Novel quinolizidinyl derivatives as antiarrhythmic agents

Article abstract of DOI:10.1021/jm060878m

Eighteen analogues of lidocaine, mexiletine, and procainamide were synthesized, replacing their aminoalkyl chains with the rigid and cumbersome quinolizidine nucleus. The target compounds were tested for antiarrhythmic, inotropic, and chronotropic effects

Full text of DOI:10.1021/jm060878m

334
J. Med. Chem. 2007, 50, 334-343
Novel Quinolizidinyl Derivatives as Antiarrhythmic Agents
Iana Vazzana,† Roberta Budriesi,‡ Emanuela Terranova,† Pierfranco Ioan,‡ Maria Paola Ugenti,‡ Bruno Tasso,†
Alberto Chiarini,‡ and Fabio Sparatore*,†
Dipartimento di Scienze Farmaceutiche, UniVersita` di GenoVa, Viale Benedetto XV 3, 16132 GenoVa, and Dipartimento di Scienze
Farmaceutiche, UniVersita` di Bologna, Via Belmeloro 6, 40126 Bologna, Italy
ReceiVed July 26, 2006
Eighteen analogues of lidocaine, mexiletine, and procainamide were synthesized, replacing their aminoalkyl
chains with the rigid and cumbersome quinolizidine nucleus. The target compounds were tested for
antiarrhythmic, inotropic, and chronotropic effects on isolated guinea pig (gp) heart tissues and to assess
calcium antagonist activity. Most compounds exhibited from moderate to high antiarrhythmic activity, and
compounds 7, 9, and 19 were more active and potent than quinidine and lidocaine, while producing only
modest inotropic, chronotropic, and vasorelaxant effects. These compounds were studied on spontaneously
beating Langendorff-perfused gp heart. While quinidine and amiodarone produced a dose-dependent
prolongation of all the ECG intervals, compounds 7, 9, and 19, even at concentrations 10-20 times higher
than EC50 for the antiarrhythmic activity, only moderately prolonged the PR and QT intervals, leaving
unchanged the QRS complex. Ether 7 deserves further investigations due to its interesting cardiovascular
profile.
Chart 1. Structure of Some Antiarrhythmic Agents
Introduction
Arrhythmia is an abnormality of cardiac rhythm affecting
about 4% of the population over 60 and about 9% over 80.1
Arrhythmias are commonly induced or accompanied by heart
disease. However, arrhythmias may occur in patients with no
clinically apparent heart disease but with other diseases and
under certain drug therapies. Although the therapeutic treatment
of the underling disorders is placed in the foreground, arrhyth-
mias represent an actual danger to the patients.2 However, acute,
as well as long-term, treatment of arrhythmias is often limited
by side effects such as cardiac depression caused by drugs.3
Antiarrhythmic drugs are able to suppress dysrhythmic cardiac
activity whatever their modes of action. Several attempts of
classification according to clinical criteria were made in the
1970s, but none of them were generally accepted.4-6 Currently,
the most common and extensively described characterization
of antiarrhythmic drugs is based on their fundamental electro-
physiological effects. As a matter of fact, antiarrhythmic drugs
display their activity through several different mechanisms and,
on the basis of their electrophysiological properties, have been
arranged by Vaughan Williams in four classes.7-10 The first
class of sodium channel blockers was further divided into three
subclasses depending on the length of the depolarized phase.
It is worth noting that in each class compounds with quite
different molecular structures are present. However, compounds
exhibiting only minimal or moderate differences in their
structures may belong to distinct classes or subclasses. Thus,
lidocaine and pilsicainide belong to the Ib and Ic classes,
respectively, while the simple introduction of a substituent on
the amino group of procainamide (class Ia) produces acecainide
and sematilide, both collocated in class III (Chart 1). On the
other hand, amiodarone, rightly included in class III for its
repolarization lengthening activity, also exhibits class Ib and,
to minor extents, class II and IV antiarrhythmic effects.
Therefore, medicinal chemists should feel encouraged to
search for better antiarrhythmic drugs.11 After so many years
from its introduction in therapy, lidocaine (class Ib) still
continues to suggest the design of new analogues to overcome
its drawbacks (cardiac depression and CNS disturbances),
expecially in long-term treatment of arrhythmias, where amio-
darone and other agents with multiple mechanisms of action
are finding wider acceptance.
On this ground, a first set of novel lidocaine-like antiarrhyth-
mic agents, corresponding to the general formula 1, were
described by Sparatore and Sparatore in 1995 (Chart 2).12 These
compounds are characterized by the presence of a bulky, highly
lipophilic, and strongly basic quinolizidine moiety. Indeed the
replacement of the various flexible dialkylaminoalkyl chains,
which characterize many drugs, with such a rigid and cumber-
some moiety could either compromise the interaction with the
biological structure responsible for the activity or give place to
a very selective one.
In fact, some of the mentioned compounds, especially 2 and
3, resulted endowed with strong antiarrhythmic activity in in
vitro and in vivo assays. Despite its structural analogy with
lidocaine and pilsicainide, compound 2 was devoid of local
* To whom correspondence should be addressed. Phone: +39 0103538359.
Fax: +39 0103538358. E-mail: sparator@unige.it.
† Universita` di Genova.
‡ Universita` di Bologna.
10.1021/jm060878m CCC: $37.00 © 2007 American Chemical Society
Published on Web 12/24/2006

Quinolizidinyl DeriVatiVes as Antiarrhythmic Agents
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2 335
Chart 2. Structures of Antiarrhythmic Homolupinanoylanilides
A second and larger group of compounds is formed by the
lupinyl (1R,9aR)-(octahydro-2H-quinolizin-1-yl)methyl (7-11)
and epi-lupinyl (1S,9aR)-(octahydro-2H-quinolizin-1-yl)methyl
(12-16) ethers of 2,6-dimethyl 4-substituted phenols, which
more closely recall the compounds of Matyus et al.
The third group is formed by the analogues of procainamide,
acecainide, and sematilide, whose diethylaminoethyl chain is
replaced by the lupinyl moiety, to which the 4-nitrobenzoyl
derivative is added. Compounds 17 and 18 were previously
described by Sparatore many years ago,15 but the antiarrhythmic
activity was not studied at that time; our interest in this kind of
compound was restored by the observed good antiarrhythmic
activity of N-lupinyl-4-amino-5-chloro-2-methoxybenzamide,
which was prepared by Iusco et al. as an analogue of metoclo-
pramide.16
Each of the three groups of compounds shares a peculiar
aromatic substructure with lidocaine (ArNH-CO-CH2-),
mexiletine and amiodarone (Ar-O-CH2-), and procainamide
(Ar-CONH-CH2-), respectively, but all of them share the
“bulkiness” of the respective strongly basic moiety with
quinidine (quinolizidine versus quinuclidine nucleus).
anesthetic activity and hence of sodium channel blocking
activity. Compound 2 also lacked â-adrenergic and calcium
channel blocking activities. On the other hand, compound 3
exhibited some local anesthetic and calcium channel blocking
activities and was moderately able to displace [3H]batrachotoxin
from the sodium channel of rat brain preparations (IC50 ) ∼3.7
µM); therefore, the strong antiarrhythmic activity must result
from these combined mechanisms.
Chemistry
Compounds 2, 4, and 6 were prepared by reacting the
homolupinanoyl chloride hydrochloride with the suitable aro-
matic amines in CHCl3 solution and in the presence of N,N-
diisopropylethylamine. The homolupinanoyl chloride hydro-
chloride[(1S,9aR)-(octahydro-2H-quinolizin-1-yl)ethanoylchloride]
was prepared as described by Sparatore et al.17,18 The noncom-
mercially available 2,6-dimethyl-4-nitroaniline was prepared
according to Wepster indications,19 while 4-amino-3,5-dimeth-
ylbenzophenone was obtained by adapting the indications given
by Staskun20 for other aminobenzophenones. The compound was
similarly prepared by Artini et al.,21 but no details were given,
and the mp was quite different. Compound 4 was reduced
catalytically, and the resulting amine was treated with mesyl
chloride to give 5 (Scheme 1).
At present, the interest in compounds possessing a multiple
mode of action is steadily increasing, and the overlapping of
the useful characteristics of class III with those of class Ib, which
could balance the proarrhythmic risk inherent to the former, is
particularly pursued.10,13
Over the past few years, Matyus et al.14 described a set of
(2,6-dimethylphenoxy)alkylamines, embodying the structural
features of mexiletine (class Ib) and amiodarone (class III), and
which, indeed, exhibited excellent antiarrhythmic effects through
the simultaneous block of sodium and potassium channels (Chart
3).
Pursuing our research on antiarrhythmic agents, we now
describe three series of new quinolizidine derivatives embodying
some of the mentioned structural elements (Chart 4).
The first group (4-6) represents the extension of the
previously described12 N-homolupinanoylanilines 1, with the
introduction of nitro, mesylamino, and benzoyl residues on the
benzene ring which are present in the phenoxyalkylamines of
Matyus et al.14
Ethers 7, 8, and 11 and 12, 13, and 16 were prepared by
reacting chlorolupinane22 (1-(chloromethyl)octahydro-(1R,9aR)-
2H-quinolizine) and epi-chlorolupinane23 (1-(chloromethyl)-
octahydro-(1S,9aR)-2H-quinolizine), respectively, with suitable
phenols in ethanolic sodium hydroxide solution (Scheme 2).
Chart 3. Structures of Some Antiarrhythmic Phenoxyalkylamines

336 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2
Vazzana et al.
Chart 4. Structures of Novel Quinolizidine-Derived Antiarrhythmic Agents
Scheme 1a
a Reagents and conditions: (a) i-Pr2EtN, CHCl3, ∆; (b) H2/Pd-C, EtOH, rt; (c) MsCl, CHCl3, rt.
Scheme 2a
a Reagents and conditions: (a) NaOH, EtOH, ∆; (b) H2/Pd-C, EtOH, rt; (c) MsCl, Pyr, rt.
The required 3,5-dimethyl-4-hydroxybenzophenone was ob-
tained by Ruminski24 by decarboxylation of 2-(3,5-dimethyl-
4-hydroxybenzoyl)benzoic acid, followed by several purification
steps. To avoid this complex preparation, 2,6-dimethylphenol
was condensed with benzoic acid in poly(phosphoric acid) at
180 °C; this method, though still requiring repeated purification,

Quinolizidinyl DeriVatiVes as Antiarrhythmic Agents
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2 337
Scheme 3a
a Reagents and conditions: (a) Et3N, PhCH3, ∆; (b) H2/Pd-C, EtOH, rt; (c) Ac2O, H2O/CH3COOH, ∆; (d) MsCl, CHCl3, rt.
Table 1. Antiarrhythmic Activity of Compounds
appears somewhat advantageous. The nitro derivatives 8 and
13 were reduced catalytically, and amines 9 and 14 were reacted
max % increase of
threshold of ac-arrhythmia
with mesyl chloride to give 10 and 15, respectively (Scheme
after pretreatment with
b
EC50
95% conf
2).
compd
compdsa (M ( SEM)
(µM)
lim (µM)
Compound 17 was prepared, as already described,15 by
amiodarone
lidocaine
procainamide
quinidine
2
4
5
10 ( 0.5* c
34 ( 2.6*
11 ( 0.4*
69 ( 0.4*
78 ( 2.5*
inactivec
reacting 4-nitrobenzoyl chloride with lupinylamine22 ((1-ami-
nomethyl)octahydro-(1S,9aR)-2H-quinolizine). Catalytic reduc-
tion of 17 gave amino compound 18, which with acetic
anhydride and mesyl chloride gave 19 and 20, respectively
(Scheme 3).
10.26
5.71
8.44-12.46
4.38-7.46
inactived
6
7
8
9
55 ( 3.0*
176 ( 10.2* d
27 ( 1.0*
166 ( 5.6* c
60 ( 3.2* e
64 ( 1.5*
35 ( 2.1* d
23 ( 1.7* f
38 ( 1.6* d
5 ( 0.2c
104 ( 2.6*
18 ( 1.3* c
28 ( 1.7* d
92 ( 3.7*
34 ( 2.1* c
4.81
0.46
4.38-5.27
0.30-0.69
All structures were supported by elemental analyses and
spectral data.
0.97
1.25
6.75
0.78-1.33
1.02-1.48
5.41-8.40
Pharmacology
10
11
12
13
14
15
16
17
18
19
20
The pharmacological profile of all compounds was tested for
antiarrhythmic activity on isolated guinea pig left atria driven
at 1 Hz, on guinea pig isolated left and right atria to evaluate
their inotropic and chronotropic effects, respectively, and on
K+-depolarized guinea pig aortic strips to assess calcium
antagonist activity.
The Langendorff-perfused guinea pig heart was used to assay
the whole cardiac activity of compounds 7, 9, and 19 and of
the reference quinidine. Compounds were checked at increasing
doses to evaluate changes in heart rate (chronotropic activity)
and in electrocardiogram (ECG) signals: PR, QRS, QT.
Data were analyzed using Student’s t test and are presented
as the mean (M) ( SEM.25 Since the drugs were added in a
cumulative manner, only the significance (P < 0.05) between
the control and the experimental values at each concentration
is indicated by an asterisk.
19.87
0.47
15.46-25.54
0.29-0.76
a Increase of the threshold of ac-arrhythmia: increase in the current
strength of a 50 Hz alternating current required to produce arrhythmia in
guinea pig left atria driven at 1 Hz in the presence of each tested compound
at 5 × 10-5 M. An asterisk indicates P < 0.05. b Calculated from log
concentration-response curves (Probit analysis according to Litchfield and
Wilcoxon25 with n ) 6-8). When the maximum effect was <50%, the
EC50 values were not calculated. c At 10-4 M. d At 10-5 M. e At 5 × 10-6
M. f At 10-6 M.
parameters. The data are reported in Table 3 compared to those
of the reference compound quinidine.
The potency of drugs defined as EC50, EC30, and IC50 was
evaluated from log concentration-response curves (Probit
analysis according to Litchfield and Wilcoxon26 or using
GraphPad Prism software25,27) in the appropriate pharmacologi-
cal preparations.
Considering that Na+ channel inhibitory antiarrhythmic drugs
such as quinidine or lidocaine increase the threshold of
ac-arrythmia, we used a simple experimental procedure which,
in contrast to chemical methods, minimizes the damage to the
myocardium (see the Experimental Section).28,29
Results and Discussion
Indeed, quinidine and lidocaine clearly increased the threshold
of ac-arrhythmia, procainamide was moderately active, and
amiodarone was almost inactive up to 100 µM (+10%). The
new compounds show different effects on the threshold of ac-
arrythmia. With only three exceptions (4, 5, and 15), all
compounds exerted from moderate to high activity, and most
of them at a concentration of 50 µM were more active than
lidocaine and procainamide and comparable or superior to
quinidine; in particular, ethers 7 and 9 exhibited 176% and 166%
increased thresholds of ac-arrythmia versus 69% with quinidine.
Moreover, six compounds exhibit the maximal activity (23-
Compounds 2 and 4-20 reported in Chart 4, and amiodarone,
lidocaine, procainamide, and quinidine as reference drugs, were
evaluated in vitro for antiarrhythmic activity on isolated guinea
pig left atria driven at 1 Hz and for influence on the
cardiovascular parameters inotropy and chronotropy and on
vascular smooth muscle. The results of these assays are collected
in Tables 1 and 2.
Simultaneously for the most interesting compounds 7, 9, and
19, the cardiovascular profile was extended to the Langendorff-
perfused guinea pig heart to detect the influence on ECG

338 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2
Vazzana et al.
Table 2. Influences of Tested Compounds on Cardiovascular Parameters
% decrease
(M ( SEM)
EC50 of inotropic
negative activity
EC30 of chronotropic
negative activity
negative
inotropic
activitya
negative
vasorelaxant
activityd
(M ( SEM)
c
c
chronotropic
EC50
95% conf
lim (µM)
EC30
95% conf
lim (µM)
comp
activityb
(µM)
(µM)
amiodarone
lidocaine
procainamide
quinidine
30 ( 2.6* e
88 ( 3.0* f
92 ( 1.4* i
71 ( 3.6*
95 ( 1.2*
79 ( 0.5*
86 ( 3.4* e
88 ( 2.6*
25 ( 1.9* k
91 ( 4.2* e
83 ( 2.9* g
83 ( 3.7* f
94 ( 0.3*
68 ( 4.9* m
83 ( 3.4* l
91 ( 3.4* l
64 ( 2.7* f
64 ( 1.5* f
74 ( 1.7* f
95 ( 3.8* e
86 ( 0.8* l
92 ( 3.6*
72 ( 4.5*
29 ( 0.9* g,h
9 ( 0.6h
5.57
4.93-6.02
3 ( 0.1
14 ( 0.9*
3 ( 0.2
30 ( 1.6*
5 ( 0.1
4 ( 0.3
0.017
0.014
3.38
0.62
3.46
0.31
0.76
0.012-0.024
0.011-0.017
2.69-4.25
0.42-0.94
2.69-4.49
0.27-0.43
0.51-1.12
86 ( 0.5* j
8 ( 0.1f,h
25 ( 1.4*
8 ( 0.2h
3.99
3.81-4.06
2
4
5
6
7
24 ( 1.5*
31 ( 0.3*
26 ( 1.4* j
16 ( 0.9*
15 ( 1.0*
30 ( 2.1*
17 ( 0.7*
21 ( 1.5*
19 ( 0.9* j
32 ( 2.1* j
23 ( 2.1*
38 ( 0.9* j
18 ( 0.9*
3 ( 0.2*
3 ( 0.2*
1 ( 0.1*
70 ( 1.7* j
19 ( 0.8l
66 ( 2.4* j
38 ( 2.7*
12 ( 0.7
3.98
3.15-4.42
8
9
8.39
0.29
0.031
0.16
0.014
0.60
0.63
0.14
0.16
0.038
0.46
0.059
0.25
7.88-8.93
0.086-0.71
0.023-0.042
0.10-0.26
0.009-0.019
0.32-0.65
0.44-0.91
0.083-0.89
0.13-0.19
0.027-0.045
0.32-0.71
0.041-0.087
0.15-0.38
23.66
21.15-24.05
10
11
12
13
14
15
16
17
18
19
20
41 ( 3.4* j
13 ( 0.6l
25 ( 1.6* g
44 ( 2.5*
34 ( 0.7*
75 ( 3.4* j
28 ( 2.1*
13 ( 0.8h
14 ( 1.4j
4 ( 0.2
6.63
6.02-7.13
a Activity: decrease in the developed tension in isolated guinea pig left atria at 5 × 10-5 M, expressed as the percentage change from the control (n )
4-6). The left atria were driven at 1 Hz. An asterisk indicates P < 0.05. b Activity: decrease in the atrial rate in guinea pig spontaneously beating isolated
right atria at 10-4 M, expressed as the percentage change from the control (n ) 6-8). The pretreatment heart rate ranged from 170 to 195 beats/min. An
asterisk indicates P < 0.05. c Calculated from log concentration-response curves (Probit analysis according to Litchfield and Wilcoxon25 with n ) 6-8).
When the maximum effect was <50%, the EC50 inotropic, EC30 chronotropic, and IC50 vasorelaxant values were not calculated. d Activity: percent inhibition
of calcium-induced contraction on K+-depolarized guinea pig aortic strips at 10-4 M. The 10-4 M concentration gave the maximum effect for most compounds.
An asterisk indicates P < 0.05. e At 10-4 M. f At 10-6 M. g At 5 × 10-6 M. h Positive chronotropic effect. i At 5 × 10-7 M. j At 5 × 10-5 M. k At 5 × 10-8
M. l At 10-5 M. m At 10-7 M.
Table 3. Influence of 7, 9, and 19 on ECG Parameters Evaluated in the Spontaneously Beating Langendorff-Perfused Guinea Pig Heart in Comparison
with That of Reference Compounds Quinidine and Amiodaronee
compd
param
basal
10-9
M
10-8
M
10-7
M
10-6
M
10-5
M
amiodarone
HRa
PRb
187 ( 11
55 ( 4
187 ( 15
54 ( 3
180 ( 13
55 ( 5
171 ( 12
55 ( 2
164 ( 9*
56 ( 6
132 ( 8*
58 ( 7
QRSc
QTd
HRa
PRb
20 ( 1.5
161 ( 8
165 ( 13
52 ( 2
20 ( 1.7
161 ( 7
160 ( 10
53 ( 4
20 ( 1.9
161 ( 11
153 ( 11
54 ( 2
21 ( 2
22 ( 2.1
174 ( 10
123 ( 12*
60 ( 3*
35 ( 1*
200 ( 11*
206 ( 5*
68 ( 3*
30 ( 4
200 ( 6*
180 ( 5*
66 ( 4*
32 ( 2
24 ( 1.8
182 ( 7*
107 ( 9*
70 ( 4*
39 ( 4*
229 ( 15*
203 ( 7*
70 ( 6*
31 ( 3
210 ( 7*
175 ( 3*
70 ( 6*
32 ( 3
169 ( 9
140 ( 10
57 ( 1*
31 ( 2*
182 ( 10*
210 ( 4
66 ( 5
quinidine
QRSc
QTd
HRa
PRb
24 ( 1
25 ( 2
27 ( 1
158 ( 5
218 ( 10
60 ( 3
158 ( 5
218 ( 6
60 ( 2
170 ( 8
215 ( 3
64 ( 4
7
9
QRSc
QTd
HRa
PRb
30 ( 1
30 ( 2
30 ( 3
28 ( 1
155 ( 6
200 ( 1
58 ( 2
155 ( 4
192 ( 3*
58 ( 3
160 ( 5
190 ( 2*
60 ( 4
170 ( 7
184 ( 3*
64 ( 3
QRSc
QTd
HRa
PRb
33 ( 1
30 ( 2
33 ( 3
31 ( 1
156 ( 2
180 ( 6
61 ( 3
160 ( 1
180 ( 5
62 ( 2
170 ( 5*
175 ( 3
65 ( 4
182 ( 4*
170 ( 2
70 ( 5
194 ( 3*
166 ( 3*
72 ( 7*
34 ( 2
205 ( 9*
165 ( 2*
75 ( 5*
33 ( 4
19
QRSc
QTd
32 ( 1
161 ( 3
34 ( 2
170 ( 2
33 ( 1
182 ( 5*
32 ( 3
190 ( 4*
195 ( 6*
204 ( 1*
a HR ) heart rate calculated from the RR interval on the ECG signal. b PR ) atrioventricular conduction time (ms). c QRS ) intraventricular conduction
time (ms). d QT ) duration of the ventricular action potential (ms). e Each value corresponds to the mean ( SEM. An asterisk indicates P < 0.05.
176%) at concentrations of 1, 5, and 10 µM, which are,
respectively, 50, 10, and 5 times lower than the concentration
at which lidocaine and quinidine exhibit their maximal activity
(34% and 69%).
For compounds exhibiting an increase of the threshold of ac-
arrhythmia higher than 50%, the EC50 values were calculated
(Table 1). These data indicate that most compounds were not
only more efficacious, but also more potent than reference drugs.
Particularly, compounds 7, 9, and 19 (EC50 ) 0.46 µM, EC50
) 0.97 µM, and EC50 ) 0.47 µM, respectively) were 22.30,
The observation of antiarrhythmic activity in all the studied
subsets of compounds indicates that the characterizing, rigid,
and cumbersome quinolizidine ring is itself well tolerated by
the biological structures involved in the regulation of heart
activity and may be responsible for the higher activity of 2 and
18 with respect to lidocaine and procainamide, respectively. On
the other hand, the effects of the substituents introduced in the
para position of the aromatic moieties appear to be quite variable
in the different subsets. This statement is supported in particular
by the comparison of the antiarrhythmic activity of the lupinyl
and epi-lupinyl ethers. Thus, the nitro group reduced the potency
in the former (8) while it increased it in the latter (13); the
10.58, and 21.83 times more potent than quinidine (EC50
10.26 µM).
)

Quinolizidinyl DeriVatiVes as Antiarrhythmic Agents
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2 339
opposite effect was observed for the mesylamino, which
increased the potency of the lupinyl ether 10, but abolished the
activity in the epi-lupinyl ether 15.
and QT intervals, leaving the QRS complex unchanged and
reducing the heart rate by only 7-12%. Amiodarone increased
the QT interval by only 13% at 10 µM concentration, which is,
however, 10 times lower than that able to produce a perceptible
increase of the threshold of ac-arrhythmia (see Table 1).
Moreover, amiodarone, like quinidine, at that concentration
produced a strong (-29%) reduction of the heart rate. Therefore,
compounds 7, 9, and 19 compared favorably also with amio-
darone.
Thus, these effects partially obscured the meaning of the steric
disposition of the quinolizidine ring relative to the aromatic
moiety in the two subsets of ethers 7-11 and 12-16. It is well-
known that stereoselective active sites are present on cardiac
sodium channels,30,31 which, for instance, preferentially bind
the R-(-)-enantiomer of mexiletine.32 Similarly, the R-enanti-
omers of the chiral sodium channel blockers mepivacaine,
ropivacaine, and bupivacaine exhibit a consistently higher
cardiotoxicity than the S-enantiomers.33 An even larger and
consistent difference of activity should have been expected for
the epimeric group of compounds 7-11 and 12-16.
In the N-(4-substituted benzoyl)lupinylamine group the an-
tiarrhythmic potency was lowest for the mesyl derivative, while
among the diethylaminoethylamino derivatives the sematilide
was much more potent than acecainide and procainamide.34
However, the lupinyl analogue of acecainide (19) was definitely
more active than the analogues of procainamide and sematilide.
To better evaluate the pharmacological profile of the inves-
tigated antiarrhythmic agents, the comparison of their influence
on additional cardiovascular parameters with that elicited by
the four reference compounds (Table 2) was deemed necessary.
Thus, compounds 2 and 4-20 were evaluated for (a) inotropic
activity on isolated guinea pig driven left atria, (b) chronotropic
activity on isolated guinea pig spontaneously beating right atria,
and (c) vasorelaxant activity expressed as inhibition of calcium-
induced contraction on K+-depolarized guinea pig aorta strips.
All compounds, with the exception of 7, decreased the
developed tension on driven left atria (Table 2), and compounds
12, 10, 17, and 19 were in that order the most potent, with EC50
in the range 0.014-0.059 µM, while lidocaine exhibited EC50
) 0.017 µM. Only two compounds (4, 8) had EC50 higher than
that of quinidine (3.38 µM); the remaining compounds exhibited
EC50 in the range 0.14-0.76 µM. As already observed by Busch
et al.,35 amiodarone did not influence the contractile force. A
weak negative inotropic activity was observed for compound 7
only at very low concentration (50 nM).
Conclusion
The studied quinolizidine derivatives represent, on the whole,
an interesting novel group of antiarrhythmic agents, among
which very high activity and potency may be found, as in
compounds 7, 9, and 19, which compared favorably with
amiodarone, lidocaine, procainamide, and quinidine. The dif-
ferent structural features which characterize the investigated
compounds seem to play an interwoven role, making it difficult
to define structure-activity relationships.
Compound 7 exhibits an interesting cardiovascular profile,
being endowed with significant antiarrhythmic activity and
potency with only modest negative inotropic and chronotropic
effects and vasorelaxant activity that may even be clinically
profitable, while more pronounced effects could induce second-
ary arrhythmias through discharges from the controlled focus.
Therefore, compound 7 deserves further investigations.
Experimental Section
Chemistry. Melting points were taken in open glass capillaries
on a Bu¨chi apparatus and were uncorrected. 1H NMR spectra were
recorded in CDCl3 or DMSO-d6 on a Varian Gemini 200 spec-
trometer. Chemical shifts (δ) are reported in parts per million from
the peak for internal Me4Si. Values of the coupling constants (J)
are reported in hertz. Lup ) lupinyl (octahydroquinolizin-1-
ylmethyl) residue, and Q ) octahydroquinolizine ring. Thin-layer
chromatography (TLC) was performed on silica gel plates [Merck
(G F254)] and the spots were observed under UV light or were
visualized with I2 vapor. Column chromatography (CC) was
performed by using silica gel 60 (Merck) or basic alumina (Across).
Elemental analyses were performed on a Carlo Erba EA 1110
CHNS-O instrument in the Microanalysis Laboratory of the
Department of Pharmaceutical Sciences of the University of
Genova.
Intermediates. The required 2,6-dimethylaniline, 2,6-dimeth-
ylphenol, 2,6-dimethyl-4-nitrophenol, and 4-nitrobenzoyl chloride
were purchased from Aldrich. 2,6-Dimethyl-4-nitroaniline was
prepared according to the Wepster method.19 The known benzophe-
none derivatives of Schemes 1 and 2 [(4-amino-3,5-dimethylphe-
nyl)phenylmethanone and (3,5-dimethyl-4-hydroxyphenyl)phenyl-
methanone] were prepared as follows.
The negative chronotropic activity (Table 2) detected on
spontaneously beating right atria was not particularly marked,
being practically comparable to that of quinidine (EC30 ) 3.99
µM) for two compounds (6, 16), 5.93-fold less potent for
compound 8, and definitely minor for all the remaining
compounds. In this in vitro model, amiodarone, like quinidine,
decreased cardiac frequency (EC30 ) 5.57 µM). It is also notable
that compounds 2, 5, and 18 showed positive chronotropic
activity as observed for lidocaine and procainamide.
(4-Amino-3,5-dimethylphenyl)phenylmethanone. A mixture of
2,6-dimethylaniline (2.52 mL, 20 mmol), benzoic acid (5.2 g, 42.5
mmol), and poly(phosphoric acid) (PPA; 40 g) was heated at 180
°C and stirred for 30 min under nitrogen. The reaction mixture
was diluted with 16 mL of water, heated again at 180 °C for 40
min, and then poured into 300 mL of water. The acidic suspension
was extracted several times with CH2Cl2. The organic phase was
washed with 1 N NaOH solution and then with water, dried over
Na2SO4, and evaporated to dryness. The residue was taken up in
dry ether, leaving behind some N-benzoyl-2,6-dimethylaniline (460
mg, mp 157-158 °C). The ether solution was evaporated, and the
residue was crystallized from EtOH/H2O (3:1) and further purified
by CC (SiO2, CH2Cl2) to obtain 1.04 g (23%) of the title compound.
Even more modest was the vasorelaxant activity (Table 2),
as expressed by the inhibition of calcium-induced contraction
on K+-depolarized (80 mM) guinea pig aortic strips, with
inhibition values in the range 15-38% at 50-100 µM, as was
also observed for quinidine and lidocaine. Compounds 2, 4, and
18-20 were practically inactive, as were procainamide and
amiodarone.
The most interesting antiarrhythmic compounds 7, 9, and 19
were studied on Langendorff retrogradely perfused guinea pig
spontaneously beating heart36 and compared with amiodarone
and quinidine. While increasing concentrations of quinidine up
to its EC50 for antiarrhythmic activity (10.26 µM) produced a
substantial dose-dependent prolongation of all the ECG intervals
and a 35% reduction of the heart rate, high concentrations of
the novel compounds, even 10-22 times higher than their EC50
for antiarrhythmic activity, only moderately prolonged the PR
1
Mp: 136-137.5 °C (lit.21 85-86 °C). H NMR (CDCl3): δ 2.26
(s, 6H, 2CH3); 4.10 (br s, 2H, NH2, collapses with D2O); 7.42-
7.60 (m, 5H, aromatic protons); 7.70-7.80 (m, 2H, aromatic
protons). Anal. (C15H15NO) C, H, N. Some unreacted 2,6-
dimethylaniline was recovered from the acidic aqueous solution.

340 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2
Vazzana et al.
(3,5-Dimethyl-4-hydroxyphenyl)phenylmethanone. A mixture
of 2,6-dimethylphenol (2.26 g, 20 mmol), benzoic acid (5.2 g, 42.5
mmol), and PPA (40 g) was heated at 180 °C and stirred for 30
min. The mixture was diluted with 16 mL of water, further heated
at 165 °C for 40 min, and finally poured into 200 mL of water and
extracted with CH2Cl2. The organic phase was extracted three times
with 1 N NaOH solution and then with water; from the organic
phase 2.76 g of an oil composed mainly of 2,6-dimethylphenyl
benzoate was recovered. The basic solution was acidified and
extracted with CH2Cl2; the dichloromethane solution was extracted
repeatedly with 5% NaHCO3 solution to remove unreacted benzoic
acid and then with water, dried (Na2SO4), and evaporated to dryness
to give 1.54 g of the required compound.
The crude 2,6-dimethylphenyl benzoate (2.76 g) was mixed with
20 g of PPA and heated at 180 °C for 20 min to promote the para
transposition of the benzoyl group. Working up the mixture as
above, a further crop of 0.51 g of the required compound was
obtained. The two fractions were joined and chromatographed on
silica gel eluting with CH2Cl2; 1.75 g (39% yield) of the title
compound was obtained. Mp: 136-138 °C. 1H NMR (CDCl3): δ
2.31 (s, 6H, 2CH3); 5.22 (s,1H, OH, collapses with D2O); 7.42-
7.68 (m, 5H, aromatic protons); 7.74-7.80 (m, 2H, aromatic
protons). Anal. (C15H14O2) C, H.
N-Homolupinanoylanilines 2, 4, and 6: General Method. A
mixture of the suitable amine and diisopropylethylamine (6.4 mol
each) in 10 mL of CHCl3 was added drop by drop to a solution of
1.61 g (6.4 mmol) of homolupinanoyl chloride hydrochloride17,18
in 15 mL of ethanol-free CHCl3 (passed on basic alumina). The
mixture was refluxed for 5 h under a slow stream of dry N2. The
solvent was removed in vacuo and the residue taken up in water
(100 mL). From the resulting turbid solution (pH 4-5) the unreacted
weakly basic amine was extracted with CHCl3, which however
extracted also some of the hydrochloride of the desired compounds,
expecially 6 (solution A). The aqueous solution was basified and
extracted again with chloroform; after evaporation of the solvent
the residue was rinsed with dry ether, leaving the main portion of
the expected amide. Additional portions of the amides were
recovered from solution A: the solvent was evaporated and the
residue chromatographed repeatedly on basic alumina (1:40) eluting
with chloroform.
(0.4 g, 1.27 mmol) was dissolved in EtOH-free CHCl3 (7 mL) and
treated with mesyl chloride (0.1 mL, 1.29 mmol) diluted with CHCl3
(5 mL). The mixture was stirred for 6 h at rt and then evaporated
to dryness. The residue was chromatographed several times on basic
alumina eluting with CHCl3 containing increasing quantities of
MeOH up to 1%. Finally, 0.130 g (26%) of 5 was obtained. Mp:
178-181 °C. 1H NMR (CDCl3): δ 1.20-2.96 [m, 18H of Lup +
2 superimposed s at 2.18 (6H, 2CH3) and 2.95 (3H, CH3SO2)];
5.95 (br s, 1H, NHSO2CH3, collapses with D2O); 6.82 (s, 2H,
aromatic protons); 7.05 (s, 1H, CONH, collapses with D2O). Anal.
(C20H31N3O3S) C, H, N, S.
Lupinyl (7, 8, 11) and epi-Lupinyl (12, 13, 16) Ethers: General
Method. The suitable phenol (4.5-6 mmol) was dissolved in EtOH
(2-2.5 mL) and treated with 6 N NaOH solution (0.75-1 mL)
and then with 4.5-6 mmol of chlorolupinane21 or epi-chlorolupi-
nane.22 The mixture was heated in an Aldrich pressure tube at 140
°C for 10-12 h with magnetic stirring. After cooling, the solvent
was evaporated in vacuo, and the residue was taken up in water
and strongly alkalinized. The mixture was extracted with ether and
the organic phase dried (Na2SO4) and evaporated to dryness. In
the case of lupinyl ethers the residue was crystallized from dry
ether, while in the case of epi-lupinyl ethers the residue was distilled
under reduced pressure (0.04 Torr) to eliminate the unreacted epi-
chlorolupinane (bp 90-100 °C, air bath temperature) and then
crystallized from dry ether, unless otherwise stated.
Data for (1R,9aR)-1-[(2,6-Dimethylphenoxy)methyl]octahy-
dro-2H-quinolizine (7). Low melting point crystals. Yield: 15%.
1H NMR (CDCl3): δ 1.05-2.30 (m, 14H of Q, with superimposed
s at 2.2, 6H, 2CH3); 2.70-2.85 (m, 2H, HR near N of Q); 3.75-
3.95 (m, 2H, CH2O); 6.75-6.95 (m, 3H, aromatic protons). Anal.
(C18H27NO) C, H, N. The following are data for the hydrochloride.
Mp: 247-255 °C dec (EtOH + Et2O). Anal. (C18H27NO‚HCl) C,
H, N.
Data for (1R,9aR)-1-[(2,6-Dimethyl-4-nitrophenoxy)methyl]-
octahydro-2H-quinolizine (8). Red-orange crystals. Mp: 88-89
°C. Yield: 48%. 1H NMR (CDCl3): δ 1.10-2.30 (m, 14H of Q);
2.36 (s, 6H, 2CH3); 2.78-2.92 (m, 2H, HR near N of Q); 3.92-
4.12 (m, 2H, CH2O); 7.93 (s, 2H, aromatic protons). Anal.
(C18H26N2O3) C, H, N.
Data for (1R,9aR)-{3,5-Dimethyl-4-[(octahydro-2H-quino-
lizin-1-yl)methoxy]phenyl}phenylmethanone (11). Orange crys-
Data for N-(2,6-Dimethylphenyl)-2-(1S,9aR)-(octahydro-2H-
quinolizin-1-yl)acetamide (2). Mp: 187-187.5 °C (EtOH).
Yield: 70%.12 1H NMR (CDCl3): δ 1.18-2.92 (m with superim-
posed s at 2.24, 18H of Lup + 6H, 2CH3); 6.85 (s, 1H, CONH,
collapses with D2O); 7.05-7.18 (m, 3H, aromatic protons). Anal.
(C19H28N2O) C, H, N.
1
tals. Mp: 84-87 °C. Yield: 56%. H NMR (CDCl3): δ 1.10-
2.30 (m, 14H of Q); 2.34 (s, 6H, 2CH3); 2.78-2.92 (m, 2H, HR
near N of Q); 3.92-4.12 (m, 2H, CH2O); 7.24-7.65 (m, 5H,
aromatic protons); 7.75-7.84 (m, 2H, aromatic protons). Anal.
(C25H31NO2) C, H, N.
Data for N-(2,6-Dimethyl-4-nitrophenyl)-2-(1S,9aR)-(octahy-
dro-2H-quinolizin-1-yl)acetamide (4). Mp: 229-235 °C (absolute
Data for (1S,9aR)-1-[(2,6-Dimethylphenoxy)methyl]octahy-
dro-2H-quinolizine (12). Low melting point solid. Yield: 16%.
1H NMR (CDCl3): δ 1.00-2.40 (m, 14H of Q, with superimposed
s at 2.20, 6H, 2CH3); 2.60-2.85 (m, 2H, HR near N of Q); 3.64
(d, J ) 4.6 Hz, 2H, CH2O); 6.70-6.95 (m, 3H, aromatic protons).
Anal. (C18H27NO) C, H, N. The following are data for the
hydrochloride. Mp: 235-240 °C dec (EtOH + Et2O). Anal.
(C18H27NO‚HCl) C, H, N.
1
EtOH). Yield: 42%. H NMR (CDCl3): δ 1.24-2.92 (m with
superimposed s at 2.33, 18H of Lup + 6H, 2CH3); 7.22 (s, 1H,
CONH, collapses with D2O); 7.97 (s, 2H, aromatic protons). Anal.
(C19H27N3O3) C, H, N.
Data for {3,5-Dimethyl-4-[N-(1S,9aR)-(octahydro-2H-quino-
lizin-1-yl)acetyl]aminophenyl}phenylmethanone (6). Mp: 98-
102 °C (benzene/pentane). Yield: 26%. 1H NMR (CDCl3): δ 1.20-
2.95 (m, 18H of Lup + superimposed s at 2.29, 6H, 2CH3); 7.10
(s,1H, CONH, collapses with D2O); 7.45-7.65 (m, 5H, aromatic
protons); 7.78-7.90 (m, 2H, aromatic protons). Anal. (C26H32N2O2)
C, H, N.
Data for (1S,9aR)-1-[(2,6-Dimethyl-4-nitrophenoxy)methyl]-
octahydro-2H-quinolizine (13). Yellow crystals. Mp: 72-73 °C
1
(pentane). Yield: 25%. H NMR (CDCl3): δ 1.10-2.30 (m, 14H
of Q); 2.36 (s, 6H, 2CH3); 2.78-2.92 (m, 2H, HR near N of Q);
3.92-4.12 (m, 2H, CH2O); 7.93 (s, 2H, aromatic protons). Anal.
(C18H26N2O3) C, H, N.
N-(4-Amino-2,6-dimethylphenyl)-2-(1S,9aR)-(octahydro-2H-
quinolizin-1-yl)acetamide. The nitro compound 4 (0.48 g, 1.4
mmol) and 10% Pd on activated carbon (0.1 g) were suspended in
40 mL of EtOH and hydrogenated at room temperature (rt) and
atmospheric pressure. The catalyst was filtered and the solvent
removed in vacuo to yield white crystals (0.44 g, quantitative yield).
Data for (1S,9aR)-{3,5-Dimethyl-4-[(octahydro-2H-quinolizin-
1-yl)methoxy]phenyl}phenylmethanone (16). Oil. Yield: 22%.
1H NMR (CDCl3): δ 1.10-2.20 (m, 14H of Q); 2.33 (s, 6H, 2CH3);
2.82-2.95 (m, 2H, HR near N of Q); 3.79 (d, J ) 4.4 Hz, 2H,
CH2O); 7.40-7.64 (m, 5H, aromatic protons); 7.74-7.83 (m, 2H,
aromatic protons). The following are data for the hydrochloride.
Mp: 242-244 °C (EtOH). Anal. (C25H31NO2‚HCl) C, H, N.
(1R,9aR)-1-[(4-Amino-2,6-dimethylphenoxy)methyl]-octahy-
dro-2H-quinolizine (9). The nitro derivative 8 (0.91 g, 2.9 mmol)
was dissolved in 50 mL of EtOH mixed with 10% Pd on activated
carbon (90 mg) and hydrogenated at rt and atmospheric pressure.
1
Mp: 147-148 °C. H NMR (CDCl3): δ 1.20-2.94 (m, 18H of
Lup + superimposed s at 2.14, 6H, 2CH3); 3.45 (br s, 2H, NH2,
collapses with D2O); 6.41 (s, 2H, aromatic protons); 6.70 (s, 1H,
CONH, collapses with D2O). Anal. (C19H29N3O) C, H, N.
N-(2,6-Dimethyl-4-mesylaminophenyl)-2-(1S,9aR)-(octahydro-
2H-quinolizin-1-yl)acetamide (5). The foregoing amino compound

Quinolizidinyl DeriVatiVes as Antiarrhythmic Agents
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2 341
The catalyst was filtered and the solvent removed in vacuo, leaving
white crystals (0.77 g, 93% yield). Mp: 94-97 °C. 1H NMR
(CDCl3): δ 1.20-2.40 (m, 14H of Q + s at 2.21, 6H, 2CH3); 2.78-
2.90 (m, 2H, HR near N of Q); 3.40 (br s, 2H, NH2, collapses with
D2O); 3.80-4.00 (m, 2H, CH2O); 6.36 (s, 2H, aromatic protons).
Anal. (C18H28N2O) C, H, N.
solution of the amino compound 18 (0.7 g, 2.43 mmol) in CHCl3
(EtOH-free, 7 mL). The mixture was stirred at rt for 1 h and then
evaporated to dryness. The residue was taken up in water (7 mL),
and the solution was neutralized with 10% Na2CO3 solution and
extracted with ether, which removed some unreacted amino
compound. By careful correction of the pH, a precipitate was
obtained that was extracted with CHCl3. After drying (Na2SO4),
the solvent was evaporated, and the residue was chromatographed
on alumina (1:30) eluting with CHCl3. Compound 20 was finally
crystallized from pentane, 0.29 g (33% yield). Mp: 179-184 °C.
1H NMR (DMSO-d6): δ 1.10-2.00 (m, 14H of Q); 3.05 (s, 3H,
CH3SO2); 2.70-2.85 (m, 2H, HR near N of Q); 3.30-3.50 (m,
2H, CH2NH); 7.18-7.90 (m, 4H, aromatic protons); 8.30-8.42 (m,
1H, NHCH2, collapses with D2O); 10.05 (br s, 1H, NHSO2CH3,
collapses with D2O). Anal. (C18H27N3O3S) C, H, N, S.
(1R,9aR)-1-[(2,6-Dimethyl-4-mesylaminophenoxy)methyl]-oc-
tahydro-2H-quinolizine (10). The foregoing amino compound 9
(0.50 g, 1.73 mmol) was dissolved in pyridine (7 mL) and treated
with mesyl chloride (0.13 mL, 1.7 mmol). The mixture was stirred
for 10 min at rt and then refluxed for 1.5 h. After cooling, the
mixture was poured onto ice, and 0.1 N NaOH solution was added
until pH ≈ 8 was reached and then thoroughly extracted with CH2-
Cl2. The solvent was evaporated, and the oily residue was distilled
under reduced pressure to remove the pyridine. The residue was
chromatographed on alumina (1:30) eluting with chloroform. Thus,
230 mg of 10 (37% yield) was obtained. Mp: 126-127 °C (Et2O).
1H NMR (CDCl3): δ 1.20-2.20 (m, 14H of Q); 2.27 (s, 6H, 2CH3);
2.80-2.94 (m, 2H, HR near N of Q); 2.98 (s, 3H, SO2CH3); 3.80-
4.00 (m, 2H, CH2O); 6.40 (br s, 1H, NHSO2CH3, collapses with
D2O); 6.89 (s, 2H, aromatic protons). Anal. (C19H30N2O3S) C, H,
N, S.
(1S,9aR)-1-[(4-Amino-2,6-dimethylphenoxy)methyl]octahydro-
2H-quinolizine (14). The nitro derivative 13 was reduced as
described for the epimeric 8; the catalyst was filtered and the solvent
evaporated to leave an oil that crystallized when rinsed with pentane
(0.63 g, 94% yield). Mp: 56-58 °C. 1H NMR (CDCl3): δ 1.20-
2.20 (m, 14H of Q); 2.20 (s, 6H, 2CH3); 2.80-2.92 (m, 2H, HR
near N of Q); 3.40 (br s, 2H, NH2, collapses with D2O); 3.65 (d, J
) 4.6 Hz, 2H, CH2O); 6.36 (s, 2H, aromatic protons). Anal.
(C18H28N2O) C, H, N.
(1S,9aR)-1-[(2,6-Dimethyl-4-mesylaminophenoxy)methyl]oc-
tahydro-2H-quinolizine (15). The foregoing amino compound 14
was treated with mesyl chloride as described for the preparation of
the mesylamino derivative 10. The separation of the mesylamino
derivative 15 from the starting amine was quite laborious; thus,
only a 13% yield of pure 15 was obtained. Mp: 157-158 °C (Et2O).
1H NMR (CDCl3): δ 1.20-2.20 (m, 14H of Q); 2.27 (s, 6H, 2CH3);
2.80-2.95 (m, 2H, HR near N of Q); 2.99 (s, 3H, SO2CH3); 3.69
(d, J ) 4.4 Hz, 2H, CH2O); 6.89 (s, 2H, aromatic protons); the
signal due to NHSO2 was not detected. Anal. (C19H30N2O3S) C,
H, N, S.
Pharmacology. 1. Guinea Pig Atrial Preparations. Female
guinea pigs (300-400 g) were sacrificed by cervical dislocation.
After thoracotomy the heart was immediately removed and washed
by perfusion through the aorta with oxygenated Tyrode solution
of the following composition (mM): 136.9 NaCl, 5.4 KCl, 2.5
CaCl2, 1.0 MgCl2, 0.4 NaH2PO4‚xH2O, 11.9 NaHCO3, and 5.5
glucose. The physiological salt solution (PSS) was buffered at pH
7.4 by saturation with 95% O2/5% CO2 gas, and the temperature
was maintained at 35 °C. Isolated guinea pig heart preparations
were used, spontaneously beating right atria and left atria driven
at 1 Hz. For each preparation, the entire left and right atria were
dissected from the ventricles, cleaned of excess tissue, and hung
vertically in a 15 mL organ bath containing the PSS continuously
bubbled with 95% O2/5% CO2 gas at 35 °C, pH 7.4. The contractile
activity was recorded isometrically by means of a force transducer
(FT 0.3, Grass Instruments Corp., Quincy, MA) using Power Lab
software (AD-Instruments Pty Ltd., Castle Hill, Australia). The left
atria were stimulated by rectangular pulses of 0.6-0.8 ms duration
and about 50% threshold voltage through two platinum contact
electrodes in the lower holding clamp (Grass S88 stimulator). The
right atrium was in spontaneous activity. After the tissue was beating
for several minutes, a length-tension curve was determined, and
the muscle length was maintained, which elicited 90% of the
maximum contractile force observed at the optimal length. A
stabilization period of 45-60 min was allowed before the atria were
used to test the compounds. During the equilibration period, the
bathing solution was changed every 15 min and the threshold
voltage was ascertained for the left atria. Atrial muscle preparations
were used to examine the inotropic and chronotropic activities of
the compounds (0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, and 100 µM)
dissolved in PSS. During the construction of cumulative dose-
response curves, the next higher concentration of the compounds
was added only after the preparation reached a steady state.
2. Alternating Current Induced Arrhythmia in Guinea Pig
Left Atrial Preparations.28,29 Left atria from guinea pigs were fixed
to platinum wire electrodes and placed between two parallel
platinum field electrodes. The preparations were mounted vertically
in a 15 mL organ bath containing a modified Krebs-Henselait
solution of the following composition (mM): 118 NaCl, 4.7 KCl,
2.5 CaCl2, 1.2 MgSO4, 1.2 NaH2PO4‚xH2O, 24.9 NaHCO3, and 10.1
glucose. The solution was buffered at pH 7.4 by saturation with
95% O2/5% CO2 gas, and the temperature was maintained at 35
°C. The preparations were connected to a force transducer. Isometric
force contraction was recorded using Power Lab software. Ar-
rhythmias were induced by application of sinusoidal alternating
current (50 Hz) of increasing strength to the isolated heart
preparations, 2 and 4 mA/(cm2‚s) to induce arrhythmia correspond-
ing to an increase in the field intensity of 0.3 V/(cm‚s). The current
strength i (mA/cm2) at which extra beats occurred was called the
“threshold of ac-arrhythmia”.37 The preparations were allowed to
stabilize for 60 min. Then concentration-response curves for the
threshold of ac-arrhythmia were recorded by cumulative application
of compounds to the bathing solution every 30 min.
4-Nitro-N-[(1S,9aR)-(octahydro-2H-quinolizin-1-yl)methyl]-
benzamide (17). Compound 17 was repared as previously de-
1
scribed.15 Mp: 121 °C (EtOH/H2O). H NMR (CDCl3): δ 1.20-
2.30 (m, 14H of Q); 2.90-3.05 (m, 2H, HR near N of Q); 3.65-
3.75 (m, 2H, CH2NH); 7.90-8.05 and 8.15-8.35 (2 m, 2H each,
aromatic para substitution); 9.90 (br s, 1H, NH, collapses with D2O).
The following are data for the hydrochloride. Mp: 154-158 °C
dec (absolute EtOH/Et2O). Anal. (C17H23N3O3‚HCl) C, H, N.
4-Amino-N-[(1S,9aR)-(octahydro-2H-quinolizin-1-yl)methyl]-
benzamide (18). Compound 18 was prepared as previously
described,15 by reduction of 17 in the presence of 10% palladium
on activated carbon instead of PtO2. Mp: 193-194 °C (EtOH).
Anal. (C17H25N3O) C, H, N.
4-Acetylamino-N-[(1S,9aR)-(octahydro-2H-quinolizin-1-yl)m-
ethyl]benzamide (19). Acetic anhydride (1.5 mL) was added to a
solution of the foregoing amino compound 18 (0.7 g, 2.4 mmol) in
2 mL of a mixture of acetic acid and water (1:1), and the mixture
was heated in a boiling water bath for 5 min. After cooling, the
solution was neutralized with 10% Na2CO3 solution, and the
precipitate was collected and washed with water. After drying, 0.67
1
g (84% yield) of 19 was obtained. Mp: 230-231 °C. H NMR
(DMSO-d6): δ 1.10-2.00 (m, 14H of Q); 2.06 (s, 3H, CH3CO);
2.66-2.82 (m, 2H, HR near N of Q); 3.35-3.48 (m, 2H, CH2-
NH); 7.55-7.80 (m, 4H, aromatic protons); 8.25-8.40 (m, 1H,
NHCH2, collapses with D2O); 10.15 (s, 1H, NHCOCH3, collapses
with D2O). Anal. (C19H27N3O2) C, H, N.
4-Mesylamino-N-[(1S,9aR)-(octahydro-2H-quinolizin-1-yl)m-
ethyl]benzamide (20). A solution of mesyl chloride (0.2 mL, 2.6
mmol) in CHCl3 (EtOH-free) (7 mL) was added dropwise to a
3. Guinea Pig Aortic Strips. The thoracic aorta was removed
and placed in Tyrode solution of the following composition
(mM): 118 NaCl, 4.75 KCl, 2.54 CaCl2, 1.20 MgSO4, 1.19 KH2-

342 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2
Vazzana et al.
(4) Lown, B.; Calvert, A. F.; Armington, R.; Ryan, M. Monitoring for
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PO4, 25 NaHCO3, and 11 glucose. The solution was equilibrated
with 95% O2/5% CO2 gas at pH 7.4. The vessel was cleaned of
extraneous connective tissue. Two helicoidal strips (10 mm × 1
mm) were cut from each aorta beginning from the end most
proximal to the heart. Vascular strips were then tied with surgical
thread (6-0) and suspended in a jacketed tissue bath (15 mL)
containing aerated pharmacological salt solution (PSS) at 35 °C.
Aortic strips were subjected to a resting force of 1 g. The strips
were secured at one end to a force displacement transducer (FT
0.3, Grass Instruments Corp.) to monitor changes in the isometric
contraction and washed every 20 min with fresh PSS for 1 h after
the equilibration period; guinea pig aortic strips were contracted
by washing in PSS containing 80 mM KCl (equimolar substitution
of K+ for Na+). After the contraction reached a plateau (about 45
min) the compounds (0.1, 0.5, 1, 5, 10, 50, and 100 µM) were
added cumulatively to the bath, allowing for any relaxation, to
obtain an equilibrated level of force. Addition of the drug vehicle
had no appreciable effect on K+-induced contraction (PSS for all
compounds).
4. Guinea Pig Isolated Perfused Heart According to Lan-
gendorff. Female guinea pigs (300-400 g) were killed by cervical
dislocation. The hearts were quickly removed and rapidly perfused
through the aorta at constant flow (11-12 mL min-1 g-1) with a
modified Krebs-Henselait solution with the following composition
(mM): 128 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 15 NaHCO3,
1.2 KH2PO4, 11.1 glucose, and 2 sodium pyruvate. The solution
was bubbled with a gas mixture (95% O2/5% CO2) (pH 7.4-7.5)
and maintained at 37 °C. A perfusion pressure of 50-60 mmHg
was obtained at this flow rate. The addition of pyruvate to the
medium has been shown to confer the same metabolic and
functional features of the heart in situ to the isolated heart.38
A
stabilization period of 30 min was given to the heart under normal
ECG conditions to keep the frequency of spontaneous beating hearts
constant. Surface ECG was recorded by means of two electrodes,
one placed near the initial portion of the anterior intraventricular
artery and the other on the left ventricular free wall. The main ECG
intervals (PR ) atrioventricular conduction time; QRS ) intra-
ventricular conduction time; QT ) duration of ventricular action
potential) were measured. The drug-induced changes in conduction
velocity of the AV node and ventricular myocardium were
calculated as changes in the reciprocal of the PR interval and QRS
interval, respectively.39 The compounds were added with increasing
concentrations (0.001, 0.01, 0.1, 1, and 10 µM). During the building
of concentration-response curves, the next higher concentration
of the compounds was added only after the preparation reached a
steady state (about 30 min).
The potency of the drugs, defined as the EC50 value, was
evaluated from log concentration-response curves (n ) 6-8)
(GraphPad) in the appropriate pharmacological preparations.27 All
data are presented as the mean ( SEM.25
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(25) GraphPad Prism 4.03; GraphPad Software Inc.: San Diego, CA,
http://www.graphpad.com.
(26) Tallarida, R. J.; Murray, R. B. Manual of Pharmacologic Calculations
with Computer Programs, 2nd ed.; Springer-Verlag: New York,
1987.
Acknowledgment. Financial support from the Italian MIUR
and from the Universities of Bologna and Genova are gratefully
acknowledged. We thank Mr. O. Gagliardo for performing the
elemental analyses.
Supporting Information Available: Elemental analysis data
for all compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
(27) GraphPad Prism 3.02; GraphPad Software Inc.: San Diego, CA,
http://www.graphpad.com.
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