Synthesis and evaluation of in vivo activity of diphenylhydantoin basic derivatives

Article abstract of DOI:10.1016/j.ejmech.2004.05.008

During the search for antiarrhythmic agents among amide derivatives of phenytoin, compound 7 {3-ethyl-1-[2-hydroxy-3-(4-phenyl-piperazin-1-yl)-propyl]- 2,4-dioxo-5,5-diphenyl-imidazolidine} was selected as it showed antiarrhythmic as well as antihypertens

Full text of DOI:10.1016/j.ejmech.2004.05.008

European Journal of Medicinal Chemistry 39 (2004) 1013–1027
www.elsevier.com/locate/ejmech
Original article
Synthesis and evaluation of in vivo activity of diphenylhydantoin basic
derivatives
Tomasz Dyla¸g a,1, Małgorzata Zygmunt b, Dorota Macia¸g b, Jadwiga Handzlik a,
Marek Bednarski b, Barbara Filipek b, Katarzyna Kiec´-Kononowicz a,*
a Department of Technology and Biotechnology of Drugs, Collegium Medicum of the Jagiellonian University, Medyczna 9, Pl 30-688 Kraków, Poland
b Laboratory of Pharmacological Screening, Collegium Medicum of the Jagiellonian University, Medyczna 9, Pl 30-688 Kraków, Poland
Received 18 March 2003; received in revised form 21 May 2004; accepted 24 May 2004
Abstract
During the search for antiarrhythmic agents among amide derivatives of phenytoin, compound 7 {3-ethyl-1-[2-hydroxy-3-(4-phenyl-
piperazin-1-yl)-propyl]-2,4-dioxo-5,5-diphenyl-imidazolidine} was selected as it showed antiarrhythmic as well as antihypertensive activity.
Treating this compound as a lead, new derivatives 8–19 were synthesised, differing in piperazine phenyl ring substitution (2-, 3-, 4-Cl,
2-CH3O) as well as in hydantoin N3 alkyl chain (ethyl, ethyl acetate or ethyl 2-propionate). The obtained compounds in form of hydrochlorides
7a–19a were examined for prophylactic antiarrhythmic and antihypertensive properties. Compounds containing ethyl 2-propionate moiety
(17a, 18a) exhibited the highest antihypertensive properties. Water-soluble compounds, containing 2-methoxyphenylpiperazine group (11a,
19a), showed strong antiarrhythmic properties in adrenaline-induced arrhythmia; compound 9a {1-[3-(4-(3-chloro-phenyl)-piperazin-1-yl)-
2-hydroxy-propyl]- 3-ethyl-2,4-dioxo-5,5-diphenyl-imidazolidine hydrochloride} exhibited the highest antiarrhythmic activity in barium
chloride arrhythmia model.
© 2004 Elsevier SAS. All rights reserved.
Keywords: Diphenylhydantoin derivatives; Antiarrhythmic activity; Hypotensive activity; Anticonvulsant activity; Microwave
1. Introduction
A number of phenytoin structure modifications have been
performed to improve its therapeutic properties [3–7]. One
direction of research led to the change of phenytoin character
from acidic to basic, i.e. ropitoin, active in various types of
arrhythmia models after oral and intravenous administration
[3]. A series of amide phenytoin derivatives were obtained
(Fig 1) and examined, including RH-7, exhibiting high pro-
phylactic antiarrhythmic activity in chloroform and barium
chloride-induced arrhythmia after intravenous injection,
being less active in adrenaline-induced arrhythmia. As the
action of RH-7 is similar to that of class Ia antiarrhythmic
drugs, introduction of hydroxyethylpiperazine substituent
into position 3 of the hydantoin ring was followed by a
change in the mechanism of action. RH-7, possessing
lidocaine-like anaesthetizing activity, is bereft of CNS activ-
ity and is less toxic than phenytoin.A lack of the activity after
oral administration was its main disadvantage [5]. Among
others, KF-2 was a promising one in a group of compounds
with 3-amine-2-propanol substituent in position 1 of the
hydantoin ring and ethyl or ethyl acetate in position 3. The
Phenytoin DPH – 5, 5 – diphenylhydantoin (Fig 1) was the
starting structure for all presented compounds. It is fre-
quently used as anticonvulsant drug, however, it is also ap-
plied in treatment of heart rhythm disturbances caused by
intoxication with Digitalis glycosides. Phenytoin is a blocker
of inactivated sodium channels and shortens the action po-
tential in heart muscle cells and, therefore – according to
Vaughan-Williams classification – it is a member of class Ib
antiarrhythmic drugs. Side effects, including CNS – central
nervous system complaints (ataxia, nystagmus or mental
confusion) [1,2], are considered as the major drawbacks of
phenytoin treatment, which have to be faced.
* Corresponding author. tel./fax +48-12-6570488.
E-mail address: mfkonono@cyf-kr.edu.pl (K. Kiec´-Kononowicz).
1 Present address: Neurobiochemistry Group, Faculty of Chemistry, Ja-
giellonian University, Ingardena 3, Pl 30-060 Kraków, Poland
0223-5234/$ - see front matter © 2004 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2004.05.008

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T. Dyla¸g et al. / European Journal of Medicinal Chemistry 39 (2004) 1013–1027
Fig. 1. Structures of some basic derivatives of phenytoin (DPH).
compound prevented symptoms of barium chloride-induced
arrhythmia in 66% of animals, although it was less active in
adrenaline-induced arrhythmia. In both cases, however,
KF-2 prolonged the survival time of animals. KF-4 was
another active compound [8]. These compounds, possessing
a hydroxypropyl fragment characteristic for beta-blockers,
display a mechanism of action similar to that of antiarrhyth-
mic Ia group. For the obtained compounds, however, 3-ethyl-
5,5-diphenylhydantoin derivative also comprising a phenyl-
piperazine substituent 7a (Fig 2) revealed the highest
Fig. 2. Synthesis of basic arylpiperazine hydantoin derivatives 7a–19a.

T. Dyla¸g et al. / European Journal of Medicinal Chemistry 39 (2004) 1013–1027
1015
activity. This compound, which antiarrhythmic activity was
confirmed in both barium chloride and adrenaline – induced
arrhythmias, was additionally examined for its hypotensive
action. It proved to diminish both systolic and diastolic blood
pressure in normotensive rats after intraperitoneal adminis-
tration in a statistically significant manner. In this way, it was
confirmed that introduction of arylpiperazine moiety into the
molecule endowed it with hypotensive action. Analysis of
structure and activity of a series of compounds, used either in
therapy or as selective a1-AR ligands in pharmacology, jus-
tifies hypothesis that a likely a1-adrenergic receptor antago-
nist was obtained. Compound 7a comprises also hydroxy-
propyl side chain, typical for beta-blockers.
Interesting properties of 7a prompted us to obtain and
analyse pharmacological activity of compounds being deri-
vatives of that structure. We decided to estimate influence of
the substitution pattern of phenylpiperazine phenyl ring in
the compounds on hypotensive action, since this substituent
may account for interaction with adrenergic receptors. The
presence of hydroxypropyl chain suggested possible
b-blocking activity, which should be desirable for antiar-
rhythmic action.
15.5 hours (method A). Time consumption as well as the
necessity to use a solvent that impedes isolation of product
from the reaction liquid are the major drawbacks of this
method. In the other method, mixed substrates were irradi-
ated in a standard domestic microwave oven (method B)
previously melting of some reagents (method B*). In this
method, without having to use a solvent for conducting the
synthesis, simple crystallisation of the reaction mixture from
ethanol afforded pure product. Rapidity and efficiency are
the supreme benefits of the method shorter than ten-minute
irradiation suffices to obtain products with yields substanti-
ally higher than in the same reactions performed in the
toluene environment. Also the purification was much easier
than in traditional method [9–11]. To improve the solubility
of free bases 7–19, which was desirable for pharmacological
screening purposes, compounds were converted into corres-
ponding hydrochlorides 7a–19a. Compounds 7–11 were tur-
ned into salts by addition of concentrated hydrochloric acid
and heating until its excess evaporated. The presence of
easily hydrolysing ester bonds in compounds 12–19 preven-
ted the use of liquid acid and, therefore, they were solved in
dry ethanol and saturated with gaseous hydrogen chloride. In
spite of converting the bases into hydrochlorides, the solubi-
lity of the compounds (with the exception of compounds 11a
and 19a) was not high enough to allow intravenous adminis-
tration. As indicated by elemental analyses, in all cases,
monohydrochloride salts were obtained. For the pharmaco-
logical screening purposes, all compounds were used as
mixtures of enantiomers.
Keeping that in mind, we designed and synthesized a
series of derivatives of 7a differing in the substitution of
piperazine phenyl ring as well as in hydantoin N3 alkyl chain.
The structurally modified compounds 8a–19a have been
examined for their influence on normal ECG and antiarrhyth-
mic and antihypertensive action. In addition, anticonvulsant
activity and neurological toxicity for some compounds were
evaluated.
3. Pharmacology
2. Chemistry
Obtained compounds were examined in vivo. The investi-
gation included effects on normal electrocardiogram, in-
fluence on blood pressure in normotensive rats and prophy-
lactic antiarrhythmic action in animal models of heart rhythm
disturbances caused by intravenous injection of either ba-
rium chloride or adrenaline. The obtained compounds were
tested as hydrochlorides, which possessed different water
solubility. Thus, the two ways of administrations were used.
Most of the compounds were examined after i.p. administra-
tion because their water solubility was low. Only two com-
pounds were soluble enough for more profitable intravenous
administration.
Protection against rhythm disturbances caused by injec-
tion of arrhythmogens (barium chloride or adrenaline) de-
picts the ability of a compound to act as a potential antiar-
rhythmic agent. The two arrhythmogenes act via different
mechanisms. Barium chloride takes effect by decrease of the
outward potassium currents, whereas adrenaline stimulates
b-adrenergic receptors.
The preparation of basic diphenylhydantoin derivatives
was accomplished with the route depicted in Fig 2.
The first step of synthetic work was based on an introduc-
tion of substituents into position 3 of hydantoin ring, which
was performed by heating of respective bromo- or chlo-
roalkyl derivatives with DPH. This process was performed at
elevated temperature in two phase-catalytic conditions in the
presence of sodium or potassium carbonate and benzyl-
triethylammonium chloride (TEBA) as phase-transfer cata-
lyst and afforded compounds 1–3.
The aim of the second step was to introduce reactive
2,3-epoxypropyl moiety into the 1 position of hydantoin.
This step involved phase-transfer catalysed reactions of com-
pounds 1–3 with epichlorhydrine used in 20% excess. The
reaction was performed at room temperature with the use of
TEBA and sodium carbonate and led to the formation of
compounds 4–6.
In the following step, the epoxy bridge was cleaved with
secondary amines: phenylpiperazine or its 2-, 3-, 4-chloro
and 2-methoxy derivatives to obtain compounds 7–19. The
first technique to achieve this goal included heating of
equimolar quantities of substrates in toulene for 6.5–
Improvement of antiarrhythmic and antihypertensive pro-
perties of phenytoin derivatives can lead to a decrease in
their CNS interactions. Treating anticonvulsant activity as a
CNS interaction symptom, three of the obtained compounds

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T. Dyla¸g et al. / European Journal of Medicinal Chemistry 39 (2004) 1013–1027
Table 1
Effects of an intravenous injection of the investigated compounds on heart rate and ECG intervals in anaesthetised male Wistar rats
Compound
Dose
(mg/kg)
50
Parameters
Time of observation (min)
0
1
5
15
11a
Beats per minute
341.1 11.4
49.4 2.3
75.7 2.4
18.6 0.7
324.0 9.4
60.4 3.6
70.2 2.6
15.4 0.2
303.1 14.3*
59.1 4.2*
76.9 3.0
19.1 1.1
260.0 15.4****
66.8 2.8
85.4 6.0*
16.8 0.9
326.8 16.0
57.7 3.2
77.1 3.0
19.1 0.7
271.0 15.4***
66.4 3.5
77.8 3.6
15.8 0.7
327.8 16.8
59.4 2.4*
74.3 3.7
20.0 1.0
284.0 7.5*
63.4 2.3
72.4 2.7
15.4 0.5
50
P-Q
50
Q-T
50
QRS
19a
50
Beats per minute
50
P-Q
Q-T
QRS
50
50
The data are the means of six experiments S.E.M. Statistical analyses were performed using a one-way ANOVA test
* P < 0.05; ** P < 0.02; *** P < 0.01; **** P < 0.001
were tested by the Antiepileptic Drug Development (ADD)
Program. The investigation containing Phase I of the evalua-
tion involved three tests: maximal electroshock (MES), sub-
cutaneous penthylenetetrazole (scMet), and neurologic toxi-
city [12].
pound 11a slightly decreased the number of cardiac beats in
1st minute and prolonged PQ interval in 1st and 15th minutes
of the test. Compound 19a showed significant decrease of
cardiac beats number for the duration of observation and
slightly prolonged QT interval in first minute after injection.
Some compounds tested intraperitoneally (Table 2), espe-
cially 9a and 10a, did not significantly affect the normal ECG
but other compounds had selective effects inducing de-
creased rate of cardiac beats in 60th (8a), prolonged PQ
intervals in 15th (18a), 30th and 60th minutes (17a, 18a),
prolonged QT interval in 15th (12a, 16a), 30th (16a) and
4. Results
The influence of compounds 11a and 19a on normal rats
ECG was tested after i.v. administration (Table 1). Com-
Table 2
Effects of an intraperitoneal injection of the investigated compounds on heart rate and ECG intervals in anaesthetised male Wistar rats
Compound
Dose
(mg/kg)
50
Parameters
Time of observation (min)
0
15
30
60
8a
Beats per minute
420.0 14.9
49.2 2.0
58.8 2.3
17.2 1.2
367.2 32.0
53.0 2.4
75.0 4.3
19.3 0.4
332.7 29.5
52.5 1.5
73.5 2.4
22.0 2.9
308.9 17.1
56.5 4.7
72.5 2.5
17.5 0.0
330.7 17.3
51.2 2.9
67.5 4.8
19.6 0.7
344.7 9.1
53.0 1.8
81.7 1.7
17.3 0.8
381.3 18.2
40.8 0.8
76.0 6.8
20.0 0.6
397.3 16.8
51.2 3.0
67.6 6.1
17.6 1.0
330.9 29.8
56.3 3.0
79.3 2.6
19.7 0.6
334.8 34.4
52.5 1.0
68.5 3.0
21.5 3.0
298.9 6.3
56.5 2.9
81.5 3.0**
16.5 0.5
313.1 15.0
53.6 2.1
77.0 1.9*
20.0 0.6
345.5 6.8
55.3 0.7
81.7 1.7
17.3 0.8
344.1 20.1
46.4 1.0***
88.4 8.5
20.8 0.8
382.0 15.3
49.6 1.9
68.0 3.7
17.2 1.2
320.8 33.1
56.3 3.0
81.6 4.0
19.7 1.0
331.4 36.2
55.0 0.6
70.0 2.4
20.5 1.9
305.5 4.4
54.5 2.6
76.5 2.4
16.0 0.1**
302.4 18.9
55.6 2.0
77.5 1.0**
20.8 0.8
350.4 13.4
56.3 1.0*
76.7 2.1
17.0 0.9
334.3 16.0
46.8 1.4***
86.0 6.8
20.8 0.8
368.2 15.6*
50.8 3.1
50
P-Q
50
Q-T
74.0 5.4*
17.2 1.2
50
QRS
9a
50
Beats per minute
318.4 39.9
57.7 3.9
50
P-Q
50
Q-T
87.3 6.8
50
QRS
20.7 0.7
10a
12a
16a
17a
18a
50
Beats per minute
325.4 36.01
56.0 1.4
50
P-Q
50
Q-T
74.0 4.8
50
QRS
22.0 2.8
50
Beats per minute
320.1 13.9
58.5 4.3
50
P-Q
50
Q-T
79.0 1.0
50
QRS
16.1 0.1**
293.8 24.8
59.2 2.0*
80.5 0.5***
21.6 0.7
50
Beats per minute
50
P-Q
50
Q-T
50
QRS
50
Beats per minute
372.6 15.0
58.3 1.0***
76.7 2.1
50
P-Q
50
Q-T
50
QRS
17.7 1.0
50
Beats per minute
333.2 12.8
50.0 1.7****
50
P-Q
Q-T
QRS
50
88.8
3.4
50
20.8 0.8
The data are the means of six experiments S.E.M. Statistical analyses were performed using a one-way ANOVA test
* P < 0.05; ** P < 0.02; *** P < 0.01; **** P < 0.001

T. Dyla¸g et al. / European Journal of Medicinal Chemistry 39 (2004) 1013–1027
1017
Fig. 3
.
Hypotensive activity of compounds tested after i. p. administration in anaesthetised normotensive rats.
60th (8a, 16a) minutes of observation. Only compound 12a
demonstrated statistically significant prolongation of QRS
complex (in 30th and 60th minutes after i.p. administration).
The obtained compounds were evaluated for antihyper-
tensive properties after intraperitoneal or intravenous admin-
istration in anaesthetised Wistar rats. Compounds of low
water solubility (7a–10a, 12a, 16a–18a) were tested for 90
(70 in case of 7a) minutes after i.p. administration. The lead
compound 7a significantly decreased both systolic (max.
12%) and diastolic (max 8.7%) blood pressure at 30th minute
of the measurement. Furthermore, three of i.p. tested com-
pounds (8a, 17a, 18a) showed statistically significant de-
crease of rat systolic blood pressure prolonged to the end of
observation. Especially, compound 17a significantly de-
creased systolic pressure (5.7–9.8%) from 5th till 90th min-
utes of the observation. The compounds 9a, 17a, 18a de-
pressed diastolic blood pressure (7.2–16.4%) lasting for 40–
60 minutes of the test (Fig 3).
Influence of water soluble compounds 11a and 19a on rat
blood pressure was assessed for 60 minutes after i.v. injection
in dose of 2.5 mg/kg. In addition, compound 19a was tested
in dose of 10 mg/kg (used also in antiarrhythmic activity
test). Both compounds affected the normal blood pressure of
animals only for 1–3 minutes after administration. During
this time, compound 19a in higher dose (10 mg/kg) showed
undesirable sudden decrease of both systolic and diastolic
pressure causing death of 33% of investigated animals. Com-
pounds 11a and 19a (2.5 mg/kg) did not possess hypotensive
property demonstrating just one weak statistically significant
decrease of rat blood pressure (systolic and diastolic) in 2nd
minute of the measured time (Fig 4).
Elementary antiarrhythmic activity of obtained com-
pounds was evaluated for anesthetized rats, using the adrena-
line and barium chloride models of arrhythmia.
For examined rats, intravenous injections of high dose of
barium chloride (32 mg/kg) caused sinus bradycardia and a
rapid ventricular extrasystoles, ventricular tachycardia and
ventricular fibrillation (100%), which led to the death of all
animals within 3-5 minutes (Fig 5). The lead compound 7a
i.p. administrated showed similar activity in both barium
chloride and adrenaline-induced arrhythmia tests. In barium
chloride induced arrhythmia models, compound 7a inhibited
premature ventricular beats in 50% of tested animals, pre-
vented occurrence of ventricular fibrillation in 75% and also
diminished arrhythmogen caused mortality to 25%. In
adrenaline arrhythmia model (Fig 6), compound 7a de-
creased the number of sinal extrasystoles (44%) and pro-
tected against death all of the tested rat population. The
results are shown in Fig 7 and Fig 8.
Among obtained compounds only one (9a) was more
effective in BaCl2-induced arrhythmia test than 7a. This
compound 9a decreased number of extrasystoles (50%), to-
tally reduced incidence of ventricular fibrillation and pro-
tected against death 100% of tested animals. Significant
Fig. 4. Hypotensive activity of compounds tested after i. v. administration in anaesthetised normotensive rats.

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T. Dyla¸g et al. / European Journal of Medicinal Chemistry 39 (2004) 1013–1027
Fig. 5
.
Heart rhythm disturbances caused by intravenous injection of high dose of barium chloride (32 mg/kg i.v.). The surface ECG lead II with paper speed
50 mm/s and display amplification 1 mV/cm are shown.
activity was shown by i.v. injected compound 11a, prevent-
ing the appearance of barium chloride arrhythmia in 50% of
animals which also diminished their mortality to 25%. Com-
pounds 12a and 16a in dose of 50 mg/kg i.p. caused the
return of sinal rhythm and prolonged the survival time for
more than 60% of tested rats. Compounds 8a, 10a, 17a, 18a
possessed weak antiarrhythmic properties after i.p. adminis-
tration, slightly reducing number of barium chloride induced
rhythm disturbances and mortality. The lowest activity was
demonstrated by compound 19a, which was not active at
dose of 10 mg/kg i.v. in 100% of tested animals following
their death within 2–4 minutes after BaCl2 administration
(Fig 7).
Rapid intravenous injection of adrenaline at dose of
20 µg/kg caused anesthetized reflex bradycardia (100%),
atrioventricular disturbances, ventricular and supraventricu-
lar extrasystoles (94%), bigeminy (50%) which led to death
of approx. 50% of animals within 10 5 minutes.
The compound 19a (10 mg/kg i.v.) was the most active in
adrenaline test, preventing the appearance of adrenaline-

T. Dyla¸g et al. / European Journal of Medicinal Chemistry 39 (2004) 1013–1027
1019
Fig. 6
. Heart rhythm disturbances caused by rapid intravenous injection of adrenaline ( 20 µg/kg i.v.). The surface ECG lead II with paper speed 50 mm/s and
display amplification 1 mV/cm are shown.

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T. Dyla¸g et al. / European Journal of Medicinal Chemistry 39 (2004) 1013–1027
The conducted preliminary studies showed that some of
the compounds slightly decreased the heart rate, lengthened
the time of duration of P-Q and Q-T intervals. The strongest
effects were produced by compounds 8a, 11a, 12a, 16a, 17a,
18a and 19a, which reduced significantly the heart rate (8a,
11a, 19a), prolonged the P-Q (11a, 16a, 17a, 18a) and Q-T
(8a, 12a, 16a, 19a) intervals, without affecting the QRS
complex; only compound 12a shortened the QRS complex.
Compounds of class Ib reveal little effect on phase 0 (normal
tissues) and have negligible or shortening effect on repolar-
ization. Thus, class Ib antiarrhythmic drugs markes no signi-
ficant influence on P-Q and QRS and tends to decrease Q-T
interval [13-15]. The presented group of new compounds
rather prolonged Q-T interval (Table I, Table 2), being simi-
lar to class Ia [8,13-15]. In contrast to class Ia, these com-
pounds did not significantly prolong and sometimes even
shorten (12a) QRS complex. In case of compounds 8a, 16a,
19a, the prolongation of Q-T and lack of influence on QRS
complex can indicate some similarities to drugs of class III of
Vaughan-Williams classification.[1,13-15].
Other compounds (9a, 10a) marked no significant effect
on P-Q, QRS and Q-T intervals. Especially, the electrocar-
diographic changes, observed after administration of 10a,
were similar to those observed for phenytoin, which usually
exerts no significant effect on P-Q and QRS duration, whe-
reas the Q-T is unaltered or slightly shortened [15].
An analysis of the results shows that the type and position
of substituent in phenylpiperazine phenyl ring as well as the
substituent in 3 position of hydantoin moiety play an impor-
tant role in the pharmacological activity of the investigated
compounds.
Fig. 7. Prophylactic antiarrhythmic activity in barium-induced arrhythmia.
induced arrhythmia symptoms in 100% of tested rats. The
other active compound, namely 11a, decreased number of
adrenaline induced cardiac rhythm disturbances and mortal-
ity to 25%. Among compounds administrated intraperito-
neally, two of them diminished adrenaline induced premature
ventricular beats (9a, 10a) for more than 50% of investigated
rats. The lowest activity in adrenaline induced model of
arrhythmia was shown by compound 12a (Fig 8).
Antiepileptic properties of compounds 9a, 10a and 16a
were tested in Phase I of ADD Program. Compounds 9a and
16a were devoid of anticonvulsant activity in the used tests
and did not show neurological toxicity. Compound 10a was
active in ScMet test in dose of 100 mg/kg after 0.5 hour and
demonstrated neurological toxicity in dose of 100 mg/kg and
300 mg/kg after 0.5 hour and 4 hours, respectively.
For three isomers 8a, 9a, 10a, ortho (8a), meta (9a) and
para (10a) chlorophenylopiperazine derivatives of lead com-
pound 7a, a significant influence of the chloride position on
expected pharmacological properties was observed . The
para isomer demonstrated weak antiarrhythmic activity, lack
of hypotensive property and significant influence on normal
rat ECG. Results of Phase I of anticonvulsant assay ascribe
this para-chloro isomer to I class antiepileptic drugs, accen-
tuating its clear neurological toxicity. This finding suggests
that para position of substituent in phenylpiperazine ring
may cause undesirable interaction with CNS and does not
enhance expected antiarrhythmic and hypotensive efficacy.
Pharmacological test results for ortho and meta isomers are
in agreement with this hypothesis. The meta-chloro deriva-
tive (9a) shows neither anticonvulsant activity nor neurolo-
gical toxicity in the Phase I anticonvulsant assay but displays
the highest antiarrhythmic properties among considered iso-
mers. Especially, in whole series of described compounds,
the 9a is the most potent in barium chloride-induced arrhyth-
mia tests. This may suggest that meta substituent in phenyl-
piperazine phenyl moiety is profitable for antiarrhythmic
action in barium chloride arrhythmia model.
5. Discussion
The introduction of new moieties into 1- and 3- position of
hydantoin ring changed pharmacological properties of phe-
nytoin which belongs to class Ib of antiarrhythmic agents.
The ventricular tachyarrhythmias, produced by barium
chloride, are most severe and hard to influence. The mecha-
nism by which barium chloride exerts its arrhythmogenic
Fig. 8
mia.
. Prophylactic antiarrhythmic activity in adrenaline-induced arrhyth-

T. Dyla¸g et al. / European Journal of Medicinal Chemistry 39 (2004) 1013–1027
1021
action is complex and not fully understood. It was postulated
that – in Purkinje fibers of the sheep – the changes caused by
barium ions are mainly brought about by an increase in
sodium inward current [16]. Barium ions decrease the
outward potassium currents, blocking the ultra-rapid delayed
rectifier K+ channels and inwardly rectifying K+ channels
(Kir), pacemaker K+ channels (IK), background K+ channels
(IK1), ACh-induced K+ current (IK,ACh), calcium-activated
K+ channels (ICa) and ATP-sensitive K+ channels ( IK,ATP),
fatty acids-activated K+ channels (IAA) [17,18]. Blocking the
potassium channels by barium ions causes rapid diastolic
depolarization and initiation of spontaneous repetitive acti-
vity by mechanisms of early afterdepolarizations and spon-
taneous phase 4 depolarization (abnormal automaticity).As a
consequence of its K+ chanel-blocking action, barium pro-
longs Q-T interval (Fig 5) [19]. It is known that local anes-
thetic antiarrhythmic drugs (lidocaine, phenytoin), blocking
Na+ channel, reduce the Na+ current and abolish early- and
delayed after depolarization (EADs and DADs).
Some of the obtained compounds (especially 7a, 9a, 12a,
16a) delayed the onset time of this arrhythmia, reduced the
incidence of ventricular tachycardia and ventricular fibrilla-
tion, prolonged the survival time and prevented or dimin-
ished mortality caused by BaCl2.
Compounds 19a, 11a, 9a, 10a diminished or prevented
the appearance of adrenaline-induced arrhythmia symptoms.
Adrenaline is an agonist of b-adrenergic receptors (b-AR),
which are located also in heart muscle tissue. b-adrenergic
stimulation is one of the most important regulatory mecha-
nisms of ions channel function. In the heart, the stimulation
activity. On contrary, the water insoluble compounds 17a as
well as its ortho-chloro analogue 18a, are the most potent
hypotensive agents among all tested series but display weak
antiarrhythmic activity. The third pair of analogue com-
pounds, namely phenylpiperazine (12a) and 2-methoxy-
phenylpiperazine (16a) derivatives of 3-hydantoin ethyl ace-
tate, is insoluble in water. Both compounds are almost
equiactive in the tests showing very low antiarrhythmic ac-
tion, and are bereft of hypotensive activity. Considering a
behaviour of three pairs of phenylpiperazine and
2-methoxyphenylpiperazine analogues, it appears that not
simply presence of ortho-methoxy group but rather increased
water solubility caused by this group enhances antiarrhyth-
mic and decreases hypotensive properties of the compounds.
Although the increase of antiarrhythmic property of soluble
ortho-methoxyphenylpiperazine derivatives is in agreement
with other works [21], the fall in hypotensive properties is a
strange inverse.
According to the in vivo results, a substituent in 3 position
of hydantoin ring is also related to the pharmacological
efficacy. Replacement of lead ethyl group (7a) with ethyl
2-propionate (17a) improves its antihypertensive activity but
introduction of ethyl acetate (12a) enhances neither antihy-
pertensive nor antiarrhythmic properties of the compound.
The similar trend is seen for 2-chlorophenylpiperazine deri-
vatives (8a, 18a). Derivatives of 2-methoxyphenylpiperazine
(11a, 16a, 19a) demonstrated similar behaviour in case of
adrenaline induced arrhythmia test. The water insoluble ethyl
acetate derivative 16a is significantly less active than the
ethyl one (11a), which in turn is slightly less active than the
compound 19a, possessing ethyl 2-propionate moiety.
Searching for effective antiarrhythmic drugs, it is needful
to assess a protection against arrhythmogens as well as an
influence of investigated compounds on ECG components.
Especially, an influence of a compound on Q-T interval is an
important criterion/issue. Lengthening of the Q-T interval
causes no symptoms but both the congenital and the aquired
form of the long Q-T syndrom (LQTS) can precipitate life-
threatening polymorphic ventricular tachycardia known as
torsade de pointes. Thus, three obtained compounds 9a, 11a
and 19a are particularly promising in the search for novel
antiarrhythmic agents. Those compounds displayed the
strongest antiarrhythmic activity in barium chloride- (9a) or
adrenaline (11a, 19a) induced arrhythmia models and they
hardly influenced the ECG components. Compound 19a dis-
played weak statistically significant prolongation of Q-T
interval only in the very first minute after administration.
Compound 11a did not cause significant lengthening of Q-T
interval, while the 9a one caused no statistically significant
change of any ECG component. It can suggest lack of proar-
rhytmic properties of these compounds.
of b-AR increases the magnitude of ICa-L, If, ICl , IKur, IKATP
,
Ist and these actions are mediated by cAMP-dependent pro-
tein kinase [17,18]. b-adrenergic stimulation, via voltage-
dependent Ca+, IKur and also Na+ channels, increases the
probability of a variety of supraventricular and ventricular
arrhythmias. Based on our results, we hypothesize that the
antiarrhythmic effects of these tested compounds are prob-
ably related to the arrest of the intraflow of Na+ and/or Ca2+
in the cardiac cells and the depression of their cardiac auto-
arrhythmicity and conductivity. As a consequence of over-
dose of adrenaline in rats, prolongation of P-Q, QRS and Q-T
intervals with concomitant decrease of heart rate was obser-
ved (Fig 6) [20].
In case of compound 11a, the introduction of methoxy
group in ortho position of lead phenyl ring significantly
improves its antiarrhythmic property in adrenaline model but
deprives the compound of antihypertensive potency. This
change of pharmacological properties may be evoked by
increase of water solubility. The similar trend is seen for
compounds 17a and 19a, possessing ethyl propionate moiety
in 3 position of hydantoin ring, and phenylpiperazine (17a)
or ortho-methoxyphenylpiperazine (19a) at the end of hy-
droxypropyl chain. The water soluble compound 19a
demonstrates the strongest antiarrhythmic efficacy against
adrenaline arrhythmogen among all the presented com-
pounds and is an obstacle to expected antihypertensive
The presented work is an introduction into the new family
of phenylpiperazine derivatives of 5,5-diphenylhydantoin.
The performed, on preliminary level, pharmacological tests,
divided the derivatives of lead 7a into five groups of different
activities: hypotensive (17a, 18a), antiarrhythmic in barium

1022
T. Dyla¸g et al. / European Journal of Medicinal Chemistry 39 (2004) 1013–1027
chloride arrhythmia model (9a), antiarrhythmic in adrenaline
model (19a, 11a), anticonvulsant (10a) and compounds of
low activity in presented tests (8a, 12a, 16a).
distilled epichlorohydrin (4.65 g, 50 mmol) in acetone
(25 ml) was added dropwise. After being stirred for the next
7.5 h, the precipitate was removed by filtration and the filtrate
was condensed to yield oily residue which was purified by
means of column chromatography (mobile phase: CHCl3).
Fractions containing product were concentrated in vacuo to
afford 6 in the form of oil, which was used for further
reactions.
6. Experimental
1H-NMR and H,H-COSY spectra were recorded on Va-
rian Mercury VX 300 MHz PFG instrument in CDCl3 at
ambient temperature. For H,H-COSY experiment of 12, 4 re-
petitions of 256 increments were performed. Chemical shifts
are given in parts per million relative to tetramethylsilane,
coupling constants are given in Hz. IR spectra were recorded
on a Jasco FT/IR-410 apparatus using KBr pellets and are
reported in cm–1. For column chromatography Merck silica
gel 60 (Merck) of seed diameter 0.063–0.200 mm [mesh
70–230 ASTM] was used. Thin-layer chromatography was
performed on pre-coated Merck silica gel 60 F254 alu-
minium plates, the used solvent systems were: (A)
toluene/acetone/methanol 5:5:1; (B) chloroform/ethyl ace-
tate 1:1. Melting points are uncorrected. Analyses indicated
by the symbols of the elements or functions were wi-
thin 0.4% of the theoretical values unless stated otherwise.
IR spectra were recorded for hydrochloride salts, NMR
spectra were recorded for free bases unless stated otherwise.
6.1.3. Synthesis of N1-phenylpiperazinealkyl-N3-alkyl-5,5-
diphenylhydantoin derivatives (7a–19a)
Commercially available monohydrochloride salts of N-2-
and N-3-chlorophenylpiperazine as well as N-4-chloro-
phenylpiperazine dihydrochloride were converted into free
bases according to the method described by Pawłowski et al.
[22].
6.1.3.1. 3-Ethyl-1-[2-hydroxy-3-(4-phenylpiperazin-1-yl)-
propyl]-2,4-dioxo-5,5-diphenylimidazolidine hydrochloride
(7a). Method B: A mixture of 4 (1.68 g, 5 mmol) and
N-phenylpiperazine (0.81 g, 5 mmol) was prepared in a
flat-bottomed 50 ml flask covered with a small inverted
funnel. The flask along with a water-filled beaker were pla-
ced in a standard household microwave oven and irradiated
(300W) for 3 minutes altogether. The presence of the product
was observed on TLC plates after increasing the power and
so irradiation was continued for the next 2 minutes at 450 W.
Longer heating did not cause any increase of the product spot
(TLC) intensity. The reaction mixture was crystallized from
EtOH to afford 1.42 g of pure 7. M.p. 136–138 ºC; yield 57%,
TLC: Rf(A) = 0.75; 1H-NMR (CDCl3, TMS) d (ppm) of base
7: 1.27 (t, J = 7.14, 3H, CH2–CH3), 2.05 (dd def., 1H,
N–CH2(a)–CHOH), 2.20–2.27 (m, 1H, N–CH2(b)–CHOH),
2.28–2.48 (m, 4H, 2 x (CH2)–N–CH2–CHOH), 3.06 (t,
J = 4.94, 4H, 2 x Ph–N–(CH2)–), 3.00–3.18 (m, 1H, CHOH),
3.40–3.59 (m, 2H, CHOH–CH2–N), 3.60–3.75 (m, 3H,
CH2–CH3, CHOH), 6.80–6.92 (m, 3H, Ar–H2, Ar–H4,
Ar–H6 Ph-piper), 7.20–7.34 (m, 6H, 2 x Ar–H2, 2 x Ar–H6
DPH;Ar–H3, Ar–H5 Ph-piper), 7.36–7.46 (m, 6H, 2 xAr–H3,
2 x Ar–H4, 2 x Ar–H5 DPH). Concentrated hydrochloric acid
was added to solid 7 and the mixture was evaporated to
dryness. The residue was recrystallized from EtOH to quan-
titatively afford pure hydrochloride salt 7a, m.p. 245–248 ºC.
IR: 3467 (OH), 3339 (OH), 2973 (CH), 2160, 1766 (C2=O),
1710 (C4=O), 1597 (Ar).
6.1. Chemistry
6.1.1. Synthesis of N3-substituted 5,5-diphenylhydantoin
derivatives (1–3)
Compounds 1–2 were prepared according to the proce-
dure described earlier [7,8].
6.1.1.1. 2-(2,5-Dioxo-4,4-diphenylimidazolidin-1-yl)-pro-
pionic acid ethyl ester (3). A mixture of DPH (25.2 g,
0.1 mol), TEBA (3 g, 0.013 mol), potassium carbonate (40 g)
and acetone (500 ml) was refluxed for 1 hour. Next, a solution
of ethyl 2-bromopropionate (18.0 g, 0.1 mol) in 50 ml ace-
tone was added dropwise and the whole was refluxed for the
next 3 h. The solid residue was removed from the reaction
mixture, the filtrate was condensed and the oily residue was
recrystallized from EtOH to give 3 at 81.7% yield, m.p.
92–94 ºC, Rf(B) = 0.82.
6.1.2. Synthesis of N3- and N1-substituted
5,5-diphenylhydantoin derivatives (4–6)
Compounds 4–5 were prepared according to the proce-
dure described earlier [7,8].
6.1.3.2. 1-[3-(4-(2-Chlorophenyl)-piperazin-1-yl)-2-hydroxy-
propyl]-3-ethyl-2,4-dioxo-5,5-diphenylimidazolidine hydro-
chloride (8a). Method A:A mixture of 4 (1.68 g, 5 mmol) and
N-2-chlorophenylpiperazine (0.98 g, 5 mmol) in toluene
(15 ml) was refluxed for 10 h under TLC control. The solvent
was evaporated. The precipitate was washed with EtOH and
crystallized from the same solvent to obtain 1.44 g of 8; m.p.
131–133 ºC; yield 54%; TLC: Rf(A) = 0.75; 1H-NMR of base
(CDCl3, TMS) d (ppm): 1.27 (t, J = 7.14, 3H, CH2–CH3),
2.06 (dd def., 1H, N–CH2(a)–CHOH), 2.23 (dd def., 1H,
6.1.2.1. 2-[3-(2,3-Epoxypropyl)-2,5-dioxo-4,4-diphenylimi-
dazolidin-1-yl]-propionic acid ethyl ester (6). A suspension
of 3 (14.8 g, 42 mmol), TEBA (1.26 g, 5.5 mmol) and
potassium carbonate (17 g) in acetone (120 ml) was stirred at
room temperature for 30 minutes, then, a solution of freshly

T. Dyla¸g et al. / European Journal of Medicinal Chemistry 39 (2004) 1013–1027
1023
N–CH2(b)–CHOH, 2.17–2.28 (m, 4H, 2 x (CH2)–N–CH2–
CHOH), 2.93 (br.s, 4H, 2 x Ph–N–(CH2)), 3.02–3.16 (m, 1H,
CHOH), 3.38–3.58 (m, 2H, CHOH–CH2–N), 3.65 (m, 3H,
CH2–CH3, CHOH), 6.90–7.00 (m, 2H, Ar–H4, Ar–H6 Ph-
piper), 7.15–7.24 (m, 2H, Ar–H3, Ar–H5 Ph-piper), 7.24–
7.37 (m, 4H, 2 x Ar–H2, Ar–H6 DPH), 7.37–7.47 (m, 6H, 2 x
Ar–H3, Ar–H4, Ar–H5 DPH).
8a was obtained by the same method as 7a; m.p. 255–258
ºC; IR: 3413, 3243 (OH), 2958, 2919, 2839 (CH), 2552
(NH+), 1769 (C2=O), 1713 (C4=O), 1589 (Ar); 1H-NMR of
hydrochloride (CDCl3, TMS) d (ppm): 1.25 (t, J = 7.14, 3H,
CH2–CH3), 2.54–2.96 (m, 3H, N–CH2–CHOH, CHOH),
2.96–3.16 (m, 2H, CHOH–CH2–N), 3.18–3.38 (m, 2H, 2 x
Ph–N–(CH2(a))–), 3.40–3.60 (t def, 4H, 2 x Ph–N–(CH2(b)),
2 x CH2(a)–NH+–CH2–CHOH), 3.60–3.78 (m, 4H, 2 x
CH2(b)–NH+–CH2–CHOH, CH2–CH3), 5.27 (br s, 1H, OH),
6.95–7.04 (t, 2H, J = 7.42, Ar–H4, Ar–H6 Ph-piper), 7.20–
7.28 (m, 1H, Ar–H5 Ph-piper), 7.36 (d, J = 7.97, 1H, Ar–H3
Ph-piper), 7.10–7.50 (m, 10H, Ar–H DPH), 11.78 (br s, 1H,
NH+).
N-4-chlorophenylpiperazine (0.90 g, 4.6 mmol) as substra-
tes. Column chromatography (CHCl3:AcOEt 1:1) and crys-
tallization form EtOH afforded 0.60 g of 10; yield 24%.
Method B*: Compound 4 (2.67 g, 7.9 mmol) and N-4-
chlorophenylpiperazine (1.56 g, 7.9 mmol) were placed in a
flat-bottomed flask covered with an inverted funnel and mel-
ted by irradiating (450 W) twice for 1 minute. The whole was
then irradiated for the next 50 s (300 W). A product was
precipitated by 96% EtOH from the glassy residue and re-
crystallized from the same solvent to give 2.17 g of 10; m.p.
139–141 ºC; yield 52%; TLC Rf(A) = 0.70; 1H-NMR
(CDCl3, TMS) d (ppm): 1,26 (t, J = 7.14, 3H, CH2–CH3),
2.03 (dd, J1 = 12.36, J2 = 4.94, 1H, N–CH2(a)–CHOH), 2.22
(dd, J1 = 8.79, J2 = 1.64, 1H, N–CH2(b)–CHOH), 2.24–2.46
(m, 4H, 2 x (CH2)–N–CH2–CHOH), 3.03 (t def., 4H, 2 x
Ph–N–(CH2)–), 3.03–3.16 (m, 1H, CHOH), 3.38–3.60 (m,
2H, CHOH–CH2–N), 3.62 (m, 1H, CHOH), 3.67 (q, J = 7.14,
2H, CH2–CH3), 6.76–6.80 (m, 2H, Ar–H2, Ar–H6 Ph-piper),
7.14–7.22 (m, 2H, Ar–H3, Ar–H5 Ph-piper), 7.24–7.34 (m,
4H, 2 x Ar–H2, 2 x Ar–H6 DPH), 7.39–7.42 (m, 6H, 2 x
Ar–H3, 2 x Ar–H4, 2 x Ar–H5 DPH).
6.1.3.3. 1-[3-(4-(3-Chlorophenyl)-piperazin-1-yl)-2-hydro-
xypropyl]-3-ethyl-2,4-dioxo-5,5-diphenylimidazolidine
hydrochloride (9a). Method A: The reaction followed the
route used to obtain 8, with the use of 4 (1.55 g, 4.6 mmol)
and equimolar quantity of N-3-chlorophenylpiperazine
(0.90 g). After a part of the solvent was evaporated, the
reaction mixture was separated by column chromatography
(CHCl3/AcOEt 1:1). Fractions containing product were poo-
led and condensed to obtain the oily residue. Its recrystalli-
zation from 99.8% EtOH furnished 0.80 g of 9; yield 33%.
Method B*: A mixture of 4 (2.05 g, 6.2 mmol) and
N-3-chlorophenylpiperazine (1.20 g, 6.2 mmol) was prepa-
red in a flat-bottomed flask covered with a small inverted
funnel. The whole was irradiated (450 W) to melt into a
homogenous liquid and then irradiation was continued for
2 minutes (300 W). The reaction progress was controlled
with TLC. The glasslike mass was crystallized from EtOH to
obtain 2.14 g of 9; yield 65%; m.p. 139–141 ºC; TLC:
10a was obtained by the same method as 7a; m.p. 260–
262 ºC; IR: 3561, 3388, 3245 (OH), 2937, 2829 (CH), 2576,
2461 (NH+), 1772 (C2=O), 1712 (C4=O), 1599 (Ar).
6.1.3.5. 3-Ethyl-1-[2-hydroxy-3-(4-(2-methoxyphenyl)-pipe-
razin-1-yl)-propyl]-2,4-dioxo-5,5-diphenylimidazolidine
hydrochloride (11a). Method A: A mixture of 4 (1.68 g,
5 mmol) and N-2-methoxyphenylpiperazine (0.96 g,
5 mmol) in toluene (15 ml) was stirred at reflux for 6.5 h. The
solvent was evaporated. Washing with diethyl ether and re-
crystallization from ethanol did not afford crystals. Concen-
trated hydrochlorid acid was added to the oily residue and the
mixture was evaporated to dryness. The residue was recrys-
tallized from EtOH to afford hydrochloride 11a; m.p. 212–
216 ºC; yield 38.2 %; TLC: Rf(A) = 0.67; IR: 3527, 3394,
3275 (OH), 2962 (CH), 2832, 2662, 2583, 2457 (NH+), 1769
1
(C2=O), 1713 (C4=O), 1594 (Ar); H-NMR of hydrochlo-
1
ride: (CDCl3, TMS) d (ppm): 1.21–1.28 (m, 5H, CH2–CH3
and 1/2 CH3–CH2–OH), 2.52–2.86 (m, 3H, NH+–CH2–
CHOH, CHOH), 2.98–3.12 (d, J = 10.71, 2H, CHOH–CH2–
N), 3.25–3.58 (m, 6H, H piper), 3.59–3.76 (m, 5H, H piper,
CH2–CH3, 1/2 CH3–CH2–OH), 3.87 (s, 3H, OCH3), 5.3 (s,
1H, OH), 6.84–6.96 (m, 3H, Ar–H3, Ar–H5, Ar–H6 Ph-
piper), 7.02–7.11 (m, 1H, Ar–H4 Ph-piper), 7.18–7.32 (m,
4H, 2 x Ar–H2, 2 x Ar–H6 DPH), 7.38–7.50 (m, 6H, 2 x
Ar–H3, 2 x Ar–H4, 2 x Ar–H5 DPH), 11.61 (br s, 1H, NH+).
Rf(A) = 0.78; H-NMR (CDCl3, TMS) d (ppm): 1.27 (t,
J = 6.87, 3H, CH2–CH3), 2.03 (dd, J1 = 12.36, J2 = 4.94, 2H,
N–CH2(a)–CHOH), 2.16–2.44 (m, 5H, N–CH2(b)–CHOH,
2 x (CH2)–N–CH2–CHOH), 3.06 (t, J = 4,94, 4H, 2 x Ph–N–
(CH2)–), 3.01–3.16 (m, 1H, CHOH), 3.40–3.58 (m, 2H,
CHOH–CH2–N), 3.58–3.75 (m, 3H, CH2–CH3, CHOH),
6.68–6.83 (m, 3H, Ar–H2, Ar–H4, Ar–H6 Ph-piper), 7.14 (t,
J = 7.97, 1H, Ar–H5 Ph-piper), 7.22–7.34 (m, 4H, 2 xAr–H2,
2 x Ar–H6 DPH), 7.36–7.45 (m, 6H, 2 x Ar–H3, 2 x Ar–H4,
2 x Ar–H5 DPH).
6.1.3.6. {3-[2-Hydroxy-3-(4-phenylpiperazin-1-yl)-propyl]-
2,5-dioxo-4,4-diphenylimidazolidin-1-yl}-acetic acid ethyl
ester hydrochloride(12a). Method A: A mixture of 5 (1.97 g,
5 mmol) and N-phenylpiperazine (0.81 g, 5 mmol) in toluene
(15 ml) was refluxed for 9.5 h. After solvent evaporation,
double recrystallization from ethanol afforded 2.13 g of ana-
lytically pure 12; m.p. 95–99 ºC; yield 64%; TLC
9a was obtained by the same method as 7a; m.p. 230–232
ºC; IR: 3323, 3244 (OH), 2918 (CH), 2549, 2457 (NH+),
1770 (C2=O), 1711 (C4=O), 1595 (Ar).
6.1.3.4. 1-[3-(4-(4-Chlorophenyl)-piperazin-1-yl)-2-hydro-
xypropyl]-3-ethyl-2,4-dioxo-5,5-diphenylimidazolidine hy-
drochloride (10a). Method A: Synthesis was analogous to
that of 9, but with the use of 4 (1.55 g, 4.6 mmol) and
1
Rf(A) = 0.73; Rf(B) = 0.27; H-NMR (CDCl3, TMS) d

1024
T. Dyla¸g et al. / European Journal of Medicinal Chemistry 39 (2004) 1013–1027
(ppm): 1.22 (t, J = 7.14, 3H, CH2–CH3), 1.29 (t, J = 7.14, 3H,
CH3–CH2–OH), 2.03–2.14 (dd def, 1H, N–CH2(a)–CHOH),
2.19–2.26 (m, 1H, N–CH2(b)–CHOH), 2.27–2.50 (m, 4H, 2 x
(CH2)–N–CH2–CHOH), 3.05 (t, J = 4.94, 4H, 2 x Ph–N–
(CH2)–), 3.00–3.10 (m, 1H, CHOH), 3.52 (d, J = 6.32, 2H,
CHOH–CH2–N), 3.69 (q def., 2H, CH3–CH2–OH), 4.24 (q,
J = 7.14, 2H, CH2–CH3), 4.32 (s, 2H, N–CH2–COO), 6.80–
6.90 (m, 3H, Ar–H2, Ar–H4, Ar–H6 Ph-piper), 7.20–7.28 (m,
2H, Ar–H3, Ar–H5 Ph-piper), 7.32–7.50 (m, 10H, Ar–H
DPH). Ascription of 1H-NMR signals to particular groups of
protons was facilitated by H,H-COSY spectrum.
Dry ethanol (20 ml) was added to a 1 g of 12. The
suspension was saturated with dried gaseous hydrogen chlo-
ride until acidic pH was obtained and, then, the solid was
separated by filtration to quantitatively obtain hydrochloride
12a; m.p. 236–238 ºC; yield 64%; IR: 3256 (OH), 2979
(CH), 2500, 2441 (NH+), 1776 (C2=O), 1748 (C=O ester),
1725 (C4=O), 1597 (Ar).
1.33 (m, 4H, CH2–CH3 + CH3–CH2–OH), 2.08 (dd def., 1H,
N–CH2(a)–CHOH), 2.16–2.24 (m, 1H, N–CH2(b)–CHOH),
2.24–2.45 (m, 4H, 2 x (CH2)–N–CH2–CHOH), 3.03–3.06
(m, 5H, 2 x Ph–N–(CH2), CHOH), 3.50 (d, J = 6.32, 1H,
CHOH–CH2–N), 3.72 (q, J = 7.14, 1H, CH3–CH2–OH), 4.25
(q, J = 7.14, 2H, CH2–CH3), 4.32 (s, 2H, N–CH2–COO),
6.68–6.83 (m, 3H, Ar–H2, Ar–H4, Ar–H6 Ph-piper), 7.14 (t,
J = 7.97, 1H, Ar–H5 Ph-piper), 7.38–7.45 (m, 10H, Ar–H
DPH).
14a was obtained by the same method as 12a; m.p. 235–
238 ºC; IR: 3420, 3254 (OH), 2979, 2927, 2851 (CH), 2544,
2445, 2364 (NH+), 1775 (C2=O), 1745 (C=O ester), 1719
(C4=O), 1596 (Ar).
6.1.3.9. {3-[3-(4-(4-Chlorophenyl)-piperazin-1-yl)-2-hydro-
xypropyl]-2,5-dioxo-4,4-diphenylimidazolidin-1-yl}-acetic
acid ethyl ester hydrochloride (15a). Method B: The synthe-
sis followed the method used to obtain 14. As substrates 5
(1.65 g, 4.2 mmol) and N-4-chlorophenylpiperazine (0.82 g,
4.2 mmol) were used to furnish 1.35 g of 15; m.p. 115–118
ºC; yield 54%; TLC: Rf(A) = 0.77; 1H-NMR (CDCl3, TMS)
d (ppm): 1.29 (t, J = 7.14, 3H, CH2–CH3), 2.08 (dd def., 2H,
N–CH2(a)–CHOH), 2.21 (dd def., 2H, N–CH2(b)–CHOH),
2.27–2.45 (m, 4H, 2 x (CH2)–N–CH2–CHOH), 2.97–3.02
(m, 4H, 2 x Ph–N–(CH2)–), 2.96–3.12 (m, 1H, CHOH), 3.51
(d, J = 6.32, 2H, CHOH–CH2–N), 4.25 (q, J = 7.14, 2H,
CH2–CH3), 4.32 (s, 2H, N–CH2–COO), 6.78 (d, J = 9.06,
2H, Ar–H2, Ar–H6 Ph-piper), 7.18 (d, J = 8.79, 2H, Ar–H3,
Ar–H5 Ph-piper), 7.36–7.44 (m, 10H, 2 x Ar–H DPH).
15a was obtained by the same method as 12a; m.p. 223–
225 ºC; IR: 3431, 3210 (OH), 2985, 2937, 2843 (CH), 2522,
2442 (NH+), 1775 (C2=O), 1740 (C=O ester), 1718 (C4=O),
1598 (Ar).
6.1.3.7. {3-[3-(4-(2-Chlorophenyl)-piperazin-1-yl)-2-hydro-
xypropyl]-2,5-dioxo-4,4-diphenylimidazolidin-1-yl}-acetic
acid ethyl ester hydrochloride (13a). Method B: In a flat-
bottomed flask
5 (3.28 g, 8.32 mmol) and N-2-
chlorophenylpiperazine (1.64 g, 8.32 mmol) were mixed and
melted on a heating jacket. The flask was then covered with
an inverted funnel and irradiated in a domestic microwave
oven (450 W) for consecutive 5 and 4 minutes; additionally, a
beaker with water was present in the oven to avoid overhea-
ting of the system. The crude product was isolated by column
chromatography (CHCl3/AcOEt 1:1) and recrystallized from
EtOH to furnish 1.68 g of 13; m.p. 106–109 ºC; yield 34%;
TLC: Rf(A) = 0.80; Rf(B) = 0.21; 1H-NMR (CDCl3, TMS) d
(ppm): 1.30 (t, J = 7.14, 3H, CH2–CH3), 2.11 (dd def., 1H,
N–CH2(a)–CHOH), 2.19–2.26 (m, 1H, N–CH2(b)–CHOH),
2.29–2.52 (m, 4H, 2 x (CH2)–N–CH2–CHOH), 2.93 (br s,
4H, 2 x Ph–N–(CH2)–), 3.00–3.12 (m, 1H, CHOH), 3.42–
3.60 (m, 2H, CHOH–CH2–N), 4.26 (q, J = 7.14, 2H, CH2–
CH3), 4.32 (s, 2H, N–CH2–COO), 6.92–6.99 (m, 2H, Ar–H4,
Ar–H6 Ph-piper), 7.16–7.23 (m, 1H, Ar–H5 Ph-piper), 7.31–
7.36 (dd, J1 = 7.79, J2 = 1.65, 1H,Ar-H3 Ph-piper), 7.37–7.45
(m, 10H, Ar–H DPH).
6.1.3.10. {3-[2-Hydroxy-3-(4-(2-methoxyphenyl)-piperazin-
1-yl)-propyl]-2,5-dioxo-4,4-diphenylimidazolidin-1-yl}-ace-
tic acid ethyl ester hydrochloride (16a). Method A: A
mixture of
5 (1.97 g, 5 mmol) and 2-methoxy-
phenylpiperazine (0.96 g, 5 mmol) in toluene (15 ml) was
refluxed for 10 hours altogether. The solvent was evaporated
from the reaction mixture and the residue was crystallized
twice from EtOH to give 0.71 g of analytically pure 16; yield
55%.
13a was obtained by the same method as 12a; m.p. 198–
210 ºC; IR: 3587, 3437, 3218 (OH), 2986, 2961, 2841 (CH),
2522, 2443 (NH+), 1776 (C2=O), 1748 (C=O ester), 1719
(C4=O), 1589 (Ar).
Method B*: The synthesis followed, in general, the
method used to obtain 10. The substrates 5 (3.94 g, 10 mmol)
and N-2-methoxyphenylpiperazine (1.92 g, 10 mmol) were
melted by three-minute irradiation (450 W) and were further
irradiated (300 W) for 1 minute. Crystallization from EtOH
furnished 4.45 g of 16; m.p. 137–138 ºC; yield 76%, TLC:
6.1.3.8. {3-[3-(4-(3-Chlorophenyl)-piperazin-1-yl)-2-hydro-
xypropyl]-2,5-dioxo-4,4-diphenylimidazolidin-1-yl}-acetic
acid ethyl ester hydrochloride (14a). Method B: Compound
5 (1.62 g, 4.1 mmol) and N-3-chlorophenylpiperazine
(0.81 g, 4.1 mmol) were melted on a heating jacket to obtain
oily mass and, then, they were irradiated twice for 5 minutes
in a domestic microwave oven (450 W, under cover of an
inverted funnel). The oily reaction mixture was crystallized
from EtOH to obtain 1.50 g of 14; m.p. 80–83 ºC; yield 62%;
TLC: Rf(A) = 0.84; 1H-NMR (CDCl3, TMS) d (ppm): 1.20–
1
Rf(A) = 0.62; H-NMR (CDCl3, TMS) d (ppm): 1.29 (t,
J = 7.14, 3H, CH2–CH3), 2.09 (dd def., 2H, N–CH2(a)
–
CHOH), 2.17–2.24 (m, 2H, N–CH2(b)–CHOH), 2.30–2.55
(m, 4H, 2 x (CH2)–N–CH2–CHOH), 2.94 (br s, 4H, 2 x
Ph–N–(CH2)–), 3.02–3.12 (m, 1H, CHOH), 3.51 (t def., 2H,
CHOH–CH2–N), 3.61 (br s, 1H, CHOH), 3.85 (s, 3H,
OCH3), 4.25 (q, J = 7.14, 2H, CH2–CH3), 4.32 (s, 2H,

T. Dyla¸g et al. / European Journal of Medicinal Chemistry 39 (2004) 1013–1027
1025
N–CH2–COO), 6.82–6.93 (m, 3H, Ar–H3, Ar–H5, Ar–H6
Ph-piper), 6.95–7.02 (m, 1H, Ar–H4 Ph-piper), 7.39–7.45
(m, 10H, 2 x Ar–H DPH).
16a was obtained by the same method as 12a; m.p. 240–
242 ºC, IR: 3195 (OH), 2957, 2829 (CH), 2451 (NH+), 1775
(C2=O), 1751 (C=O ester), 1721 (C4=O), 1593 (Ar).
6.1.3.13. 2-{3-[2-Hydroxy-3-(4-(2-methoxyphenyl)-pipera-
zin-1-yl)-propyl]-2,5-dioxo-4,4-diphenylimidazolidin-1-yl}-
propionic acid ethyl ester hydrochloride (19a). Method B:
The reaction followed the method used to obtain 14 with
substrates 6 (2.05 g, 5 mmol) and N-2-methoxyphenyl-
piperazine (0.96 g, 5 mmol). The reaction mixture was puri-
fied by column chromatography (CHCl3/AcOEt 1:1) to give
19 as oil, to which dry ethanol (25 ml) was added. The
mixture was saturated with dried gaseous hydrogen chloride
until precipitation in acidic pH took place and, then, the solid
was separated by filtration to obtain hydrochloride 19a; m.p.
192–196 ºC; yield 24%; TLC: Rf(A) = 0.75; IR: 3611, 3434,
3263 (OH), 2948, 2835 (CH), 2662, 2591, 2450 (NH+), 1773
6.1.3.11. 2-{3-[2-Hydroxy-3-(4-phenylpiperazin-1-yl)-pro-
pyl]-2,5-dioxo-4,4-diphenylimidazolidin-1-yl}-propionic
acid ethyl ester hydrochloride (17a). Method A: A suspen-
sion of 6 (3.40 g, 8.3 mmol) and N-phenylpiperazine (1.21 g,
7.5 mmol) in toluene (16.5 ml) was heated at reflux for 8 h.
After the solvent evaporation, the residue was purified by
column chromatography (CHCl3/AcOEt 1:1) to obtain
1.94 g of 17; m.p. 99–102 ºC; yield 45%; TLC: Rf(A) = 0.77;
1H-NMR (CDCl3, TMS) d (ppm): 1.23 (t, J = 7.14, 3H,
CH2–CH3), 1.65 (d, J = 7.42, 3H, CH–CH3), 2.02–2.12 (m,
1
(C2=O), 1740 (C=O ester), 1720 (C4=O), 1593 (Ar); H-
NMR of hydrochloride (CDCl3, TMS) d (ppm): 1.27 (q,
J = 7.15, 3H, CH2–CH3), 1.62–1.70 (m, 3H, CH–CH3),
2.55–2.90 (m, 3H, NH+–CH2–CHOH, CHOH), 2.95–3.15
(m, 2H, CHOH–CH2–N), 3.27–3.79 (m, 8H, H-piper), 3.87
(s, 3H, OCH3), 4.20–4.28 (m, 2H, CH2–CH3), 4.79–4.89 (m,
1H, CH–CH3), 5.37 (br s, 1H, OH), 6.85–6.96 (m, 3H,
Ar–H3, Ar–H5, Ar–H6 Ph-piper), 7.01–7.11 (m, 1H, Ar–H4
Ph-piper), 7.25–7.37 (m, 4H, 2 x Ar–H2, 2 x Ar–H6 DPH),
7.38–7.51 (m, 6H, 2 x Ar–H3, 2 x Ar–H4, 2 x Ar–H5 DPH),
11.49 (br s, 1H, NH+).
2H, N–CH2(a)–CHOH), 2.16–2.25 (m, 2H, N–CH2(b)
–
CHOH), 2.25–2.41 (m, 4H, 2 x (CH2)–N–CH2–CHOH),
3.01–3.07 (m, 5H, 2 x Ph–N–(CH2), CHOH), 3.46–3.60 (m,
2H, CHOH–CH2–N), 3.58 (s, 1H, CH2–CHOH–CH2), 4.14–
4.28 (m, 2H, CH2–CH3), 4.85 (q, J = 7.42, 1H, CH–CH3),
6.80–6.90 (m, 3H, Ar–H2, Ar–H4, Ar–H6 Ph-piper), 7.21–
7.28 (m, 2H, Ar–H3, Ar–H5 Ph-piper), 7.30–7.46 (m, 10H,
Ar–H DPH).
17a was obtained by the same method as 12a; m.p. 187–
192 ºC; IR: 3255 (OH), 2993, 2942 (CH), 2428 (NH+), 1771
(C2=O), 1744 (C=O ester), 1713 (C4=O), 1597 (Ar).
6.2. Pharmacology
6.2.1. Chemicals
Adrenaline (Adrenalinum hydrochloride, Polfa), barium
chloride (Barium chloratum, POCh), sodium chloride (Na-
trium chloratum, POCh), heparin (Heparinum natrium,
Polfa), methylcellulose (Methylcellulose, Fluka), thiopental
(Thiopentone, Biochemie GmbH).
6.1.3.12. 2-{3-[3-(4-(2-Chlorophenyl)-piperazin-1-yl)-2-
hydroxypropyl]-2,5-dioxo-4,4-diphenylimidazolidin-1-yl}-
propionic acid ethyl ester hydrochloride (18a). Method A:
A suspension of 6 (2.83 g, 6.93 mmol) and N-2-
chlorophenylpiperazine (1.21 g, 6.15 mmol) in toluene
(15 ml) was refluxed for 15.5 h. After evaporation of the
solvent, the reaction mixture was purified by column chro-
matography (CHCl3/AcOEt 1:1) to give 1.48 g of 18 in the
form of oil. A trial of recrystallization form EtOH did not
afford crystals. To the oil dry ethanol (25 ml) was added and
the mixture was saturated with dried gaseous hydrogen chlo-
ride until precipitation in acidic pH, then the solid was
removed by filtration to obtain hydrochloride 18a; m.p. 215–
218 ºC; yield 32%; TLC: Rf(A) = 0.74; IR: 3231 (OH), 2924
(CH), 2442 (NH+), 1772 (C2=O), 1717 (C4=O), 1588 (Ar).
1H-NMR of hydrochloride (CDCl3, TMS) d (ppm): 1.26–
1.32 (m, 3H, CH2–CH3), 1.64–1.80 (m, 6H CH–CH3), 2.58–
2.90 (m, 3H, NH+–CH2–CHOH, CHOH), 2.90–3.20 (m, 2H,
CHOH–CH2–N), 3.20–3.81 (m, 8H, H-piper), 4.20–4.29 (m,
2H, CH2–CH3), 4.80–4.91 (m, 1H, CH–CH3), 5.32 (d,
J = 4.12, 1H, OH), 7.01–7.08 (m, 2H, Ar–H4, Ar–H6 Ph-
piper), 7.20–7.50 (m, 12H, Ar–H DPH, Ar–H3, Ar–H5 Ph-
piper), 11.57 (br s, 1H, NH+).
6.2.2. Animals
Experiments were carried out on male Wistar rats (130–
380 g). Animals were housed in standard cages, under a 12 h
light-dark cycle at the temperature of 20–24 ºC and relative
humidity of 60%. The animals had free access to standard
pellet food and water.
6.2.3. Statistical analysis
The data are expressed as means SEM. The statistical
significance was calculated using a one-way ANOVA test.
Differences were considered significant when p < 0.05.
6.2.4. Influence on normal electrocardiogram in vivo
Physiological ECG recordings were taken from rats
anaesthetised with thiopental (60 mg/kg i.p.) and, afterwards,
an examined compound was injected. Compounds 8a–10a,
12a and 16a–18a were suspended in 0.5% methylcellulose
and administered intraperitoneally at the dose of 50 mg/kg.
ECG parameters were collected after 15, 30 and 60 minutes
of administration. Compounds 11a and 19a were dissolved in
physiological saline and injected intravenously at the dose of
10 mg/kg, the ECG recordings were taken after 1, 5 and
10 minutes after administration of 11a.

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T. Dyla¸g et al. / European Journal of Medicinal Chemistry 39 (2004) 1013–1027
6.2.5. Antiarrhythmic activity
taneous pentylenetetrazole (scMet) tests in mice within the
framework of the Anticonvulsant Screening Project at the
National Institute of Health, Bethesda, MD, USA. The test,
performed in male Carworth Farm No. 1 (CF 1) mice, used
small groups of animals (1–8). Compounds were suspended
in 30% polyethylene glycol 400 for intraperitoneal adminis-
tration and tested at 30 minutes and 4 hours following doses
30, 100 and 300 mg/kg.
In the MES test, an electrical stimulus of 0.2 s in duration
(50 mA) delivered via coroneal electrodes primed with an
electrolite solution containing an anaesthetic agent.
6.2.5.1. Barium chloride – induced arrhythmia[23]. The
arrhythmia was evoked in rats anaesthetised with thiopental
(60 mg/kg i.p.) by i.v. injection of barium chloride
(32 mg/kg, in a volume of 1 ml/kg). Compounds 8a–10a, 12a
and 16a–18a were suspended in 0.5% methylcellulose and
administered i.p. at the constant dose of 50 mg/kg, 60 minu-
tes prior to arrhythmogen administration. Compounds 11a
and 19a were injected i.v. as saline physiological solution at
the dose of 10 mg/kg, 15 minutes prior to arrhythmogen.
Overdose of BaCl2 induced progressively increasing dis-
turbances of cardiac rhythm, associated with premature ven-
tricular beats, ventricular tachycardia and ventricular fibrilla-
tion, leading to death within 2-5 minutes (Fig 5).
The ScMet utilised
a dose of penthylenetetrazol
(85 mg/kg) administrated subcutaneously at the anticipated
time of testing.
In the minimal neurotoxicity test, toxicity induced by the
compounds was detected using the standardised rotorod test.
Untreated control mice, when placed on a 6 r.p.m. rotation
rod could maintain their equilibrium for a prolonged period
of time. Neurological impairment can be demonstrated by
the inability of a mouse to maintain equilibrium for one
minute in each of three succesive trials [25,26].
Attenuation or lack of disturbances and the decreased
mortality after BaCl2 administration were accepted as a cri-
terion of the antiarrhythmic effect of the compound.
6.2.5.2. Adrenaline – induced arrhythmia. Cardiac arrhyth-
mia was induced according to Szekeres [23] by i.v. adminis-
tration of adrenaline (20µg/kg in a volume of 1 ml/kg) to
anaesthetised rats (thiopenthal 60 mg/kg i.p.) The routes of
administration and doses of the analyzed compounds were
identical to those in case of barium chloride – induced ar-
rhythmia experiment.
Rapid intravenous injection of adrenaline induced reflex
bradycardia, blocks, premature ventricular beats (VBs), ven-
tricular tachycardia with ventricular and supraventricular ec-
topic (4 or more successive VBs) for 100% of the animals,
and frequently, even ventricular fibrillation and death for
50 % of the animals of the control group (Fig 6).Arrhythmias
were analysed according to the Lambeth Convention [24].
The lack of extrasystoles and of heart rhythm disturban-
ces, in comparison with the control group, were accepted as
criteria of the antiarrhythmic activity.
Acknowledgments
The authors would like to thank Professor James Stables
for providing pharmacological data through theAntiepileptic
Drug Development Program National Institutes of Health in
Bethesda. We thank Mrs. Maria Kaleta for her technical
assistance. This work was partly supported by Polish State
Committee of Scientific Research, grant No. BBN
501/P/162/F.
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