.11
Ack n ow led gm en t. We thank the Canadian Insti-
tutes of Health Research (Grant No. MT-8892) for
financial support of this research and for a fellowship
to R.S. The authors also acknowledge the excellent
technical assistance of C.-A. McEwen, P. Nicholl, and
C. Mapplebeck.
Nitr ic Oxid e Relea se. In vitro nitric oxide release was
assayed using a modification of the previously reported
procedure.15 The test compound (0.0075 mmol) was added to
a thoroughly mixed solution of 0.1 M phosphate buffer (pH
7.4) and acetonitrile (1:1, v/v) (1.5 mL) containing N-acetyl-
cysteamine (1 equiv per -ONO2 moiety), or without 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% phos-
phoric acid (10 mL) in distilled water (final volume, 100 mL)]
for 10 min at 25 °C. After dilution of this solution with water
to a volume of 5 mL, ultraviolet absorbance was measured at
540 nm. Solutions of 3-24 µM sodium nitrite were used to
prepare a nitrite absorbance 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
absorbance versus concentration curve.
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2-Nitrooxyethyl Enantiomers
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J M010394K