Vasorelaxation by new hybrid compounds containing dihydropyridine and pinacidil-like moieties

Article abstract of DOI:10.1021/jm990443h

The synthesis and pharmacological properties of a novel type of vasorelaxant hybrid compounds are described. The investigated compounds originate from fluorinated 4-aryl-1,4-dihydropyridines, which are known calcium channel blockers, and/or from fluorinated analogues of pinacidil, which is an opener of ATP-sensitive potassium channels. In particular, we studied the most potent hybrid, 2,6-dimethyl-3,5-dicarbomethoxy-4-(2- difluoromethoxy-5-N-(N'-cyano-N'-1,2,2-trimethyl-propylguanidyl)-phenyl)-1,4- dihydropyridine (4a), together with its parent compounds, the dihydropyridine 1b and the pinacidil analogue 3. In isolated rat mesenteric arteries, micromolar concentrations of 4a relaxed contractions exerted by K⁺- depolarization or by norepinephrine. The latter effect was sensitive to the potassium channel blocker glibenclamide. Micromolar 4a also inhibited [³H](+)-isradipine and [³H]P1075 binding to rat cardiac membranes, and it blocked L-type calcium channels expressed in a mammalian cell line. The respective parent compounds lb and 3 were always more potent and more selective regarding calcium channel or potassium channel interaction, respectively. In contrast, 4a combined both effects within the same concentration range, indicating that it may represent a lead structure for a novel class of pharmacological hybrid compounds.

Full text of DOI:10.1021/jm990443h

5266
J . Med. Chem. 1999, 42, 5266-5271
Va sor ela xa tion by New Hybr id Com p ou n d s Con ta in in g Dih yd r op yr id in e a n d
P in a cid il-Lik e Moieties
Lev M. Yagupolskii,† Wolfram Antepohl,‡ Ferruh Artunc,§ Renate Handrock,‡ Boris M. Klebanov,†
Irina I. Maletina,† Bent Marxen,‡ Kirill I. Petko,† Ulrich Quast,§ Anna Vogt,‡ Carolin Weiss,‡ J utta Zibold,§ and
Stefan Herzig*,‡
Institute of Organic Chemistry, National Academy of Sciences of Ukraine, 5 Murmanskaya Str., 253660 Kiev-94, Ukraine,
Department of Pharmacology, University of Cologne, Gleueler Strasse 24, 50931 Ko¨ln, Germany, and Department of
Pharmacology, University Tu¨bingen, Wilhelmstrasse 56, 72074 Tu¨bingen, Germany
Received August 30, 1999
The synthesis and pharmacological properties of a novel type of vasorelaxant hybrid compounds
are described. The investigated compounds originate from fluorinated 4-aryl-1,4-dihydropy-
ridines, which are known calcium channel blockers, and/or from fluorinated analogues of
pinacidil, which is an opener of ATP-sensitive potassium channels. In particular, we studied
the most potent hybrid, 2,6-dimethyl-3,5-dicarbomethoxy-4-(2-difluoromethoxy-5-N-(N′′-cyano-
N′-1,2,2-trimethyl-propylguanidyl)-phenyl)-1,4-dihydropyridine (4a ), together with its parent
compounds, the dihydropyridine 1b and the pinacidil analogue 3. In isolated rat mesenteric
arteries, micromolar concentrations of 4a relaxed contractions exerted by K+-depolarization
or by norepinephrine. The latter effect was sensitive to the potassium channel blocker
glibenclamide. Micromolar 4a also inhibited [3H](+)-isradipine and [3H]P1075 binding to rat
cardiac membranes, and it blocked L-type calcium channels expressed in a mammalian cell
line. The respective parent compounds 1b and 3 were always more potent and more selective
regarding calcium channel or potassium channel interaction, respectively. In contrast, 4a
combined both effects within the same concentration range, indicating that it may represent
a lead structure for a novel class of pharmacological hybrid compounds.
Sch em e 1
In tr od u ction
The 4-aryl-1,4-dihydropyridine derivatives (1,4-DHP),
for example nifedipine (1a ) and foridon (1b), are effec-
tive L-type calcium channel blockers (Scheme 1). They
are used widely in medical practice for the treatment
of hypertension because of their vasorelaxant properties.
A similar effect is exerted by the so-called potassium
channel openers, for example, pinacidil (2). Several
types of openers of ATP-sensitive potassium channels
have attracted the attention of medicinal chemists over
the past several years. We have previously found a
hypotensive effect exhibited by a less toxic fluorine-
containing benzene analogue (3) of pinacidil that we
named flocalin.1,2 A rationale for combination therapy
(as frequently used in hypertensive patients) as well as
for the design of hybrid compounds is a common main
pharmacological effect (here: vasodilation) and a diver-
gent side effect profile. Furthermore, blockade of L-type
calcium channels together with potassium channel
activation may even lead to a combined antianginal and
antiischemic action, respectively, due to the proposed
role of ATP-sensitive potassium channels in cardiac
ischemic preconditioning.3
Sch em e 2
2): hybrids of calcium channel blocker and potassium
channel activator (4), as racematic mixtures, and a
hybrid of potassium channel activator and presumed
weak calcium channel activator (according to ref 4)
containing a cyano group instead of the alkoxycarbonyl
group in 5-position of dihydropyridine ring (5), which
represents a mixture of four stereoisomers. We describe
our attempts to elucidate whether the test hybrid
compounds type 4 and 5 are active as vasodilators. More
specifically, we elucidated whether compound 4a indeed
interacts with L-type calcium channels and whether it
behaves like a potassium channel opener.
In this work we have synthesized hybrid compounds
that contain the structural elements of calcium channel
modulators and a potassium channel activator-flocalin
(3). We have obtained two types of preparations (Scheme
* Corresponding author. Phone: ++49 221 478 6064. Fax: ++49
221 478 5022. E-mail: stefan.herzig@uni-koeln.de.
† National Academy of Sciences of Ukraine.
‡ University of Cologne.
§ University of Tu¨bingen.
10.1021/jm990443h CCC: $18.00 © 1999 American Chemical Society
Published on Web 11/23/1999

Synthesis of Vasorelaxant Hybrid Compounds
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 25 5267
Sch em e 3
Sch em e 5
Sch em e 4
Ta ble 1. Vasorelaxation of Rat Mesenteric Arteries by Test
Compoundsa
contractile
stimulus
log (EC50
)
p value (vs
compd
mean ( SEM norepinephrine)
N
1b
K+-depolarization
-8.75 ( 0.13
0.011
6
6
6
5
6
5
10
9
9
6
norepinephrine (NE) -8.20 ( 0.14
NE + glibenclamide
-8.44 ( 0.12
-5.13 ( 0.09
ns
0.00001
3
K+-depolarization
norepinephrine (NE) -6.54 ( 0.13
NE + glibenclamide
-6.12 ( 0.21
-6.43 ( 0.11
0.076
ns
4a
K+-depolarization
norepinephrine (NE) -6.70 ( 0.12
NE + glibenclamide
K+-depolarization
K+-depolarization
-6.20 ( 0.08
-6.02 ( 0.06
-5.63 ( 0.05
0.002
4b
5
6
a
Vasorelaxation was measured by cumulative addition of the
test compounds after the respective contractile stimulus (second
column). Significance was checked (two-tailed t test) for a given
compound between log(EC50) values obtained with norepinephrine
(10-5 M) versus K+-depolarization (120 mM) or versus norepi-
nephrine in the presence of glibenclamide (3 × 10-6 M).
Ch em istr y
The starting o-difluoromethoxybenzaldehyde (6) was
synthesized by difluoromethylation of salicylic alde-
hyde.5 The nitration of 6 afforded 2-difluoromethoxy-5-
nitrobenzaldehyde (7) in 90% yield. Aldehyde 7 was
introduced in modified Hantzsch reaction (Scheme 3)
to give 1,4-DHP with alkoxycarbonyl groups in 3,5-
positions of dihydropyridine ring (8a -c) and also 1,4-
DHP with one methoxycarbonyl and one cyano group
(9).
Reduction of nitro group by hydrogen/Raney Ni (for
compounds 8) or by Fe in hydrochloric acid (for com-
pound 9) led to corresponding amino derivatives 10 or
11 (available in situ) that were converted into isothio-
cyanates 12 and 13 by treating of diethylthiocarbamoyl
chloride or thiophosgen (Scheme 4).
When treated with stoichiometric equivalents of pi-
nacolylamine, isothiocyanates 12 and 13 gave thioureas
14 and 15, respectively. Thioureas 14 were converted
into carbodiimides 16 by action of the system CCl4-
Et3N-PPh3, whereas thiourea 15 was dehydrosulfur-
izated into carbodiimide 17 by mercury oxide. Com-
pounds 4 and 5 were prepared by treatment of carbodi-
imides 16 and 17 with cyanamide in the presence of
diisopropylethylamine (Scheme 5).
ation of resistance arteries. The potency, gauged by the
log(EC50) value, depended on the precontracting stimu-
lus (Table 1). As expected for a dihydropyridine, 1b was
potent in relaxing K+-constricted arteries, and signifi-
cantly less potent in case of norepinephrine contractions.
The reverse was true, again as expected, for the potas-
sium channel opener 3. The hybrid compounds 4a , 4b,
and 5 were all less potent when tested with K+-
depolarization, indicating weaker calcium channel block
compared with 1b. Compound 5 and its parent dihy-
dropyridine did not show functional evidence of calcium
channel agonism (N ) 4, not shown). Therefore, we
focused our attention on the most potent hybrid com-
pound, 4a . This compound was nearly equally potent
(p > 0.05) with regard to relaxation of K+-induced
versus norepinephrine-induced contractions, unlike its
parent compounds 1b and 3. Like the potassium chan-
nel opener 3, relaxation by 4a of norepinephrine-
contracted arteries was sensitive to the blocker of ATP-
sensitive potassium channels, glibenclamide (p < 0.05).
Thus, functional evidence exists that both calcium
channel blockade and potassium channel opener action
may be present-at pharmacologically active concentra-
tions-in the hybrid 4a .
P h a r m a cologica l Resu lts a n d Discu ssion
Bin d in g Exp er im en ts. To substantiate the interac-
tion of the test compounds with L-type calcium channels
or ATP-sensitive potassium channels respectively, com-
Va sor ela xa tion in Mesen ter ic Ar ter ies. All tested
compounds exerted a concentration-dependent relax-

5268 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 25
Yagupolskii et al.
F igu r e 2. Inhibition of barium currents in CHO cells trans-
fected with the R1C-b calcium channel subunit by 4a . Cells were
depolarized to +10 mV from a holding potential of either -80
mV (bottom traces) or -30 mV (top traces). Data were obtained
before and after application of compound 4a (10-5 M) as
indicated. Scale bars denote 20 ms and 200 pA, respectively.
Ta ble 2. Calcium Channel Inhibition by the Test Compoundsa
concn
(M)
holding
potential (mV)
peak current (%)
mean × SEM
compd
N
0
0
-80
-30
-80
-30
-80
-30
-80
-30
-80
-30
-80
-30
-80
-30
71.4 ( 5.0
17.2 ( 3.2
47.4 ( 3.5
7.8 ( 2.0
9.2 ( 1.4
0.9 ( 0.3
58.2 ( 4.3
10.8 ( 1.6
26.7 ( 3.0
2.4 ( 0.6
60.4 ( 4.7
9.2 ( 1.2
38.2 ( 4.6
2.5 ( 0.9
10
10
7
7
4
4
6
6
6
6
7
7
7
1b
10-8
10-8
10-5
10-5
10-5
10-5
10-4
10-4
10-6
10-6
10-5
10-5
3
4a
F igu r e 1. Competition binding experiments in rat cardiac
membranes. Test compounds were coincubated with [3H](+)-
isradipine (top) as a calcium channel ligand and with [3H]-
P1075 as a potassium channel ligand. Data (mean ( SEM)
are from N ) 3-5 independent experiments. IC50 values are
from nonlinear regression. Slope factors were close to unity.
7
Barium currents were measured in CHO cells transfected with
the R1C-b calcium channel subunit. Peak current was measured at
the optimum test potential, and initial stable values obtained at
a holding potential of -80 mV were set at 100%. Data in the first
two data rows are from time-matched drug-free control experi-
ments, indicating slight time-dependent rundown at a holding
potential of -80 mV and a pronounced steady-state inactivation
at -30 mV.
petition binding assays were performed using classical
high-affinity ligands ([3H](+)-isradipine and [3H]P1075,
respectively) in rat cardiac membranes (Figure 1). [3H]-
(+)-Isradipine binding was inhibited concentration-
dependently, with rank order of potency of 1b . 4a >
3. This is the same order of potency as found for
vasorelaxation in depolarized arteries, corroborating the
idea of calcium channel inhibition for 1b, the hybrid 4a ,
and interestingly for the pinacidil analogue 3. This is
in contrast to the absence of significant calcium channel
blockade reported6 for the prototype pinacidil (2). Bind-
ing of [3H]P1075 was also inhibited in a competitive,
concentration-dependent manner, but the rank order of
potency was reversed: 3 . 4a > 1b. The novel potas-
sium channel opener 3 was slightly more potent than
2, included here for comparison. Compound 1b inhibited
[3H]P1075 binding only at very high concentrations.
Importantly, the hybrid compound interacts with both
binding sites within the same concentration range.
These data further validate our assumption that va-
sorelaxation by 4a is due to both calcium channel and
potassium channel interaction.
forming subunit expressed in arterial smooth muscle7
was used. As depicted in Figure 2, one drug concentra-
tion was checked in each experiment at two holding
potentials (-80 mV and -30 mV). We chose equimolar
(10-5 M) and equieffective (approximately half-maximal
block at the -30 mV holding potential: 10-8 M 1b, 10-6
M 4a , and 10-5 M 3) concentrations of 1b, 3, and 4a .
As shown in Table 2, all three compounds inhibited
calcium channel currents voltage8- and concentration-
dependently, with a rank order of potency (1b . 4a >
3) similar to the dihydropyridine binding experiments.
In summary, the novel hybrid compound 4a displays
the pharmacological properties expected from its ances-
tor chemical constituents. Like 1b, it relaxes depolarized
arteries, inhibits dihydropyridine binding, and blocks
calcium channels. Like 3, it relaxes agonist-contracted
vessels, it acts in a glibenclamide-sensitive manner, and
it inhibits binding of a ligand at the potassium channel
opener site. Importantly, the two pharmacological ef-
fects of 4a are exerted within the same concentration
range. It still has to be tested which stereoisomer(s)
possess the pharmacological activities. Whether the
hybrid mode of action represents a beneficial therapeu-
tic strategy also remains to be determined.
Ca lciu m Ch a n n el In h ibition . Since inhibition of
dihydropyridine binding can be found with blockers and
activators of calcium channel, we sought direct evidence
for calcium channel block by the hybrid compound 4a .
The cloned R1C-b isoform of the calcium channel pore-

Synthesis of Vasorelaxant Hybrid Compounds
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 25 5269
8a (4.12 g, 0.01 mol), Raney nickel (0.5 g, 0.01 mol), and
methanol (40 mL) was stirred for 6 h at 50-55 °C under an
atmosphere of hydrogen. After filtration the warm solution was
concentrated to about 25% of volume. The precipitated (after
12 h at 0 °C) solid was filtered off, washed with cold methanol
(5 mL), and dried to afford the analytically pure 10a (3.43 g,
Exp er im en ta l Section
All reagents and solvents were of reagent grade or were
purified by standard methods before use. Melting points were
determined in open capillaries and are given uncorrected.
Proton nuclear magnetic resonance spectra (1H NMR) were
recorded on a Varian VXR-300 spectrometer, and chemical
shifts are reported in parts per million (ppm) relative to
tetramethylsilane (TMS, 0.00 ppm). Infrared spectra were
obtained on a UR-20 infrared spectrophotometer.
1
90%): mp 203-205 °C; H NMR (CDCl3) δ 2.40 (s, 6H), 3.50
(s, 6H), 3.54 (s, 2H), 4.96 (s, 1H), 5.62 (s, 1H), 6.59 (t, 1H, J )
72 Hz), 6.90-7.20, 7.80-7.90 (m, 3H). Anal. (C18H20F2N2O5)
C, H, N.
2-Diflu or om eth oxy-5-n itr oben za ld eh yd e (7). o-Difluo-
romethoxybenzaldehyde (6) (17.2 g, 0.1 mol) was added drop-
wise during 1 h to a stirred mixture of sulfuric acid (d ) 1.84)
(25 mL) and fumic nitric acid (d ) 1.52) (25 mL) at -10 to -5
°C. The mixture was slowly heated to room temperature and
was poured into crushed ice after 1 h. The precipitate was
filtered off, washed with water, and dissolved in ether (100
mL). The ether solution was washed with water (50 mL), 5%
solution of NaHCO3 (2 × 50 mL), and water (50 mL) and dried
with MgSO4. Evaporation of solvent afforded product 7 (19.5
g, 90%): mp 41-43 °C, pure enough for the next synthesis.
Recrystallization of a sample from hexane afforded the ana-
2,6-Dim eth yl-3,5-d ica r boeth oxy-4-(2-d iflu or om eth oxy-
5-a m in op h en yl)-1,4-d ih yd r op yr id in e (10b). Prepared from
8b (4.38 g, 0.01 mol) by the procedure described in the
preparation of 10a , except that the reaction was held in
ethanol. Yield 3.54 g (87%): mp 147-148 °C; 1H NMR (CDCl3)
δ 1.22 (t, 6H, J ) 7 Hz), 2.31 (s, 6H), 3.55 (s, 2H), 4.06 (q, 4H,
J ) 7 Hz), 5.01 (s, 1H), 5.90 (s, 1H), 6.60 (t, 1H, J ) 73 Hz),
7.05-7.15, 7.90-8.00 (m, 3H). Anal. (C20H24F2N2O5) C, H, N.
2,6-Dim eth yl-3,5-dicar bom eth oxy-4-(2-diflu or om eth oxy-
5-isot h iocya n a t op h en yl)-1,4-d ih yd r op yr id in e (12a ). A
mixture of amino derivative 10a (3.8 g, 0.01 mol), diethyl-
thiocarbamoyl chloride (1.7 g, 0.011 mol), and anhydrous
dichloroethane (50 mL) was stirred and heated to reflux for 2
h. After cooling the resulting solution was filtered through
short column (5 cm) filled with silica gel (40-100 µm) and then
eluted with methylene chloride. Evaporation of solvent and
recrystallization from CCl4 afforded product 12a (2.76 g,
1
lytically pure product: mp 45 °C; H NMR (CDCl3) δ 7.37 (t,
1H, J ) 72 Hz), 7.61-7.65; 8.51-8.67 (m, 3H). Anal. (C8H5F2-
NO3) C, H, N.
2,6-Dim eth yl-3,5-dicar bom eth oxy-4-(2-diflu or om eth oxy-
5-n itr op h en yl)-1,4-d ih yd r op yr id in e (8a ). A mixture of 7
(6.5 g, 0.03 mol), methyl 3-aminocrotonate (3.45 g, 0.03 mol),
methyl acetoacetate (3.5 g, 0.03 mol), and methanol (20 mL)
was stirred and heated to reflux for 8 h. The mixture was
cooled, and after 12 h at 0 °C the yellow crystals formed were
filtered off, recrystallized from 2-propanol, and dried to afford
the product 7a (8.5 g, 69%): mp 187-189 °C; 1H NMR (CDCl3)
δ 2.33 (s, 6H), 3.60 (s, 6H), 5.41 (s, 1H), 6.08 (s, 1H), 6.67 (t,
1H, J ) 73 Hz), 7.25-7.38, 8.01-8.25 (m, 3H). Anal.
(C18H18F2N2O7) C, H, N.
2,6-Dim eth yl-3,5-d ica r boeth oxy-4-(2-d iflu or om eth oxy-
5-n itr op h en yl)-1,4-d ih yd r op yr id in e (8b). Prepared from 7
(6.5 g, 0.03 mol), ethyl 3-aminocrotonate (3.9 g, 0.03 mol), and
ethyl acetoacetate (3.85 g, 0.03 mol) in ethanol by the
procedure described in the preparation of 8a . Yield 10.24 g
(78%): mp 191-192 °C; 1H NMR (CDCl3) δ 1.22 (t, 6H, J ) 7
Hz), 2.37 (s, 6H), 4.07 (q, 4H, J ) 7 Hz), 5.31 (s, 1H), 6.20 (s,
1H), 6.52 (t, 1H, J ) 72 Hz), 7.70-7.75, 8.10-8.30 (m, 3H).
Anal. (C20H22F2N2O7) C, H, N.
1
65%): mp 150-151 °C; H NMR (CDCl3) δ 2.25 (s, 6H), 3.41
(s, 6H), 5.16 (s, 1H), 5.68 (s, 1H), 6.45 (t, 1H, J ) 72 Hz), 6.92-
7.25 (m, 3H); IR (KBr) 1130 (CF), 1705 (CdO), 2060 (NdCd
S), 3300 (NH) cm-1. Anal. (C19H18F2N2O5S) C, H, N.
2,6-Dim eth yl-3,5-d ica r boeth oxy-4-(2-d iflu or om eth oxy-
5-isoth iocya n a top h en yl)-1,4-d ih yd r op yr id in e (12b). Pre-
pared from 10b (4.1 g, 0.01 mol) by the procedure described
in the preparation of 10a . Yield 2.98 g (66%): mp 166-167
°C; 1H NMR (CDCl3) δ 1.14 (t, 6H, J ) 7 Hz), 2.24 (s, 6H),
4.00 (q, 4H, J ) 7 Hz), 5.17 (s, 1H), 5.70 (s, 1H), 6.43 (t, 1H,
J ) 72 Hz), 6.90-7.19 (m, 3H); IR (KBr) 1125 (CF), 1710 (Cd
O), 2050 (NdCdS), 3300 (NH) cm-1. Anal. (C21H22F2N2O5S)
C, H, N.
2,6-Dim e t h yl-3-ca r b om e t h oxy-5-cya n o-4-(2-d iflu o-
r om e t h oxy-5-isot h iocya n a t op h e n yl)-1,4-d ih yd r op yr i-
d in e (13). To a vigorously stirred mixture of 9 (3.8 g, 0.01
mol), Fe sawdust (4 g, 0.07 mol), acetone (10 mL), and water
(10 mL) was added dropwise concentrated (d ) 1.17) hydro-
chloric acid (5 mL). The reaction mixture was stirred at 50 °C
for 3 h and then filtered, and the solid was washed with water
(20 mL). The solution of thiophosgen (1.3 g, 0.011 mol) in
methylene chloride (5 mL) was added dropwise at ambient
temperature. The resulting precipitate was dissolved in tolu-
ene (25 mL). The toluene solution was stirred and heated to
reflux with Al2O3 (1 g). Absorbent was filtered off, and the hot
solution was diluted with hexane (25 mL) and cooled to afford
the pure light yellow 13 (2.0 g, 51%): mp 182-183 °C; 1H NMR
(CDCl3) δ 2.20 (s, 3H), 2.38 (s, 3H), 3.58 (s, 3H), 5.20 (s, 1H),
5.90 (s, 1H), 6.70 (t, 1H, J ) 73 Hz), 7.00-7.90 (m, 3H); IR
(KBr) 1120 (CF), 1715 (CdO), 2060 (NdCdS), 2210 (CN), 3300
(NH) cm-1. Anal. (C18H15F2N3O3 S) C, H, N.
2,6-Dim et h yl-3-ca r b om et h oxy-5-ca r b oet h oxy-4-(2-d i-
flu or om eth oxy-5-n itr op h en yl)-1,4-d ih yd r op yr id in e (8c).
Prepared from 7 (2.17 g, 0.01 mol), methyl 3-aminocrotonate
(1.15 g, 0.01 mol), and ethyl acetoacetate (1.30 g, 0.01 mol) in
methanol (7 mL) by the procedure described in the preparation
of 8a except that the reaction mixture was refluxed for 10 h.
Recrystallization of the product from ethanol and then from
1
toluene/hexane 1:1 gave 2 g (46%) of 8c: mp 153-155 °C; H
NMR (CDCl3) δ 1.19 (t, 3H, J ) 7 Hz), 2.32 (s, 3H), 2.34 (s,
3H), 3.59 (s, 3H), 4.04 (q, 2H, J ) 7 Hz), 5.34 (s, 1H), 5.86 (s,
1H), 6.61 (t, 1H, J ) 72 Hz), 7.10-7.20, 8.05-8.20 (m, 3H).
Anal. (C19H20F2N2O7) C, H, N.
2,6-Dim e t h yl-3-ca r b om e t h oxy-5-cya n o-4-(2-d iflu o-
r om eth oxy-5-n itr oph en yl)-1,4-dih ydr opyr idin e (9). A mix-
ture of 7 (4.35 g, 0.02 mol), methyl 3-aminocrotonate (2.3 g,
0.02 mol), 3-aminocrotonitrile (1.64 g, 0.02 mol), and acetic
acid (15 mL) was stirred and heated to reflux for 2 h. The
mixture was cooled, poured into water (150 mL), and stirred
vigorously for 0.5 h. The precipitate was filtered off, washed
with water, and dissolved in toluene (25 mL). The toluene
solution was stirred and heated to reflux with Al2O3 (1 g).
Absorbent was filtered off, and the hot solution was diluted
with hexane (25 mL) and cooled to afford the pure light yellow
8a (3.94 g, 57%): mp 191-192 °C; 1H NMR (CDCl3) δ 2.48 (s,
3H), 2.41 (s, 3H), 3.56 (s, 3H), 5.16 (s, 1H), 6.09 (s, 1H), 6.67
(t, 1H, J ) 72 Hz), 7.50-8.10 (m, 3H); IR (KBr) 1110 (CF),
1715 (CdO), 2210 (CN), 3300 (NH) cm-1. Anal. (C17H15F2N3O5)
C, H, N.
2,6-Dim eth yl-3,5-dicar bom eth oxy-4-(2-diflu or om eth oxy-
5-N-(N′-1,2,2-tr im eth ylp r op ylth iou r ed yl)p h en yl)-1,4-d i-
h yd r op yr id in e (14a ). To a stirred suspension of 12a (2.12
g, 5 mmol) in ethanol (10 mL) was added pinacolylamine (0.7
g, 7 mmol) dropwise at ambient temperature. The resultant
solution was evaporated in vacuo. Crude product was dissolved
in CCl4 (20 mL), and the solution was stirred vigorously at
-10 to -5 °C for 8-10 h until formation of the first crystals,
and then it was allowed to stay at 0 °C for 3 days. Filtration
and air-drying of the resultant precipitate gave analytically
1
pure 14a (2.53 g, 97%): mp 185-186 °C; H NMR (CDCl3) δ
0.74 (s, 9H), 1.01 (d, 3H, J ) 5 Hz), 2.23 (s, 6H), 3.53 (s, 6H),
4.30 (m, 1H), 5.23 (s, 1H), 5.61 (d, 1H, J ) 4 Hz), 5.91 (s, 1H),
6.49 (t, 1H, J ) 74 Hz), 7.00-7.30 (m, 3H), 7.75 (s, 1H). Anal.
(C25H33F2N3O5S) C, H, N.
2,6-Dim eth yl-3,5-dicar bom eth oxy-4-(2-diflu or om eth oxy-
5-a m in op h en yl)-1,4-d ih yd r op yr id in e (10a ). A mixture of

5270 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 25
Yagupolskii et al.
2,6-Dim eth yl-3,5-d ica r boeth oxy-4-(2-d iflu or om eth oxy-
5-N-(N′-1,2,2-tr im eth ylp r op ylth iou r ed yl)p h en yl)-1,4-d i-
h yd r op yr id in e (14b). Prepared from 12b (2.26 g, 5 mmol)
by the procedure described in the preparation of 14a . Yield
2.54 g (92%): mp 160-161 °C; 1H NMR (CDCl3) δ 0.75 (s, 9H),
1.02 (d, 3H, J ) 5 Hz), 1.14 (t, 6H, J ) 7 Hz), 2.22 (s, 6H),
4.00 (q, 4H, J ) 7 Hz), 4.30 (m, 1H), 5.21 (s, 1H), 5.61 (d, 1H,
J ) 4 Hz), 5.69 (s, 1H), 6.51 (t, 1H, J ) 74 Hz), 6.90-7.20 (m,
3H), 7.40 (s, 1H). Anal. (C27H37F2N3O5S) C, H, N.
2,6-Dim et h yl-3-ca r b om et h oxy-5-cya n o-4-(2-d iflu or o-
m eth oxy-5-N-(N′-1,2,2-tr im eth ylp r op ylth iou r ed yl)p h en -
yl)-1,4-d ih yd r op yr id in e (15). Prepared from 13 (1.97 g, 5
mmol) by the procedure described in the preparation of 14a ,
except that the crystallization time lasted two weeks. Yield
1.13 g (46%): mp171-172 °C; 1H NMR (CDCl3) δ 0.78 (s, 9H),
1.00 (d, 3H, J ) 5 Hz), 2.29 (s, 6H), 5.24 (m, 1H), 5.32 (s, 1H),
5.65 (d, 1H, J ) 4 Hz), 5.72 (s, 1H), 6.51 (t, 1H, J ) 74 Hz),
6.90-7.25 (m, 3H), 7.45 (s, 1H). Anal. (C24H30F2N4O3S) C, H,
N.
washout and complete relaxation, the vessel was exposed to
the other contractile stimulus (K+-depolarization, or norepi-
nephrine as described) for 1 h to check the stability of the
preparation. After washout, arteries were allowed to recover
for about 20 min, before the same long-term stimulus (K+-
depolarization or norepinephrine, respectively) was repeated
for a second time. After contraction reached a stable value,
test compounds were added cumulatively (usually eight con-
centrations at half-logarithmic intervals) to obtain a complete
concentration-response curve. In some experiments, the
concentration-response curve was done in the presence of
glibenclamide (3 × 10-6 M). Under all conditions, the test
compounds exerted a complete or near-complete (>90%) va-
sorelaxation in the investigated concentration range. We
therefore analyzed the concentration-response curve using a
logistic equation with two free parameters, i.e., Y ) 100%/{1
+ (X/EC50)H}, where Y is percent relaxation, X is the concen-
tration, EC50 is the concentration causing half-maximal re-
laxation, and H is a slope factor. EC50 values were determined
in every experiment. Data are given as mean log(EC50) values.
Bin d in g Exp er im en ts. Binding studies were performed
using homogenates from rat cardiac ventricle prepared as
previously described.10,11 As a calcium channel ligand, [3H](+)-
isradipine (NEN) was used; and for studying the potassium
channel opener site, [3H]P1075 (Amersham) was employed.
Equilibrium binding was measured after incubating the ra-
dioligand together with the indicated concentrations of test
compounds for 1 h at 32 °C (50 mM Tris‚HCl, pH 7.4) or at 37
°C (in mM: NaCl 139, KCl 5, MgCl2 25, CaCl2 1.25, HEPES
20, pH 7.4, supplemented with creatine phosphokinase 50
u/mL, creatine phosphate 20 mM, and Na2ATP 3 mM) for
calcium channel and potassium channel binding, respectively.
Separation of bound and free ligand was accomplished by rapid
filtration (rinsing with ice cold H2O, or Tris buffer, respec-
tively), and bound radioactivity was determined in triplicate
assays by liquid scintillation counting. Nonspecific binding was
determined in excess of unlabeled ligand (10-5 M of nitren-
dipine or of unlabeled P1075, respectively). Data are given as
percentages of specific binding (amounting to >90% of total
[3H](+)-isradipine binding, and 56 ( 2% of total [3H]P1075
binding, respectively).
Electr op h ysiology. Calcium channel currents were mea-
sured in the Chinese hamster ovary (CHO) cell line stably
transfected with the pore-forming R1C-b subunit cloned from
rabbit lung.7 Cells were grown in 35 mm polystyrene dishes
serving as a bath chambers. They were used 1-3 days after
plating. Patch clamp measurements of whole-cell barium
currents (Axopatch 1B, Axon) were done at room temperature,
using borosilicate pipets containing (mM) CsCl 120, MgCl2 3,
MgATP 5, EGTA 10, and HEPES 5, at pH 7.4. The bath
contained (mM) NaCl 120, BaCl2 10.8, MgCl2 1, CsCl 5.4,
glucose 10, and HEPES 10 at pH 7.4. Test pulses of 100 ms
duration were delivered at 10 s intervals. The experimental
protocol was as follows: a current-voltage relationship was
constructed using a holding potential of -80 mV. The test
voltage eliciting maximum currents (+10 to +30 mV) was then
used throughout. When stable currents were recorded ()
100%) using this protocol (-80 mV to test potential), the
holding potential was switched to -30 mV. Then a compound
was added until a steady-state effect was obtained. Finally,
the holding potential was returned to -80 mV. Drug-free
control experiments were done using the same time schedule
and voltage protocol.
2,6-Dim eth yl-3,5-dicar bom eth oxy-4-(2-diflu or om eth oxy-
5-N-(N′′-cyan o-N′-1,2,2-tr im eth ylpr opylgu an idyl)ph en yl)-
1,4-d ih yd r op yr id in e (4a ). A mixture of 14a (2.1 g, 4 mmol),
triphenylphosphine (1.2 g, 5 mmol), triethylamine (1.5 mL),
and CCl4 (20 mL) was stirred and heated to reflux for 8 h and
then evaporated to an oil. The oil was chromatographed on a
column of silica gel (40-100 µm) in CCl4 to afford 16a pure
enough for the next synthesis. It was mixed with cyanamide
(0.84 g, 20 mmol), diisopropylethylamine (0.26 g, 2 mmol), and
hexane (2 mL), stirred for 5 h, and allowed to stay for 12 h at
ambient temperature. The resulting solid was recrystallized
1
from methanol to afford 4a (1.0 g, 48%): mp 211-212 °C; H
NMR (acetone-d6) δ 0.87 (s, 9H), 1.08 (d, 3H, J ) 5 Hz), 2.30
(s, 6H), 3.56 (s, 6H), 3.85 (m, 1H), 5.32 (s, 1H), 6.93 (t, 1H, J
) 74 Hz), 7.06-7.36 (m, 3H), 7.39 (d, 1H, J ) 4 Hz), 8.06 (s,
1H), 8.18 (s, 1H). Anal. (C26H33F2N5O5) C, H, N.
2,6-Dim eth yl-3,5-d ica r boeth oxy-4-(2-d iflu or om eth oxy-
5-N-(N′′-cyan o-N′-1,2,2-tr im eth ylpr opylgu an idyl)ph en yl)-
1,4-d ih yd r op yr id in e (4b). Prepared from 14b (2.2 g, 4 mmol)
by the procedure described in the preparation of 4a . Yield 0.98
g (44%): mp 120-121 °C; 1H NMR (CDCl3) δ 0.77 (s, 9H), 1.01
(d, 3H, J ) 5 Hz), 1.15 (t, 6H, J ) 7 Hz), 2.24 (s, 6H), 4.00 (q,
4H, J ) 7 Hz), 4.33 (m, 1H), 5.24 (s, 1H), 5.65 (d, J ) 4 Hz,
1H), 5.75 (s, 1H), 6.50 (t, 1H, J ) 73 Hz), 6.95-7.27 (m, 3H),
7.41 (s, 1H). Anal. (C28H37F2N5O5) C, H, N.
2,6-Dim et h yl-3-ca r b om et h oxy-5-cya n o-4-(2-d iflu or o-
m eth oxy-5-N-(N′′-cyan o-N′-1,2,2-tr im eth ylpr opylgu an idyl)-
p h en yl)-1,4-d ih yd r op yr id in e (5). A mixture of 15 (1.0 g, 2
mmol), yellow HgO (1.3 g, 5 mmol), and toluene (40 mL) was
boiled with simultaneous azeotrope distillation for 3 h while
the reaction mixture was concentrated to about 25% of volume.
After cooling, the mixture was filtered through a short column
(1 cm) filled with silica gel (40-100 µm), eluted with toluene
(10 mL), and evaporated to afford 16. Further procedure
described in the preparation of 4a afforded 5 (0.20 g, 20%):
1
mp 235-236 °C; H NMR (CDCl3) δ 0.78 (s, 9H), 1.05 (d, 3H,
J ) Hz), 2.25 (s, 6H), 3.45 (s, 3H), 4.30 (m, 1H), 5.22 (s, 1H),
5.65 (d, 1H, J ) 5 Hz), 5.80 (s, 1H), 6.51 (t, 1H, J ) 74 Hz),
6.97-7.20 (m, 3H), 7.38 (s, 1H); IR (KBr) 1110 (CF), 1710 (Cd
O), 2200-2210 (CN), 3300 (NH) cm-1. Anal. (C25H30F2N6O3)
C, H, N.
Dr u g Effects in Mesen ter ic Ar ter ies. Small mesenteric
arteries (third branch) were isolated from adult Wistar rats
and mounted in a small vessel myograph9 as described.10 An
isometric force of ∼2 mm segments was recorded online
(MacLab/4e, MacIntosh Perfoma 6200) at a resting length of
90% of the value obtained at a transmural pressure of 100
mmHg. The solution was gassed (95% O2/5% CO2) and
maintained at 37 °C. The composition (mM) was NaCl 137,
KCl 2.7, CaCl2 1.8, NaHCO3 12, MgCl2 1.1, NaH2PO4 0.11,
glucose 5.5. Vessels were first precontracted briefly by K+-
depolarization (KCl raised to 120 mM, with corresponding
reduction of NaCl) or by norepinephrine (NE, 10-5 M, given
at a slightly elevated KCl of 10 mM). The integrity of the
endothelium was checked by applying carbachol (10-5 M). After
Ack n ow led gm en t. We gratefully acknowledge Dr.
Franz Hofmann (Munich) for providing the transfected
CHO cell line, Mrs. Elke Hippauf (Cologne) for skillful
help with binding experiments, and Birgit Anwald,
J ohanna von Beust, and Robert J aekel for contributing
some data on mesenteric arteries.
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