Aminoderivatives of cycloalkanespirohydantoins: Synthesis and biological activity

Article abstract of DOI:10.1016/S0014-827X(01)01198-3

3-Aminocycloalkanespiro-5-hydantoins were synthesized and their biological activity was studied. In contrast to hydantoins, these compounds failed to induce either anticonvulsive effects in the central nervous system or inhibitory effects on cholinergic contractions in the enteric nervous system. However, they exerted well pronounced, atropinsensitive, contractile effects on the guinea-pig ileum longitudinal muscle preparations. Structure-activity relationships established allow the assumption that: (i) the reduction of the ring size in the molecule of the spirohydantoins leads to an increase in the potency of the respective analogue to induce contractile effect; (ii) the introduction of -NH₂ in position 3 increases the ability of all the compounds studied to exert contractions; (iii) the enlargement of the ring leads to: (1) an increase of the degree of desensitization of the preparations; and (2) a decrease (except 1a) of the potency of the analogues to exert contractile effects.

Full text of DOI:10.1016/S0014-827X(01)01198-3

Il Farmaco 57 (2002) 189–194
www.elsevier.com/locate/farmac
Aminoderivatives of cycloalkanespirohydantoins: synthesis and
biological activity
Emilia Naydenova a,*, Nevena Pencheva b, Julita Popova a, Neyko Stoyanov c,
Maria Lazarova b, Boris Aleksiev c
a Uni6ersity of Chemical Technology and Metallurgy, bl6d. Kl.Ohridski 8, 1756 Sofia, Bulgaria
b Institute of Physiology, Bulgarian Academy of Sciences, Acad. G. Bonche6 Street, bl. 23, 1113 Sofia, Bulgaria
c College of Technology, Razgrad 7200, Bulgaria
Received 28 September 2001; accepted 26 October 2001
Abstract
3-Aminocycloalkanespiro-5-hydantoins were synthesized and their biological activity was studied. In contrast to hydantoins,
these compounds failed to induce either anticonvulsive effects in the central nervous system or inhibitory effects on cholinergic
contractions in the enteric nervous system. However, they exerted well pronounced, atropinsensitive, contractile effects on the
guinea-pig ileum longitudinal muscle preparations. Structure–activity relationships established allow the assumption that: (i) the
reduction of the ring size in the molecule of the spirohydantoins leads to an increase in the potency of the respective analogue
to induce contractile effect; (ii) the introduction of ꢀNH2 in position 3 increases the ability of all the compounds studied to exert
contractions; (iii) the enlargement of the ring leads to: (1) an increase of the degree of desensitization of the preparations; and (2)
´
a decrease (except 1a) of the potency of the analogues to exert contractile effects. © 2002 Editions scientifiques et me´dicales
Elsevier SAS. All rights reserved.
Keywords: Anticonvulsive activity; Contractile effect; Hydrazine reaction; NH-group reactivity
1. Introduction
biological activity, including the role of the amino
group, both in the central and in the enteric nervous
systems.
Hydantoins and cycloalkanespirohydantoins are the
subject of extensive investigations due to their biologi-
cal properties [1]. The anticonvulsive activity is the
strongest expressed in hydantoins. It has been found
that this activity can be increased on introducing amino
groups in the hydantoin ring [2]. Some cycloalkane-
spiro-5-hydantoins also have modest anticonvulsive ef-
fect. It has been observed that enlargement of the
cycloalkane ringsize increases the effect [3]. Many
derivatives of these compounds have been synthesized
and studied [4], but there are no data about the in-
fluence of the amino group in the hydantoin ring on
their biological activity. The objectives of the present
study are: (i) the synthesis of 3-aminoderivatives of the
cycloalkanespiro-5-hydantoins with 5-, 6-, 7-, 8- and
12-membered rings and (ii) the investigation of their
2. Results and discussion
2.1. Synthesis and characteristics of
cycloalkanespiro-5-hydantoins and aminoderi6ati6es
The cycloalkanespiro-5-hydantoins 1a–5a were ob-
tained from the corresponding cyclic ketones after the
Bucherer–Lieb reaction [5,6]. Modifying a previous
method [7], we transformed them into 3-aminoderiva-
tives by means of NH2NH2·H2O (Scheme 1).We ran the
reaction for 1 h with 98% NH2NH2·H2O and then for 3
h in refluxing with 80% NH2NH2·H2O (yields over
90%). The correction of the concentration was done by
adding the corresponding amount of H2O to the reac-
tion mixture without interrupting the heating. For ob-
taining 3-aminocyclododecanespiro-5-hydantoin, the
* Corresponding author.
E-mail address: emilia@uctm.edu (E. Naydenova).
´
0014-827X/02/$ - see front matter © 2002 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
PII: S0014-827X(01)01198-3

190
E. Naydeno6a et al. / Il Farmaco 57 (2002) 189–194
corresponding cyclododecanespiro-5-hydantoin was dis-
solved in ethanol at high temperature, and
NH2NH2·H2O was added. The duration of this reaction
(MH)+ for all the compounds. The spectral data of the
starting cycloalkanespiro-5-hydantoins and their
aminoderivatives are presented in Table 1.
a
was 10 h, and a yield of 60% was obtained. All synthe-
1
sized compounds were analyzed by means of IR-, H
2.2. Biological acti6ity
NMR-, 13C NMR- and mass-spectra. The results of the
spectral analysis not previously reported showed that
only the NH-group at position 3 in the hydantoine ring
took part in the reaction. The increased reactivity at
position 3 of the starting cycloalkanespiro-5-hydantoins
is due to the presence of two carbonyl groups. As a
result of their influence the signal of the N-3 proton on
The 3-aminoderivatives of the cycloalkanespiro-5-hy-
dantoins investigated (1b–5b) did not show an anticon-
vulsive activity using the pentylenetetrazole seizure and
electroconvulsive shock seizure models. Moreover, the
compounds 2b, 3b and 5b induced seizures. This finding
is in agreement with the decreased anticonvulsive effects
of cycloalkanespiro-5-hydantoins as compared to that
of hydantoins and their analogues [1]. The observed
failure of 3-aminoderivatives of cycloalkanespiro-5-hy-
dantoins, which was studied, to induce any antiseizure
activity in the brain provoked further investigation on
the enteric nervous system. The guinea pig ileum longi-
tudinal muscle preparation is a classical model for
investigation of exogenously applied drugs on the neu-
rotransmitter interactions. To more fully characterize
the compounds we followed their effects on both
1
the H-NMR spectrum appears in a weaker field (ꢀ10
ppm), while the signal for the N-1 proton appears at
ꢀ8 ppm. When aminoderivatives were obtained, the
signal at ꢀ10 ppm disappeared, but the one at ꢀ8
ppm was preserved. This confirms the generation of a
monoderivative at position 3. The two protons of the
introduced ꢀNH2 group were detected as an intensive
peak at 4.6–4.8 ppm. The structures were confirmed by
mass-spectral analysis, a chemical ionization with NH3
being applied. The most intense peak (100%) was
Scheme 1. Synthesis of 3-aminoderivatives of the cycloalkanspiro-5-hydantoins.
Table 1
Physical and analytical data of the synthesized cycloalkanespiro-5-hydantoins and their aminoderivatives
Product
Yield (%)
m.p. (°C)
1H NMR, l (ppm)
IR (KBr) wmax
(cm−1
MS (m/z)
)
1a [6]
90
95
95
91
87
80
95
90
90
205–205.5
219–220
213–215
240–241
278–279
140–141
166–166.5
162–163
175–176
191–191.5
(CD3COCD3) 1.84–2.11 (8H, m),
7.3 (1H, s), 10.4 (1H, s)
(DMSO) 1.6–1.96 (10H, m), 8.2
(1H, s), 10.3 (1H, s)
(DMSO) 1.6–2.31 (12H, m), 8.3
(1H, s), 10.4 (1H, s)
(DMSO) 1.5–2.11 (14H, m), 8.2
(1H, s), 10.4 (1H, s)
(DMSO) 1.29–1.98 (22H, m), 7.95
(1H, s), 10.44 (1H, s)
(CDCl3) 1.7–2.3 (8H, m), 4.06 (2H, 3320, 3200, 1788,
s), 6.7 (1H, s) 1738, 1612
(DMSO) 1.2–1.6 (10H, m), 4.6 (2H, 3320, 3200, 1775,
s), 8.5 (1H, s)
(CDCl3) 1.5–2.18 (12H, m), 4.13
(2H, s), 7.4 (1H, s)
3215, 3074, 1788,
1737
3198, 3070, 1775,
1727
3183, 3080, 1782,
1730
3250, 3183, 1765,
1713
154 (M)+
168 (M)+
182 (M)+
196 (M)+
252 (M)+
169 (M)+
183 (M)+
C7H10N2O2
2a [6]
C8H12N2O2
3a [6]
C9H14N2O2
4a [6]
C
10H16N2O2
5a [3]
3183, 3063, 1772,
1728
C14H24N2O2
1b [7]
C7H11N3O2
2b* [7]
C8H13N3O2
3b [7]
C9H15N3O2
4b [7]
C10H17N3O2
5b C14H25N3O2 60
1732, 1610
3320, 3200, 2500,
1770, 1730, 1610
(Cl, NH3, 100 °C): (MH)+ 198,
(MH3)+ 200, (M)+ 197
(Cl, NH3, 100 °C): (MH)+ 212
(M)+ 211
(CDCl3) 1.5–2.1 (14H, m), 3.9 (2H, 3310, 3220, 1782,
s), 5.6 (1H, s)
(DMSO) 1.17–1.8 (22H, m), 4.62
(2H, s), 7.9 (1H, s)
1730, 1615
3300, 3266, 2857,
1773, 1727, 1660
(Cl, NH3, 100 °C): (MH)+ 268,
(MH3)+ 270, (M)+: 267
2b*: 13C NMR l 175.4 (C4), 156.1 (C2), 59.6 (C5), 33.6 (C6, C10), 24.6 (C8), 21.0 (C7, C9).

E. Naydeno6a et al. / Il Farmaco 57 (2002) 189–194
191
tractions before the application of the test substances
were not observed. Cumulative or noncumulative addi-
tion of all compounds studied at concentrations in-
creasing from 1 pM to 1 mM at 1–2 min intervals, did
not exert any inhibitory effect on evoked contractions.
Moreover, at concentration over 1 mM some com-
pounds such as 3a, 2b and 1b induced pronounced,
dose dependent tonic contractile effects (Fig. 1). These
results indicate that the compounds were unable to
inhibit contractions, which according to parameters of
the stimulation are mainly cholinergic by nature. This
assumption closely corresponds with our data for ab-
sence of anticonvulsive activity of the compounds
tested. However, despite the evoked release of acetyl-
choline during electrically-stimulated responses, the
compounds reveal their ability to increase the tonic
contractile activity of the preparations. The longitudi-
nal layer of segments isolated from guinea-pig ileum
showed pronounced spontaneous mechanical activity.
After the equilibration period, the activity was charac-
terized by a steady state tone and spontaneous contrac-
tions with amplitude of 0.3590.11 g (25 preparations
from 10 animals). All compounds applied at concentra-
tions of 1 pM to 1 mM caused concentration-dependent
contractile effects in the spontaneous activity of the
smooth muscle preparations. The effect of compound
2b is shown at Fig. 2. The contractile effects comprised
a tonic and a phasic component expressed as a total
contraction. These effects were followed using cumula-
tive or non-cumulative manners of application depen-
ding on the type of the desensitization of the
preparations. Desensitization was achieved in separate
experiments by applying the compounds in a non-cu-
mulative way at a concentration of 1 mM, a contact
time of 5–7 min, and without intervening washings.
When intervals of 25–30 min, or 20 min, or 15 min, or
Fig. 1. Longitudinal layer of guinea-pig ileum. Tonic contractile
effects of 3a, 2b and 1b on the electrically-evoked (single pulses, 0.5
ms duration, 40 V, 0.1 Hz frequency) contractions; (ꢀ) application;
(ꢁ) washing.
electrically-stimulated and spontaneous mechanical ac-
tivity of the isolated preparations. Electrical field stimu-
lation (0.5 ms duration, 40 V, 0.1 Hz, frequency)
elicited well-expressed, twitch-like contractions of the
longitudinal layer of guinea pig ileum. The mean ampli-
tude of the responses before treatment was 1.2690.14
g (calculated in 20 preparations isolated from eight
animals). During stimulation, neither the amplitude of
the contractions nor the tone of the preparations were
considerably changed. Significant differences between
the amplitudes of the control electrically evoked con-
Fig. 2. Longitudinal layer of guinea-pig ileum. Dose dependent contractile effect of 2b on the spontaneous mechanical activity; (ꢀ) application;
(ꢁ) washing.

192
E. Naydeno6a et al. / Il Farmaco 57 (2002) 189–194
Table 2
Longitudinal layer of guinea pig ileum. EC50 (mM) values of the contractile effects produced by cyclopentanespiro-5-hydantoin (1a), cyclohexane-
spiro-5-hydantoin (2a), cycloheptanespiro-5-hydantoin (3a), 3-aminocyclopentanespiro-5-hydantoin (1b), 3-aminocyclohexanespiro-5-hydantoin
(2b) and 3-aminocycloheptanespiro-5-hydantoin (3b), on the spontaneous mechanical activity. The maximum contractile effects (g) and the type
of desensitization are also presented.
The values for EC50 and maximum contractile effect are the mean9SEM; the number of observations are at least 6. Asterisks indicate significant
difference vs. the corresponding value for the compound without -NH2 (*pB0.05 and **pB0.01). Designations for the type of the desensitization:
(-), very poor or absent; (+), poor; (++), middle; and (+++), powerful.
10 min (with washings) between single doses of com-
pounds were sufficient to prevent desensitization, it
was determined, respectively, as: powerful, middle,
poor and very poor or absent. Comparing the EC50
values of the respective concentration–response curves
(Table 2) it could be assumed that: (i) the reduction
of the ring size in the molecule of the spirohydantoins
leads to an increase in the potency of the respective
analogue to induce contractile effect; the most potent
are 1b and 2b; and (ii) the introduction of ꢀNH2 in
position 3 increases the ability of all the compounds
studied to exert contractions; this effect is statistically
insignificant between 2a and 2b, statistically signifi-
cant (PB0.05) between 3a and 3b, and maximal
(PB0.01) between 1a and 1b. The parameters of
maximal contractile effect, expressed in absolute val-
ues (g), provide information concerning the efficacy
of the compounds. All compounds (except 3a and 3b)
induced high amplitude contractile effects. The substi-
tution with amino group subsequently does not
change at 3a, decreases at 2a, and increases twice the
efficacy at 1a of the respective compounds. The en-
largement of the ring lead to: (1) an increase of the
degree of desensitization of the preparations; and (2)
a decrease (except 1a) of the potency of the analogues
to exert contractile effects. Our recent (unpublished)
data showed that the acylation of the amino group of
the spirohydantoins with an amino acid, or dipeptides
eliminated the contractile effects of the respective
compounds. The cholineblocker atropine, applied at a
concentration of 3 mM, reduced or absolutely blocked
the contractile effects of all compounds tested. This
finding suggests that contractile effects of the spirohy-
dantoins and their aminoderivatives on spontaneous
activity of guinea-pig ileum longitudinal muscle
preparations, could be due to an interaction with
cholinergic neurotransmission which probably pro-
voked the release of acetylcholine [8].
3. Experimental
Infrared spectra were recorded by the use of
Perkin–Elmer FTIR-1600 spectrophotometer. For
recording the NMR spectra, a Bruker WM-250 spec-
trometer was used at 250 and 62.9 MHz. Mass-spec-
tra were recorded using 5890-plus mass spectrometer
(Hewlett Packard). All melting points were deter-
mined using a Kofler apparatus without correction.
Analytical TLC plates were purchased from E.
Merck: silica gel 60F-254, aluminum backed. The
plates were developed with ninhydrin, Cl2-o-tolidine
or UV light (254 nm). The following chromatography
systems were used: CHCl3/MeOH, 9:1; CHCl3/
MeOH/AcOH, 9:2:1; n-BuOH/AcOH/H2O, 3:1:1.

E. Naydeno6a et al. / Il Farmaco 57 (2002) 189–194
193
3.1. Preparation of cycloalkanespiro-5-hydantoins
(1a–5a) by Bucherer–Lieb synthesis
3.4.2. Electrocon6ulsi6e shock
An electroshock stimulation (monophase rectangular
pulses with a current intensity of 50 mA, single phase
duration 1 ms; stimulation frequency of 50 Hz and a
trial duration of 0.2 s) was applied by means of silver
corneal electrodes inducing clonic–tonic seizures. A
sham electroshock was applied. The percentage of the
tonic seizure was calculated.
The 3-aminocyclohexanespiro-5-hydantoin, 3-amino-
cycloheptanespiro-5-hydantoin, and 3-amino-cyclodo-
decanespiro-5-hydantoin were administered i.p. at the
following doses: 400 mg/kg, 600 mg/kg, 30 min before
pentylenetetrazole or electroconvulsive shock.
The data of both seizure intensity and latency to first
seizure were assessed by multifactor ANOVA. The data
of mortality and the percentage of the tonic seizures
were analyzed by Fisher’s Exact Probability Two Tailed
test.
To a solution of cycloalkanone (290 mmol) in etha-
nol (225 ml) and water (200 ml) were added sodium
cyanide (28.5 g, 438 mmol) and ammonium carbonate
(119 g, 1.13 mol). The mixture was refluxed for 6 h with
stirring. After dilution with water, the cooled mixture
was acidified with concentrated hydrochloric acid. The
crude
cycloalkanespiro-5-hydantoin
precipitated
overnight upon cooling at 5 °C. Pure compound was
crystallized from water as colorless crystals.
3.2. Synthesis of 3-aminocycloalkanespiro-5-hydantoins
(1b–4b)
The corresponding cycloalkanespiro-5-hydantoin
(0.03 mol) was refluxed with 10 ml (0.2 mol) of 98%
NH2NH2·H2O for 1 h, and with 80% NH2NH2·H2O for
3.5 h (the correction of the concentration was done by
adding the corresponding amount of H2O to the reac-
tion mixture without interrupting the heating). The
reaction mixture was cooled, then poured over a small
amount of crushed ice. Upon standing, the product
crystallized slowly and was filtered, washed with a
minimum of cold water, dried, and recrystallized from
water or aqueous ethanol. Results are recorded in
Table 1.
3.5. Biological acti6ity in the enteric ner6ous system
3.5.1. Smooth muscle preparations
Male guinea pigs (250–300 g) were used in the
experiments. A segment of the ileum (excluding the
portion 15 cm proximal to the ileocaecal junction)
10–15 cm long, was isolated and the intraluminal con-
tents were removed with aerated Krebs solution con-
taining (mM): NaCl 120, KCl 5.9, NaHCO3 15.4,
NaH2PO4 1.2, MgCl2 2.5 and glucose 11.5. In order to
retain the myenteric plexus intact, segments of the
ileum approximately 2.0 cm long were cut out.
3.3. Synthesis of
3-aminocyclododecanespiro-5-hydantoin (5b)
3.5.2. Spontaneous mechanical acti6ity
5 g (0.02 mol) of the cyclododecanespiro-5-hydantoin
(5a) were dissolved in hot ethanol (25 ml) then 10 ml
(0.2 mol) 98% NH2NH2·H2O were added. The optimal
duration of the reaction was 10 h. The compound was
obtained according to the above method as a white
solid: Anal. Calc. for C14H25N3O2: C, 62.92; H, 9.36; N,
15.73. Found: C, 62.95; H, 9.30; N, 15.71%.
The isolated segments were mounted along the axis
of the longitudinal layer in 3 ml organ baths, contain-
ing Krebs solution, continuously aerated with 95% O2
and 5% CO2 at 36.5 °C. The spontaneous mechanical
activity as well as the compound- or electrically-evoked
responses of the longitudinal layer were followed under
isometric conditions after standard calibration of a
mechanoelectrical transducer (Experimetria Ltd., Hun-
gary) connected to a recording device TZ 4620 (Labo-
ratorni Pristroje, Praha) at a tension equivalent to a
load of 0.5 g. There was a 60 min equilibration period
before application of the compounds. Concentration–
response curves were constructed for each of the com-
pounds. The EC50 values, presented as means9SEM
and significance of the differences (Student’s t-test at
PB0.05 and PB0.01) were calculated using computer
programs [10]. The concentration–response curves were
further followed in the presence of cholinoblocker at-
ropine (3 mM) which remained in the organ bath before
retesting the compounds for 10 min.
3.4. Biological acti6ity on the central ner6ous system
Experimental seizure models (pentylenetetrazole
seizures and electrocon6ulsi6e shock)
The experiments were carried out on male Wistar
mice (10 mice per group), weighing 18–20 g.
3.4.1. Pentylenetetrazole seizure model
Pentylenetetrazole was injected subcutaneously in a
dose of 85 mg/kg. This dose was chosen on the basis of
preliminary experiments in which moderate seizures
were produced in the majority of animals. The effect
was evaluated by the differences between the control
and the experimental group of mice in seizure intensity
determined by a six point scale [9]. The animals were
observed 1 h after pentylenetetrazole injection.
3.5.3. Electrical stimulation
Electrical field stimulation was applied by a pair of
ring wire platinum electrodes (0.45 mm thick), to

194
E. Naydeno6a et al. / Il Farmaco 57 (2002) 189–194
duction of hydantoin derivatives, Ger. Patent. 1,173,102 (1963);
Chem. Abstr. 61 (1964) 9504e.
[4] W. Oldfield, C.H. Cashin, The chemistry and pharmacology of a
series of cycloalkanespiro-5-hydantoins, J. Med. Chem. 8 (1965)
239–249.
induce neurogenic contraction of the smooth muscle
preparations. Stimulation with single rectangular pulses
(generated by a stimulator ST-02, Experimetria MM,
Hungary) of supramaximal voltage, 75 mA current, 0.5
ms duration, and 0.1 Hz frequency was applied. Since
the electrically stimulated release of neurotransmitters
by specific activation of the neural structures in the
intestine depends on the stimulus frequency [11], we
used stimulation with a relatively low frequency to
provoke responses, mainly cholinergic by nature.
8
[5] H. Bucherer, V. Lieb, Uber die bildung substituierter hydantoine
aus aldehyden and ketonen. Synthese von hydantoinen, J. Prakt.
Chem. 141 (1934) 5–43.
[6] J. Tsang, B. Schmied, R. Nyfeler, M. Goodman, Peptide sweet-
eners. 6. Structural studies on the C-terminal amino acid of
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[7] A. Wildonger, M. Winstead, 3-Aminospirohydantoins, J. Med.
Chem. 10 (1967) 981–982.
[8] W.D. Paton, A. Zar, The origin of acetylcholine released from
guinea-pig intestine and longitudinal muscle strip, J. Physiol.,
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[9] K.S. Rusinov, M. Lazarova, S. Atanassova-Shopova, On certain
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Acknowledgements
This study was supported by Grants X-596 and
L-637 from the Ministry of Education and Science.
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