Synthesis, hypnotic properties and molecular modeling studies of 1,2,7,9-tetraaza-spiro[4.5]dec-2-ene-6,8,10-triones

Article abstract of DOI:10.1016/j.ejmech.2011.07.058

1,3-Dipolar cycloaddition reaction of nitrilimines with 5-arylidene-1,3-dimethyl-2,4,6-pyrimidinetriones 1a-i afforded 7,9-dimethyl-1,3,4-triaryl-1,2,7,9-tetraaza-spiro[4.5]dec-2-ene-6,8,10-triones 3a-k in a high regioselective manner. Single crystal X-ra

Full text of DOI:10.1016/j.ejmech.2011.07.058

European Journal of Medicinal Chemistry 46 (2011) 4964e4969
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journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, hypnotic properties and molecular modeling studies
of 1,2,7,9-tetraaza-spiro[4.5]dec-2-ene-6,8,10-triones
,
a
b
c
Adel S. Girgis a , Hanaa Farag , Nasser S.M. Ismail , Riham F. George
*
a Pesticide Chemistry Department, National Research Centre, Dokki, Cairo 12622, Egypt
b Pharmaceutical Chemistry Department, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt
c Pharmaceutical Chemistry Department, Faculty of Pharmacy, Cairo University, Cairo, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
1,3-Dipolar cycloaddition reaction of nitrilimines with 5-arylidene-1,3-dimethyl-2,4,6-pyrimidinetriones
1aei afforded 7,9-dimethyl-1,3,4-triaryl-1,2,7,9-tetraaza-spiro[4.5]dec-2-ene-6,8,10-triones 3aek in
a high regioselective manner. Single crystal X-ray study of 3d added a conclusive support for the assigned
structure. Potentiating effects of the synthesized compounds 3aek (at a dose of 25 mg/kg body weight)
on hypnotic action of sodium thiopental (at a dose of 75 mg/kg body weight i.p.) were investigated
in vivo using Albino mice according to the standard method. Most of the tested compounds revealed
promising hypnotic potentiating effects especially compounds 3k and 3e that could be nominated as
short-acting hypnotics. A hypothesis of molecular modeling study, including fitting of the synthesized
compounds into 3D-pharmacophore using Discovery Studio 2.5 software and their docking into opti-
mized homology model of GABAA-a1 showed good results consistent with the observed pharmacological
properties.
Received 8 March 2011
Received in revised form
26 July 2011
Accepted 30 July 2011
Available online 12 August 2011
Keywords:
5-Arylidene-1,3-dimethyl-2,4,6-
pyrimidinetriones
Hydrazonoyl halides
1,2,7,9-Tetraaza-spiro[4.5]dec-2-ene-6,8,10-
triones
Ó 2011 Elsevier Masson SAS. All rights reserved.
Hypnotics
Molecular modeling
1. Introduction
ultra-short acting barbiturate, to induce sleep prior to administra-
tion of the agents that are necessary for maintaining anesthesia
Many efforts to develop novel CNS-acting drugs especially,
sedative/hypnotic drugs with increased potency and reduced side
effects, have been made in order to assure the availability of suit-
able alternatives for the treatment of sleep-disorders. Problems
with sleeping in the general population are associated with a year-
to-year increase in the aging rate and mental disorders including
depression, neurosis and dementia. The current clinical approach
employs different varieties of benzodiazepine derivatives that are
used as hypnotic agents for those having difficulties in falling asleep
and maintaining sleep. However, these agents are often associated
with adverse effects such as amnesia, nightmares and prolonged
sleepiness [1,2]. Additionally, surgical procedures require the
administration of several intravenous drugs to ensure hypnosis,
analgesia, relaxation and control of visceral reflex responses. The
use of intravenous drugs adds flexibility and permits the adminis-
tration of lower doses of inhalational anesthetic agents. General
anesthesia most often is initiated by an injection of thiopental, an
during the surgical procedure. Ultra-short acting barbiturates have
an important place in the practice of anesthesiology [3]. Most of
anxiolytic, hypnotic, muscle-relaxant and anticonvulsant active
agents act allosterically to influence central
g-aminobutyric acid
(GABA)-mediated neurotransmission [4]. GABA mediates the
inhibitory neurotransmission in brain via GABAA and the GABAB
receptors [5e7]. GABAA receptor has been implicated in a number
of neurological diseases and ligands associated with it have been
identified as potential therapeutic agents [8,9]. Hence, the phar-
macotherapy of various neurological and psychiatric diseases such
as generalized anxiety disorders, sleep disturbances, muscle
spasms and seizure disorders involves the modulation of GABA-
mediated synaptic transmission in CNS [10e14].
In the present work it is intended to investigate synthesis of
tetraaza-spiro[4.5]dec-2-ene-6,8,10-trione analogs utilizing 1,3-
dipolar cycloaddition reaction of nitrilimines to 1,3-dimethyl-2,4,
6-pyrimidinetriones (barbiturate derivatives) possessing exocyclic
olefinic linkage at the 5-position. Needless to say that, the newly
synthesized spiro-containing heterocycles are still having the
barbiturate nucleus so, still possessing most of the pharmacolo-
gical properties belong to this ring system especially, the unique
* Corresponding author. Tel.: þ202 0120447199; fax: þ202 33370931.
E-mail address: girgisas10@yahoo.com (A.S. Girgis).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.07.058

A.S. Girgis et al. / European Journal of Medicinal Chemistry 46 (2011) 4964e4969
4965
sedative/hypnotic effects. Recent publication describing the anti-
convulsant and sedative effects of 5-substituted bromopyrazolinic
spirobarbiturates [15] also supported our assumption directed
toward developing promising bioactive targets through the present
study. A hypothesis of molecular modeling study will be also
considered in the present work to explore the intermolecular
interactions taking place between the test set of the synthesized
compounds and the target protein that may assist in validating the
observed pharmacological properties and distinguishing the
attained structureeactivity relationships.
triones 3aek rather than their regio-isomeric forms 2,3,7,9-
tetraaza-spiro[4.5]dec-1-ene-6,8,10-triones 4a-k explaining the
high regioselectivity of the conducted reactions under the applied
conditions (Scheme 1). The structures of 3aek were assigned based
on spectroscopic (IR, 1H NMR, 13C NMR, MS) and elemental analyses
data.
IR spectra of 3aek reveal a strong carbonyl stretching vibration
band at
n
¼ 1685e1697 cmꢀ1
.
1H NMR spectra of 3aek exhibit the
pyrazolinyl H-4 as a sharp singlet signal at
d
¼ 5.23e5.81. The
appearance of this characteristic signal at the mentioned chemical
shift value region is consistent with many other similar reported
spiro-containing ring systems supporting the assigned regio-
isomeric form especially, 20,40-dihydro-20,40,50-triaryl-spiro[2H-
indene-2,3’-[3H]pyrazole]-1,3-diones which reveal their pyrazolinyl
2. Results and discussion
2.1. Chemistry
H-40 signal at
dioxa-1,2-diaza-spiro[4.5]dec-2-ene-6,10-diones that exhibit their
pyrazolinyl H-4 at
¼ 5.33e5.68 [17]. Additionally, the methyl
d
¼ 5.14e5.48 [16] and 1,3,4-triaryl-8,8-dimethyl-7,9-
Reaction of 5-arylidene-1,3-dimethyl-2,4,6-pyrimidinetriones
1aei with nitrilimines (generated in situ via triethylamine dehy-
drohalogenation of the corresponding hydrazonoyl chlorides 2a, b)
in refluxing benzene afforded single products as exhibited by TLC.
The structures of the isolated products were established to be 7,9-
dimethyl-1,3,4-triaryl-1,2,7,9-tetraaza-spiro[4.5]dec-2-ene-6,8,10-
d
groups attached to the pyrimidinyl nitrogens (N-7, N-9) are observed
as singlet signals at
3d (as a representative example) adds a good support for the
established structure, revealing the methine (HC-4) and spiro
d
¼ 2.65e2.98, 3.45e3.51. 13C NMR spectrum of
O
N
H3C
N
R
O
O
CH3
(1)
PhCCl=NNHR' (2)
C6H6, TEA
R
Ph
R
R'
O
N
4
O
N
7
H3C
O
H3C
O
N
1
N
N
N
5
N
R'
Ph
O
N
O
9
CH3
CH3
(3)
(4)
1a, R = Ph
1b, R = 4-ClC6H4
3a, R = R' = Ph
3b, R = Ph, R' = 3-ClC6H4
3c, R = 4-ClC6H4, R' = Ph
1c, R = 2,4-Cl2C6H3
1d, R = 4-FC6H4
3d, R = 4-ClC6H4, R' = 3-ClC6H4
3e, R = 2,4-Cl2C6H3, R' = Ph
3f, R = 4-FC6H4, R' = Ph
1e, R = 4-H3CC6H4
1f, R = 4-H3COC6H4
1g, R = 4-(H3C)2NC6H4
1h, R = 2-thienyl
3g, R = 4-H3CC6H4, R' = Ph
3h, R = 4-H3COC6H4, R' = Ph
3i, R = 4-(H3C)2NC6H4, R' = Ph
3j, R = 2-thienyl, R' = Ph
1i, R = 5-methyl-2-furanyl
2a, R' = Ph
3k, R = 5-methyl-2-furanyl, R' = Ph
2b, R' = 3-ClC6H4
Scheme 1. Synthetic route of compounds 3aek.

4966
A.S. Girgis et al. / European Journal of Medicinal Chemistry 46 (2011) 4964e4969
carbons (C-5) at
d
¼ 63.7, 77.9, respectively which are in good accord
that a few of the synthesized compounds enhanced (shorten) the
induction time (time elapsed between injection of sodium thio-
pental and loss of the righting reflex) relative to the control
experiment (induction time ¼ 2.66 min.), which are 3j, 3b, 3f, 3e
and 3d (induction time, 1.79, 1.85, 2.00, 2.12 and 2.30 min.,
respectively). However, most of the tested compounds revealed
promising hypnotic potentiating effects (i.e. increased the sleeping
time relative to the control experiment, which is the time interval
between loss and recuperation of the righting reflex), especially
compounds 3k, 3e, 3f, 3d and 3g (% increase in sleeping time
relative to the control experiment, 904.0, 501.2, 358.6, 340.9 and
311.3, respectively).
Structureeactivity relationships based on the observed sodium
thiopental-induced hypnotic potentiations of the synthesized
compounds indicated that, introduction of a m-chloro substituent at
the phenyl group attached to the 1-position of 1,2,7,9-tetraaza-spiro
[4.5]dec-2-ene-6,8,10-triones intensively enhanced the hypnotic
potentiating properties as exhibited in pairs 3a, 3b (% increase in
sleeping time relative to the control experiment, 1.3, 192.0, respec-
tively) and 3c, 3d (% increase in sleeping time relative to the control
experiment, 20.0, 340.9, respectively). Additionally, the type of
substitution of the phenyl group oriented at the 4-position of the
constructed heterocycles seems a controlling factor for exhibiting the
total observed pharmacological properties. It has been noticed that,
attachment of the phenyl group located at the 4-position of the
synthesized heterocycles with an electron-donating function, greatly
decreases the potentiating hypnotic action. The stronger the electron-
donating property of that substituent, the decrease in the observed
sleeping period as exhibited in compounds 3h, 3i and 3g (% increase in
sleeping time relative to the control experiment, ꢀ20.3, 299.4 and
311.3, respectively). Otherwise, attachmentof the phenyl group located
at the 4-position of the prepared heterocycles with an electron-
withdrawing residue enhances greatly the observed potentiating
hypnotic effects as exhibited in compounds 3c, 3e and 3f (% increase in
sleeping time relative to the control experiment, 20.0, 501.2 and 358.6,
respectively). Meanwhile, introduction of a furanyl function at the 4-
position of the synthesized compound seems much more promising
for constructing a hypnotic active agent compared to the case of
adopting a thienyl moiety, as exhibited in pairs 3j and 3k (% increase in
sleeping time relative to the control experiment, ꢀ25.8, 904.0,
respectively).
with many other reported similar structures [16e21]. The carbonyl
carbons are observed at
carbons attached to N-7, N-9 are appeared at
spectra of the other synthesized analogues revealed similar obser-
vations (c.f. Section 4). Mass spectral data (EI-MS) of the synthesized
compounds 3a, cee, gek add good support for the assigned struc-
ture revealing the corresponding parent ion peak with considerable
relative intensity value (c.f. Section 4).
Single crystal X-ray study of 3d allows confirmation of the
established structure (Fig. 1). Theoretical calculations were pro-
cessed by both AM1 and PM3 methods to compare the observed
geometric parameters “geometric parameters obtained experi-
mentally through single crystal X-ray studies and theoretically
calculated ones with both AM1 and PM3 methods are presented in
Table 1 of supplementary material”. The geometries of 3d were
optimized by the molecular mechanics force field (MMþ) followed
by either semi-empirical AM1 [22] or PM3 [23,24] methods
implemented in the HyperChem 8.0 package. The structures were
fully optimized without fixing any parameters, thus bringing all
geometric variables to their equilibrium values. The energy mini-
mization protocol employed the PolakeRibiere conjugated
gradient algorithm. Convergence to a local minimum was achieved
when the energy gradient was ꢁ0.01 kcal molꢀ1. The RHF method
was used in the spin pairing for the two semi-empirical tools
[25,26].
d
¼ 150.6, 163.8, 166.1, while the methyl
d
¼ 29.1, 30.1. 13C NMR
2.2. Sodium thiopental-induced hypnosis potentiations of the
synthesized compounds
Potentiating effects of the synthesized compounds 3aek on the
hypnotic action of sodium thiopental were investigated in vivo
using Albino mice according to the reported standard method
[15,27,28]. From the observed results (Table 1), it has been noticed
Toxicological studies of the most promising prepared hypnotic
active agents (3deg, i and k) were performed using LD50 standard
method in mice [29] at a dose of 250, 500 and 750 mg/kg (body
weight) “that is, 10e30 fold of the used hypnotic effective dose
(25 mg/kg body weight)”. It has been noticed that no mortality rates
oranytoxic symptoms were observed after 24 h post-administrations
explaining the safe behavior of the tested compounds at the experi-
mentally applied doses.
A hypothesis of molecular modeling study including fitting the
synthesized compounds into a generated 3D-pharmacophore and
their docking into optimized homology model of GABAA-a1 was
reported in the supplementary material.
3. Conclusion
From all the above it could be concluded that, 1,3-dipolar
cycloaddition reactions of nitrilimines with 5-arylidene-1,3-
dimethyl-2,4,6-pyrimidinetriones 1aei proceed regioselectively
affording 7,9-dimethyl-1,3,4-triaryl-1,2,7,9-tetraaza-spiro[4.5]dec-
2-ene-6,8,10-triones 3aek in good yields (71e81% yield). The
constructed heterocycles possess promising hypnotic potentiating
effects especially, compounds 3k and 3e that could be nominated as
short-acting hypnotics. A hypothesis of molecular modeling study
was undertaken for exploring the intermolecular interactions
Fig. 1. ORTEP projection of single crystal X-ray diffraction of 3d.

A.S. Girgis et al. / European Journal of Medicinal Chemistry 46 (2011) 4964e4969
4967
Table 1
Sodium thiopental-induced hypnosis potentiations of the synthesized compounds.
Entry
Compound
R
R0
Induction time (min ꢃ SE mean)
Sleeping time (min ꢃ SE mean)
% Increase in sleeping time
1
2
3
4
5
6
7
8
Control
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
e
e
2.66 ꢃ 0.23
3.00 ꢃ 0.39
1.85 ꢃ 0.20*
3.47 ꢃ 0.53*
2.30 ꢃ 0.12
2.12 ꢃ 0.27
2.00 ꢃ 0.22
2.92 ꢃ 0.11
5.25 ꢃ 0.37*
2.91 ꢃ 0.10
1.79 ꢃ 0.26*
2.66 ꢃ 0.27
21.81 ꢃ 1.81
e
Ph
Ph
Ph
3-ClC6H4
Ph
3-ClC6H4
Ph
Ph
Ph
Ph
Ph
22.09 ꢃ 6.58
1.3
63.68 ꢃ 16.02
26.17 ꢃ 6.04
192.0
20.0
4-ClC6H4
4-ClC6H4
2,4-Cl2C6H3
4-FC6H4
4-H3CC6H4
4-H3COC6H4
4-(H3C)2NC6H4
2-thienyl
5-methyl-2-furanyl
96.15 ꢃ 12.39*
131.13 ꢃ 21.15*
100.02 ꢃ 24.59*
89.71 ꢃ 21.73*
17.38 ꢃ 1.65
340.9
501.2
358.6
311.3
ꢀ20.3
299.4
ꢀ25.8
904.0
9
10
11
12
87.10 ꢃ 34.42*
16.19 ꢃ 1.43
Ph
Ph
218.97 ꢃ 30.37*
*Statistically significant from the control at p < 0.05.
taking place between the test set of the synthesized compounds
and the target protein which revealed good results consistent with
the observed pharmacological properties.
130.4, 133.1, 133.8, 143.4 (arom. C), 146.7 (C-3), 150.0, 163.3, 165.8
(C]O). Anal. Calcd. for C26H21ClN4O3 (472.94): C, 66.03; H, 4.48; N,
11.85. Found: C, 66.14; H, 4.57; N, 12.08.
4. Experimental
4.1.3. 4-(4-Chlorophenyl)-7,9-dimethyl-1,3-diphenyl-1,2,7,9-
tetraaza-spiro[4.5]dec-2-ene-6,8,10-trione (3c)
Melting points were recorded on a Stuart SMP3 digital melting
point apparatus. IR spectra (KBr) were recorded on a JASCO 6100
FT-IR spectrophotometer. 1H NMR spectra were recorded on a Var-
ian MERCURY 300 MHz spectrometer. 13C NMR spectra were
recorded on JEOL AS 500 (125 MHz) and Varian MERCURY 300
(75 MHz) spectrometers. Mass spectra were recorded on a Shi-
madzu GCMS-QP 1000 EX (EI, 70 eV) spectrometer. The starting
compounds 1aei [30e34] and 2a, b [35,36] were prepared
according to the previously reported procedures.
Reaction time 7 h, colorless crystals from n-butanol, mp
230e232 ꢂC, yield 76%. IR: nmax/cmꢀ11695 (C]O),1595,1494 (C]N,
C]C). 1H NMR (CDCl3):
d
2.76 (s, 3H, CH3), 3.50 (s, 3H, CH3), 5.25 (s,
1H, H-4), 6.90e7.50 (m, 14H, arom. H). 13C NMR (DMSO-d6):
d
28.4,
29.4 (CH3), 62.9 (HC-4), 77.3 (C-5 “spiro-carbon”), 113.7, 119.8, 120.5,
120.6, 126.3, 128.5, 128.6, 128.89, 128.95, 130.1, 131.2, 132.3, 133.5,
141.9 (arom. C), 145.2 (C-3), 150.0, 163.5, 165.8 (C]O). MS: m/z (%)
472 (M, 45), 474 [(M þ 2), 16], 367 (36), 366 (35), 310 (5), 309 (12),
282 (8), 281 (4). Anal. Calcd. for C26H21ClN4O3 (472.94): C, 66.03; H,
4.48; N, 11.85. Found: C, 65.89; H, 4.37; N, 12.03.
4.1. Reaction of 1aei with hydrazonoyl chlorides 2a, b (general
procedure)
4.1.4. 1-(3-Chlorophenyl)-4-(4-chlorophenyl)-7,9-dimethyl-3-
phenyl-1,2,7,9-tetraaza-spiro[4.5]dec-2-ene-6,8,10-trione (3d)
Reaction time 10 h, colorless crystals from n-butanol, mp
249e251 ꢂC, yield 79%. IR: nmax/cmꢀ1 1693 (C]O), 1589, 1478 (C]
A mixture of equimolar amounts of 1aei (5 mmol) and the
corresponding 2a, b in dry benzene (25 ml) containing triethyl-
amine (7.5 mmol) was boiled under reflux for the appropriate time.
The reaction mixture was filtered while hot to remove triethyl-
amine hydrochloride then, evaporated till dryness under reduced
pressure and the remaining residue was triturated with methanol
(5 ml) so, the separated solid material was collected and crystal-
lized from a suitable solvent affording the corresponding 3aek.
N, C]C). 1H NMR (CDCl3):
d
2.76 (s, 3H, CH3), 3.50 (s, 3H, CH3), 5.23
(s, 1H, H-4), 6.43e7.50 (m, 13H, arom. H). 13C NMR (DMSO-d6):
29.1, 30.1 (CH3), 63.7 (HC-4), 77.9 (C-5 “spiro-carbon”), 112.6,
d
114.2, 120.1, 127.1, 129.2, 129.3, 130.0, 130.3, 131.1, 131.8, 132.7, 134.2,
134.3, 143.8 (arom. C), 147.1 (C-3), 150.6, 163.8, 166.1 (C]O). MS:
m/z (%) 506 (M, 59), 508 [(M þ 2), 48], 510 [(M þ 4), 17], 367 (100),
366 (77), 310 (20), 309 (28), 282 (19), 281 (15). Anal. Calcd. for
C26H20Cl2N4O3 (507.38): C, 61.55; H, 3.97; N, 11.04. Found: C, 61.49;
H, 3.84; N, 11.25.
4.1.1. 7,9-Dimethyl-1,3,4-triphenyl-1,2,7,9-tetraaza-spiro[4.5]dec-
2-ene-6,8,10-trione (3a)
Reaction time 7 h, colorless crystals from n-butanol, mp
214e216 ꢂC, yield 82%. IR: nmax/cmꢀ11687 (C]O),1594,1496 (C]N,
4.1.5. 4-(2,4-Dichlorophenyl)-7,9-dimethyl-1,3-diphenyl-1,2,7,9-
tetraaza-spiro[4.5]dec-2-ene-6,8,10-trione (3e)
C]C). 1H NMR (CDCl3):
d
2.66 (s, 3H, CH3), 3.51 (s, 3H, CH3), 5.29 (s,
1H, H-4), 6.89e7.53 (m, 15H, arom. H). 13C NMR (DMSO-d6):
d
28.3,
Reaction time 9 h, yellow crystals from ethanol, mp 193e195 ꢂC,
29.4 (CH3), 64.1 (HC-4), 77.5 (C-5 “spiro-carbon”),113.6,119.6,120.8,
126.3, 128.4, 128.5, 128.7, 128.8, 128.9, 129.5, 130.4, 133.3, 141.9
(arom. C),145.3 (C-3),150.0,163.5,166.1 (C]O). MS: m/z (%) 438 (M,
73), 333 (51), 332 (41), 276 (9), 275 (17), 248 (13), 247 (9). Anal.
Calcd. for C26H22N4O3 (438.49): C, 71.22; H, 5.06; N,12.78. Found: C,
71.43; H, 5.20; N, 12.94.
yield 71%. IR: nmax/cmꢀ1 1696 (C]O), 1593, 1496 (C]N, C]C). 1H
NMR (CDCl3):
d
2.89 (s, 3H, CH3), 3.45 (s, 3H, CH3), 5.81 (s, 1H, H-4),
28.5, 29.3
6.91e7.48 (m, 13H, arom. H). 13C NMR (DMSO-d6):
d
(CH3), 59.8 (HC-4), 76.6 (C-5 “spiro-carbon”), 113.8, 120.1, 126.1,
127.9, 128.8, 129.0, 129.2, 129.4, 129.6, 133.3, 133.9, 134.7, 141.7
(arom. C), 144.5 (C-3), 150.0, 163.3, 165.6 (C]O). MS: m/z (%) 506
(M, 32), 508 [(M þ 2), 25], 510 [(M þ 4), 6], 401 (21), 400 (12), 344
(3), 343 (5), 316 (5), 315 (4). Anal. Calcd. for C26H20Cl2N4O3 (507.38):
C, 61.55; H, 3.97; N, 11.04. Found: C, 61.43; H, 3.93; N, 10.98.
4.1.2. 1-(3-Chlorophenyl)-7,9-dimethyl-3,4-diphenyl-1,2,7,9-
tetraaza-spiro[4.5]dec-2-ene-6,8,10-trione (3b)
Reaction time 9 h, colorless crystals from n-butanol, mp
156e158 ꢂC, yield 81%. IR: nmax/cmꢀ1 1690 (C]O), 1590, 1480 (C]
4.1.6. 4-(4-Fluorophenyl)-7,9-dimethyl-1,3-diphenyl-1,2,7,9-
tetraaza-spiro[4.5]dec-2-ene-6,8,10-trione (3f)
N, C]C). 1H NMR (CDCl3):
d
2.65 (s, 3H, CH3), 3.51 (s, 3H, CH3), 5.28
(s, 1H, H-4), 6.45e7.53 (m, 14H, arom. H). 13C NMR (DMSO-d6):
28.3, 29.5 (CH3), 64.3 (HC-4), 77.6 (C-5 “spiro-carbon”), 112.0,
113.6, 119.4, 120.6, 126.6, 128.48, 128.53, 128.8, 129.2, 129.4, 130.0,
Reaction time 7 h, colorless crystals from n-butanol, mp
d
208e210 ꢂC, yield 75%. IR: nmax/cmꢀ11686 (C]O),1597,1500 (C]N,
C]C). 1H NMR (CDCl3):
d 2.75 (s, 3H, CH3), 3.50 (s, 3H, CH3), 5.27 (s,

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A.S. Girgis et al. / European Journal of Medicinal Chemistry 46 (2011) 4964e4969
1H, H-4), 6.90e7.51 (m, 14H, arom. H). Anal. Calcd. for C26H21FN4O3
(456.48): C, 68.41; H, 4.64; N, 12.27. Found: C, 68.19; H, 4.51; N,
12.08.
(C-3), 153.1, 163.3, 165.8 (C]O). MS: m/z (%) 442 (M, 52), 337 (58),
336 (58), 280 (3), 279 (9), 252 (4), 251 (2). Anal. Calcd. for
C25H22N4O4 (442.48): C, 67.86; H, 5.01; N, 12.66. Found: C, 67.98; H,
5.24; N, 12.74.
4.1.7. 7,9-Dimethyl-1,3-diphenyl-4-(4-methylphenyl)-1,2,7,9-
tetraaza-spiro[4.5]dec-2-ene-6,8,10-trione (3g)
4.2. Single crystal X-ray crystallographic data of 3d
Reaction time 10 h, colorless crystals from n-butanol, mp
175e177 ꢂC, yield 80%. IR: nmax/cmꢀ1 1692 (C]O), 1597, 1496 (C]N,
Full crystallographic details, excluding structure factors have been
deposited at Cambridge Crystallographic Data Centre (CCDC) as
supplementary publication number CCDC 828750. For X-ray crystal-
lographic studies, compound 3d was recrystallized as prismatic
colorless crystals from n-butanol. The crystallographic data were
collected at T ¼ 298 K on a Kappa CCD Enraf Nonius FR 590 diffrac-
tometer using a graphite monochromator with Mo-Ka radiation
C]C). 1H NMR (CDCl3):
d
2.32 (s, 3H, ArCH3), 2.68 (s, 3H, NCH3), 3.50
(s, 3H, NCH3), 5.26 (s,1H, H-4), 6.85e7.53 (m,14H, arom. H).13C NMR
(DMSO-d6): 20.6 (AreCH3), 28.3, 29.4 (NCH3), 64.0 (HC-4), 77.5 (C-
d
5 “spiro-carbon”), 113.6, 119.6, 120.5, 126.3, 128.5, 128.8, 128.87,
128.97, 129.3, 130.2, 130.4, 138.1, 142.0 (arom. C), 145.5 (C-3), 150.1,
163.6, 166.1 (C]O). MS: m/z (%) 452 (M, 81), 347 (62), 346 (63), 290
(9), 289 (19), 262 (12), 261 (7). Anal. Calcd. for C27H24N4O3 (452.52):
C, 71.67; H, 5.35; N, 12.38. Found: C, 71.52; H, 5.15; N, 12.55.
(
l
¼ 0.71073 Å). The crystal structures were determined by SIR92 [37]
and refined by maXus [38] (Bruker Nonius, Delft and MacScience,
Japan). Chemical formula C26H20Cl2N4O3, Mr ¼ 507.377, orthorhombic,
crystallizes in space group Pna21. Cell lengths “a ¼ 11.4832(3),
4.1.8. 7,9-Dimethyl-1,3-diphenyl-4-(4-methoxyphenyl)-1,2,7,9-
tetraaza-spiro[4.5]dec-2-ene-6,8,10-trione (3h)
b ¼ 16.4905(5), c ¼ 12.4679(3) Å”. Cell angles “
a
¼ 90.00,
b
¼ 90.00,
g
¼ 90.00ꢂ”, V ¼ 2360.97(11) Å3, Z ¼ 4, Dc ¼ 1.427 mg/m3,
q values
Reaction time 9 h, colorless crystals from n-butanol, mp
229e231 ꢂC, yield 77%. IR: nmax/cmꢀ1 1685 (C]O), 1597, 1496 (C]
2.910e26.022ꢂ, absorption coefficient
m
(Mo-Ka) ¼ 0.31 mmꢀ1
,
N, C]C). 1H NMR (CDCl3):
d
2.73 (s, 3H, NCH3), 3.50 (s, 3H, NCH3),
3.78 (s, 3H, OCH3), 5.25 (s, 1H, H-4), 6.81e7.53 (m, 14H, arom. H). 13
NMR (DMSO-d6): 28.4, 29.4 (NCH3), 55.1 (OCH3), 63.7 (HC-4), 77.5
F(000) ¼ 1048. The unique reflections measured 2621 of which 1627
C
reflections with threshold expression I > 3s(I) were used in the
d
structural analysis. Convergence for 316 variable parameters by least-
squares refinement on F2 with w ¼ 1/[
s
2(F20) þ 0.10000F20]. The final
(C-5 “spiro-carbon”), 113.6, 113.8, 119.5, 120.7, 125.0, 126.3, 128.5,
128.75, 128.86,130.4, 130.7,142.0, 145.5 (arom. C), 150.1 (C-3), 159.3,
163.7, 166.2 (C]O). MS: m/z (%) 468 (M, 59), 363 (35), 362 (46), 306
(4), 305 (12), 278 (8), 277 (5). Anal. Calcd. for C27H24N4O4 (468.52):
C, 69.22; H, 5.16; N, 11.96. Found: C, 69.27; H, 5.19; N, 12.07.
agreement factors were R ¼ 0.037 and wR ¼ 0.067 with a goodness-of-
fit of 1.491.
4.3. Potentiation of hypnotic effect of sodium thiopental
4.1.9. 7,9-Dimethyl-4-(4-dimethylaminophenyl)-1,3-diphenyl-
1,2,7,9-tetraaza-spiro[4.5]dec-2-ene-6,8,10-trione (3i)
Potentiating effects of the synthesized compounds 3aek on
hypnotic action of sodium thiopental was conducted according to
the reported standard method [15,27,28]. Albino mice (z20 g)
were divided into 12 groups of 6 animals each. Administration of
the tested compounds (3aek) emulsified in Tween 80/normal
saline in a dose of 25 mg/kg body weight were given intraperito-
neally. The control group was given vehicle only. One hour later all
mice were administered with sodium thiopental (75 mg/kg body
weight i.p.) in physiological saline. Induction time (time elapsed
between injection of sodium thiopental and loss of the righting
reflex) and sleeping time (time interval between loss and recu-
peration of the righting reflex) were recorded. Data were collected,
checked, revised and analyzed. Quantitative variables from normal
distribution were expressed as means ꢃ SE “standard error”. The
significant difference between groups was tested by using one-way
ANOVA available in SPSS 11 followed by post-hoc test and the
chosen level of significance was p < 0.05.
Reaction time 10 h, colorless crystals from n-butanol, mp
208e210 ꢂC, yield 83%. IR: nmax/cmꢀ1 1697 (C]O), 1604, 1496 (C]
N, C]C). 1H NMR (CDCl3):
d
2.73 (s, 3H, NCH3), 2.94 [s, 6H, N(CH3)2],
3.49 (s, 3H, NCH3), 5.23 (s,1H, H-4), 6.70e7.55 (m,14H, arom. H). 13
NMR (DMSO-d6): 28.4, 29.3 (NCH3), 64.5 (HC-4), 77.7 (C-5 “spiro-
C
d
carbon”), 111.6, 113.5, 119.4, 119.6, 120.6, 126.4, 128.4, 128.6, 128.9,
130.1, 130.7, 142.1, 145.7 (arom. C), 150.1 (C-3), 150.2, 163.8, 166.4
(C]O). MS: m/z (%) 481 (M, 85), 376 (39), 375 (95), 318 (5), 291 (9),
290 (5). Anal. Calcd. for C28H27N5O3 (481.56): C, 69.84; H, 5.65; N,
14.54. Found: C, 69.97; H, 5.82; N, 14.57.
4.1.10. 7,9-Dimethyl-1,3-diphenyl-4-(2-thienyl)-1,2,7,9-tetraaza-
spiro[4.5]dec-2-ene-6,8,10-trione (3j)
Reaction time 9 h, colorless crystals from n-butanol, mp
191e193 ꢂC, yield 86%. IR: nmax/cmꢀ11686 (C]O),1596,1497 (C]N,
C]C). 1H NMR (CDCl3):
d
2.85 (s, 3H, CH3), 3.51 (s, 3H, CH3), 5.57 (s,
% Increase in sleeping time was expressed as percentage
increase in sleeping durations in treated animal groups relative to
the control group (Table 1).
1H, H-4), 6.80e7.57 (m, 13H, arom. H). 13C NMR (DMSO-d6):
d
28.6,
29.4 (CH3), 58.8 (HC-4), 77.4 (C-5 “spiro-carbon”), 113.6, 119.8,
120.5, 126.3, 127.0, 128.2, 128.5, 128.9, 129.6, 130.4, 134.7, 141.9
(arom. C),144.9 (C-3),150.1,163.3, 165.8 (C]O). MS: m/z (%) 444 (M,
62), 446 [(M þ 2), 7], 339 (56), 338 (74), 282 (7), 281 (16), 254 (10),
253 (5). Anal. Calcd. for C24H20N4O3S (444.52): C, 64.85; H, 4.54; N,
12.60. Found: C, 65.07; H, 4.60; N, 12.76.
Td ꢀ Tc
%increase in sleeping time ¼
ꢄ 100
Tc
where, Tc and Td are the sleeping times for the control and drug-
treated animal groups, respectively.
4.1.11. 7,9-Dimethyl-1,3-diphenyl-4-(5-methyl-2-furanyl)-1,2,7,9-
tetraaza-spiro[4.5]dec-2-ene-6,8,10-trione (3k)
4.4. LD50 determination
Reaction time 10 h, colorless crystals from n-butanol, mp
169e171 ꢂC, yield 81%. IR: nmax/cmꢀ11688 (C]O),1594,1492 (C]N,
The toxicological behavior of the most promising prepared
hypnotic active agents (3deg, i and k) were studied using the
standard known LD50 method in mice [29]. Albino mice weighing
20e25 g were divided into 21 groups of 6 animals each. Adminis-
trations of the tested compounds (3deg, i and k) emulsified in
Tween 80/normal saline at doses of 250, 500 and 750 mg/kg (body
C]C). 1H NMR (CDCl3):
d
2.25 (s, 3H, furanyl CH3), 2.98 (s, 3H,
NCH3), 3.51 (s, 3H, NCH3), 5.41 (s, 1H, H-4), 5.89e7.59 (m, 12H,
arom. H). 13C NMR (DMSO-d6):
13.1 (AreCH3), 28.8, 29.4 (NCH3),
d
58.3 (HC-4), 76.3 (C-5 “spiro-carbon”), 107.1, 112.7, 113.5, 119.7,
120.8, 126.0, 128.5, 128.9, 130.6, 141.9, 142.9, 144.5 (arom. C), 150.2

A.S. Girgis et al. / European Journal of Medicinal Chemistry 46 (2011) 4964e4969
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formula:
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ðzxdÞ
LD50 ¼ Dm
ꢀ
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where Dm ¼ the largest dose which killed all animals, z ¼ mean of
dead animals between two successive groups, d ¼ the constant
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Acknowledgment
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This study was supported financially by the Science and Tech-
nology Development Fund (STDF), Egypt, Grant No. 1357. Thanks
are also to Pharmacology Department, NRC, Cairo, Egypt, for
allowing performance of pharmacological activity screening.
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Appendix. Supplementary information
The supplementary data associated with this article can be
found in the online version at doi:10.1016/j.ejmech.2011.07.058.
These data include MOL files and InChiKeys of the most important
compounds described in this article.
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