Effects of novel synthesized pyridothiazines on various guinea pig heart muscle preparations

Article abstract of DOI:10.1248/bpb.27.1364

Calcium channel blockers have become important tools in the treatment of cardiovascular disorders and other diseases. Hybridization of well established calcium antagonist subclasses was an attempt to optimize their pharmacological profile. The intension o

Full text of DOI:10.1248/bpb.27.1364

1364
Biol. Pharm. Bull. 27(9) 1364—1370 (2004)
Vol. 27, No. 9
Effects of Novel Synthesized Pyridothiazines on Various Guinea Pig Heart
Muscle Preparations
Bettina SCHADE,a Thomas ERKER,b Manuela WEBER,b and Christian STUDENIK*,a
a Department of Pharmacology and Toxicology, University of Vienna; and b Department of Pharmaceutical Chemistry,
University of Vienna; Althanstrasse 14, A-1090 Vienna, Austria. Received February 9, 2004; accepted May 28, 2004
Calcium channel blockers have become important tools in the treatment of cardiovascular disorders and
other diseases. Hybridization of well established calcium antagonist subclasses was an attempt to optimize their
pharmacological profile. The intension of this study was to investigate the electrophysiological properties of
MM 10 and MM 11 two newly synthesized compounds structurally closely related to KT-362 (5-[3-[[2-(3,4-
dimethoxyphenyl)ethyl]amino]-1-oxopropyl]-2,3,4,5-tetrahydro-1,5-benzothiazepine fumarate) in various iso-
lated guinea pig heart muscle preparations by means of the conventional intracellular microelectrode tech-
nique. MM 10 (2,3-dihydro-1-[N-[2-(3,4-dimethoxyphenyl)ethyl]-N-methylaminoacetyl]-1H-pyrido[2,3-b][1,4]-
thiazine fumarate) and MM 11 (2,3-dihydro-1-[N-[2-(3,4-dimethoxyphenyl)ethyl]-N-methylaminopropionyl]-1H-
pyrido[2,3-b][1,4]thiazine fumarate) exerted very similar effects though the action of MM 11 was more pro-
nounced. Whereas action potential amplitude and maximum upstroke velocity (Vmax) in papillary muscle, left
atria and spontaneously beating Purkinje fibers was not affected by the compounds in a concentration range
from 3 to 30 mmol/l, action potential duration at 90% time to repolarization was significantly prolonged in a con-
centration-dependent manner. Action potential duration at 20% time to repolarization was decreased in sponta-
neously beating Purkinje fibers and remained unchanged in papillary muscles and left atria. In sinoatrial nodes
both compounds reduced rate of activity, action potential amplitude, maximum upstroke velocity and slope of
slow diastolic depolarization while time to repolarization was prolonged. In 3 out of 6 experiments with sponta-
neously beating Purkinje fibers, MM 11 (30 mmol/l) led to the occurrence of early afterdepolarizations with a
take off potential between ꢀ50 and ꢀ60 mV. All observed effects were completely reversible during washout with
drug-free physiological salt solution. From these results it was concluded that both compounds in addition to
their calcium antagonistic properties might depress repolarizing potassium currents. In contrast to the mother
compound KT-362 they do not seem to affect the fast sodium inward current. Replacement of the benzothi-
azepine nucleus by a pyridothiazine structure may weaken or even eliminate sodium channel blocking ability.
Shortening of the side chain might result in a general loss in activity.
Key words calcium channel antagonist; microelectrode technique; guinea pig heart muscle; KT-362 derivative; 1,4-pyrido-
thiazine
Since the early work of Fleckenstein1) calcium entry
structurally closely related to KT-362 (Fig. 1) with regard to
a possible structure–activity relationship.
blockers have become important therapeutical tools. As a re-
sult of their ability to reduce the influx of calcium ions
through voltage dependent channels substances like vera-
pamil or diltiazem are effective in the treatment of essential
hypertension and angina pectoris as well as they are useful
therapeutics in migraine and cardiac arrhythmia.2)
Hybridization of calcium antagonist subclasses like ben-
zothiazepines and phenylalkylamines was an attempt to in-
crease beneficial and/or minimize undesirable side effects.
The compound KT-362 (5-[3-[[2-(3,4-dimethoxyphenyl)-
ethyl]amino]-1-oxopropyl]-2,3,4,5-tetrahydro-1,5-benzothi-
azepine fumarate) represents such a hybrid containing struc-
tural elements of verapamil as well as of diltiazem (Fig. 1).
Inter alia KT-362 was found to be effective in the suppression
of digitalis and adrenaline induced arrhythmia,3) to reduce is-
chemia-reperfusion injury in dogs4) and to exert cardiopro-
tective effects in the stunned canine myocardium.5) As a re-
sult, KT-362 is currently undergoing clinical trials in Japan
as an antiarrhytmic agent with additional clinical potential in
treating myocardial ischemia and hypertension.6) Thus it was
of interest to investigate the electrophysiological properties
of MM 10 (2,3-dihydro-1-[N-[2-(3,4-dimethoxyphenyl)eth-
yl]-N-methylaminoacetyl]-1H-pyrido[2,3-b][1,4]thiazine fu-
marate) and MM 11 (2,3-dihydro-1-[N-[2-(3,4-dimethoxy-
phenyl)ethyl]-N-methylaminopropionyl]-1H-pyrido[2,3-
b][1,4]thiazine fumarate), two newly synthesized substances
Fig. 1. Chemical Structures of KT-362, MM 10 and MM 11
* To whom correspondence should be addressed. e-mail: christian.studenik@univie.ac.at
© 2004 Pharmaceutical Society of Japan

September 2004
1365
MATERIALS AND METHODS
analysis results were within ꢁ0.4% of the theoretical values
for the formulas given. Solutions in organic solvents were
dried over anhydrous sodium sulfate.
Preparations Guinea pigs of either sex (240—320 g)
were killed by a blow on the neck and their hearts were re-
moved rapidly via a midsternal thoracotomy. The left atrium
was excised from the heart. Right atrium was further dis-
sected. Only the area containing the sinoatrial node (approxi-
mate size of 0.5ꢀ0.5 cm) was used for experiments. Papil-
lary muscles were isolated from the right ventricle after re-
moving Purkinje fibers to prevent spontaneous activity. Purk-
inje fibers were carefully removed from the left ventricle,
only spontaneously beating preparations were used for exper-
iments.
Experimental Setup Preparations were mounted in a
continuous-flow chamber. Papillary muscles and left atria
were continuously stimulated with square wave impulses de-
livered by an anapulse stimulator and an isolation unit
(World Precision Instruments, New Haven, CT, U.S.A.) at a
frequency rate of 1 Hz. Membrane potentials and maximum
upstroke velocities (Vmax) were measured with respect to a
grounded bath by using glass microelectrodes of 10—28 MW
resistance filled with 3 M KCl and two amplifiers (M 701 and
M 707 Microprobe System, World Precision Instruments,
New Haven, CT, U.S.A.). Action potentials were recorded
with a dual beam storage oscilloscope (Tektronix Inc.,
Beaverton, OR, U.S.A.). Photos of membrane potentials were
taken every five minutes (Nihon Kohden Camera PC-2A,
Nihon Kohden, Tokyo, Japan) and signals analyzed after
magnification.
Drugs and Solutions During the experiments prepara-
tions were perfused with a modified Tyrode’s solution con-
taining (in mmol/l) NaCl 136.9, KCl 2.7 (for Purkinje fibers)
or 5.4 (for left atria, papillary muscles and sinoatrial nodes),
MgCl2 1.05, NaH2PO4 0.42, CaCl2 1.8 and glucose 5.0. The
solution was continuously bubbled with a mixture of 95% O2
and 5% CO2 at a temperature of 37ꢁ1 °C.
The investigated compounds MM 10 and MM 11 were
synthesized at the Department of Pharmaceutical Chemistry,
University of Vienna.7) Drugs were dissolved in modified Ty-
rode’s solution, stock solutions were prepared freshly every
day.
2,3-Dihydro-1H-pyrido[2,3-b][1,4]thiazine (1) Com-
pound 1 was synthesized as reported by El-Subbagh et al.7)
Chloroacetyl-2,3-dihydro-1H-pyrido[2,3-b][1,4]thiazine
(2) To a solution of compound 1 (0.152 g, 1 mmol) in dry
THF (5 ml) triethylamine (0.101 g, 1 mmol) and chloroacetyl
chloride (0.170 g, 1.5 mmol) were added. The reaction mix-
ture was stirred for 3 h at room temperature. After this the
solvent was removed under reduced pressure. The residue
was diluted with water and extracted three times with
dichloromethane. The combined organic layers were dried
over Na2SO4, filtered, and concentrated to give the crude
product, which was purified by chromatography (toluene–
ethylacetate, v/vꢂ6 : 4) to give an oil. This was recrystallized
from dil. ethanol to give 2 as pale yellow needles: mp, 87—
1
90 °C; yield, 0.169 g, 74%. H-NMR (CDCl3) d: 3.33 (2H, t,
Jꢂ5.1 Hz, CH2), 4.04 (2H, t, Jꢂ5.1 Hz, CH2), 4.20 (2H, s,
CH2Cl), 7.08 (1H, dd, Jꢂ7.9 Hz, Jꢂ4.7 Hz, Ar-H), 7.63 (1H,
s br, Ar-H), 8.30 (1H, d, Jꢂ4.7 Hz, Ar-H). 13C-NMR (CDCl3)
d: 28.2, 40.9, 119.1, 131.8, 132.6, 147.4, 165.4. MS m/z:
230/228 (Mꢃ), 179, 153/151. Anal. Calcd for C9H9ClN2OS:
C, 47.27; H, 3.97; N, 12.25. Found: C, 47.06; H, 3.84; N,
11.98.
1-[[N-[2-(3,4-Dimethoxyphenyl)ethyl]methylamino]-
acetyl]-2,3-dihydro-1H-pyrido[2,3-b][1,4]thiazine (3) To
a solution of compound 2 (0.228 g, 1 mmol) in dry ethanol
(8 ml) triethylamine (0.111 g, 1.1 mmol) and 2-(3,4-
dimethoxyphenyl)-N-methyl-1-ethylamine (0.195 g, 1 mmol)
were added. The reaction mixture was refluxed for 3 h. After
this the solvent was removed under reduced pressure. The
residue was diluted with water and extracted three times
with dichloromethane. The combined organic layers were
dried over Na2SO4, filtered, and concentrated to give a crude
product, which was purified by chromatography (dichloro-
methane–methanol, v/vꢂ20 : 1) to give an oil. Yield: 0.265 g,
1
68%. H-NMR (CDCl3) d: 2.37 (3H, s, NCH3), 2.69 (4H,
s, 2CH2), 3.16—3.35 (4H, m, 2CH2), 3.84 (3H, s, OCH3),
3.86 (3H, s, OCH3), 3.91—3.99 (2H, m, CH2), 6.68—6.81
(3H, m, Ar-H), 6.96 (1H, dd, Jꢂ8.1 Hz, Jꢂ4.7 Hz, Ar-H),
7.65 (1H, s br, Ar-H), 8.25 (1H, d, Jꢂ4.7 Hz, Ar-H).
13C-NMR (CDCl3) d: 28.6, 31.8, 33.1, 41.7, 53.2, 55.5, 55.6,
59. 5, 61.4, 111.0, 111.7, 118.7, 125.0, 132.6, 132.7, 133.9,
137.5, 146.8, 147.0, 148.5, 171.1. MS m/z: 388 (Mꢃꢃ1),
236, 165. Anal. Calcd for C20H25N3O3S: C, 61.99; H, 6.50;
N, 10.84. Found: C, 61.70; H, 6.39; N, 10.71.
Experimental Protocols After an equilibration period of
at least one hour preparations were impaled and action poten-
tials recorded for 30 min in drug-free Tyrode’s solution. After
this control period drugs were added at increasing concentra-
tions until a steady state was reached followed by a washout
period of 60 min. Continuous impalements were maintained
throughout each experiment.
Statistics Data are presented as meansꢁS.D. and evalu-
ated statistically using Student’s t-test for unpaired observa-
tions. A p value less than 0.05 was considered to indicate a
significant change.
1-[[N-[2-(3,4-Dimethoxyphenyl)ethyl]methylamino]-
acetyl]-2,3-dihydro-1H-pyrido[2,3-b][1,4]thiazine Fuma-
rate (MM 10) To a solution of compound 3 (0.388 g,
1 mmol) and fumaric acid (0.116 g, 1 mmol) in dry ethanol
(10 ml) dry ether was added gradually until a slight precipita-
tion could be detected. This mixture was stored at 0 °C for
20 h. After this the precipitated solid was filtered to give MM
10 as white needles: mp, 119—121 °C; yield, 0.342 g, 68%.
Anal. Calcd for C24H29N3O7S: C, 57.24; H, 5.80; N, 8.34.
Found: C, 57.04; H, 5.65; N, 8.25.
Chemical Syntheses The syntheses of MM 10 and MM
11 is shown in Fig. 2. Melting points were determined on a
Kofler hot stage apparatus and are uncorrected. The H- and
1
13C-NMR spectra were recorded on a Varian UnityPlus 200
(200 MHz and 50 MHz). Chemical shifts are reported in d
values (ppm) relative to Me4Si line as internal standard and J
values are reported in Hertz. Mass spectra were obtained by a
Shimadzu GC/MS QP 1000 EX or Hewlett Packard (GC:
5890; MS: 5970) spectrometer. The obtained elemental
Acryloyl-2,3-dihydro-1H-pyrido[2,3-b][1,4]thiazine (4)
To a solution of compound 1 (0.152 g, 1 mmol) in dry THF
(5 ml) triethylamine (0.101 g, 1 mmol) and chloropropionyl
chloride (0.317 g, 2.5 mmol) were added. The reaction mix-

1366
Vol. 27, No. 9
ture was stirred for 3 h at room temperature. After this the
solvent was removed under reduced pressure. The residue
was diluted with water and extracted three times with
dichloromethane. The combined organic layers were dried
over Na2SO4, filtered, and concentrated to give a crude prod-
uct, which was purified by chromatography (dichlorome-
thane–methanol, v/vꢂ10 : 1) to give an oil. This was recrys-
tallized from dil. ethanol to give 4 as white needles: mp,
137—139 °C; yield, 0.130 g, 63%. 1H-NMR (CDCl3) d: 3.32
(2H, t, Jꢂ5.5 Hz, CH2), 4.14 (2H, t, Jꢂ5.1 Hz, CH2), 5.73—
5.81 (1H, m, CH), 6.43—6.50 (2H, m, CH), 7.03 (1H, dd,
Jꢂ8.0 Hz, Jꢂ4.7 Hz, Ar-H), 7.33 (1H, d, Jꢂ8.0 Hz, Ar-H),
8.29 (1H, dd, Jꢂ4.7 Hz, Jꢂ1.5 Hz, Ar-H). 13C-NMR
(CDCl3) d: 28.9, 40.1, 118.8, 127.9, 129.6, 132.7, 133.4,
146.9, 152.1, 164.4. MS m/z: 206 (Mꢃ), 152, 137, 55. Anal.
Calcd for C10H10N2OS: C, 58.23; H, 4.89; N, 13.58. Found:
C, 58.02; H, 4.76; N, 13.31.
Fig. 2. Chemical Syntheses of MM 10 and MM 11
(10 mmol/l) and to 306.25ꢁ8.54 ms (30 mmol/l) (nꢂ5,
pꢄ0.05), whereas no significant change in action potential
duration at 20% time to repolarization (APD20) could be ob-
served. Membrane resting potential (MRP) as well as action
potential amplitude (APA) and maximum rate of rise (Vmax
were not affected in experiments with MM 10.
1-[3-[N-[2-(3,4-Dimethoxyphenyl)ethyl]methylamino]-
propionyl]-2,3-dihydro-1H-pyrido[2,3-b][1,4]thiazine (5)
To a solution of compound 4 (0.206 g, 1 mmol) in dry
ethanol (8 ml) 2-(3,4-dimethoxyphenyl)-N-methyl-1-ethyl-
amine (0.195 g, 1 mmol) was added. The reaction mixture
was refluxed for 3 h. After this the solvent was removed
under reduced pressure. The residue was diluted with water
and extracted three times with dichloromethane. The com-
bined organic layers were dried over Na2SO4, filtered, and
concentrated to give a crude product, which was purified by
chromatography (toluene–ethylacetate–triethylamin, v/v
)
Used in the same concentrations MM 11 did not exert any
effect on action potential amplitude, maximum upstroke ve-
locity and membrane resting potential. Action potential dura-
tion at 50% time to repolarization was prolonged from
195.0ꢁ9.13 to 210.0ꢁ10.8 ms (3 mmol/l) and to 212.5ꢁ
10.41 ms (30 mmol/l) (nꢂ4, pꢄ0.05) as well as at 90% time
to repolarization from 212.25ꢁ4.79 to 246.25ꢁ14.36 ms
(3 mmol/l) and to 262.5ꢁ9.57 ms (30 mmol/l) (nꢂ4, pꢄ0.05)
prolonged in a concentration-dependent manner. All ob-
served effects occurred immediately after drug application
reached maximum in approximately 30 min and where com-
pletely reversible during washout with drug-free Tyrode’s so-
lution. Original recordings are shown in Fig. 2.
Effects on Action Potential of Left Atria Similar to ex-
periments with papillary muscles action potential duration at
50% time to repolarization was clearly prolonged from
44.75ꢁ5.5 to 51.0ꢁ4.62 ms (3 mmol/l), to 58.0ꢁ5.72 ms
(10 mmol/l) and to 66.75ꢁ7.68 ms (30 mmol/l) (nꢂ4, pꢄ
0.05) as well as at 90% time to repolarization from 97.0
ꢁ2.16 to 113.25ꢁ3.95 ms (3 mmol/l) (nꢂ5, pꢄ0.05), to
130.0ꢁ4.08 ms (10 mmol/l) and to 147.5ꢁ6.54 ms (30
mmol/l) (nꢂ4, pꢄ0.05), during exposure to MM 10. On the
other hand the compound did not produce any change in
APD20, membrane resting potential, action potential ampli-
1
ꢂ6 : 3 : 1) to give a pale yellow oil. Yield: 0.258 g, 64%. H-
NMR (CDCl3) d: 2.21 (3H, s, NCH3), 2.52—2.79 (8H, m,
4CH2), 3.36 (2H, t, Jꢂ5.4 Hz, CH2), 3.86 (3H, s, OCH3),
3.87 (3H, s, OCH3), 3.95—4.10 (2H, m, CH2), 6.76 (1H, s,
Ar-H), 6.69 (1H, d, Jꢂ7.8 Hz, Ar-H), 6.77 (1H, d, Jꢂ7.8 Hz,
Ar-H), 7.01 (1H, dd, Jꢂ8.0 Hz, Jꢂ4.7 Hz, Ar-H), 7.41 (1H,
s br, Ar-H), 8.28 (1H, dd, Jꢂ4.7 Hz, Jꢂ1.3 Hz, Ar-H). 13C-
NMR (CDCl3) d: 28.6, 31.9, 33.1, 41.7, 53.2, 55.5, 55.6,
59.5, 61.4, 110.9, 111.7, 118.7, 125.0, 132.6, 132.6, 133.9,
137.5, 146.8, 147.0, 148.5, 171.1. MS m/z: 402 (Mꢃ), 250,
207. Anal. Calcd for C21H27N3O3S: C, 62.82; H, 6.78; N,
10.47. Found: C, 62.59; H, 6.75; N, 10.20.
1-[3-[N-[2-(3,4-Dimethoxyphenyl)ethyl]methylamino]-
propionyl]-2,3-dihydro-1H-pyrido[2,3-b][1,4]thiazine
fumarate (MM 11) To a solution of compound 5 (0.402 g,
1 mmol) and fumaric acid (0.116 g, 1 mmol) in dry ethanol
(10 ml) dry ether was added gradually until a slight precipita-
tion could be detected. This mixture was stored at 0 °C for
20 h. After this the precipitated solid was filtered to give MM
11 as white needles: mp, 62 °C; yield, 0.460 g, 89%. Anal.
Calcd for C25H31N3O7S: C, 58.01; H, 6.04; N, 8.12. Found:
C, 57.93; H, 5.89; N, 8.03.
tude or Vmax
.
Similar results could be obtained with MM 11 (3 mmol/l
and 30 mmol/l). In these experiments action potential ampli-
tude, Vmax and membrane resting potential remained un-
changed, whereas the investigated substance produced signif-
icant prolongation in action potential duration at 50% time to
repolarization was prolonged from 42.5ꢁ2.9 to 55.75ꢁ
1.2 ms (3 mmol/l) and to 71.67ꢁ2.89 ms (30 mmol/l) (nꢂ4,
pꢄ0.05) as well as at 90% time to repolarization from
96.25ꢁ9.46 to 123.75ꢁ8.54 ms (3 mmol/l) and to 161.67ꢁ
10.41 ms (30 mmol/l) (nꢂ4, pꢄ0.05). In contrast to experi-
ments with MM 10 APD20 was as well prolonged from
21.25ꢁ2.5 to 28.75ꢁ2.5 ms (3 mmol/l) and to 33.33ꢁ2.89
ms (10 mmol/l) (nꢂ4, pꢄ0.05). Both substances started to
exert their effects soon after application, maximum effects
could be measured after approximately 30 min and were
RESULTS
Effects on Action Potential of Papillary Muscles Ap-
plication of MM 10 in concentrations of 3 mmol/l, 10 mmol/l
and 30 mmol/l led to significant prolongation of action poten-
tial duration at 50% time to repolarization from 237.5ꢁ6.45
to 260.0ꢁ4.08 ms (3 mmol/l), to 267.5ꢁ2.98 ms (10 mmol/l)
and to 268.75ꢁ4.79 ms (30 mmol/l) (nꢂ5, pꢄ0.05) as well
as at 90% time to repolarization from 258.78ꢁ10.31 to
280.0ꢁ13.5 ms (3 mmol/l) (nꢂ5, pꢄ0.05), to 293.0ꢁ8.9 ms

September 2004
1367
Fig. 3. Left Hand Panel: Transmembrane Action Potentials of a Guinea Pig Papillary Muscle in Control (A), after the Addition of 3 mmol/l MM 10 (B),
after the Addition of 10 mmol/l MM 10 (C), after the Addition of 30 mmol/l MM 10 (D) and during Washout (E). Right Hand Panel: Transmembrane Action
Potentials in Control (A), after the Addition of 3 mmol/l MM 11 (B), after the Addition of 30 mmol/l MM 11 (C) and during Washout (D)
The preparation was constantly driven at 1 Hz. Upper trace shows membrane action potential and lower trace the upstroke spike of the action potential (Vmax).
completely reversible during washout. Figure 3 shows origi-
nal recordings.
concentration of 30 mmol/l MM 10 significantly reduced rate
of activity from 186.0ꢁ24.0 to 140.0ꢁ33.05 beats/min
(nꢂ3, pꢄ0.05) and slope of slow diastolic depolarization
from 69.0ꢁ3.61 to 41.67ꢁ7.64 mV/s (nꢂ3, pꢄ0.05) in
sinoatrial nodes. Maximum diastolic potential was decreased
from ꢅ59.6ꢁ1.77 to ꢅ53.7ꢁ2.05 mV (nꢂ3, pꢄ0.05) as
well as action potential amplitude from 73.33ꢁ2.89 to
61.67ꢁ5.77 mV (nꢂ3, pꢄ0.05) and Vmax from 16.67ꢁ5.77
to 10.0ꢁ4.36 V/s (nꢂ3, pꢄ0.05). On the other hand the
compound produced significant prolongation of 50% time to
repolarization from 98.33ꢁ2.89 to 123.33ꢁ5.77 ms (nꢂ3,
pꢄ0.05) and 90% time to repolarization from 132.0ꢁ8.19 to
181.67ꢁ10.41 ms (nꢂ3, pꢄ0.05) whereas 20% time to repo-
larization was not affected.
In the same concentration MM 11 exerted similar but
more pronounced effects. In a concentration of 30 mmol/l
MM 11 significantly reduced rate of activity from 192.0ꢁ0
to 114.0ꢁ15.87 beats/min (nꢂ3, pꢄ0.05) and slope of slow
diastolic depolarization from 62.67ꢁ2.52 to 38.33ꢁ2.89
mV/s (nꢂ3, pꢄ0.05) in sinoatrial nodes. Maximum diastolic
potential was decreased from ꢅ60.0ꢁ0 to ꢅ48.33ꢁ2.31 mV
(nꢂ3, pꢆ0.05) as well as action potential amplitude from
70.33ꢁ2.89 to 52.33ꢁ4.62 mV (nꢂ3, pꢄ0.05) and Vmax
from 20.67ꢁ1.15 to 8.0ꢁ2.0 V/s (nꢂ3, pꢄ0.05). 50% time
to repolarization increased from 90.0ꢁ0 to 112.0ꢁ3.46 ms
(nꢂ3, pꢄ0.05) and 90% time to repolarization from
118.33ꢁ2.89 to 185.33ꢁ0.58 ms (nꢂ3, pꢄ0.05) whereas
20% time to repolarization was not affected.
Effects on Action Potential of Spontaneously Beating
Purkinje Fibers In spontaneously beating Purkinje fibers
MM 10 led to a significant prolongation of APD90 from
356.25ꢁ32.5 to 400.0ꢁ41.79 ms (3 mmol/l) and to 475.0ꢁ
59.02 ms (10 mmol/l) (nꢂ4, pꢄ0.05). A concnetration of
30 mmol/l increased APD90 411.67ꢁ12.58 to 566.67ꢁ14.53
(nꢂ4, pꢄ0.05). In addition 30 mmol/l MM 10 produced a
significant decrease in APD20 from 130.0ꢁ17.32 to 90.0ꢁ
17.32 ms as well as in slope of slow diastolic depolarization
(SSDD) from 13.33ꢁ2.31 to 6.67ꢁ1.53 mV/s and rate of ac-
tivity (RA) from 49.67ꢁ5.96 to 35.0ꢁ6.24 beats/min (nꢂ4,
pꢄ0.05). Action potential amplitude, Vmax and maximum di-
astolic potential (MDP) were not affected by MM 10.
Exposure to MM 11 led to a concentration-dependent pro-
longation of action potential duration at 50% time to repolar-
ization from 195.0ꢁ48.22 to 236.67ꢁ55.08 ms (3 mmol/l,
nꢂ3) and to 263.33ꢁ58.59 ms (30 mmol/l, nꢂ3) as well
as at 90% time to repolarization from 285.0ꢁ40.93 to
376.67ꢁ64.29 ms (3 mmol/l, nꢂ3) and to 406.67ꢁ81.45 ms
(30 mmol/l, nꢂ3, pꢄ0.05). Slope of slow diastolic depolar-
ization and rate of activity were significantly decreased at a
concentration of 30 mmol/l from 12.0ꢁ2.0 to 8.0ꢁ2.0 mV/s
and (nꢂ3, pꢄ0.05) and from 44.0ꢁ4.58 to 33.0ꢁ3.0
beats/min (nꢂ3, pꢄ0.05). In 3 out of 6 experiments MM 11
in a concentration of 30 mmol/l led to the occurrence of early
afterdepolarizations with a take-off potential between ꢅ50
and ꢅ60 mV in 4 to 6 min after drug application. Early after-
depolarizations and all other observed effects were com-
pletely reversible during washout. Original recordings from
action potential and early afterdepolarizations are shown in
Fig. 4.
In the same concentration MM 11 exerted similar but
more pronounced effects. In these experiments effects as well
occurred soon after drug application reached maximum in
approximately 30 min and were completely reversible during
washout with drug-free physiological salt solution.
Effects on Action Potential of Sinoatrial Nodes In a

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Fig. 4. Left Hand Panel: Transmembrane Action Potentials of a Left Atrium in Control (A), after the Addition of 3 mmol/l MM 10 (B), after the Addition
of 10 mmol/l MM 10 (C), after the Addition of 30 mmol/l MM 10 (D) and during Washout (E). Right Hand Panel: Transmembrane Action Potentials in Con-
trol (A), after the Addition of 3 mmol/l MM 11 (B), after the Addition of 30 mmol/l MM 11 (C) and during Washout (D)
The preparation was constantly driven at 1 Hz. Upper trace shows membrane action potential and lower trace the upstroke spike of the action potential (Vmax).
Fig. 5. Left Hand Panel: Transmembrane Action Potentials of a Guinea Pig Spontaneously Beating Purkinje Fibre in Control (A), after the Addition of
30 mmol/l MM 10 (B) and during Washout (C). Right Hand Panel: Transmembrane Action Potentials in Control (A), after the Addition of 3 mmol/l MM 11
(B), after the Addition of 30 mmol/l MM 11 (C) and during Washout (D)
Upper trace shows membrane action potential and lower trace the upstroke spike of the action potential (Vmax).

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Fig. 6. Left Hand Panel: Transmembrane Action Potentials of a Guinea Pig Sinoatrial Node in Control (A), after the Addition of 30 mmol/l MM 10 (B) and
during Washout (C). Right Hand Panel: Transmembrane Action Potentials in Control (A), after the Addition of 30 mmol/l MM 11 (B) and during Washout
(C)
Upper trace shows membrane action potential and lower trace the upstroke spike of the action potential (Vmax).
DISCUSSION
activation of fast sodium channels does not seem to play any
role.12,13) Application of MM 10 and MM 11 led to signifi-
Although the effects of MM 11 were more pronounced cant decreases in action potential amplitude and Vmax as well
both compounds exerted very similar electrophysiological as in slope of slow diastolic depolarization and rate of activ-
profiles. Since phase 0 of action potential of papillary mus- ity, as it is expected for calcium channel antagonists.
cles, left atria and Purkinje fibers is generated by fast influx
Though experimental data from these experiments do not
of sodium ions, action potential amplitude and Vmax are good provide direct evidence concerning the influence of MM 10
indicators of fast inward current INa.8) This current seems not and MM 11 on depolarizing and/or repolarizing currents, fol-
to be affected by the investigated substances considering the lowing conclusions may be reasonable: (i) fast sodium cur-
fact that neither MM 10 nor MM 11 produced any change in rent seems not to be affected, (ii) Isi is decreased as well as
APA or Vmax of these preparations. While the action potential (iii) one or more repolarizing potassium currents. According
duration is maintained by a balance between influx of cal- to the model of January and Riddle14,15) the delay in repolar-
cium ions (Isi) during the plateau phase and the activation of ization caused by MM 11 may provide for the “conditioning
several repolarizing outward potassium currents like IK phase” preceeding the activation of a depolarizing inward
and/or IK1.9,10) So prolongation of action potential duration as current and finally leading to the occurrence of early afterde-
it was caused by MM 10 and MM 11 in papillary muscles polarizations.
and left atria as well as in Purkinje fibers may be due to ei-
In contrast to its derivatives KT-362 led to a concentration-
ther an increase in Isi or a decrease in outward current. The dependent reduction in action potential amplitude and Vmax in
first possibility is rather unlikely for both compounds exerted guinea pig ventricular muscle.16) In isolated guinea pig hearts
negative inotropic effects in isolated guinea pig papillary KT-362 produced a concentration-dependent decrease in
muscles.11) Depression of potassium currents and concomi- atrial rate with an EC50 of 20 mmol/l.17) In addition to an inhi-
tant prolongation of action potential duration at 90% time to bition of the L-type calcium current Yuan-Na and cowork-
repolarization may as well provide an explanation for the de- ers18) found a decrease in peak sodium current in guinea pig
crease in rate of activity that could be observed in experi- ventricular myocytes in a concentration range from 10 to
ments with Purkinje fibers. Additionally, MM 11 was found 30 mmol/l. In the same study KT-362 inhibited the delayed
to depress slope of slow diastolic depolarization in these rectifier potassium current and the inward rectifier potassium
preparations, probably due to a decrease in pacemaker cur- current. The compound is as well considered to act in a ryan-
rent.
odine-like manner as an inhibitor of intracellular calcium re-
The fact that action potential duration at 20% and 50% lease.19,20) Concerning the structure–activity relationship the
time to repolarization was shortened or not affected by MM results of this study gave rise to the assumption, that the re-
10 and MM 11 can be attributed to a decrease in Isi in part placement of the benzothiazepine nucleus by a pyridothi-
overlapped by the simultaneous delay in repolarization. This azine ringsystem leads to a weakening or even a loss of
assumption is supported by the results obtained in experi- sodium channel blocking ability whereas calcium antagonis-
ments with sinoatrial nodes. In this preparation phase 0 and tic characteristics as well as depressing effects on potassium
phase 1 of transmembrane action potential are mainly depen- currents are preserved. Shortening of the side chain may ac-
dent on depolarizing slow inward calcium current whereas count for the fact that the effects exerted by MM 11, particu-

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Arch. Pharm. Pharm. Med. Chem., 332, 19—24 (1999).
8) Weidmann S., J. Physiol. (London), 127, 213 (1955).
9) Sakmann B., Trube G., J. Physiol. (London), 347, 641—657 (1984).
10) Sanguinetti M. C., Jurkiewicz N. K., J. Gen. Physiol., 96, 195—215
(1990).
11) Schade B., Studenik C., Biol. Pharm. Bull., 22, 683—686 (1999).
12) Yamagishi S., Sano T., Proc. Jpn. Acad., 42, 1194—1196 (1966).
13) Kreitner D., J. Mol. Cell. Cardio., 7, 655—662 (1975).
14) January C. T., Riddle J. M., Cir. Res., 64, 977—990 (1989).
15) January C. T., Riddle J. M., Salata J. J., Circ. Res., 62, 563—571
(1988).
16) Kodama I., Wakabayashi S., Toyama J., Shibata S., Yamada K., J. Car-
diovasc. Pharmacol., 11, 687—693 (1988).
17) Buljubasic N., Marijic J., Stowe D. F., Gross G. J., Kampine J. P.,
Bosnjak Z. J., J. Cardiovasc. Pharmacol., 18, 594—604 (1991).
18) Yuan-Na C., Tatsuko K., Makoto A., J. Pharmacol. Exp. Ther., 270,
851—857 (1994).
19) Kodama I, Shibata S., J. Pharmacol. Exp. Ther., 258, 332—338
(1991).
20) Eskinder H., Hillard C. J., Wilke R. A., Gross G. J., J. Cardiovasc.
Pharmacol., 13, 502—507 (1989).
larly those on repolarization, were more pronounced than
that performed by MM 10. Whether the investigated 1,4-
pyridothiazines in accordance with the mother compound
KT-362 act on intracellular calcium stores and which ion cur-
rents they affect in particular is left to further investigations.
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