5,7,8-trimethyl-benzopyran and 5,7,8-trimethyl-1,4-benzoxazine aminoamide derivatives as novel antiarrhythmics against ischemia-reperfusion injury

Article abstract of DOI:10.1021/jm801228h

6-Hydroxy-5,7,8-trimethyl-benzopyran derivatives and 5,7,8-trimethyl-1,4- benzoxazine analogues substituted by the lidocaine pharmacophore aminoamide functionality at C4 or N4, respectively, were synthesized and evaluated against arrhythmias associated wi

Full text of DOI:10.1021/jm801228h

2328
J. Med. Chem. 2009, 52, 2328–2340
5,7,8-Trimethyl-benzopyran and 5,7,8-Trimethyl-1,4-benzoxazine Aminoamide Derivatives as
Novel Antiarrhythmics against Ischemia-Reperfusion Injury
,‡
Eftychia N. Koini,† Panagiota Papazafiri,‡ Athanasios Vassilopoulos, Maria Koufaki,† Zolta´n Horva´th,| Istva´n Koncz,§
La´szlo´ Vira´g,§ Gy J. Papp,§,| Andra`s Varro´,§,| and Theodora Calogeropoulou*,†
Institute of Organic and Pharmaceutical Chemistry, National Hellenic Research Foundation, 48 Vas. Constantinou AVenue, 116 35 Athens, Greece,
Department of Animal and Human Physiology, School of Biology, UniVersity of Athens, Panepistimiopolis, 15784 Athens, Greece, Department of
Pharmacology and Pharmacotherapy, UniVersity of Szeged, Szeged Dom ter 12 H-6720, Hungary, Research Unit for CardioVascular Pharmacology,
Hungarian Academy of Sciences, Szeged Hungary
ReceiVed September 29, 2008
6-Hydroxy-5,7,8-trimethyl-benzopyran derivatives and 5,7,8-trimethyl-1,4-benzoxazine analogues substituted
by the lidocaine pharmacophore aminoamide functionality at C4 or N4, respectively, were synthesized and
evaluated against arrhythmias associated with ischemia-reperfusion injury. The antiarrhythmic effect of
substitutents at positions C2 and C6 was examined. Six out of the 11 new derivatives, exhibited arrhythmia
scores 1.0-1.3 versus the control (4.5 ( 1.2), which was also reflected to the % premature beats (0.5-3.9),
control (13.7 ( 3.6). Selected compounds were further studied by a conventional microelectrode method.
2-Diethylamino-1-(5,7,8-trimethyl-2-phenyl-2,3-dihydro-benzo[1,4]oxazin-4-yl)-ethanone (50) and the trolox-
inspired 4-(2-diethylamino-acetyl)-2,5,7,8-tetramethyl-3,4-dihydro-2H-benzo[1,4]oxazine-2-carboxylic acid
ethyl ester (62) suppress reperfusion arrhythmias and reduce malondialdehyde (MDA) content, leading to
a fast recovery of the heart after ischemia-reperfusion. They exhibit combined class IB and class III
antiarrhythmic properties, which constitutes them as promising compounds for further studies because, due
to their multichannel “amiodarone like” effect, less proarrhythmic complications can be expected.
Introduction
stores.7 Electrophysiological balance requires precise control of
sarcolemmal ion channels and exchangers, many of which are
regulated by phosphatidylinositol(4,5)bisphosphate, which is the
immediate precursor of inositol(1,4,5)trisphosphate (IP3), a
regulator of intracellular Ca2+ signaling and, therefore, a
potential contributor to arrhythmogenesis by altering Ca2+
homeostasis.7b Recent studies have shown that both R1-adre-
noceptor subtypes (R1A-AR and R1B-AR) can provide protection
from IP3 generation and arrhythmogenesis in early postischemic
reperfusion through different mechanisms.7b,c
Acute myocardial ischemia causes highly arrhythmogenic
changes in cardiac electrical properties1,2 that strongly promote
ventricular tachycardias and ventricular fibrillation and lead to
a high incidence of sudden death in minutes to hours.2
Reperfusion through thrombolysis or percutaneous angioplasty
(primary PTCA) is standard treatment in impending acute
myocardial infarction.3 While early reperfusion of the heart is
essential in preventing further tissue damage due to ischemia,
reintroduction of blood flow can expedite the death of vulner-
able, but still viable, myocardial tissue, by initiating a series of
events involving both intracellular and extracellular mechanisms.4,5
The first few minutes of reperfusion constitute a critical phase,
as lethal tissue injury in addition to that already developed during
ischemia may be initiated. The manifestations of reperfusion
injury include arrhythmia, reversible contractile dysfunction-
myocardial stunning, endothelial dysfunction, and cell death.
Reperfusion injury of the myocardium is a complex phenomenon
consisting of several independent etiologies.6 The mechanisms
proposed to contribute include oxygen free radical formation,
calcium overload, neutrophil-mediated myocardial and endot-
helial injury, progressive decline in microvascular flow to the
reperfused myocardium, and depletion of high energy phosphate
A variety of compounds have been investigated in different
experimental models of myocardial ischemia-reperfusion.8
These include oxygen free radical scavengers, antioxidants,
calcium-channel blockers, inhibitors of neutrophils, nitric oxide,
adenosine-related agents, inhibitors of the renin-angiotensin
system, endothelin receptor antagonists, Na+/H+ exchange
agents, mitochondrial KATP channel openers, and antiapoptotic
agents. All of these categories of biologically active agents have
been demonstrated to protect from reperfusion injury determined
as limitation of infarct size, improved myocardial and endothelial
function, and reduced incidence of arrhythmias.
It is now well recognized that arrhythmia is the main
manifestation of ischemia and reperfusion myocardial dysfunc-
tion.9 In particular, reperfusion following a certain period of
ischemia induces ventricular arrhythmias, such as ventricular
tachycardia and ventricular fibrillation,10 which are different
from coronary-evoked arrhythmias.11 Lidocaine, a Na+ channel
blocker, is often used as an antiarrhythmic drug in ischemia-
reperfusion situations.12 Besides having antiarrhythmic effects,
lidocaine may protect myocardium not only against ischemic
but also against reperfusion injury by affecting intracellular
concentrations of sodium13,14 and calcium15,16 during ischemia
and reperfusion, by protecting cellular membranes against long-
chain acylcarnitines17 and reactive oxygen species,18 and perhaps
* To whom correspondence should be addressed. Phone: +3010 7273833.
Fax: +30107273818. E-mail: tcalog@eie.gr.
†
Institute of Organic and Pharmaceutical Chemistry, National Hellenic
Research Foundation.
‡
Department of Animal and Human Physiology, School of Biology,
University of Athens.
§
Department of Pharmacology and Pharmacotherapy, University of
Szeged.
|
Research Unit for Cardiovascular Pharmacology, Hungarian Academy
of Sciences.
Current address: Genetics of Development, Disease Branch, National
Institute of Diabetes, Digestive, Kidney Diseases, National Institutes of
Health, Bethesda, Maryland, Maryland 20892.
10.1021/jm801228h CCC: $40.75
2009 American Chemical Society
Published on Web 03/23/2009

Benzopyrans and Benzoxazine Aminoamide DeriVatiVes
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2329
Figure 1. Design of new 5,7,8-trimethylbenzopyran- and 5,7,8-trimethyl-1,4-benzoxazine aminoamide derivatives.
by blocking calcium channels.19,20 Lidocaine reduces myocardial
ischemia-reperfusion injury in isolated rat heart21,22 and in
vivo.23-27
19 gave the corresponding anilines, which spontaneously
cyclized to benzoxazinones 20 and 21, respectively. Reduction
of the nitro group in compound 24 was effected using CuCl/
NaBH4 in ethanol34 to give benzoxazinone 25 after spontaneous
cyclization. Treatment of 20, 21, and 25 with a mixture of acetic
acid/hydrogen peroxide/hydrogen chloride in petroleum ether
afforded the 6-chloro-benzoxazinones 22, 23, and 26. The
synthesis of 2,3,5-trimethyl-6-nitro hydroquinone (27) was
effected by hydroxylation of 2,3,5-trimethyl-6-nitro-phenol (17)
using K2S2O8 in 10% aqueous NaOH. Nitration of 2,3,5-
trimethylhydroquinone afforded the corresponding quinone
derivative instead of the 2,3,5-trimethyl-6-nitro hydroquinone.
Efforts to nitrate 1,4-dimethoxy-2,3,5-trimethyl phenol resulted
in deprotection and oxidation to the quinone, as previously
observed.35 Protection of the hydroxyl groups of 27 using
dimethylsulfate in the presence of K2CO3 in acetone afforded
the dimethoxy derivative 28, which upon selective deprotection
In our previous studies involving antiarrhythmic antioxi-
dants,28 the 6-hydroxy benzopyran ring of vitamin E and the
pharmacophore diethylamino amide moiety of class I antiar-
rhythmics, procainamide and lidocaine, were combined in a
single molecular entity. Among the new analogues, the lidocaine
derivatives (I) were the most potent in suppressing reperfusion
arrhythmias. As a continuation of our efforts in the field of novel
cardioprotective agents against arrhythmias associated with
ischemia-reperfusion injury,29 we focused our studies on two
classes of analogues. The first comprises 6-hydroxy-5,7,8-
trimethyl-benzopyran derivatives substituted by the lidocaine
aminoamide functionality at C-4 (II), while the second encom-
passes 5,7,8-trimethyl-1,4-benzoxazine analogues in which the
lidocaine aminoamide functionality constitutes part of the
heterocyclic system (III). In addition, we studied the antiar-
rhythmic effect of alkyl or aryl substitutents at position C2 as
well as of substituents at position C6. (Figure 1). Selected
compounds were further studied by a conventional microelec-
trode method in order to get insight into their cellular mode of
action.
36
using BF3 ·S(CH3)2 complex in CH2Cl2 yielded 4-methoxy-
2,3,5-trimethyl-6-nitro-phenol (29). Alkylation with 2-bromo-
2-phenyl methyl acetate, as above for compound 24, afforded
analogue 30, which upon reduction using CuCl/NaBH4 in
ethanol34 and spontaneous cyclization gave benzoxazinone 31.
The final aminoamide derivatives 46-52 were obtained as
described in Scheme 4. Thus, reduction of benzoxazinones
20-23, 25, 26, and 31 with BF3 ·Et2O and NaBH4 in THF, at
0-5 °C, to the corresponding benzoxazines 32-38, followed
by acylation by bromoacetylbromide in the presense of triethy-
lamine in CH2Cl2 to give bromoamides 39-45 and subsequent
treatment with diethylamine in toluene produced the final
aminoamides 46-52, respectively. The synthesis of compounds
62 and 63, which can be envisaged as derivatives of trolox, the
water soluble derivative of Vitamin E, was accomplished as
depicted in Scheme 5. Alkylation of 17 using 2-bromo-2-methyl-
malonic acid diethyl ester to afford 53 was followed by catalytic
hydrogenation to the corresponding aniline, which spontaneously
cyclized to benzoxazinone 54. Treatment of 54 with a mixture
of acetic acid/hydrogen peroxide/hydrogen chloride in petroleum
ether afforded the 6-chloro-benzoxazinone 55. The desired
benzoxazine derivatives 58 and 59 could not be obtained by
treatment with BF3 ·Et2O and NaBH4 or BH3 ·THF due to the
concomitant reduction of the ester functionality at C2. We were
able to circumvent this problem through formation of the
corresponding thioamides 56 and 57 by treatment of 54 and
55, respectively, with Lawesson reagent37 followed by desul-
phurization using Ra/Ni to afford benzoxazines 58 and 59,
respectively. Acylation by bromoacetylbromide in the presence
of triethylamine in CH2Cl2 gave bromoamides 60 and 61, and
Chemistry
The synthesis of compounds 13-15, 46-52, 62, and 63 is
depicted in Schemes 1-5. More specifically, condensation of
3,6-dihydroxy-2,4,5-trimethyl acetophenone with acetone or
2-octanone in the presence of pyrrolidine and molecular sieves
in ethanol gave chromanones 1 and 2, respectively, while
condensation of 3,6-dihydroxy-2,4,5-trimethyl acetophenone
with benzaldehyde in the presence of piperidine, boric acid, and
silica gel in DMF30 afforded 3. Compounds 1-3 upon treatment
with hydroxylamine in pyridine were converted to oximes 4-6,
which were in turn reduced to the corresponding amines 7-9
using TiCl4/NaBH4 in DME.31 Analogues 7-9 were acylated
using bromoacetyl chloride in the presence of NaHCO3 in THF/
H2O to afford bromoamides 10-12, which reacted with diethy-
lamine in toluene32 to produce the desired 4-aminobenzopyran
derivatives 13-15. The preparation of 1,4-benzoxazinone
derivatives 20-23, 25, 26, and 31 is depicted in Schemes 2
and 3. Thus, treatment of 2,3,5-trimethylphenol (16) with NaNO3
in the presence of HCl and a catalytic amount of La(NO3)3 in
a biphasic system (water-ether)33 gave 2,3,5-trimethyl-6-nitro-
phenol (17), which was alkylated with the appropriate R-bro-
moester in the presense of Cs2CO3 and a catalytic amount of
TBAI to give ethers 18, 19, and 24. Hydrogenation of 18 and

2330 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Koini et al.
Scheme 1a
a Reagents and conditions: (a) CH3COR (R ) CH3 or C6H13), pyrrolidine, EtOH, molecular sieves, 50 °C; (b) PhCHO, H3BO3/piperidine, DMF, reflux;
(c) NH2OH·HCl, pyridine, 50 °C; (d) TiCl4, NaBH4, DME; (e) bromoacetyl chloride, NaHCO3, THF/H2O; (f) Et2NH, toluene, 40 °C.
Scheme 2a
a Reagents and conditions: (a) NaNO3, La(NO)3, HCl, Et2O; (b) R1R2C(Br)COOCH2CH3, Cs2CO3, TBAI, DMF, 40 °C; (c) H2, Pd/C, EtOH, 50 °C; (d)
AcOH-H2O2-conc HCl, PE, reflux; (e) PhCH(Br)COOCH3, Cs2CO3, TBAI, DMF; (f) NaBH4, CuCl, EtOH, reflux.
Scheme 3a
a Reagents and conditions: (a) K2S2O8, 10% aq NaOH; (b) (CH3O)2SO2, K2CO3, TBAI, acetone, reflux; (c) BF3 ·S(CH3)2, CH2Cl2, 0 °C; (d)
PhCH(Br)COOCH3, Cs2CO3, TBAI, DMF; (e) NaBH4, CuCl, EtOH, reflux.
subsequent treatment with diethylamine in toluene produced the
final aminoamides 62 and 63, respectively.
racemic mixture, or Ra-Ni, which afforded the desired enan-
tiomer of 26b but in low yield (∼40%). We were pleased to
find that reduction of 65 using Fe in the presence of NH4Cl39
followed by spontaneous cyclization afforded benzoxazinone
26b, in 70% yield, which upon reduction with BH3 ·SMe2
yielded benzoxazine 37b. The desired amino amide 51b was
obtained after acylation by bromoacetylbromide in the presence
of triethylamine in CH2Cl2 and subsequent treatment with
diethylamine in toluene.
The enantioselective synthesis of (R)-(-)-1-(6-chloro-5,7,8-
trimethyl-2-phenyl-2,3-dihydro-benzo[1,4]oxazin-4-yl)-2-diethy-
lamino-ethanone (51b) is described in Scheme 6. Chlorination
of 2,3,5-trimethyl-6-nitrophenol (17) afforded 4-chloro-2,3,5-
trimethyl-6-nitrophenol (64), which upon Mitsunobu reaction
using (S)-(+)-R-hydroxy phenyl methyl acetate in the presence
of triphenylphosphine and DEAD either at room temperature
or at 40 °C afforded the desired R-enantiomer 65 but in low
yield (∼18%). The yield was dramatically increased when the
reaction was performed in an ultrasonic bath (∼75%).38 The
next step was the reduction of the nitro group in 65 followed
by spontaneous cylization to afford (R)-(-)-6-chloro-5,7,8-
trimethyl-2-phenyl-2H-benzoxazine-3(4H)-one (26b). To this
end, initially we employed CuCl in the presence of NaBH4,
which afforded benzoxazinone 26b but unfortunately as a
Results and Discussion
The ability of the new analogues 13-15, 46-52, and 62 to
suppress reperfusion arrhythmias and to inhibit lipid peroxida-
tion was evaluated using the Krebs perfused Langendorff
model40 on isolated rat heart preparations (Tables 1 and 2). The
results were obtained from 3-5 independent experiments. The
compounds were present at the last 5 min of ischemia and during

Benzopyrans and Benzoxazine Aminoamide DeriVatiVes
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2331
Scheme 4a
a Reagents and conditions: (a) BF3 ·Et2O, NaBH4, THF, 0-5 °C; (b) Bromoacetyl bromide, Et3N, CH2Cl2; (c) Et2NH, toluene, 40 °C.
Scheme 5a
a Reagents and conditions: (a) CH3C(Br)(COOCH2CH3)2, Cs2CO3, TBAI, DMF, 40 °C; (b) H2, Pd/C, EtOH, 50 °C; (c) AcOH-H2O2-HCl, PE, reflux; (d)
Lawesson reagent, toluene 110 °C; (e) Raney/nickel, EtOH-H2O; (f) BrCH2COBr, Et3N, CH2Cl2; (g) Et2NH, toluene, 40 °C.
Table 1. Antiarrhythmic and Antioxidant Activity of the New Analoguesa
compd MDA (ng/g wet tissue) arrhythmia score premature ventricular beats PVB (%) ventricular tachycardia (VT) ventricular fibrillation (VF)
13
14
15
46
47
48
49
50
51
52
62
control
lidocaine
NEb
NE
9 ( 3 (30 µM) 8 ( 5 (100 µM)
10 ( 3.5 (30 µM) 9 ( 4 (100 µM)
4.3 ( 2.58c (1 µM)
3.4 ( 2.25c(1 µM)
128.6 ( 42.0
152.0 ( 29.0
160.0 ( 1.0c
163.0 ( 16.0
143.0 ( 52
120.0 ( 20.0c
118.3 ( 2.0e
136.0 ( 32.0
88.6 ( 4.0e
230.0 ( 32.0
160.0 ( 40.0
3.6 ( 0.86
1.3 ( 0.08c
5.1 ( 1.3
1.56 ( 0.24
0.84 ( 0.16
1.6 ( 0.64e(1 µM)
1.0 ( 0.13
1.0 ( 0.09e
5.7 ( 1.00
1.0 ( 0.01e
1.0 ( 0.05e
1.0 ( 0.03e
1.0 ( 0.02e
4.5 ( 1.24
1.0 ( 0.04
3.9 ( 0.28c(1 µM)
2.1 ( 2.59c(1 µM)
3.68 ( 0.21
0.5 ( 0.07e(1 µM)
1.0 ( 0.05e (1 µM)
2.8 ( 0.84d (1 µM)
3.0 ( 0.35d (1 µM)
13.0 ( 3.6 (1 µM)
3.1 ( 1.0 (1 µM)
a n ) 3-5. b NE ) not examined. c *p < 0.05, versus control. d **p < 0.01, versus control. e ***p < 0.001, versus control.
reperfusion. Arrhythmia scores were calculated for the first 10
min of reperfusion and were quantified according to the Lambeth
Convention guidelines41 by the following scoring system: hearts
with premature ventricular beats (PVBa) less than 5% were
given a score of 1, with PVB more than 5% or bigeminy/salvos
a score of 2, ventricular tachycardia (VT) a score of 3, transient
ventricular fibrillation (VF) a score of 4, and sustained VF a
score of 5. Ventricular fibrillations lasting more than 1 min were
considered as sustained. The arrhythmia score in the absence
of compound was 4.5 ( 1.24 and was mainly due to premature
beats (13.0 ( 3.6). Lidocaine had an arrhythmia score of 1.0 (
0.04 and was also due to premature beats (3.1 ( 1.0).
Peroxidation of membrane phospholipid polyunsaturated fatty
acids is considered a major mechanism of the damage occurring
on reperfusion of the myocardium after a prolonged period of
ischemia. Lipid peroxides are unstable and decompose to form
a series of compounds including reactive carbonyl compounds.
Malondialdehyde (MDA) is the final product of lipid peroxi-
dation, and it has been found in the blood of patients after
reperfusion of the myocardium.42 Thus, malondialdehyde quan-
titation by the thiobarbituric acid test (TBA test) has been used
as indicator of lipid peroxidation.43 Malondialdehyde (MDA)
levels were measured at the end of reperfusion (ng/g wet tissue)
(Tables 1,2), and the reduction of MDA levels was an indication
of the antioxidant activity of the compounds under study.
a Abbreviations: APA, action potential amplitude, APD50, action potential
duration measured at 50% repolarization; APD90, action potential duration
measured at 90% repolarization; MDA, malondialdehyde; PVB, premature
ventricular beats; RMP, resting membrane potential, Vmax, maximal rate of
depolarization; VF, ventricular fibrillation, VT, ventricular tachycardia.

2332 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Koini et al.
Table 2. Antiarrhythmic and Antioxidant Activity of the Racemic Mixture of Compound 51 and Its Enantiomers, 51a and 51b, Tested at 1 µM
Concentrationa
compd MDA (ng/g wet tissue) arrhythmia score premature ventricular beats PVB (%) ventricular tachycardia (VT) ventricular fibrillation (VF)
51
51a
51b
control
lidocaine
118.3 ( 2.0b
106.0 ( 6.0b
153.0 ( 34.0
230.0 ( 32.0
160.0 ( 40.0
1.0 ( 0.05b
4.3 ( 1.34
1.0 ( 0.14b
4.5 ( 1.24
1.0 ( 0.04
1.0 ( 0.05b
1.5 ( 1.32c
0.80 ( 0.07b
13.0 ( 3.6
3.1 ( 1.0
2.64 ( 0.32
0.3 ( 0.08
a n ) 3-5. b ***p < 0.001, versus control. c **p< 0.01, versus control.
The C6 chloro-substituted analogue of 50 (compound 51)
reduced PVBs to a lesser extent than 50 (PVB ) 1.0 ( 0.05)
but possessed slightly higher antioxidant activity (118.3 ( 2.0
ng/g wet tissue). In addition, both compounds resulted in very
good recovery of the heart. Analogue 52 is substituted by a
methoxy group at C6 and caused a substantial decrease of PVBs
and MDA in comparison to both the control and lidocaine (PVB
) 2.8 ( 0.84 and MDA ) 136 ( 32.0 ng/g wet tissue).
However, when compared to 50 and 51, the presence of the
methoxy group was less advantageous than chlorine or hydrogen.
Because compound 51 was not only very potent in suppress-
ing reperfusion arrhythmias but also was the most potent
antioxidant (MDA 118.3 ( 2.0 ng/g wet tissue), it was of
interest to investigate whether the two enantiomers of 51
possessed different activity than the racemate. Thus we em-
ployed chiral HPLC (DAICEL-CHIRACEL OD, mobile phase
hexane/2-propanol 90/10, flow rate 2.4 mL/min) for the separa-
tion of the racemic mixture (Figure 2). The first eluting
enantiomer (t1 ) 18.8 min) 51a ([R]2D0) +41.3°) reduced the
MDA levels more than 50% (106.0 ( 6.0) and the PVBs to 1.5
( 1.32 but it caused tachycardia and fibrillation (Table 2). The
second eluting enantiomer (t2 ) 22.2 min) 51b ([R]2D0) - 42.8°)
almost totally suppressed reperfusion arrhythmias PVB (%) )
0.80 ( 0.07, reduced MDA (153.0 ( 34.0), not as efficiently
as 51a, and resulted in very good recovery of the heart. To
determine the absolute stereochemistry of the active enantiomer
51b, we performed chiral synthesis, as depicted in Scheme 6,
and it was found that compound 51b was the R-enantiomer. It
must be noted that even though the two enantiomers differed
in activity, the racemic mixture was equally active as 51b. This
has been previously reported for the racemate of the ꢀ-adrenergic
blocker and class III antiarrhythmic, sotalol and its dextroro-
tatory isomer (D-sotalol), which were both equally effective in
increasing cardiac action potential durations.44
The antiarrhythmic and the antioxidant capacity of the trolox
resembling derivative 62 were also measured using the same
conditions (Table 1). This compound was found to possess the
highest antioxidant capacity as reduced the level of MDA to
approximately 40% of the control (p < 0.001), while its
antiarrhythmic activity was similar to that of compounds 50-52.
To get an insight on the mechanism of action of the potent
5,7,8-trimethyl-1,4-benzoxazine derivatives, we employed the
conventional microelectrode technique to study their effects on
the action potential parameters. More specifically, we selected
the nonsubstituted at C2 and C6 compounds 46 and 47, which
bear two methyl groups at C2, 49, which is the 6-chloro
derivative of 47, 50, which has a phenyl substituent at C2, and
the two trolox-resembling derivatives 62 and 63.
Figure 2. Chiral HPLC separation of racemic 51.
The activity of compounds 13-15, 46-52, and 62 against
reperfusion arrhythmias and lipid peroxidation is presented in
Table 1. Six out of the 11 new compounds induced only
premature beats and therefore were given the arrhythmia score
1. When compared to the control value (13.0 ( 3.6), these
compounds reduced significantly the occurrence of PVBs,
although to a different extent.
In more detail, within the first group, the 6-hydroxy-5,7,8-
trimethyl-benzopyran-4-aminoamide compounds 13 and 14 were
evaluated at 30 and 100 µM and were found to possess low
activity slightly suppressing premature beats. The levels of MDA
were not measured because the compounds were not very potent.
Conversely, introduction of a phenyl group at C2 resulted in
increased activity and compound 15 at concentration of 2 µM
was found to reduce premature beats to 4.3 ( 2.6 with respect
to the control (13.0 ( 3.6) and MDA levels by 45%. However,
tachycardia was observed as a side effect. (Table 1). On the
contrary, the 5,7,8-trimethyl-1,4-benzoxazine derivatives 46-52,
tested at 2 µM (Table 1), were found to possess significant
antioxidant and antiarrhythmic activity. The C2 and C6 unsub-
stituted derivative 46 reduced premature beats to 3.4 ( 2.25
with an arrhythmia score of 1.3 ( 0.1 versus the control (4.5
( 1.24) and MDA levels by 34%. Introduction of chlorine at
C6, compound 48, reduced MDA levels by 29% and premature
beats to 3.9 ( 0.28 with an arrhythmia score of 1.0 ( 0.09.
The 2,2-dimethyl derivative 47 significantly suppressed
premature beats (1.6 ( 0.64) but caused tachycardia and
ventricular fibrillation, resulting in an arrhythmia score of 5.1
( 1.3. The presence of a chlorine atom at C6 in 47 (compound
49) caused also reduction of PVBs (2.1 ( 2.59) and suppressed
tachycardia but increased fibrillation (arrhythmia score 5.7 (
1.00). The antioxidant capacity of 47 and 49 was similar.
The 2-phenyl substituted analogues 50-52 exhibited the
highest antioxidant and antiarrhythmic activity of all the
compounds of the present study. Thus, derivative 50 decreased
MDA levels by approximately 48% and PVBs to 0.5 ( 0.07
(control PVB (%) ) 13 ( 3.6 and lidocaine PVB (%) ) 3.1 (
1.0).
The cellular electrophysiological effects of analogues 46, 47,
49, 50, 62, and 63 were investigated using rabbit right ventricular
papillary muscle at 5 µM. As control, we employed the class
I/B drug mexiletine, which is an orally active lidocaine analogue,
and the class III antiarrhythmic sotalol, at 20 µM concentration
(Figure 3). The relation of the in vitro cellular action potential
and in vivo ECG measurements is illustrated in Figure 4. The

Benzopyrans and Benzoxazine Aminoamide DeriVatiVes
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2333
Scheme 6a
a Reagents and conditions: (a) (S)-(+)-R-Hydroxy-phenyl-acetic acid methyl ester, Ph3P, DEAD, THF, ultrasound; (b) Fe, NH4Cl, EtOH/H2O, 85 °C; (c)
BH3 ·SMe2, THF; (d) BrCH2COBr, Et3N, CH2Cl2; (e) Et2NH, toluene, 40 °C.
13 and 14, or in analogues that caused ventricular tachycardia
and/or ventricular fibrillation, 47 and 49, respectively. Con-
versely, the presence of a phenyl group at C2 resulted in potent
compounds in both the 5,7,8-trimethyl-1,4-benzoxazine and the
5,7,8-trimethyl-benzopyran series. This effect was more pro-
nounced within the 1,4-benzoxazine analogues because all
2-phenyl substituted derivatives 50-53 resulted in very good
recovery of the heart after ischemia-reperfusion. The trolox-
inspired-1,4-benzoxazinic derivative 62, reduced reperfusion
arrhythmias, and resulted in an extremely low MDA level, even
lower than the nonperfused heart. We cannot give a reasonable
explanation for this result based on the biological experiments
performed so far.
The presence of a C6-chloro-substituent in 5,7,8-trimethyl-
1,4-benzoxazines did not affect the antioxidant capacity and the
arrhythmia score with respect to the nonchlorinated congeners,
but it resulted in differences on the action potential parameters.
A possible explanation could be differences in membrane
binding and permeation due to halogenation, as previously
reported.50
As a general observation, the 5,7,8-trimethyl-1,4-benzox-
azine scaffold resulted in more potent antiarrhythmic com-
pounds when compared to the 5,7,8-trimethyl-benzopyran-
substituted derivatives. This is not unexpected because the
2H-1,4-benzoxazine-3-(4H)-one and 3,4-dihydro-2H-1,4-ben-
zoxazine systems have been studied extensively for building
natural and designed biologically active compounds, which
span from herbicides, fungicides, cardiovascular agents, KATP
channel openers, compounds against diabetes, neuropro-
tectants, and agents against anxiety and depression.51-53
Thus, the above-mentioned heterocycles can be considered
as privileged scaffolds for the development of potential new
drugs. To the best of our knowledge, the 5,7,8-trimethyl-
1,4-benzoxazine moiety has not been utilized yet as a
template for the design of new antiarrhythmics. The presence
of the three methyl substituents renders the system so electron
rich that the corresponding 6-hydroxy-2-alkyl- or 6-hydroxy-
2-aryl-5,7,8-trimethyl-1,4-benzoxazines were not stable54 in
contrast to the 6-methoxy-derivative 52.
Concerning the effect of the new analogues on action
potential parameters, we cannot draw specific structure-activity
relationships, however it is evident that the compounds that
suppressed reperfusion arrhythmias and did not induce
ventricular tachycardia and/or ventricular fibrillation possess
a multichannel profile. In addition, we cannot deduce any
clear trend between MDA reduction and suppression of
reperfusion arrhythmias for the compounds tested. It is
possible that our compounds affect pathways of ischemia-
reperfusion injury, which are not reflected on the MDA
produced. However, the analogues that effectively suppressed
reperfusion arrhythmias reduced MDA levels with respect
to the control.
Figure 3. Structures of known antiarrhythmic drugs.
cellular electrophysiological results are illustrated and sum-
marized in Table 3 and Figures 5 and 6. The compounds can
be divided in three groups according to their effects. Thus,
compounds 47 and 63 did not or only slightly changed the
repolarization measured as APD50 or APD90 but exerted marked
and use-dependent depression of the maximal rate of depolar-
ization (Vmax) and increase of impulse conduction time (CT),
particularly at fast stimulation frequencies. In this respect, it
has to be noted that 47 decreased Vmax only at stimulation cycle
lengths shorter than 1500-2000 ms, while, 63 decreased Vmax
at all stimulation cycle lengths studied. These effects resemble
those of mexiletine (Figure 6)45 and flecainide,46 are consistent
with class I/B and class I/C properties47 and strongly suggest
considerable fast sodium channel inhibition. Compounds 46 and
49 induced a marked reverse rate dependent prolongation of
repolarization measured as APD90 without or only slightly
changing Vmax or CT. This effect by 46 is similar to the class
III reference compound sotalol (Figure 6) and is consistent with
class III antiarrhythmic properties,48 suggesting inhibition of
one of the repolarizing cardiac potassium channels most likely
the rapid delayed rectifier outward potassium current (IKr)
flowing through the HERG channels. Compounds 50 and 62
showed considerable effect both on repolarization (APD90) and
depolarization/impulse conduction in a frequency dependent
manner. Both analogues decreased Vmax and increased CT more
at fast stimulation frequencies corresponding to cycle lengths
of less than 2000 ms than at normal or slow ones, but they
delayed repolarization reflected as increase of APD90 somewhat
more at slow than at fast stimulation frequencies. This combina-
tion of class IB and class III antiarrhythmic properties resembles
to that of amiodarone49 and suggests combined or multichannel
drug action.
The 2,2-dialkyl-substituted-4-aminobenzopyrane derivatives
13 and 14 possessed similar antiarrhythmic activity as their
5-aminobenzopyrane congeners, previously reported by our
group.28 This suggests that the position of the aminoamide
functionality in the 2,2-dialkyl-chroman derivatives is not very
critical for activity against reperfusion arrhythmias. The presence
of alkyl substituents at C2 resulted either in moderately active
compounds, in the case of the 4-amino-benzopyran derivatives
In conclusion, we have synthesized 5,7,8-trimethylbenzopyran
and 5,7,8-trimethyl-1,4-benzoxazine derivatives encompassing
the pharmacophore aminoamide functionality of lidocaine. On

2334 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Koini et al.
Figure 4. (A) Arrangement of the intracellular action potential measurement. A fine glass sharp tip microelectrode was inserted into the intracellular
space and the other (reference) electrode was placed at the extracellular environment. The potential difference between the two electrodes was
measured through an amplifier and represents the electrical activity of the ventricular papillary muscle cells. (B) Body surface electrocardiogram
(ECG), which represents the electrical activity of the whole heart and commonly used in the clinical practice. (C) Illustration of the relation between
the intracellular action potential and the ECG recordings. The ECG signal represents the average of the large number of action potentials produced
by the individual myocardial cells measured at the body surface. The P wave corresponds to the activity of the atrial cells, the QRS and T waves
the depolarization and the repolarization of the ventricular myocytes, respectively. Therefore, as the figure shows changes of the maximal rate of
depolarization (Vmax) would result changes in the QRS waves and changes in the ventricular action potential duration would result changes in the
QT interval.
Table 3. Effect of Compounds 46, 47, 49, 50, 62, and 63 and Reference Drugs Sotalol and Mexiletine on the Action Potential Parameters in Rabbit
Right Ventricular Papillary Muscle at 1 Hz Stimulation Frequency
h
compda
RMP,e mV
APA,f mV
APD50,g ms
APD90,g ms
Vmax
,
V/s
46b
-85.8 ( 0.3
-86.5 ( 0.9
-85.9 ( 1.0
-87.4 ( 0.4
-85.3 ( 2.0
-84.0 ( 2.5
-82.7 ( 3.4
-84.4 ( 2.3
-86.6 ( 0.7
-85.2 ( 1.0
-81.5 ( 3.4
-83.7 ( 2.1
-85.9 ( 1.8
-87.9 ( 1.7
-85.7 ( 2.0
-83.6 ( 0.9
111.9 ( 1.9
111.6 ( 1.7
108.4 ( 2.8
111.3 ( 0.9
116.2 ( 1.7
115.4 ( 2.1
115.1 ( 3.1
114.4 ( 2.6
110.9 ( 3.1
109.5 ( 2.0
105.1 ( 4.2
109.8 ( 0.9
109.5 ( 2.1
109.8 ( 1.7
105.9 ( 2.9
106.2 ( 1.3
192.3 ( 7.5
160.7 ( 10.7
147.1 ( 11.7
145.1 ( 9.7
171.6 ( 3.5
155.8 ( 7.7
175.6 ( 12.9
149.8 ( 7.0
161.7 ( 19.6
144.6 ( 119
107.7 ( 11.8
110.0 ( 11.7
219.9 ( 23.3
153.8 ( 11.1
141.0 ( 10.6
139.7 ( 8.5
236.8 ( 6.5
205.3 ( 9.1
179.5 ( 10.1
178.6 ( 9.3
214.4 ( 3.5
195.4 ( 7.6
210.9 ( 12.4
185.9 ( 5.2
201.2 ( 18.5
182.5 ( 10.6
159.5 ( 5.0
161.7 ( 5.3
275.0 ( 24.8
198.6 ( 10.7
180.9 ( 10.6
179.6 ( 8.7
200.2 ( 18.2
220.2 ( 15.2
175.4 ( 25.8
275.4 ( 20.8
188.4 ( 14.5
197.0 ( 9.3
174.5 ( 15.4
203.3 ( 15.2
171.9 ( 15.8
193.5 ( 8.7
184.7 ( 8.7
251.7 ( 25.9
202.4 ( 18.6
220.2 ( 21.9
190.3 ( 19.7
200.7 ( 9.5
controlb
47
control
49
control
50
control
62
control
63
control
sotalolc
control
mexiletined
control
a n ) 3. b n ) 4. c n ) 7 (reference compound). d n ) 10 (reference compound). e RMP ) resting membrane potential. f APA ) action potential amplitude.
h
g APD50, APD90 ) action potential duration measured at 50% and 90% repolarization, respectively. Vmax ) maximal rate of depolarization.
the basis of the evaluation of the antiarrhythmic and antioxidant
properties as well as the cellular cardiac electrophysiological
properties of the studied analogues 50 and 62 are promising
compounds for further studies because of their multichannel
“amiodarone like” effect, less proarrhythmic complications can
be expected than with 47, 63, 46, and 49, which seem to have
either class I or class III actions, which were responsible to the
increased mortality in the CAST2 and SWORD55 multicenter
clinical trials. However, it has to be emphasized that further
studies are needed to establish the exact mode of action of the
studied compounds because several possible potential antiar-
rhythmic mechanisms like calcium, ATP, acetylcholine ultra
rapid delayed rectifier (IKur kV 1.5) channel, or ꢀ receptor block
can not be ruled out.
Experimental Section
NMR spectra were recorded on a Bruker AC 300 spectrometer
1
1
operating at 300 MHz for H and 75.43 MHz for 13C. H NMR
spectra are reported in units of δ relative to the internal standard
of signals of the remaining protons of deuterated chloroform, at
7.24 ppm. 13C NMR shifts are expressed in units of δ relative to
CDCl3 at 77.0 ppm. 13C NMR spectra were proton noise decoupled.
All NMR spectra were recorded in CDCl3. Silica gel plates
Macherey-Nagel Sil G-25 UV254 were used for thin layer chroma-
tography. Chromatographic purification was performed with silica

Benzopyrans and Benzoxazine Aminoamide DeriVatiVes
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2335
Figure 5. Frequency dependent effect of compounds 49, 50, and 62
on the conduction time (CT), maximal rate of depolarization (Vmax),
and action potential duration (APD90) on rabbit ventricular papillary
muscles.
gel (200-400 mesh). Elemental analyses were carried out on a
Perkin-Elmer series II CHNS/O 2400 analyzer. Mass spectra were
recorded on a Varian Saturn 2000 GC-MS instrument in the EI
mode.
2,3-Dihydro-2-phenyl-6-hydroxy-2,5,7,8-tetramethyl-4H-1-
benzopyran-4-one (3). 2,4,5-Trimethyl-3,6-dihydroxyacetophenone
(1.94 g, 10 mmol) and benzaldehyde (1.04 mL, 10 mmol) were
added to a mixture of H3BO3 (0.927 g, 15 mmol), piperidine (0.25
mL, 2.5 mmol), and SiO2 (2.5 g) in DMF (20 mL). The mixture
was stirred at 120 °C overnight, and then it was cooled to room
temperature and was diluted with acetone and filtered. The filtrate
was evaporated in vacuo to afford benzopyranone 3 as viscous oil
(0.9 g, 34%), which was used without further purification in the
1
next step. H NMR (δ) 7.5-7.34 (m, 5H), 5.41 (d, J ) 12.8 Hz,
1H), 3.05-2.84 (m, 2H), 2.47 (s, 3H), 2.21 (s, 3H), 2.13 (s, 3H).
13C NMR (δ) 193.5, 158.3, 142.5, 139.3, 137.2, 129.7, 128.7, 128.4,
125.8, 124.4, 117.6, 78.5, 20.4, 14.1, 14.0, 12.2.
Figure 6. Frequency dependent effect of compounds 46, 47, and 63
and reference drugs sotalol and mexiletine on the conduction time (CT),
maximal rate of depolarization (Vmax), and action potential duration
(APD90) on rabbit ventricular papillary muscles.
2,3-Dihydro-2-phenyl-6-hydroxy-5,7,8-trimethyl-4H-1-ben-
zopyran-4-one-oxime (6). Oxime 6 was prepared according to the
procedure described for oxime 4 using benzopyranone 3, dry
pyridine (5 mL), and hydroxylamine hydrochloride (0.963 g, 13.9
dimethoxyethane was added dropwise. The mixture was stirred at
room temperature overnight. The reaction mixture was cooled to 0
°C and water was added. The mixture was made basic with the
addition of 28% aqueous ammonia and was extracted with
dichloromethane. The organic layer was extracted with brine, was
dried (Na2SO4), and the solvent was evaporated in vacuo to afford
9, which was used without further purification. Viscous oil, 0.121
1
mmol) 0.21 g, 92% yield. H NMR (δ) 7.52-7.32 (m, 5H), 4.95
(dd, J ) 12.8 Hz, J ) 3.7 Hz, 1H), 3.50 (dd, J ) 17.7 Hz, J ) 3.7
Hz, 1H), 2.90 (dd, J ) 17.7 Hz, J ) 12.2 Hz, 1H), 2.50 (s, 3H),
2.22 (s, 3H), 2.19 (s, 3H). 13C NMR (δ) 153.6, 150.2, 146.8, 140.6,
128.5, 127.9, 125.9, 125.8, 123.7, 118.5, 115.9, 77.2, 32.9, 14.7,
12.7, 12.1.
1
4-Amino-3,4-dihydro-2-phenyl-5,7,8-trimethyl-2H-1-benzopy-
ran-6-ol (9). To a solution of TiCl4 (0.17 mL, 1.53 mmol) in
dimethoxyethane (2 mL) at 0 °C was added NaBH4 (116 mg, 3.06
mmol). The mixture was stirred at 0 °C for 10 min, and
subsequently a solution of oxime 6 (0.15 g, 0.51 mmol) in 2 mL
g, yield: 84%. H NMR (δ) 7.46-7.29 (m, 5H), 5.26-4.95 (m,
1H), 4.07-4.16 (m, 1H), 2.24-2.12 (m, 11H).
N-(3,4-Dihydro-2-phenyl-6-hydroxy-5,7,8-trimethyl-2H-1-
benzopyran-4-yl)-bromoacetamide (12). To a solution of 4-amino-
3,4-dihydro-2-phenyl-5,7,8-trimethyl-2H-1-benzopyran-6-ol (9) (85

2336 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Koini et al.
mg, 0.30 mmol) in a mixture of THF/H2O 3/2 at 0 °C was added
NaHCO3 followed by the dropwise addition of bromoacetylchloride
(0.60-1.2 mmol) until completion of the reaction by TLC. The
reaction mixture was diluted with dichloromethane, and the organic
layer was washed with saturated aqueous NaHCO3, brine, and was
dried over Na2SO4. The solvent was evaporated in vacuo to afford
the desired bromoacetamide 12, which was used without further
purification. Viscous oil, 0.295 g, 73% yield. 1H NMR (δ)
7.54-7.30 (m, 5H), 5.23-4.91 (m, 1H), 4.11-4.32 (m, 1H),
3.92-3.49 (m, 2H), 2.25-1.99 (m, 11H).
6-Chloro-2,2,5,7,8-pentamethyl-2H-1,4-benzoxazin-3(4H)-
one (23). To a slurry of 2,2,5,7,8-pentamethyl-2H-1,4-benzoxazin-
3(4H)-one (21) (0.13 g, 0.59 mmol) in petroleum ether (10.5 mL)
was added a mixture of AcOH/H2O2 (30%)/HCl (37%) (0.22 mL,
3.85 mmol/ 0.43 mL, 3.85 mmol/0.15 mL, 1.48 mmol) and the
resulting mixture was refluxed for 48 h. The reaction was diluted
with ethyl acetate and the organic layer was extracted with water,
saturated aqueous NaHCO3, brine, and was dried (Na2SO4). The
solvent was evaporated in vacuo and the residue was purified by
flash column chromatrography (petroleum ether 40-60 °C/ethyl
acetate (9:1), to afford compound 23 as a white solid, 0.147 g,
N-(3,4-Dihydro-2-phenyl-6-hydroxy-5,7,8-trimethyl-2H-1-
benzopyran-4-yl)-(diethylamino)acetamide (15). To a solution of
N-(3,4-dihydro-2-phenyl-6-hydroxy-5,7,8-trimethyl-2H-1-benzopy-
ran-4-yl)-bromoacetamide (12) (113 mg, 0.28 mmol) in 7 mL
toluene at 0 °C was added diethylamine (0.07 mL, 0.70 mmol).
After stirring for 2 days at room temperature, the mixture was
extracted with 2 N HCl. The aqueous layer was made basic with 2
N NaOH and extracted with CH2Cl2. The organic layer was dried
(Na2SO4) and the solvent was evaporated in vacuo. Purification of
the residue by flash column chromatography using dichloromethane/
methanol 98:2 as elution solvent, compound 15 was obtained as a
1
98% yield; mp 234-237 °C. H NMR (δ) 8.33 (s, 1H), 2.29 (s,
6H), 2.17 (s, 3H), 1.49 (s, 6H). 13C NMR (δ): 170.4, 138.9, 130.1,
127.9, 124.5, 123.7, 118.4, 77.19, 23.3, 17.0, 13.9, 12.4. MS, m/z:
253 (100, M+), 255 (36). Anal. (C13H16ClNO2) C, H, N.
2-(2,3,5-Trimethyl-6-nitrophenoxy)-2-phenylacetic Acid Meth-
yl Ester (24). Following the procedure for 19, using 2-nitro-3,5,6-
trimethylphenol (17) (0.4 g, 2.21 mmol), cesium carbonate (1.08
g, 3.31 mmol), bromophenylacetic acid methyl ester (0.758 g, 3.31
mmol), and stirring at room temperature for 5 min, compound 24
was obtained after purification by flash column chromatography
(petroleum ether 40-60 °C/ethyl acetate (95:5)). Gummy solid,
0.88 g, 97% yield. 1H NMR (δ) 7.45-7.33 (m, 5H), 6.85 (s, 1H),
5.29 (s, 1H), 3.69 (s, 3H), 2.18 (s, 3H), 2.17 (s, 3H), 1.93 (s, 3H).
13C NMR (δ) 169.7, 147.5, 144.3, 140.9, 135.2, 129.3, 128.7, 127.9,
127.8, 127.5, 84.6, 52.5, 20.2, 17.0, 13.2. Anal. (C18H19NO5) C,
H, N.
1
gummy solid (0.086 g, 78%). H NMR (δ) 7.41-7.29 (m, 5H),
5.40-4.89 (m, 1H), 4.32-4.11 (m, 1H), 3.64-1.98 (m, 17H),
1.04-0.85 (m, 6H). 13C NMR (δ) 173.8, 150.1, 146.5, 140.8, 128.3,
127.6, 125.2, 125.1, 123.9, 118.7, 115.6, 77.1, 59.9, 48.5, 41.1,
39.8, 14.5, 12.5, 12.2, 11.8. Anal. (C24H32N2O3) C, H, N.
2-Nitro-3,5,6-trimethylphenol (17). A solution containing 2,3,5-
trimethylphenol (16) (6.0 g, 44 mmol) in diethyl ether (100 mL)
was added dropwise to a solution of sodium nitrate (3.71 g, 44
mmol) and La(NO)3 ·6H2O (0.191 g, 0.44 mmol) in 6 N HCl (72
mL) at 0 °C. The reaction mixture was stirred at room temperature
for 1 h and was then extracted with ethyl acetate. The organic layer
was washed with brine, dried (Na2SO4), and the solvent was
evaporated in vacuo. The residue was purified by flash column
chromatography (petroleum ether 40-60 °C/acetone 95:5) to afford
2-nitro-3,5,6-trimethylphenol as yellow solid (5.19 g, 65% yield):
2-Phenyl-5,7,8-trimethyl-2H-1,4-benzoxazin-3(4H)-one (25).
To a solution of 2-(2,3,5-trimethyl-6-nitrophenoxy)-2-phenylacetic
acid methyl ester (24) (0.800 g, 2.43 mmol) in absolute ethanol
(12.1 mL) at 0 °C was added CuCl (1.202 g, 12.15 mmol) followed
by NaBH4 (0.918 g, 24.3 mmol) over 5 min. The resulting mixture
was refluxed for 15 min, and the reaction was cooled to room
temperature. Water was added and the mixture was extracted with
ethyl acetate. The organic layer was washed with brine, dried over
Na2SO4, and the solvent was evaporated in vacuo. The crude product
was purified by flash column chromatography (petroleum ether
40-60 °C/acetone (9:1)) to afford compound 25 as white crystals,
1
mp 74-77 °C. H NMR (δ) 11.02 (s, 1H), 6.62 (s, 1H), 2.55 (s,
3H), 2.27 (s, 3H), 2,18 (s, 3H). 13C NMR (δ) 153.8, 145.4, 133.2,
132.9, 125.1, 124.4, 124.2, 22.5, 20.5, 11.6. MS (EI), m/z: 181 (100,
M+). Anal. (C9H11NO3) C, H, N.
1
0.5 g, 77% yield; mp 171-173 °C. H NMR (δ) 9.23 (s, 1H),
7.33-7.48 (m, 5H), 6.61 (s, 1H), 5.72 (s, 1H), 2.21 (s, 9H). 13C
NMR (δ) 166.3, 140.9, 135.5, 132.3, 128.5, 126.6, 125.1, 122.6,
122.2, 120.9, 78.0, 19.4, 16.2, 11.6; MS, m/z: 267 (10, M+), 207
(100).
2-Methyl-2-(2,3,5-trimethyl-6-nitrophenoxy)propanoic Acid
Ethyl Ester (19). To a solution of 2-nitro-3,5,6-trimethylphenol
(17) (0.4 g, 2.21 mmol) in dry DMF (7.35 mL) was added cesium
carbonate (2.16 g, 6.62 mmol), TBAI (catalytic amount), and
2-bromo-2-methyl-propionic acid ethyl ester (0.97 mL, 6.62 mmol)
and the mixture was heated at 60 °C for three days. Subsequently,
the reaction the mixture was diluted with water and extracted with
ethyl acetate. The organic layer was washed with brine and dried
(Na2SO4). The solvent was evaporated in vacuo, and the crude
product was purified by flash column chromatography (petroleum
ether 40-60 °C/acetone (95:5)) to afford compound 19 as a viscous
oil, 0.487 g, 83% yield. 1H NMR (δ) 6.78 (s, 1H), 4.19 (q, J ) 7.1
Hz, 2H), 2.18 (s, 3H), 2.13 (s, 3H), 2.05 (s, 3H), 1.40 (s, 6H), 1.28
(t, J ) 7.3 Hz, 3H). 13C NMR (δ) 173.3, 144.8, 139.7, 131.0, 127.8,
127.7, 126.8, 83.2, 61.6, 24.7, 20.3, 16.6, 13.9, 13.6. Anal.
(C15H21NO5) C, H, N.
2,2,5,7,8-Pentamethyl-2H-1,4-benzoxazin-3(4H)-one (21). To
a solution of 2-methyl-2-(2,3,5-trimethyl-6-nitrophenoxy)propanoic
acid ethyl ester (19) (0.45 g, 1.52 mmol) in dry ethanol (18.7
mL) was added 10% palladium on carbon (0.112 mg). The
suspension was stirred under 1 atm of hydrogen gas at 70 °C
for 24 h. The reaction mixture was filtered through celite, the
filtrate was concentrated in vacuo, and the crude product was
purified by flash column chromatography (petroleum ether
40-60 °C/acetone (9:1)) to afford compound 21 as a white solid,
0.283 g, 85% yield: mp 166-170 °C. 1H NMR (δ) 9.00 (s, 1H),
6.60 (s, 1H), 2.22 (s, 3H), 2.18 (s, 3H), 2.11 (s, 3H), 1.51 (s,
6H). 13C NMR (δ) 170.1, 140.4, 132.0, 124.5, 123.2, 122.8,
119.5, 77. 3, 23.5, 19.4, 15.9, 11.3. MS, m/z: 219 (100, M+).
Anal. (C13H17NO2) C, H, N.
2-Phenyl-5,7,8-trimethyl-3,4-dihydro-2H-1,4-benzoxazine (36).
To a solution of 2-phenyl-5,7,8-trimethyl-2H-1,4-benzoxazin-3(4H)-
one (25) (0.2 g, 0.75 mmol) in THF (35 mL) was added dropwise
at 0 °C a solution of boron trifluoride etherate (2.24 mmol, 0.28
mL). The reaction mixture was stirred at 0 °C for 20 min, and
subsequently NaBH4 (85 mg, 2.24 mmol) was added over 10 min.
The resulting mixture was stirred at room temperature overnight
and was diluted with ethyl acetate. The organic layer was extracted
with saturated aqueous NaHCO3 and brine and was dried (Na2SO4).
The solvent was evaporated in vacuo, and the residue was purified
by flash column chromatrography (petroleum ether 40-60 °C/ethyl
acetate (9:1)), to afford benzoxazine 36 as white crystals, 0.186 g,
1
98% yield; mp 80-82 °C. H NMR (δ) 7.50-7.34 (m, 5H), 6.59
(s, 1H), 5.10 (dd, J ) 8.5 Hz, 2.4 Hz, 1H,), 3.61 (d, J ) 11.6 Hz,
1H), 3.36-3.29 (m, 1H), 2.23 (s, 3H), 2.19 (s, 3H), 2.15 (s, 3H).
13C NMR (δ) 142.5, 139.7, 128.5, 128.4, 128.0, 126.8, 129.1, 123.3,
122.4, 120.4, 75.5, 48.2,19.2, 16.6, 11.5. MS, m/z: 253 (100, M+).
Anal. (C17H19NO) C, H, N.
2-Bromo-1-(5,7,8-trimethyl-2-phenyl-2,3-dihydro-1,4-benzox-
azin-4-yl)-ethanone (43). To a solution of 2-phenyl-5,7,8-trimethyl-
3,4-dihydro-2H-1,4-benzoxazine (36) (150 mg, 0.59 mmol) in dry
dichloromethane (12 mL) was added dropwise at 0 °C triethylamine
(0.17 mL, 1.18 mmol) and bromoacetylbromide (0.08 mL, 0.89
mmol). The resulting mixture was stirred at room temperature for
30 min and then diluted with saturated aqueous NaHCO3 and
extracted with ethyl acetate. The organic phase was washed with
saturated aqueous NaCl and was dried (Na2SO4). The solvent was
evaporated in vacuo, and the crude residue was purified by flash

Benzopyrans and Benzoxazine Aminoamide DeriVatiVes
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2337
column chromatography (petroleum ether 40-60 °C/ethyl acetate
(9:1)) to afford compound 43 as a yellowish solid, 0.174 g, 79%
yield; mp 109-111 °C. Two rotamers of compound 43 were
detected in the NMR spectra, and the corresponding peaks are
defined by an asterisk. 1H NMR (δ) 7.47-7.00 (m, 5H), 6.69 (bs,
1H), 5.73-4.88 (m, 2H), 4.64-2.78 (m, 3H), 2.28-2.10 (m, 9H).
13C NMR (δ) 167.9, 147.3, 138.1, 136.6, 135.9*, 128.8, 128.7,
128.6, 128.5*, 125.9, 125.6*, 125.5*, 123.9, 123.8*, 122.8, 78.1,
51.6*, 48.6, 27.1, 26.3*, 19.9, 19.8*, 17.2, 11.7.
Diethylamino-1-(5,7,8-trimethyl-2,3-dihydro-1,4-benzoxazin-
4-yl)-ethanone (46). Following the procedure for compound 15
using 2-bromo-1-(5,7,8-trimethyl-2,3-dihydro-1,4-benzoxazin-4-yl)-
ethanone (39) (83 mg, 0.28 mmol), compound 46 was obtained
after purification of the crude residue by flash column chromatog-
raphy (dichloromethane/methanol (95:5)). Viscous oil, 77 mg, 95%
yield. Two rotamers of compound 46 were detected in the NMR
spectra and the corresponding peaks are defined by an asterisk. 1H
NMR (δ) 6.59* and 6.58 (1H), 4.90* (ddd, J ) 13.4 Hz, 4.3 Hz,
1.2 Hz, 0.6H) and 4.65 (d, J ) 14.7 Hz, 0.4H), 4.49-4.19 (m,
2H), 3.49-3.09 (m, 2H and 0.4H), 2.80* (ddd, J ) 13.4 Hz, 4.3
Hz, 4.3 Hz, 0.6H), 2.65-2.48 (m, 4H), 2.20-2.04 (m, 9H), 1.05
(t, J ) 7.0 Hz, 2.4H), 0.89* (t, J ) 7.0 Hz, 3.6H). 13C NMR (δ)
171.9, 168.9*, 146.9, 146.2*, 135.8, 135.1*, 131.3*, 129.3, 124.8,
123.5*, 123.3, 122.9*, 122.3, 121.5*, 67.5*, 67.2, 57.8, 53.0*, 47.6,
47.2*, 44.0*, 41.7, 19.8, 19.7*, 18.8*, 17.3, 12.1*, 11.5. MS (ESI)
m/z 291.3 ([M + 1]+, 100). HRMS (FAB+) calcd for C17H27O2N2
[M + 1]+ 291.2073, found 291.2087. Anal. (C17H26N2O2) C, H,
N.
2-Diethylamino-1-(2,2,5,7,8-pentamethyl-2,3-dihydro-1,4-ben-
zoxazin-4-yl)-ethanone (47). Following the procedure for com-
pound 15 using 2-bromo-1-(2,2,5,7,8-pentamethyl-2,3-dihydro-1,4-
benzoxazin-4-yl)-ethanone (40) (90 mg, 0.28 mmol), compound
47 was obtained after purification of the crude residue by flash
column chromatography (dichloromethane/methanol (96:4)). Vis-
cous oil, 85 mg, 95% yield. Two rotamers of compound 47 were
detected in the NMR spectra and the corresponding peaks are
defined by an asterisk. 1H NMR (δ) 6.62* (s, 0.5H), 6.58 (s, 0.5H),
4.88* (d, J ) 12.8 Hz, 0.5 H), 4.45 (d, J ) 14.0 Hz, 0.5H),
3.73-3.15 (m, 2H and 0.5H), 2.83-2.65 (m, 4H), 2.59* (d, J )
12.8 Hz, 0.5 H), 2.21-2.04 (m, 9H), 1.40-1.24 (m, 6H), 1.11 (t,
J ) 7.0 Hz, 3H), 1.00 (t, J ) 7.0 Hz, 3H). 13C NMR (δ) 169.8,
168.9*, 148.3, 146.2*, 136.0, 134.9*, 130.5, 128.5*, 125.2, 123.8,
123.3*, 123.1*, 121.8, 121.6*, 80.1, 76.2*, 52.9, 52.1*, 47.7, 47.5*,
27.5, 27.1*, 26.6, 25.0*, 19.7, 19.6, 19.3, 17.4, 11.7, 11.4. MS (ESI)
m/z 319.4 ([M + 1]+, 100). HRMS (FAB+) calcd for C19H31O2N2
[M + 1]+ 319.2386, found 319.2380. Anal. (C19H30N2O2) C, H,
N.
compound 49 was obtained after purification of the crude residue
by flash column chromatography (dichloromethane/methanol (96:
4)). Viscous oil, 92 mg, 93% yield. Two rotamers of compound
49 were detected in the NMR spectra, and the corresponding peaks
are defined by an asterisk. 1H NMR (δ) 4.91* (d, J ) 12.8 Hz, 0.6
H), 4.55 (d, J ) 14.0 Hz, 0.4H), 3.63 (d, J ) 14.6 Hz, 0.4H),
3.37-3.10 (m, 2H), 2.83-2.54 (m, 4H and 0.6H), 2.32-2.10 (m,
9H), 1.40-1.24 (m, 6H), 1.08 (t, J ) 7.3 Hz, 2.4H), 0.92* (t, J )
7.0 Hz, 3.6H). 13C NMR (δ) 171.1, 170.0*, 146.8, 144.9*, 133.8,
133.0*, 129.1, 127.6*, 126.9, 126.2*, 126.1, 124.5*, 123.1, 122.5*,
80.4, 76.5*, 57.3, 53.5*, 52.9, 51.8*, 47.4, 27.3, 27.0, 26.5, 25.1,
17.7, 17.1, 16.9, 12.5, 12.3, 12.2, 11.8. MS (ESI) m/z 353.3 ([M +
1]+, 100), 355.3 ([M + 3]+, 37). HRMS (FAB+) calcd for
C19H30O2N2Cl [M + 1]+ 353.1996, found 353.2001. Anal.
(C19H29ClN2O2) C, H, N.
2-Diethylamino-1-(5,7,8-trimethyl-2-phenyl-2,3-dihydro-1,4-
benzoxazin-4-yl)-ethanone (50). Following the procedure for
compound 15 using 2-bromo-1-(5,7,8-trimethyl-2-phenyl-2,3-di-
hydro-1,4-benzoxazin-4-yl)-ethanone (43) (104 mg, 0.28 mmol),
compound 50 was obtained after purification of the crude residue
by flash column chromatography (dichloromethane/methanol (98:
2)). Viscous oil, 98 mg, 95% yield. Two rotamers of compound
50 were detected in the NMR spectra and the corresponding peaks
1
are defined by an asterisk. H NMR (δ) 7.43-7.23 (m, 5H), 6.67
(bs, 1H), 5.67-4.78 (m, 2H), 3.72-2.43 (m, 7H), 2.27-2.08 (m.,
9H), 1.11-0.83 (m, 6H). 13C NMR (δ) 172.3, 168.5*, 147.2, 146.6*,
140.8*, 138.7, 136.0, 135.3*, 131.3, 129.3*, 128.7, 128.6, 128.3,
125.8, 125.7, 124.7, 123.6, 123.5*, 122.5, 122.4*, 121.7, 79.2*,
79.0, 58.2, 53.2, 50.6, 48.4, 47.7, 47.5, 47.3*, 19.8, 19.7*, 18.9,
17.4, 12.2, 11.9*, 11.7, 11.4*. MS (ESI) m/z 367.4 ([M + 1]+,
100); HRMS (FAB+) calcd for C23H31O2N2 [M + 1]+ 367.2386,
found 367.2370. Anal. (C23H30N2O2) C, H, N.
1-(6-Chloro-5,7,8-trimethyl-2-phenyl-2,3-dihydro-1,4-benzox-
azin-4-yl)-2-diethylamino-ethanone (51). Following the procedure
for compound 15 using 2-bromo-1-(6-chloro-5,7,8-trimethyl-2-
phenyl-2,3-dihydro-1,4-benzoxazin-4-yl)-ethanone (44) (114 mg,
0.28 mmol), compound 51 was obtained after purification of the
crude residue by flash column chromatography (dichloromethane/
methanol (98:2)). Viscous oil, 109 mg, 97% yield. Two rotamers
of compound 51 were detected in the NMR spectra and the
corresponding peaks are defined by an asterisk. 1H NMR (δ)
7.41-7.21 (m, 5H), 5.67-4.80 (m, 2H), 3.71-2.03 (m, 16H),
1.13-0.82 (m, 6H). 13C NMR (δ) 172.4, 170.3, 168.7, 145.8, 145.5,
145.3, 140.6, 138.4, 134.1, 133.5, 133.3, 129.9, 129.7, 128.7, 128.6,
128.4, 127.0, 126.7, 125.8, 125.7, 125.4, 124.2, 123.8, 123.2, 123.0,
122.8, 79.3, 79.0*, 58.0, 56.5, 53.6, 50.5, 48.9, 48.3*, 47.6, 47.5,
47.3, 17.5, 17.2, 16.9, 12.7, 12.3, 11.9, 11.3. MS (ESI) m/z 401.3
([M + 1]+, 100), 403.4 ([M + 3]+, 37). HRMS (FAB+) calcd for
C23H30O2N2Cl [M + 1]+ 401.1996, found 401.1968. Anal.
(C23H29ClN2O2) C, H, N.
1-(6-Chloro-5,7,8-trimethyl-2,3-dihydro-1,4-benzoxazin-4-yl)-
2-diethylamino-ethanone (48). Following the procedure for com-
pound 15 using 2-bromo-1-(6-chloro-5,7,8-trimethyl-2,3-dihydro-
1,4-benzoxazin-4-yl)-ethanone (41) (93 mg, 0.28 mmol), compound
48 was obtained after purification of the crude residue by flash
column chromatography (dichloromethane/methanol (98:2)). Vis-
cous oil, 90 mg, quantitative yield). Two rotamers of compound
48 were detected in the NMR spectra and the corresponding peaks
1-(6-Methoxy-5,7,8-trimethyl-2-phenyl-2,3-dihydro-1,4-ben-
zoxazin-4-yl)-2-diethylamino-ethanone (52). Following the pro-
cedure for compound 15 using 2-bromo-1-(6-methoxy-5,7,8-
trimethyl-2-phenyl-2,3-dihydro-1,4-benzoxazin-4-yl)-ethanone (45)
(120 mg, 0.28 mmol), compound 52 was obtained after purification
of the crude residue by flash column chromatography (dichlo-
romethane/methanol (98:2)). Viscous oil, 0.105 g, 95% yield); Two
rotamers of compound 52 were detected in the NMR spectra, and
1
are defined by an asterisk. H NMR (δ) 4.90* (dd, J ) 13.4 Hz,
4.3 Hz, 0.6 H), 4.64 (dd, J ) 13.7 Hz, 2.7 Hz, 0.4H), 4.56-4.17
(m, 2H), 3.44-3.08 (m, 2H, and 0.4H), 2.78* (ddd, J ) 12.2 Hz,
4.3 Hz, 4.3 Hz, 0.6H), 2.65-2.43 (m, 4H), 2.30 (s, 1.8H), 2.27 (s,
1.2H), 2.23 (s, 1.8H), 2.14 (s, 1.8H), 2.10 (s, 1.2H), 2.09 (s, 1.2H),
1.04 (t, J ) 7.0 Hz, 2.4H), 0.87* (t, J ) 7.0 Hz, 3.6H). 13C NMR
(δ) 172.2, 169.2*, 145.4, 144.8*, 133.9, 133.3*, 129.8, 128.2, 126.7,
126.4*, 125.5, 123.7, 123.5*, 122.9, 67.5, 67.1*, 57.6*, 53.5, 47.6,
47.2*, 43.9, 41.6, 17.3, 17.1, 16.7, 12.5, 12.2, 12.1, 11.4. MS (ESI)
m/z 325.3 ([M + 1]+, 100), 327.3 ([M + 3]+, 39). HRMS (FAB+)
calcd for C17H26O2N2Cl [M + 1]+ 325.1683, found 325.1686. Anal.
(C17H25ClN2O2) C, H, N.
1
the corresponding peaks are defined by an asterisk. H NMR (δ)
7.43-7.21 (m, 5H), 5.65-4.75 (m, 2H), 3.69 (s, 3H), 3.68-2.38
(m, 7H), 2.28-2.04 (m, 9H), 1.12-0.82 (m, 6H). 13C NMR (δ)
172.3*, 168.4, 150.5*, 150.0, 143.8*, 143.1, 138.7, 129.1, 128.7,
128.5, 128.2, 125.8, 125.7, 124.8, 124.6, 123.3, 122.9, 122.8, 122.5,
122.1, 79.0, 78.9*, 77.8*, 60.2, 58.1, 56.6, 53.3, 50.7, 49.1, 48.4,
47.5, 47.4, 47.2, 12.7, 12.5, 12.3, 12.1, 12.0, 11.3. MS (ESI) m/z
397.4 ([M + 1]+, 100). HRMS (FAB+) calcd for C24H33O3N2 [M
+ 1]+ 397.2491, found 397.2512. Anal. (C24H32N2O3) C, H, N.
2-(2,3,5-Trimethyl-6-nitrophenoxy)-2-methylmalonic Acid Di-
ethyl Ester (53). Following the procedure for compound 19 using
2-nitro-3,5,6-trimethylphenol (17) (0.4 g, 2.21 mmol) in dry DMF
(7.35 mL), cesium carbonate (1.44 g, 4.42 mmol), TBAI (catalytic
1-(6-Chloro-2,2,5,7,8-pentamethyl-2,3-dihydro-1,4-benzoxazin-
4-yl)-2-diethylamino-ethanone (49). Following the procedure for
compound 15 using 2-bromo-1-(6-chloro-2,2,5,7,8-pentamethyl-2,3-
dihydro-benzo[1,4]oxazin-4-yl)-ethanone (42) (103 mg, 0.28 mmol),

2338 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Koini et al.
amount), and 2-bromo-2-methyl-malonic acid diethyl ester (0.84
mL, 4.42 mmol) after purification by flash column chromatography
(petroleum ether 40-60 °C/diethyl ether (9:1)), we obtained
4-(2-Diethylamino-acetyl)-2,5,7,8-tetramethyl-3,4-dihydro-2H-
1,4-benzoxazine-2-carboxylic Acid Ethyl Ester (62). Following
the procedure for compound 15 using 4-(2-bromo-acetyl)-2,5,7,8-
tetramethyl-3,4-dihydro-2H-1,4-benzoxazine-2-carboxylic acid ethyl
ester (60) (109 mg, 0.28 mmol), compound 62 was obtained after
purification of the crude residue by flash column chromatography
(dichloromethane/ethyl acetate (6:4)). Viscous oil, 85 mg, 81%
yield. The NMR spectra contain multiple signals due to the presence
of rotamers. 1H NMR (δ) 6.68-6.61 (m, 1H), 5.32-5.06 (m, 1H),
4.21-4.02 (m, 2H), 3.74-2.41 (m, 7H), 2.22-2.03 (m, 9H),
1.69-1.55 (m, 3H) 1.33-0.89 (m, 9H). 13C NMR (δ): 173.0, 172.2,
172.0, 171.9, 171.6, 169.9, 148.7, 146.6, 145.3, 136.3, 135.8, 134.6,
130.5, 128.9, 128.7, 127.3, 124.9, 124.0, 123.9, 122.6, 121.7, 121.5,
82.4, 80.1, 79.2, 62.1, 61.7, 61.5, 57.7, 57.5, 53.6, 53.1, 50.3, 50.0,
49.4, 49.3, 47.6, 47.4, 24.7, 23.0, 22.9, 19.8, 19.6, 19.4, 17.5, 17.1,
14.0, 13.9, 12.3, 12.2, 11.8, 11.6, 11.5. MS (ESI) m/z 377.4 ([M +
1]+, 100). HRMS (FAB+) calcd for C21H33O4N2 [M + 1]+ 377.2440,
found 377.2441. Anal. (C21H32N2O4) C, H, N.
Animals and Heart Preparations. Male Wistar rats weighing
about 300-350 g were housed under controlled light (12 L/12D)
and temperature with free access to food and water in compliance
with the prescriptions for the care and use of laboratory animals.
Rats were anesthetized with pentobarbital (30-40 mg per animal).
After intravenous administration of heparin, the chests were opened
and the hearts were rapidly excised and mounted on a nonrecir-
culating Langendorff perfusion apparatus. Retrogade perfusion was
established at a pressure of 90 cm H2O with an oxygenated
normothermic Krebs-Hensleit bicarbonate (KHB) buffer (25 mmol
l-1 NaHCO3, 118 mmol l-1 NaCl, 2.5 mmol l-1 CaCl2, 4.7 mmol
l-1 KCl, 1.4 mmol l-1 MgSO4, 1.2 mmol l-1 KH2PO4, pH 7.2 at
25 °C) supplemented with 11 mmol l-1 glucose and equilibrated
with 95% O2/5% CO2. The temperature of the hearts and perfusates
was maintained at 37 °C by the use of a water-jacketed apparatus.
All hearts were equilibrated for 20 min under these conditions. At
the end of the equilibration period, hearts were made ischemic for
15 min by perfusing them with the ischemic KHB (KHB with Tris-
HCl 10 mM instead of glucose and equilibrated with N2 before
use) followed by 60 min of reperfusion. The compounds were
present during the last 5 min of ischemia and during reperfusion at
a final concentration of 2 µM.
Evaluation of Antiarrhythmic Activity. Electrocardiograms
were recorded during equilibration, ischemia, and reperfusion.
Arrhythmia scores (AS) were calculated for the first 10 min of
reperfusion according to the Lambeth Convention Guidelines.41
Evaluation of Antioxidant Activity. At the end of the perfu-
sions, hearts were “freeze-clamped” between aluminum tongs,
cooled in liquid N2, and after the removal of the atria, the ventricles
were pulverized under liquid N2 and powders were stored at -80
°C. A portion of the tissue powder was analyzed for malondial-
dehyde (MDA) content by using the thiobarbituric acid assay.43
To prevent auto-oxidation of the samples, homogenization was
carried out at 4 °C in nitrogen equilibrated solution in the
presence of 0.04% butylated hydroxytoluene, 1.6% ethanol. The
values were expressed as nanomoles of TBA reactive substances
(MDA equivalent) per gram of tissue. 1,1,3,3-Tetraethoxypro-
pane(0,0.5,1.0,2.0,4.0,8.0 and 16.0 nmol) served as external
standard. Results are expressed as mean ( SEM. Differences
between groups were assessed by Student’s unpaired and
ANOVA t tests and considered significant when p < 0,05.
Conventional Microelectrode Technique. New Zealand rabbits
weighing 1.5-2.0 kg of either sex were used. Each animal was
sacrificed by cervical dislocation after an intravenous injection of
400 IU kg/L heparin. The chest was opened, the heart quickly
removed, and immediately rinsed in oxygenated modified Locke’s
solution containing (in mM): NaCl, 120; KCl, 4; CaCl2, 1.0; MgCl2,
1; NaHCO3, 22 and glucose, 11. The pH of this solution was 7.35
to 7.40 when saturated with 95% O2 and 5% CO2 at 37 °C. Tips of
papillary muscles obtained from the right ventricle were individually
mounted in a tissue chamber (volume ≈ 50 mL). Each ventricular
preparation was initially stimulated at a basic cycle length of 1000
ms (frequency ) 1 Hz), using rectangular constant current pulses
1
compound 53 as a viscous oil 0.748 g, 96% yield. H NMR (δ)
6.71 (s, 1H), 4.09 (q, J ) 7.1 Hz, 4H), 2.05 (s, 3H), 1.97 (s, 3H),
1.93 (s, 3H), 1.32 (s, 3H), 1.12 (t, J ) 7.3 Hz, 6H). 13C NMR (δ):
167.9, 145.8, 143.1, 139.9, 131.1, 128.2, 126.5, 84.4, 61.9, 19.6,
17.7, 17.6, 15.9, 13.4, 12.7. MS, m/z: 353 (8, M+), 205 (100). Anal.
(C17H23NO7) C, H, N.
2,5,7,8-Tetramethyl-3-oxo-3,4-dihydro-2H-1,4-benzoxazine-
2-carboxylic Acid Ethyl Ester (54). Following the procedure for
compound 21 using 2-(2,3,5-trimethyl-6-nitrophenoxy)-2-methyl-
malonic acid diethyl ester (53) (0.7 g, 1.98 mmol) and Pd/C (0.175
g) after purification by flash column chromatography (petroleum
ether 40-60 °C/ethyl acetate (85:15)), we obtained compound 54
1
as white crystals, 0.444 g, 81% yield; mp 124-126 °C. H NMR
(δ) 8.15 (s, 1H), 6.62 (s, 1H), 4.20-4.03 (m, 2H), 2.19 (s, 3H),
2.18 (s, 3H), 2.17 (s, 3H), 1.85 (s, 3H), 1.13 (t, J ) 7.3 Hz, 3H).
13C NMR (δ) 168.9, 164.7, 141.2, 132.3, 125.5, 123.3, 122.3, 120.3,
80.5, 62.0, 20.5, 19.3, 15.9, 13.8, 11.4. MS, m/z: 277 (61, M+),
204 (100). Anal. (C15H19NO4) C, H, N.
2,5,7,8-Tetramethyl-3-thioxo-3,4-dihydro-2H-1,4-benzoxazine-
2-carboxylic Acid Ethyl Ester (56). A mixture of 2,5,7,8-
tetramethyl-3-oxo-3,4-dihydro-2H-1,4-benzoxazine-2-carboxylic acid
ethyl ester (54) (0.2 g, 0.72 mmol), Lawesson’s reagent (0.32 g,
0.79 mmol), and toluene (0.81 mL) was refluxed under inert
atmosphere for 2 h. The resulting mixture was mixed with silica
gel and the toluene was evaporated in vacuo. The silica gel cake
was then added to a column of silica gel, and the column was eluted
with petroleum ether 40-60 °C/ethyl acetate (85:15) to afford
compound 56 as a green-yellow solid (0.205 g, yield: 97%); mp
1
114-116 °C. H NMR (δ) 9.45 (s, 1H), 6.59 (s, 1H,), 4.21-4.05
(m, 2H), 2.22 (s, 3H), 2.17 (s, 3H), 2.15 (s, 3H), 1.95 (s, 3H), 1.13
(t, J ) 7.0 Hz, 3H). 13C NMR (δ) 191.5, 168.6, 142.5, 134.4, 125.2,
123.4, 122.2, 119.5, 83.9, 62.2, 23.8, 19.6, 15.9, 13.8, 11.2. MS,
m/z: 293 (10, M +), 220 (100). Anal. (C15H19NO3S) C, H, N.
2,5,7,8-Tetramethyl-3,4-dihydro-2H-1,4-benzoxazine-2-car-
boxylic Acid Ethyl Ester (58). To a solution of 2,5,7,8-tetramethyl-
3-thioxo-3,4-dihydro-2H-1,4-benzoxazine-2-carboxylic acid ethyl
ester (56) (0.2 g, 0.68 mmol) in tetrahydrofuran (1 mL) and ethanol
(1 mL) was added Raney nickel and water (0.5 mL). After stirring
for 5 min, an additional amount of catalyst was added and water
(0.5 mL) and the reaction was stirred at room temperature for
additional 15 min. The reaction mixture was diluted with ethyl
acetate, it was filtered through celite, and the solvent was evaporated
in vacuo. The residue was purified by flash column chromatography
petroleum ether 40-60 °C/ethyl acetate (90:10) to afford compound
1
58 as white crystals (0.146 g, 82%); mp 84-87 °C. H NMR (δ)
6.49 (s, 1H), 4.13 (q, J ) 7.1 Hz, 2H), 3.75 (d, J ) 11.6 Hz, 1H),
3.14 (d, J ) 11.6 Hz, 1H), 2.16 (s, 3H), 2.15 (s, 3H), 2.05 (s, 3H),
1.56 (s, 3H), 1.17 (t, J ) 7.0 Hz, 3H). 13C NMR (δ) 172.5, 141.0,
127.7, 126.7, 123.4, 122.1, 120.2, 76.4, 61.2, 48.2, 22.6, 19.3, 16.4,
14.0, 11.4. MS, m/z: 263 (100, M+); Anal. (C15H21NO3) C, H, N.
4-(2-Bromo-acetyl)-2,5,7,8-tetramethyl-3,4-dihydro-2H-1,4-
benzoxazine-2-carboxylic Acid Ethyl Ester (60). Following the
procedure for compound 43 using 2,5,7,8-tetramethyl-3,4-dihydro-
2H-1,4-benzoxazine-2-carboxylic acid ethyl ester (58) (156 mg, 0.59
mmol), compound 60 was obtained after purification of the crude
residue by flash column chromatography (petroleum ether 40-60
°C/dichloromethane/ethyl acetate (80:15/5)). Yellowish solid, 0.201
g, 89% yield; mp 112-114 °C. The NMR spectra contain multiple
1
signals due to the presence of rotamers. H NMR (δ) 6.69-6.63
(m, 1H), 5.22-4.48 (m, 1H), 4.19-3.75 (m, 4H), 2.22-2.13 (m,
9H), 1.71-1.60 (m, 3H), 1.32-1.14 (m, 3H). 13C NMR (δ) 172.8,
171.6, 167.5, 167.2, 165.5, 148.5, 146.4, 145.1, 136.9, 136.3, 135.2,
130.8, 128.7, 128.0, 126.2, 124.9, 124.2, 124.0, 122.8, 122.7, 121.6,
120.5, 82.1, 79.5, 78.9, 62.4, 62.2, 61.6, 50.9, 49.5, 49.1, 27.9, 27.0,
26.4, 24.3, 22.8, 22.3, 19.8, 19.6, 18.2, 17.3, 16.9, 14.0, 11.5, 11.4.
Anal. (C17H22BrNO4) C, H, N.

Benzopyrans and Benzoxazine Aminoamide DeriVatiVes
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2339
Cꢀ1 expression in cardiomyocytes. J. Mol. Cell. Cardiol. 2008, 45,
679–684.
1 ms in duration. These stimuli were isolated from ground and
delivered through a bipolar platinum electrode in contact with the
preparation. Temperature of the superfusate was kept constant at
37 °C. Transmembrane potentials were recorded using conventional
microelectrode techniques. Microelectrodes filled with 3 M KCl
and having tip resistances of 5-20 MΩ were connected to the input
of a high impedance electrometer (Experimetria). The voltage output
of the amplifier was displayed on a memory oscilloscope (Tektronix
2230 100 MHz digital storage oscilloscope, Beaverton, OR) and
fed into a computer (PC, Windows XP) at a sampling rate of 10
kHz. The stimulation triggering and data acquisition were governed
by homemade (APES)/software. The resting membrane potential
(RP), conduction time (CT), action potential amplitude (APA),
maximal rate of depolarization (Vmax), and action potential duration
at 50 and 90% of (APD50/90) were automatically measured using
the software and analyzed both online and off-line. In each
experiment, baseline action potential characteristics were first
determined during continuous pacing at 1 Hz, and then pacing cycle
length was sequentially varied between 300 and 5000 ms. The 25th
action potential was measured at each cycle length, and the cycle
length was then changed so that “quasi” steady-state frequency
response relations could be generated rapidly. At least 1 h was
allowed for each preparation to equilibrate after mounting before
experimental measurements were initiated.
(8) (a) Wang, Q. D.; Pernow, J.; Sjoquist, P. O.; Ryden, L. Pharmacologi-
cal possibilities for protection against myocardial reperfusion injury.
CardioVasc. Res. 2002, 55, 25–37. (b) Grover, G. J.; Garlid, K. D.
ATP-Sensitive Potassium Channels: A Review of their Cardioprotec-
tive Pharmacology. J. Mol. Cell. Cardiol. 2000, 32, 677–695.
(9) Parrat, J. R. Inhibitors of the slow calcium current and early ventricular
arrhythmias In: Parratt, J. R., Ed.; Early Arrhythmias Resulting from
Myocardial Ischemia; Macmillan: London, 1982, 329-346.
(10) He, Z. S.; Komori, S.; Tamura, K.; Hashimoto, K. Inhibitory effect
of moricizine on reperfusion induced tachyarrhythmias in rats. A
comparison study with disopyramide and mexiletine. Jpn. Circ. J.
1992, 56, 861–865.
(11) Corr, P. B.; Pogwizd, S. M. Mechanisms contributing to arrhythmo-
genesis during early myocardial ischemia, subsequent reperfusion, and
chronic infarction. Angiology 1988, 39, 684–699.
(12) Komori, S.; Sano, S.; Li, B. H.; Matsumara, K.; Naitoh, A.; Mochizuki,
S.; Ishihara, T.; Watanabe, A.; Umetani, K.; Iiiri, H.; Hashimoto, K.;
Tamura, K. Effects of bidisomide (SC-40230), a new class I antiar-
rhythmic agent, on ventricular arrhythmias induced by coronary artery
occlusion and reperfusion in anesthetized rats; comparison with
mexiletine and dispyramide. Heart Vessel 1995, 10, 7–11.
(13) Butwell, N. B.; Ramasamy, R.; Lazar, I.; Sherry, A. D.; Malloy, C. R.
Effect of lidocaine on contracture, intracellular sodium and pH in
ischemic rat hearts. Am. J. Physiol. 1993, 264, H1884–H1889.
(14) Tani, M.; Neely, J. R. Role of intracellular Na+ in Ca2+ overload and
depressed recovery of ventricular function of reperfused ischemic rat
hearts. Possible involvement of H+-Na+ exchange. Circ. Res. 1989,
65, 1045–1056.
Acknowledgment. This work was supported in part by the
Greek General Secretariat for Research and Technology,
“Excellence in the Research Institutes Supervised by the GSRT”
EPAN 3.3.1. (2006-2009), by the Hungarian Academy of
Sciences, and by grants from the Hungarian National Research
Foundation (OTKA NI-61902) and the Hungarian Ministry of
Health (ETT-360/2006).
(15) Pike, M. M.; Kitakaze, M.; Chacko, V. P.; Marban, E. Lidocaine
prevents the increase in myocardial free calcium during ischemia and
reperfusion: an antiarrhythmic mechanism. Circulation 1987, 76, IV-
16.
(16) Saint, D. A. The cardiac persistent sodium current: an appealing
therapeutic target? Br. J. Pharmacol. 2008, 153, 1133–1142.
(17) Arakawa, J.; Hara, A.; Kokita, N. Lidocaine attenuates mechanical
and metabolic derangements induced by palmitoyl-L-carnitine in the
isolated perfused rat heart. Pharmacology 1997, 55, 259–268.
(18) Das, D. K.; Misra, H. P. Lidocaine: a hydroxyl radical scavenger and
singlet oxygen quencher. Mol. Cell. Biochem. 1992, 115, 179–185.
(19) Josephson, I. R. Lidocaine blocks Na, Ca, and K currents of chick
ventricular myocytes. J. Mol. Cell. Cardiol. 1988, 20, 593–604.
(20) Sugiyama, K.; Muteki, T. Local anesthetics depress the calcium current
of rat sensory neurons in culture. Anesthesiology 1994, 80, 1369–
1378.
Supporting Information Available: Experimental procedures
and spectroscopic data for compounds 1, 2, 4, 5, 7, 8, 10, 11, 13,
14, 18, 20, 22, 26-35, 37-42, 44, 45, 55, 57, 59, 61, 63-65, 26b,
37b, and 51b. Effect of compounds 46, 47, 49, 50, 62, and 63 and
reference drugs sotalol and mexiletine on the action potential in
rabbit right ventricular papillary muscles (Figure A). Elemental
analysis data for compounds 13-15, 17-19, 21-26, 30, 31, 36-38,
and 44-63. This material is available free of charge via the Internet
at http://pubs.acs.org.
(21) Kojima, M.; Miura, M. Protective effect of lidocaine on the ischemic-
reperfused rat heart: a phosphorus 31 nuclear magnetic resonance
study. Basic Res. Cardiol. 1993, 28, 619–628.
(22) Ohmura, T.; Muramatsu, I.; Kigoshi, S.; Noguchi, H.; Muraoka, R.;
Komoriya, Y.; Hayashi, H. Novel cardioprotective effects of TYB-
3823 on ischemic damage in the working hearts of rats: comparison
with lidocaine. J. Pharmacol. Exp. Ther. 1990, 254, 696–701.
(23) Vitola, J. V.; Forman, M. B.; Holsinger, J. P.; Atkinson, J. B.; Murray,
J. J. Reduction of myocardial infarct size in rabbits and inhibition of
activation of rabbit and human neutrophils by lidocaine. Am. Heart J.
1997, 133, 315–322.
(24) Wendland, M. F.; Saeed, M.; Kondo, C.; Derugin, N.; Higgins, C. B.
Effect of lidocaine on acute regional myocardial ischemia and
reperfusion in the cat. An in-vivo 31P magnetic resonance spectroscopy
study. InVest. Radiol. 1993, 28, 619–628.
(25) Hatori, N.; Roberts, R. L.; Tadokoro, H.; et al. Differences in infarct
size with lidocaine as compared with bretylium tosylate in acute
myocardial ischemia and reperfusion in pigs. J. CardioVasc. Phar-
macol. 1991, 18, 581–588.
(26) Lee, R.; Nitta, T.; Schmid, R. A.; Schuessler, R. B.; Harris, K. M.;
Gay, W. A. Retrograde infusion of lidocaine or L-arginine before
reperfusion reduces myocardial infarct size. Ann. Thorac. Surg. 1998,
65, 1353–1359.
(27) Lesnefsky, E. J.; Van Benthuysen, K. M.; McMurtry, I. F.; Shikes,
R. H.; Johnston, R. B.; Horwitz, L. D. Lidocaine reduces canine infarct
size and decreases release of a lipid peroxidation product. J. Cardio-
Vasc. Pharmacol. 1989, 13, 895–901.
(28) Koufaki, M.; Calogeropoulou, T.; Rekka, E.; Chryselis, M.; Papazafiri,
P.; Gaitanaki, C.; Makriyannis, A. Bifunctional Agents for Reperfusion
Arrhythmias: Novel Hybrid Vitamin E/Class I Antiarrhythmics.
Bioorg. Med. Chem. 2003, 11, 5209–5219.
(29) Koufaki, M.; Kiziridi, C.; Papazafiri, P.; Vassilopoulos, A.; Varro´,
A.; Nagy, Z.; Farkas, A.; Makriyannis, A. Synthesis and biological
evaluation of benzopyran analogues bearing class III antiarrhythmic
pharmacophores. Bioorg. Med. Chem. 2006, 14, 6666–6678.
References
(1) Janse, M. J.; Wit, A. L. Electrophysiological mechanisms of ventricular
arrhythmias resulting from myocardial ischemia and infarction. Physiol.
ReV. 1989, 69, 1049–1169.
(2) (a) The Cardiac Arrhythmia Suppression Trial (CAST) Investigators.
Preliminary Report: Effect of Encainide and Flecainide on Mortality
in a Randomized Trial of Arrhythmia Suppression after Myocardial
Infarction. N. Engl. J. Med. 1989, 321, 406-412; (b) The Cardiac
Arrhythmia Suppression Trial II Investigators. Effect of the antiar-
rhythmic agent moricizine on survival after myocardial infarction.
N. Engl. J. Med., 1992, 327, 227-233.
(3) Zareba, W. Implantable cardioverter defibrillator therapy in postinf-
arction patients. Curr. Opin. Cardiol. 2004, 19, 619–624.
(4) (a) Piper, H. M.; Garcia-Dorado, D.; Ovize, M. A. fresh look at
reperfusion injury. CardioVasc. Res. 1998, 38, 291–300. (b) An-
dreadou, I.; Iliodromitis, E. K.; Koufaki, M.; Farmakis, D.; Tsotinis,
A.; Kremastinos, D. Th. Alternative Pharmacological Interventions
that Limit Myocardial Infarction. Curr. Med. Chem. 2008, 15, 3204–
3213.
(5) Ambrosio, G.; Tritto, I. Reperfusion injury: experimental evidence
and clinical implications. Am. Heart J. 1999, 138, S69–S75.
(6) (a) Park, J. L.; Lucchesi, B. R. Mechanisms of myocardial reperfusion
injury. Ann. Thorac. Surg. 1999, 68, 1905–1912. (b) Yellon, D. M.;
Hausenloy, D. J. Myocardial Reperfusion Injury. N. Engl. J. Med.
2007, 357, 1121–1135.
(7) (a) Piper, H. M.; Meuter, K.; Scha¨fer, C. Cellular mechanisms of
ischemia-reperfusion injury. Ann. Thorac. Surg. 2003, 75, S644–S648.
(b) Woodcock, E. A. Roles of alpha1A- and alpha1B-adrenoceptors
in heart: insights from studies of genetically modified mice. Clin. Exp.
Pharmacol. Physiol. 2007, 34, 884–888. (c) Vasilevski, O.; Grubb,
D. R.; Filtz, T. M.; Yang, S.; McLeod-Dryden, T. J.; Luo, J.; Karna,
D.; Chen, J.; Woodcock, E. A. Ins(1,4,5)P(3) regulates phospholipase

2340 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Koini et al.
(30) Zhang, W. H.; Chan, W. L.; Lin, Y. H.; Szeto, Y. S.; Lin, Y. C.;
Yeung, C. H. Synthesis of hydroxyflavanones from substituted
acetophenones and benzaldehydes in the presence of silica gel, boric
acid and piperidine. Heterocycles 1997, 45, 71–75.
(31) Kano, S.; Tanaka, Y.; Sugino, E.; Hibino, S. Reduction of some
functional groups with titanium(IV) chloride/sodium borohydride.
Synthesis 1980, 695–697.
(32) Phillips, G. B.; Morgan, T. K.; Nickish, K.; Lind, J. M.; Gomez, R. P.;
Wohl, R. A.; Argentieri, T. M.; Sullivan, M. E. Synthesis and cardiac
electrophysiological activity of aryl-substituted derivatives of the class
III antiarrhythmic agent sematilide. J. Med. Chem. 1990, 33, 627–
633.
(33) Ouertani, M.; Girard, P.; Kagan, H. B. Selective Nitration of Phenols
Catalyzed by Lanthanum(III) Nitrate. Tetrahedron Lett. 1982, 23,
4315–4318.
(34) Ono, A.; Hiroi, M.; Shimazaki, K.; Reduction of aromatic nitro-
compounds by the sodium borohydride-copper(I) chloride system.;
Chem. Ind. 1984, 75.
(35) Malen, C.; Lacoste, J.-M.; Vilaine, J.-P.; Lenaers, A. Acylaminophenol
compounds. U.S. Patent 5254590, 1993.
(36) Mortensen, D. S.; Rodriguez, A. L.; Carlson, K. E.; Sun, J.;
Katzenellenbogen, B. S.; Katzenellenbogen, J. A. Synthesis and
biological evaluation of a novel series of furans: ligands selective for
estrogen receptor alpha. J. Med. Chem. 2001, 44, 3838–3848.
(37) Cava, M. P.; Leninsson, M. I. Thionation Reactions of Lawesson
Reagents. Tetrahedron 1985, 41, 5061–5087.
(38) Lepore, S. D.; He, Y. Use of Sonication for the Coupling of Sterically
Hindered Substrates in the Phenolic Mitsunobu Reaction. J. Org.
Chem. 2003, 68, 8261–8263.
dioVasc. Pharmacol. 1990, 16, 557–567. (c) Ma´tyus, P.; Varga, I.;
Rettegi, T.; Simay, A.; Ka´llay, N.; Ka´rolyha´zy, L.; Kocsis, A.; Varro´,
A.; Pe´nzes, I.; Papp, J. G. Novel antiarrhythmic compounds with
combined class IB and class III mode of action. Curr. Med. Chem.
2004, 11, 61–69.
(46) Campbell, T. J.; Vaughan Williams, E. M. Voltage- and time-dependent
depression of maximum rate of depolarisation of guinea-pig ventricular
action potentials by two new antiarrhythmic drugs, flecainide and
lorcainide. CardioVasc. Res. 1983, 17, 251–258.
(47) Campbell, T. J. Kinetics of onset of rate-dependent effects of class I
antiarrhythmic drugs are important in determining their effects on
refractoriness in guinea-pig ventricle and provide a theoretical basis
for their subclassification. CardioVasc. Res. 1983, 17, 344–352.
(48) Knilans, T. K.; Lathrop, D. A.; Na´na´si, P. P.; Schwartz, A.; Varro´, A.
Rate and concentration-dependent effects of UK-68,798, a potent new
class III antiarrhythmic, on canine Purkinje fibre action potential
duration and Vmax. Br. J. Pharmacol. 1991, 103, 1568–1572.
(49) Papp, J. G.; Ne´meth, M.; Krasso´i, I. I.; Mester, L.; Ha´la, O.; Varro´,
A. Differential Electrophysiologic Effects of Chronically Administered
Amiodarone on Canine Purkinje Fibers versus Ventricular Muscle.
J. CardioVasc. Pharmacol. Ther. 1996, 1, 287–296.
(50) Gerebtzoff, G.; Li-Blatter, X.; Fischer, H.; Frentzel, A.; Seelig, A.
Halogenation of Drugs Enhances Membrane Binding and Permeation.
ChemBioChem 2004, 5, 676–684.
(51) (a) Achari, B.; Mandal, S. B.; Dutta, P. K.; Chowdhurry, C.
Perspectives on 1,4-benzoxazines and their 2,3-dihydro derivatives.
Synlett 2004, 2449–2467. (b) Cecchetti, V.; Tabarrini, O.; Sabatini,
S. From Cromakalim to Different Structural Classes of KATP Channel
Openers. Curr. Top. Med. Chem. 2006, 6, 1049–1068.
(39) Caron, S.; Vasquez, E. Efficient Synthesis of [6-Chloro-2-(4-chlo-
robenzoyl)-1H-indol-3-yl]-acetic Acid, a Novel COX-2 Inhibitor. J.
Org. Chem. 2003, 68, 4104–4107.
(40) Heisler, B. E.; Ferrier, G. Proarrhythmic actions of flecainide in an
isolated tissue model of ischemia and reperfusion. J. Pharmacol. Exp.
Ther. 1996, 279, 317–324.
(41) Walker, M.; Curtis, M.; Hearse, D. The Lambeth Conventions:
guidelines of the study of arrhythmias in ischemia, infarction, and
reperfusion. Cardiol. Res. 1988, 22, 447–455.
(52) Ilasˇ, J.; Anderluh, P.; Dolenc, M. S.; Kikelj, D. Recent advances in
the synthesis of 2H-1,4-benzoxazin-3-(4H)-ones and 3,4-dihydro-2H-
1,4-benzoxazines. Tetrahedron 2005, 61, 7325–7348.
(53) Rybczynski, P. J.; Zeck, R. E.; Dudash, J.; Combs, D. W.; Burris,
T. P.; Yang, M.; Osborne, M. C.; Chen, X.; Demarest, K. T.
Benzoxazinones as PPARR Agonists. 2. SAR of the Amide Substituent
and In Vivo Results in a Type 2 Diabetes Model. J. Med. Chem. 2004,
47, 196–209.
(42) (a) Ceconi, C.; Cargnoni, A.; Pasini, E.; Condorelli, E.; Curello, S.;
Ferrari, R. Evaluation of phospholipid peroxidation as malondialdehyde
during myocardial ischemia and reperfusion injury. Am. J. Physiol.
Heart Circ. Physiol. 1991, 260, H1057–H1061. (b) Lefer, D. J.;
Granger, N. Oxidative Stress and Cardiac Disease. Am. J. Med. 2000,
109, 315–323.
(43) Draper, H. H.; Hardley, M. Malondialdehyde Determination as Index
of Lipid Peroxidation. Methods Enzymol. 1990, 186, 283–293.
(44) Lathrop, D. A.; Varro´, A.; Na´na´si, P. P.; Bo´di, I. I.; Takyi, E.; Pankucsi,
C. Differences in the Effects of D- and DL-Sotalol on Isolated Human
Ventricular Muscle: Electromechanical Activity after Beta-Adreno-
receptor Stimulation. J. CardioVasc. Pharmacol. Ther. 1996, 1, 65–
74.
(54) The corresponding compounds were detected by GC-MS but decom-
posed upon isolation.
(55) (a) Pratt, C. M.; Camm, A. J.; Cooper, W.; Friedman, P. L.; MacNeil,
D. J.; Moulton, K. M.; Pitt, B.; Schwartz, P. J.; Veltri, E. P.; Waldo,
A. L. Mortality in the Survival With Oral D-sotalol (SWORD) trial:
why did patients die? Am. J. Cardiol. 1998, 81, 869–876. (b) Waldo,
A. L.; Camm, A. J.; deRuyter, H.; Friedman, P. L.; MacNeil, D. J.;
Pauls, J. F.; Pitt, B.; Pratt, C. M.; Schwartz, P. J.; Veltri, E. P. Effect
of D-sotalol on mortality in patients with left ventricular dysfunction
after recent and remote myocardial infarction. The SWORD Investiga-
tors Survival With Oral D-Sotalol. Lancet 1996, 348, 7–12. (c) Waldo,
A. L.; Camm, A. J.; deRuyter, H.; Freidman, P. L.; MacNeil, D. J.;
Pitt, B.; Pratt, C. M.; Rodda, B. E.; Schwartz, P. J. Survival with oral
D-sotalol in patients with left ventricular dysfunction after myocardial
infarction: rationale, design, and methods (the SWORD trial). Am. J.
Cardiol. 1995, 75, 1023–1027.
(45) (a) Varro, A.; Elharrar, V.; Surawicz, B. Frequency-dependent effects
of several class I antiarrhythmic drugs on Vmax of action potential
upstroke in canine cardiac Purkinje fibers. J. CardioVasc. Pharmacol.
1985, 7, 482–492. (b) Varro´, A.; Lathrop, D. A. Sotalol and mexiletine:
combination of rate-dependent electrophysiological effects. J. Car-
JM801228H