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Synthesis and Smooth Muscle Calcium Channel Effects of Dialkyl 14-Dihydro-26-dimethyl-4-aryl-35-pyridinedicarboxylates Containing a Nitrone Moiety in the 4-Aryl Substituent.

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53
Synthesis and Smooth Muscle Calcium Channel Effects of Dialkyl
1,4-Dihydro-2,6-dimethyl-4-aryl-3,5-pyridinedicarboxylates
Containing
a Nitrone Moiety in the 4-Aryl Substituent
Raymond D. Ananaa),Helen Nga), Susan E. Howlettb),and Edward E. Knausa)*
a) Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2N8, and
Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7
b,
Department of Pharmacology,
Key Words: Hantzsch 1,4-dihydropyridines;nitrones; calcium channels; smooth muscle relaxation; voltage-clamp studies
Summary
A group of dialkyl 1,4-dihydro-2,6-dimethy1-4-(
3- (or 4-)[[(Z)-Noxo-N-[4-substituted-phenylmethylene(or vinylmethylene)]-)L5azdnyl]phenyl)-3,5-pyridinedicarboxylates 7a-n were synthesized. Reaction of the C-4 nitrophenyl compounds 6a-d with an
aryl Grignard reagent afforded the corresponding nitrone derivatives 7a-e. Alternatively, reaction of the aryl hydroxylamine compounds 8a-b prepared by reduction of the nitrophenyl compounds
6c-d with Zn/NH4CI, or the aryl hydroxylamine compounds 8c-d
prepared by reduction of the nitrophenyl compounds 6e-f with 5%
rhodium-on-charcoal and 65% hydrazine hydrate, with a 4-substituted-benzaldehy de, benzaldehyde or acrolein afforded the respective nitrone compounds 7f-n. In vitro calcium channel (CC)
antagonist activities were determined using the guinea pig ileum
longitudinal smooth muscle assay. This class of compounds containing a nitrone moiety on the 1,4-dihydrop?ridine C-4 phenyl
M range)
ring exhibited CC antagonist activities (10- to
relative to the reference drug nifedipine (Ic50 = 1.43 x lo-* M).
Structure-activity relationships showed that the position of the
nitrone moiety on the C-4 phenyl ring was a determinant of CC
antagonist activity where the potency order was always meta-nitrone > para-nitrone. The effect of the ester alkyl substituent was
variable depending upon whether the nitrone substituent was at the
meta orpara-position (rneta-nitrone, Et > i-Pr= Me; para-nitrone,
i-Pr> Me = Et). In the diethyl ester series of compounds having a
rneta-nitrone moiety, the difference in potency for the various
R2-nitrone substituents varied by a factor of 15-fold (Ic50 = 1.5 I
x
M range) (4-Cl-C6H4- 2 4-Me-CsH4- = C6H5to 9.84 x
2 4-02N-C6H4- 4-F,C-C6H4- >> CH2=CH-). Whole-cell voltageclamp studies using isolated guinea pig ventricular myoc tes indicated that the 4 4 3-[(Z)-N-oxo-N-(phenylmethylene)-h-azanyl]is aI
calcium
)
channel antagonist
phenyl) compound 7c (10 @
which decreased the calcium current ( 1 ~ ~ ) .
Y
Introduction
Tissue-selective 1,4-dihydropyridine (DHP) L-type voltage-dependent calcium channel modulators are important
therapeutic agents and/or potential probes to stud the structure-function relationshipof calcium channels[14! DHP calcium channel antagonists with high vascular selectivity, that
exert a minimal inotropic effect, are effective for the treatment of vasospastic disorders and hypertensi~n[~I.
Alternatively, a number of 1,4-DHP calcium channel agonists such
Arch. Pharm.Pharm. Med. Chem.
as the nitro compounds Bay K 8644 [l, (-)- 5')-enant i ~ m e r ] [ and
~ , ~PN
] 202-79 1 [2, (+)-(S)-enantiomer]t6.81have
been reported (see Figure 1). Although Bay K 8644 exhibits
a positive inotro ic effect by enhancing calcium entry into
cardiac muscler9f it simultaneouslyinduces vasoconstriction
which precludes its potential clinical utility to treat congestive heart failure (CHF)['ol. We have discovered a novel
group of (f)-isopropyl 1,4-dihydro-2,6-dimethyl-3-nitro-4(pyridinyl)-5-pyridinecarboxylateisomers 3a-c. The rac-2pyridinyl isomer 3a produced dual cardioselective calcium
channel agonisdsmooth muscle selective calcium channel
antagonist in vitro effects. In contrast, the rac-3-pyridinyl3b
and rac-4-pyridinyl 3c isomers exhibited agonist in vitro
effects on both heart and smooth muscle. The (-)-Zpyridinyl
enantiomer (-)-3a exhibited in vitro cardiac agonist and
smooth muscle antagonist properties that would be highly
desirable for the treatment of CHF["]. In earlier studies, we
also described the novel 3,5-diester compounds (+))-4[121,as
well as the (+)-4 and (-)-4 enantiomers[l3I, all of which
exhibited dual cardioselective partial calcium channel
agonist (positive inotropic)/smoothmuscle selective calcium
channel antagonist in vitro effects.
In vivo studies using rabbits showed that r a c 3 b and rac3c
produced a hypertensive effect that was qualitatively similar
to that of rac-Bay K 8644 after fbblockade with propranolol,
whereas rac3a produced a hypotensive effect like nifedipine["]. These results indicate that the position of the pyridinyl
nitrogen free electron-pair, and/or charge distributi~n['~,'~I
in the pyridinyl ring, may be important determinants of calcium channel agonist-antagonistmodulation effects. Accordingly, it was envisaged that incorporation of a nitrone
substituent [-N'(0-)=CH-] at the meta- orpara-position of a
C-4 phenyl ring on a Hantzsch 1,4-DHP would be capable of
electrostatic binding to the al-subunit binding site of the
L-type calcium channel receptor[16]. A nitrone moiety is
related electronically to a nitro group, and it has some structural similarity to the C-4 heteraryl ring system present in PN
202-79 1 (2). The ionic dipolar character of the nitrone moiety
should exclude its passage across the blood-brain barrier
thereby precluding CNS stimulant effects such as those induced by Bay K 8644[17].Although a group of compounds 5
having a nitrone moiety at the meta- or para-position of the
C-4 phenyl substituent has been described that were evaluated as platelet-aggregation inhibitors and antimetastatic
agents, no calcium channel modulation activities were reported[lgl.As part of our on-going program to design tissue
0 VCH Verlagsgesellschaft mbH, D-6945 1 Weinheim, 1997
0365-6233/97/0303-0053$17.50 +.50/0
54
Anana, Ng, Howlett, and Knaus
2
I
3a, 2-pyridinyl
3b, 3-pyridinyl
312.rlpyridinyl
T
(*)a
(+)a
H
5
(4-4
Figure 1. Structures of Bay K 8644 (1 ), PN 202-79 1 (2), isopropyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-(pyndinyl)-5-py~dinec~xyl~e
isomers (3),
isopropyl 1-[2-(4-methylphenyl)ethyl] 1,4-dihydro-2,6-dimethyl-4-(2-pyridinyl)-3,5-pyndinedicarboxylate (4). and 3- or 4-nitrone analogs of Hantzsch
1.4-dihydropyridines(5).
selective third generation calcium channel modulators, we
now report the synthesis, in vitro smooth muscle calcium
channel modulating effects and whole-cell voltage-clamp
studies using isolated guinea pig ventricular myocytes for
dialkyl 1,4-dihydro-2,6-dimethyl-4-aryl-3,5-pyridinedicarboxylates 7a-n having a nitrone moiety in the C-4 phenyl
substituent.
4
I
6a,R1 = Me, R2 = >NO2
Bb, R1 = M e , R2 = 4-No2
Be, R1 = Et. R2 = >NO2
Bd, R1 = Et. R2 = 4 4 0 2
I
7a, R1 = Me, Rz = H
7b, R1 = Me, R2 = H
7c, R1 = Et, R2 = H
7d, R1 = Et R2 = H
7e, R1 = Et. R2 = M e
Nitrone
paltion
3
P
/,
h . R = W H
8b, R = 4-NIOH
71. R = 4OzFcCgHq7g, R = 4-F3C-CgH47h. R = 4-CCCgH471. R = -CkCliz
7j, R = 4-02N-CgHq-
Chemistry
for the
A novel method developed by Bartoli et
preparation of (2)-nitrones by reaction of a nitro group with
a benzyl Grignard reagent at -70 "C in THF was employed
for the synthesis of the nitrone compounds 7a-e. Groups that
are normally highly reactive toward Grignard reagents such
as carbonyl functions of ketones and ester are completely
inert provided the Grignard reagent is added slowly at low
temperature. Thus, reaction of the C-4 nitrophenyl 1,4-DHP
compounds 6a-d with benzylmagnesium chloride, or 4methylbenzylmagnesiumchloride, in THF at -70 "C afforded
the respective target compounds 7a-e in 1 4 4 5 % chemical
yields as illustrated in Scheme 1. A similar attempt to prepare
analogs of 7 possessing the nitrone substituent at the orthoposition of the C-4 phenyl ring was unsuccessful, possibly
due to subsequent reaction of the reactive ortho-nitrone moiety with the 1,4-DHP C-3 alkyl ester substituent.
?-
+Pc.
7k,R=46CCgH471, R = -CkCttr
-
Nltrom
3
3
3
3
4
4
4
Scheme 2. Reagents and conditions:(a) Zn powder, NHdCI, 86% EtOH; (b)
absolute EtOH, anhydrous NazSOj, 4-substituted-benzaldehyde(7f-h, 7j-k)
or CHz=CHCHO (74 71). 25 "C.
stituted-benzaldehyde, or acrolein, using absolute EtOH as
solvent in the presence of anhydrous Na2S04 to remove water
formed in the reaction, to afford the respective (a-nitrone
products 7f-1 in 5-5 1% chemical yields. The condensation of
aryl hydroxylamines with aryl aldehydes produces (z)-nitrones (aryl roup from aryl aldehyde syn to nitrone oxygen)
exclusively["l. The low yield of products 7f-I in these reactions is attributed to the fact that 10-15% of unreacted nitrophenyl compound 6c or 6d was recovered, and a dimeric
product is also formed (3040%). The 'H NMR spectrum of
the dimeric product exhibited dual resonances for the C-4
phenyl, C-3 and C-5 ethyl ester, 1A-DHP C-2 and C-6 Me's,
and the NH protons. No resonance for a nitrone =CH proton
which normally appears at about 6 7.9 in compounds 7f-1 was
present. The 'H NMR spectral data are consistent with an
azoxy product 9 resulting from reaction of the hydroxylamine
intermediate product 8a or 8b with the corresponding transient nitroso (N=O) intermediate also formed during the
preparation of 8a or 8b from the nitro precursor 6c or 6d.
0-
4
3
4
3
Scheme 1. Reagents and conditions: (a) PhCH2MgCI (7a-d) or 4-Me-CsH4CHzMgCI (7e). THF, -70 "C.
The nitrone compounds 7f-1 were prepared using the alternate rocedure illustrated in Scheme 2. Accordingly, reductionlp8I of the nitrophenyl compounds 6c-d using zinc
powder and NH&l in 86% EtOH at 25 "C for 50 minutes
afforded the respective hydroxylamineproducts 8a-b. Compounds 8a-b, which were used without further purification
due to their instability, were condensed with either a 4-sub-
EtWzC;;Et
E~OZCJ@%EI
Me
I
I
9
Attempts to prepare the diisopropyl ester nitrone products
7m-n by reaction of 6e-f with benzylmagnesium chloride as
illustrated in Scheme 1 was unsuccessful since the reaction
did not proceed and unreacted 6e or 6f was recovered. In
Arch. P h a n P h a n Med. Chem. 330,53-58(1997)
55
1,4-Dihydr0-3,5-pyridinedicarboxylates
contrast, reduction of 6e and 6f to the respective hydroxylamine derivatives 8c and 8d using 5% rhodium-on-charcoal
and 65% hydrazine hydrate in THF[211proceeded smoothly.
Subsequent condensation of the intermediate hydroxylamine
derivative 8c or 8d with benzaldehyde in absolute EtOH in
the presence of anhydrous Na2S04 afforded 7m (54%) and
7n (62%), respectively as illustrated in Scheme 3. Optimal
yields of 7m and 7n were obtained when reduction of 6e or
6f to the corresponding hydroxylamine8c or 8d was allowed
to proceed to 80-90% completion prior to condensation with
benzaldehyde.
~
w,wf4.-..-'
l-p**c~
I
6e, R = 3-NO2
8c. R = 5 M H
Sf, R = 4 N 0 2
8d. R = 4-NHOH
Nitrone
position
7m,R=-CgH5
7n, R = -Gj&
3
4
Scheme 3. Reagents and conditions: (a) 5% rhodium-on-charcoal, 65% w/v
aqueous hydrazine hydrate, THF, 25 "C; (b) absolute EtOH, anhydrous
Na2S04, PhCHO, 25 "C.
Results and Discussion
The 1,4-DHP compounds 7a-n investigated possess identical C-3 and C-5 alkyl (Me, Et, i-Pr) ester substituents.
Consequently, the 1,4-DHP C-4 center is not chiral which
precludes the possibility of differential calcium channel
agonist/antagonist effects due to enantiorners[l',13].
The in vitro calcium channel antagonist activities of 7a-n,
determined as the concentration required to produce 50%
inhibition of guinea pi ileum longitudinal smooth muscle
(GPLSM) contractilit$221 are presented in Table 1. Compounds 7a-n exhibited calcium channel antagonist activities
(
to
M range) relative to the reference drug nifedipine (IC50 = 1.43x
M). The position of the nitrone moiety
on the C-4 phenyl ring (meta- orpara-) was a determinant of
calcium channel antagonist activity where the potency order
was always meta-nitrone >para-nitrone when the R' and R2
substituents were identical (7a > 7b; 7c > 7d; 7f > 7j; 7h >
7k; 7i > 71; 7m > 7n). This structure-activity relationship
(SAR) is in agreement with known SARs for 1,4-DHP calcium channel antagonists where the potency order for C-4
phenyl substituents is generally ortho > meta > para[231and
for C-4 pyridinyl isomers where 2-pyridinyl> 3-pyridinyl>
4-~yridinyl[~~I.
The effect of the alkyl ester R1-substituent
was variable depending upon whether the nitrone substituent
was located at the meta or para position of the C-4 phenyl
ring. For example, in the rneta-nitrone series, the relative
potency order was Et (7c) > i-Pr (7m) = Me (7a), whereas in
the para-nitrone series the potency profile was i-Pr (7m) >
Me (7b) = Et (7d).This latter SAR for the para-nitrone series
is in agreement with previous SARs which indicated that
larger alkyl ester substituents provide optimal calcium channel antagonist activity when the substituenton the C-4 phenyl
ring is located at the p a r a - p ~ s i t i o n [In
~ ~the
~ . diethyl ester
series of compounds possessing a meta-nitrone moiety (7c,
7+7i), the differences in potency for the various R2-nitrone
Arch Phann Pham. Med Chem 330,53-58(1997)
substituents varied by a factor of 15-fold (IC50 = 1.51 x
to 9.84 x
M range) [4-C1-c6&- (7h) 2 4-Me-CgH4- (7e)
= C6H5- (7C) 2 4-02N-C6H4- (70 > 4-F3C-C6H4- (7g 1, and
these compounds are much more potent than the R -vinyl
analog (7i, IC50 = 1.70 x lo4 M). In contrast, in the diethyl
series of compoundshaving apara-nitrone moiety (7d,7k-1),
the only compound exhibiting significant activity was the R2
= 4-02N-C6&- analog (7j, IC50 = 4.30 x
M) where the
R2-substituent profile was 4-02N-C6H4- (7j) > 4-CI-C6H4(7k), C6H5- (7d) and -CH=CH2 (7h).
The modulation of L-type voltage-sensitive calcium channels by the nitrone compound 7c in isolated guinea pig
ventricular myocytes using the whole-cell voltage-clamp
technique was investigated. The nitrone analog 7c was selected as a model compound for the voltage-clamp study
since it exhibited good in vitro calcium channel antagonist
activity, and the nitrone moiety at the meta-position is between that of the ortho- and para-positions of the C-4 phenyl
ring. The mean (+SEM) current-voltage (I-V) relations for
calcium current (Zca)elicited by depolarizing steps (200 ms)
from a membrane potential of -40 mV to potentials between
4 0 and +80 mV under control conditions and in the presence
of 1 and 10 pM concentrations of 7c are shown in Figure 2.
All solutions, including control solution, contained 0.001%
DMSO. Under control conditions,the threshold for activation
of Zca was between -40 and -30 mV and I c a reached a
maximum between -10 and 0 mV. The lower concentration
of 7c (1 pM) had little effect on I c a at all membrane potentials
7c reduced the magnitude of I c a
examined. However, 10
3
0.0
.
-0.2
c
-0.4
C
g!
L
3
0 -0.6
0 control
0 10pM7c
-0.8
I
-1.0
-60
I
I
-40
-20
II
0
I
I
I
I
20
40
60
80
Voltage (rnv)
Figure 2. Mean (fSEM) I-Vrelationships for Ica,measured as peaklcaminus
current at 200 ms, under control conditions and in the presence of 1 and
10 pM concentrations of 7c. Voltage steps were made from a membrane
potential of -40 mV. Although 1 pM 7c had very little effect on current,
10 pM 7c inhibited Ica by about 50 % at the peak of the I-V curve. Experiments were conducted in 2 mM Ca2+buffer which contained 200 pM lidocaine to inhibit Na' currents. All solutions, including control solution,
contained 0.001% DMSO. Asterisks indicate points that are significantly
different from control O-, < 0.05).
56
Anana, Ng, Howlett, and Knaus
Table 1. Physical properties and calcium channel antagonist activities of dialkyl 1,4-dihydro-2,6-dimethyl-4-(
3- (or 4-)[(Z)N-oxo-N-[4-substituted-phenylmethylene (or vinylmethylene)]-h5-azanyl]phenyl)-3.5-pyridinedicarboxylates(7a-n).
R1ofiJ@zl
Me
Cmpd
7a
7b
lc
7d
7e
7f
7g
7h
7i
7j
7k
n
7m
7n
Nifedipine
R'
R2
Me
Me
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
i-Pr
i-Pr
I
Nitrone
position
Cryst.
solvent
MP, "C
3
4
3
4
3
3
3
3
3
4
4
4
3
EtOAc-hexane
EtOAc-hexane
EtOAc-hexane
EtOAc- hexane
EtOAc-hexane
EtOAc-hexane
EtOAc- hexane
EtOAc-hexane
CH~C12-hexane
EtOAc-hexane
140-142
24S242
183-185
214-215
162-1 63
184-186
181-182
187-1 88
133-134
175-177
semi-solid
semi-solid
95-97
169-1 7 1
4
EtOAc- hexane
EtOAc- hexane
Formula[a1
%
Yield
30
40
42
45
14
5
22
21
21
42
51
48
54
62
Calcium channel
antagonist act:^^^,, ( M ) ~ '
2.21 k 0.01 x I O - ~(3)
2.77 f 0.21 x 1 0 - ~ (3)
(3)
3.69 f 0.17 x
3.53 k 0.07 x 10-~ (3)
(3)
1.54 0.03 x
6.17k0.33 x lo-* (3)
1.51 k 0.03 x
(3)
9.84 k 0.73 x
(3)
1.70 k 0.01 x 10" (3)
4.30 k 0.1I x I O - ~(3)
l.l0*0.55x 10-5(3)
4.41 ? 0.19 x lo-' (3)
1.55k0.11 x lO-'(3)
1.04 k 0.02 x 10" (3)
1.43 k 0.38 x lo4 (8)
+
[alMicroanalyticalanalysis were within f 0.4% of theoretical values for C, H and N unless otherwise indicated.
[blThemolar concentration of antagonist test compound causing a 50% decrease in the slow component, or tonic contractile response, (Icso f SEM) in guinea
M) was determined graphically from the dose-response curves. The
pig ileal longitudinal smooth muscle by the muscarinic agonist carbachol (1.6 x
number of experiments is shown in brackets.
["I12 molecule of water of hydration.
ld11/4molecule of water of hydration.
lelExactmass calculated: 398.18417; found (high resolution mass spectrum): 398.18417.
[%he product could not be recrystallized. Rf (silica gel TLC) 0.57 using EtOAc-hexane (2: 1, v/v) as development solvent.
IglTheproduct could not be recrystallized. Rf (silica gel TLC) 0.61 using EtOAc-hexane (2: 1. v/v) as development solvent.
[h11/2molecule of water of hydration. N: calcd, 6.44; found, 6.95.
"l3/4 molecule of water of hydration.
when compared to control. The threshold for activation of Zca
was not affected by 10 pM 7c, but the peak of the I-V curve
was decreased by almost half. The main effect of 7c (10 pM)
on Zca was statistically significant (p < 0.05). These results
indicate the rnetu-nitrone compound 7c is an antagonist of the
voltage-sensitive calcium channel in guinea pig ventricular
myocytes. Unfortunately, we were not able to synthesize the
ortho-nitrone isomer of 7c which would have allowed us to
determine its modulation (antagonist/agonist) effect on the
voltage-sensitive L-type calcium channel in myocytes.
Acknowledgments
We are grateful to the Medical Research Council of Canada (Grant No.
MT-8892) for financial support of this research, to the Association of
Commonwealth Universities of Canada for a Canadian Commonwealth
Scholarship Award (to R.A.) and to the Alberta Heritage Foundation for
Medical Research for a Summer Studentship Award (to H.N.). The authors
would also like to acknowledge the excellent technical assistance of C.-A.
McEwen and P. Nicholl.
Experimental
Melting points were determined using a Thomas-Hoover capilliaq apparatus and are uncorrected. 'H NMR spectra were recorded on a Bruker
AM-300 spectrometer. The assignment of exchangeable protons was confirmed by the addition of [D2]H20. Infrared spectra were acquired using a
Nicolet 5DX-FT spectrometer. Silica gel column chromatography was carried out using Merck 7734 (6S200 mesh) silica gel. Preparative silica gel
TLC was performed using Camag Kieselgel DF-5 plates, 1.0 mm in thickness. The progress of reactions was monitored using Macherey-Nagel Polygram@Sil GAJV254 plates (0.25 mm diameter). Microanalyses were within
f 0.40% of theoretical values for C, H, and N, unless otherwise stated. The
dialkyl 1,4-dihydro-2,6-dimethyl-4-(nitrophenyl)-3,5-pyridinedicarboxylates (6a-f) were prepared according to the reported procedures[261.
General Method for the Preparation of Dialkyl 1,4-Dihydro-2,6-dimethyl4-[3-(or 4-)[(Z)-N-oxo-N-(arylmethylene)-hs-a~anyl]phenyl}-3~5-pyridinedicarboxylates 7a-e.Method I
A solution of either benzylmagnesium chloride, or 4-methylbenzylmagnesium chloride, in THF (2M solution, 5 mmol) was added slowly with stirring
to a solution of the respective nitrophenyl compound (6a, 6b, 6c or 6d,
2 mmol) in dry THF (50 ml) at -70 "C under a nitrogen atmosphere. The
reaction was allowed to proceed at -70 "C until such time (0.5-5 h) that silica
gel TLC analysis indicated the respective nitrophenyl compound 6a-d was
Arch. Phrm. Phurm. Med. Chem 330,53-58 ( I 997)
57
I ,4-Dihydro-3,5-pyridinedicarboxylates
all consumed. The reaction mixture was quenched with saturated aqueous
NH4C1 (10 ml). Extraction with EtOAc (3 X 50 ml), drying the combined
EtOAc extracts (MgS04), and removal of the solvent in vacuo gave the
respective product 7. Products 7a, 7b, 7c, 7d and 7e were purified by silica
gel column chromatography using EtOAc-hexane (3:1, v/v)as eluent prior
to recrystallization from EtOAc-hexane. The mp and % yield of products
7a-e are listed in Table 1. Representative spectral data (IR,'H NMR) for
compounds 7b and 7e are provided since the spectral data for 7a, 7c and 7d
are qualitatively similar.
Diethy/I,4-Djhydro-2,6-dimethy/-4-{4-[(Z)-N-oxo-N-(vinylmethylene)hs-azanyl]phenyl]-3,5-pyridinedicarboqlate71
IR (KBr): v = 3339 cm-' (NH), 1698 (COz), 1226 (NO).- 'H NMR
(CHC13-di): 6 7.18 (d, 52.3 = 55.6 = 8.6 Hz, 2H, C-4 phenyl H-3 and H-5),
6.91(d,J2.~=J5,ti=8.6Hz,2H,C-lphenylH-2andH-6),5.91(~,1H,NH),
4.92 (s, IH, H-4). 5.69(d, J c H . c H =Hz,
~ .IH,
~ nitrone =CH-CH=CHA 4.03
(q,J=7.1 H z , ~ H , C O ~ C H ~ C3.62-3.74,3.31-3.44and2.362.56(three
H~),
m, lHeach,CH=CH2), 2.31 (s,6H,DHPC-2andC-6Me's), 1.22 (t,J=7.1
Hz, 6H, C02CH2CH3).
Dimethyl 1,4-Dihydro-2,6-dimethyl-4-{4-[(Z)-N-oxo-N-([phenylmethylene)-hs-azanyl]phenyl/-3,5-pyridinedicarboxylate
76
General Method for the Preparation of Diisopropyl I,4-DihydroIR (KBr): v = 3345 cm-' (NH), 1692 (C02). 1213 (NO).- 'H NMR
(CHCl3-di): 6 8.37-8.40 (m, 2H, C-4 phenyl H-3 and H-5). 7.88 (s, lH,
nitrone =CH), 7.61 (dd, J2.3 = J5.6 = 8.0 Hz, 2H, C-phenyl H-2 and H-6).
7.45-7.50 (m, 3H, C-phenyl H-3, H-4 and H-5). 7.41 (dd, 52.3 = J5.6 = 8.0
Hz, 2H, C-4 phenyl H-2 and H-6), 6.54 (s, IH, NH), 5.04(s, lH, DHP H-4),
3.65 (s, 6H, CO2Me), 2.05 (s, 6H, DHP C-2 and C-6 Me's).
2,6-dimethyl-4-{3-(or
4-)[(Z)-N-oxo-N-(phenylmethylene)hs-azanyl]phenyl]-3,5-pyridinedicarboxylates
7m-n.. Method 3
Aqueous hydrazine hydrate (2 mmol of 65% w/v)was added slowly to a
solution of the respective nitrophenyl compound 6e or 6f (0.80 g, 2 mmol)
in dry THF (50 ml) containing 5% rhodium-on-charcoal (20 mg) with
stirring. The reaction was allowed to proceed at 25 "C until silica gel TLC
indicated that reduction has proceeded to near completion. The reaction
mixture was filtered, water (30 ml) was added to the filtrate, and the mixture
Diethyl I,4-Dihydro-2,6-dimethyl-4-{3-[(Z)-N-oxo-N-(phenylmethylene)was extracted with ether (3 x 50 ml). The organic solution was dried
hs-azanyl]phenyl]-3,5-pyridinedicarboxylate
7e
(NazSOd), and the solvent was removed in vacuo to afford the respective
IR (KBr): v = 3335 cm-' (NH), 1699 (C02). 1230 (NO).- 'H NMR
hydroxylamine product 8c or 8d which was used immediately in the suhsequent reaction without further purification. Benzaldehyde (0.18 g, 2 mmol)
(CHCh-di): 6 8.27 (d, J4.5 = 8.0,55,6= 8.0 Hz, 2H, C-4 phenyl H-4 and H-6).
was added to a solution of either 8c or Sd, obtained above, in absolute EtOH
7.83 (s, IH, nitrone =CH), 7.70 (s, lH, C-4 phenyl H-2). 7.20-7.55 (m, 5H,
(30 ml) containing anhydrous Na2.904 (0.40 g). The reaction was allowed to
C-4 phenyl H-5, tolyl hydrogens), 5.72 (s, IH, NH), 5.08 (s, IH, DHP H-4),
4.12 (q, J = 7.0 Hz, 4H, C02CH2CH3), 2.42 (s, 3H, tolyl-Me), 2.33 (s, 6H,
proceed at 25 "C with stirring for 12 h, chloroform (100 ml) was added, the
DHP C-2 and C-6 M e ' s ) , 1.23 (t, J = 7.0 Hz,6H, CH2CH3).
solution was dried (MgS04) and the solvent was removed in vacuo. The
respective product (7m or 7n) was purified sequentially by silica gel column
chromatography using EtOAc-hexane as eluent, and then preparative silica
General Method for the Preparation of DiethylI,4-Dihydro-2,6-dimethyl- gel TLC using EtOAc-hexane (4:1, v/v) as development solvent, prior to
4-{3-(or4-)[(Z)-N-oxo-N-(substituted-methylene)-hs-azanyl]phenyl]recrystallization from EtOAc-hexane. The mp and % yield of 7m and 7n are
3,S-pyridinedicarboxylates7f-I.Method 2
listed in Table 1. The IR and 'H NMR spectral data for 7m-n are listed below.
Zinc powder (0.52 g, 7.95 mmol) and NH4CI (0.26 g, 4.8 mmol) were
added to a solution of the respective diethyl ester compound 6c or 6d (0.75 g,
2 mmol) in 86% EtOH (22 ml). The resulting mixture was stirred vigorously
at 25 "C for 50 min, the reaction mixture was filtered and the filtrate was
poured into CH2C12 (100 ml). This solution was washed with water (2 x
30 ml) and the organic fraction was dried (MgS04). Removal of the solvent
in vacuo gave the respective hydroxylamine product as an oil (8a or 8b)
which was used without further purification in the subsequent reaction. A
4-substituted-benzaldehyde,or acrolein, (2 mmol) was then added to a
solution of the respective hydroxylamine product obtained above (8a or 8b)
in absolute EtOH (30 ml) containing Na2S04 (0.4 g) under an atmosphere of
nitrogen. The reaction was allowed to proceed at 25 "C with stirring until
silica gel TLC analysis indicated that 8a or 8b had been consumed (about
20-24 h), chloroform (100 ml) was added and the solution was dried
(MgS04). Removal of the solvent in vacuo gave aresidue which was purified
by silica gel column chromatography using EtOAc-hexane (1:2, v/v; 7f-i) as
eluent, or consecutive silica gel column chromatography and then preparative
silica gel TLC chromatography using EtOAc-hexane (2: 1, v/v)as eluent and
development solvent (7j-l), respectively. Products 7f-h and 7j were recrystallized from EtOAc-hexane, and 7i was recrystallized from CHzClz-hexane.
Products 7k-1, which could not be recrystallized, were isolated as yellow
semi-solids. The % yield of products 7f-I, and mp or products 7f-j, are listed
in Table 1. Representative spectral data (IR, 'H NMR) for compounds 7k
and 71 are listed below.
Diisopropyl I,4-Dihydro-2.6-dimethyl-4-{3-[(Z)-N-oxo-N-(phenylmethylene)-h5-nzanyl]phenyl/-j.5-pyridinedi~arboxyla~e
7m
IR (KBr): v = 3338 cm-' (NH), 1697 (C02), 1225 (NO).- 'H NMR
(CHC13-di): 6 8.30-8.40 (m, 2H, C-4 phenyl H-2 and H-4). 7.87 (s, lH,
nitrone =CH), 7.67-7.70 (m, IH, C-4 phenyl H-6). 7.267.47 (m, 6H, C-4
phenyl H-5, C-phenyl hydrogens), 6.29 (s, lH, NH), 5.05 (s, lH, DHP H-4).
4.96(septet,J=6.2Hz,2H,CHMez),2.29(s, 6H,DHPC-2andC-6Me's),
1.13and 1.24(twod,J=6.2Hz,6Heach,CHMe2).
Diisopropyl 1,4-Dihydro-2,6-dimethyl-4-{4-[(Z)-N-oxo-N-(phenylmethy1ene)-h5-azanyl]phenyl]9,5-pyridinedicarboxylate7n
1R (KBr): v = 3337 cm-' (NH), 1697 (COz), 1221 (NO).- 'H NMR
(CHCI3-di): 6 8.32-8.40 (m, 2H, C-4 phenyl H-3 and H-5). 7.89 (s, IH,
nitrone =CH), 7.62 (d, J2.3 = J5.6 = 8.6 Hz, 2H, C-phenyl H-2 and H-6),
7.42-7.50 (m, 3H, C-phenyl H-3, H-4 and H-5). 7.39 (d, f2.3 Z55.6 = 8.6 Hz,
2H, C-4 phenyl H-2 and H-6), 6.20 (s, IH, NH), 5.02 (s, IH, DHP H-4), 4.96
(septet,J=6.2Hz,2H,CHMe2),2.31 (s,6H, DHPC-2andC-6Me's), 1.14
and 1.25 (two d, J = 6.2 Hz,6H each, CHMez).
Diethyl I,4-Dihydro-2,6-dimethyl-4-{4-[(Z)-N-oxo-N-(4-chlorophenylIn Vitro Calcium Channel Antagonist Assay
methylene)-hs-azanyl]phenyl~-3,5-pyridinedicarboxylate
7k
IR (KBr): v = 3340 cm-' (NH), 1692 (CO2), 1227 (NO).- 'H NMR
(CHci3-dl): 6 8.34 (d, J ~= J, ~~= 8.6
, ~H ~2, ~c -, 4 phenyl H-3 and ~ - 5 ) ,
7.87 (s, IH, nitrone =CH), 7.59 (d, J2.3 = J5.6 = 8.6 Hz, 2H, chlorophenyl H-3
and H-5). 7.40-7.52 (m, 4H, chlorophenyl H-2 and H-6, C-4 phenyl H-2 and
H-6), 6.54 (s, IH, NH), 5.03 (s, lH, DHP H-4). 4.10 (q, J = 7.1 Hz, 4H,
C O Z C H ~ C H 2.28
~ ) , (s, 6H, DHP C-2 and C-6 Me's), 1.23 (t. J = 7.1 H, 6H,
COZCH~CH~).
Arch. Pham Pham Med Ckm. 330,53-58 (1597)
The calcium channel antagonist activities of compounds 7811 were determined as the molar concentration of the test compound required to produce
50%inhibition of the muscarinic receptor-mediated (carbachol, 1 . 6 w
~7M)
Ca2+-dependentcontraction (tonic response) of guinea pig ileum Ion itudinal
smooth muscle (GPILSM) using the procedure reported previousl$zzI. The
ICso value (+ SEM) was determined graphically from the dose-response
curve.
58
Anana, Ng, Howlett, and Knaus
Whole-Cell Voltage-Clamp Studies
1. Myocyte preparation. Guinea pigs (male or female, 350-400 g, Charles
River) were injected with heparin (3.3 IU/g) 30 min prior to anesthesia
(sodium pentobarbital, 160 mgkg). Hearts were perfused retrogradely
through the aorta for 7-8 min (10-12 m l h i n ) with Ca +-free solution (mM):
120NaCI. 3.8 KCI, 1.2 KHzP04.1.2 MgS04.10 HEPES, 1 1 glucose (gassed
with 100%02, 36 "C, pH 7.4). Hearts then were perfused with Ca +-free
solution supplemented with 40 mg collagenase A (Boehringer Mannheim)
and 6 mg protease XIV (Sigma) for 4-5 min. Ventricles were minced and
myocytes were released in high K' solution (mM): 80 KOH, 50 glutamic
acid, 30 KCI, 30 KHzP04, 20 taurine, 10 HEPES, 10 glucose, 3 MgS04,
0.5 EGTA (pH 7.4 KOH). Ventricular myocytes were placed in a petri dish
in an open perfusion micro-incubator (Model PDMI-2, Medical Systems
Corp.) on the stage of an inverted microscope and superfused with a HEPESbuffered solution (mM): 145 NaCI, 4 KCI, 1 MgCIz, 10 HEPES, 10 glucose,
2 CaCIz (gassed with 100% 02,pH 7.4). All experiments were conducted in
the presence of 200 pM lidocaine (Sigma Chemical Co.) to block Na+
currents.
2. Electrophysiological recording. Recordings were made with intracellular microelectrodes (18-25 MR) filled with 2.7 M KCI. Experiments were
performed at 36 "C with discontinuous single electrode voltage clamp (sample rate 10-16 kHz). Voltage clamp protocols were generated with pClamp
software (Axon Instruments Inc.). Transmembrane current and voltage were
recorded with an Axoclamp-2A amplifier (Axon Instruments Inc.), digitized
with a Labmaster AID interface at 125 kHz (TL1-125, Axon Instruments
Inc.) and stored on hard disk for subsequent analysis. Test steps were
preceded by a 200 ms conditioning step to 0 mV from the holding potential
of -80 mV, followed by return to a membrane potential of 4 0 mV. Icawas
activated by a series of 200 ms test steps to potentials between 4 0 to +80mV,
in 10 mV increments. Current recordings were analyzed with pClamp
analysis software. The peak amplitude of Icawas measured as the difference
between the peak inward current and net current after 200 ms. The validity
of this measure of Ica was tested in separate experiments in which cells were
exposed to Ca2+channel blockers. Peak amplitude of Ica was plotted as a
function of membrane potential to construct I-V relationships. Data are
presented as means f SEM; n represents the number of myocytes sampled.
Differences between groups were assessed with two-way repeated measures
analysis of variance. Differences were considered significant whenp c 0.05.
3. Drugs. The test compound (7c) was dissolved in distilled DMSO, which
partially inhibits Ica["].To control for solvent effects, control and drug
solutions all contained the same amount of DMSO (0.001%). Cells were first
exposed to control solution for 10 min, then to 1 pM 7c for 10 min and finally
to 10 pM 7c for 10 min. Drugs were then washed out for up to 15 min,
although we found that effects of 7c on Icawere not reversed within that time.
References
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Arch. Pham. P h a m Med. Chem. 330,53-58 (1997)
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channel, dihydro, muscle, nitrone, moiety, pyridinedicarboxylate, substituents, aryl, effect, synthesis, containing, smooth, dialkyl, calcium, dimethyl
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