Synthesis, biological evaluation and molecular modeling study of thiadiazolo[3,2-a][1,3]diazepine analogues of HIE-124 as a new class of short acting hypnotics

Article abstract of DOI:10.1016/j.ejmech.2016.08.038

A new series of 6,7-dihydro-[1,3,4]thiadiazolo[3,2-a][1,3]diazepine analogues were synthesized, and biological evaluated. Compound GS-62 (33) exhibited potent in?vivo short acting hypnotic activity with onset time, duration of sleep and therapeutic index of 6.4?±?0.2, 94.8?±?5.3?min, 6.62, respectively), in comparison to thiopental sodium (6). Compounds 33 did not show any sign of acute tolerance reported with the maintenance dose of 6. Meanwhile 33 potentiated the in?vivo hypnotic effect of 6 in an equimolar amounts (0.06?mmol) combination showing an onset and duration of 7.5?±?1.3, 62.5?±?5.9?min, respectively. This combination allowed the use of lower doses of both drugs to avoid the undesirable side effects. Docking studies revealed favorable interactions and binding to BDZ binding site of the GABA_Areceptor especially with Arg87, Arg149, and Thr151 amino acid residues.

Full text of DOI:10.1016/j.ejmech.2016.08.038

Accepted Manuscript
Synthesis, biological evaluation and molecular modeling study of thiadiazolo-[3,2-a]
[1,3]diazepine analogues of HIE-124 as a new class of short acting hypnotics
Hussein I. El-Subbagh, Ghada S. Hassan, Kamal E.H. El-Taher, Shahenda M. El-
Messery, Adel S. Al-Azab, Alaa A.-M. Abdelaziz, Mohamed M. Hefnawy
PII:
S0223-5234(16)30681-X
10.1016/j.ejmech.2016.08.038
EJMECH 8834
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 16 June 2016
Revised Date: 27 July 2016
Accepted Date: 17 August 2016
Please cite this article as: H.I. El-Subbagh, G.S. Hassan, K.E.H. El-Taher, S.M. El-Messery, A.S. Al-
Azab, A.A.-M. Abdelaziz, M.M. Hefnawy, Synthesis, biological evaluation and molecular modeling study
of thiadiazolo-[3,2-a][1,3]diazepine analogues of HIE-124 as a new class of short acting hypnotics,
European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.08.038.
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ACCEPTED MANUSCRIPT
1
Synthesis, Biological Evaluation and Molecular
Leave this area blank for abstract info.
Modeling Study of Thiadiazolo[3,2-a][1,3]diazepine
Analogues of HIE-124 as a New Class of Short Acting
Hypnotics.
Hussein I. El-Subbagh,1,2* Ghada S. Hassan,1 Kamal E. H. El-Taher,3 Shahenda M. El-Messery,4 Adel S. Al-
Azab,5,6 Alaa A.-M. Abdelaziz,1,5 Mohamed M. Hefnawy.5,7
Structure of the most active short acting hypnotic GS-62 (33), onset time 6.4 ± 0.2 min, duration of
sleep 94.8 ± 5.3 min, and therapeutic index, 6.62,

ACCEPTED MANUSCRIPT
Synthesis, Biological Evaluation and Molecular Modeling Study of Thiadiazolo-
[3,2-a][1,3]diazepine Analogues of HIE-124 as a New Class of Short Acting
Hypnotics.#
Hussein I. El-Subbagh,1,2* Ghada S. Hassan,1 Kamal E. H. El-Taher,3 Shahenda M. El-Messery,4
Adel S. Al-Azab,5,6 Alaa A.-M. Abdelaziz,1,5 Mohamed M. Hefnawy.5,7
1Department of Medicinal Chemistry, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt
2Department of Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences & Pharmaceutical Industries, Future
University, Cairo 12311, Egypt
3Department of Pharmacology, College of Pharmacy, King Saud University, P.O.Box 2457, Riyadh 11451, Saudi Arabia
4Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Mansoura University, Mansoura 35516,
Egypt
5Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P.O.Box 2457, Riyadh 11451,
Saudi Arabia
6Department of Organic Chemistry, Faculty of Pharmacy, Al-Azhar University, Cairo 11884, Egypt
7Department of Analytical Chemistry, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt
Correspondence: Hussein I. El-Subbagh, Department of Medicinal Chemistry, Faculty of Pharmacy, Mansoura
University, Mansoura 35516, Egypt
E-mail: subbagh@yahoo.com.
Tel: +20-50-2247800, Fax: +20-50-2247496
Abstract
A new series of 6,7-dihydro-[1,3,4]thiadiazolo[3,2-a][1,3]diazepine analogues were synthesized, and
biological evaluated. Compound GS-62 (33) exhibited potent in vivo short acting hypnotic activity
with onset time, duration of sleep and therapeutic index of 6.4 ± 0.2, 94.8 ± 5.3 min, 6.62,
respectively), in comparison to thiopental sodium (6). Compounds 33 did not show any sign of acute
tolerance reported with the maintenance dose of 6. Meanwhile 33 potentiated the in vivo hypnotic
effect of 6 in an equimolar amounts (0.06 mmol) combination showing an onset and duration of 7.5
± 1.3, 62.5 ± 5.9 min, respectively. This combination allowed the use of lower doses of both drugs to
avoid the undesirable side effects. Docking studies revealed favorable interactions and binding to
BDZ binding site of the GABAA receptor especially with Arg87, Arg149, and Thr151 amino acids
residues.
Keywords: Synthesis, Thiadiazolodiazepine Analogues, Short acting hypnotic Agents, Molecular
Modeling Study
____________
# Part of the Patents EP 2 514 753 B1 and US 8,741,893 B2, ref. [21,22].
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1.
Introduction
Episodes of sleep disorders such as insomnia and deterioration of sleep related to ageing processes
resulted in poor day time performance and reduced quality of life. Medical treatment of insomnia
started with the introduction of the first generation such as barbital, and phenobarbitone followed by
chlordiazepoxide as the second generation of hypnotics [1,2]. Those two generations suffered from
numerous side effects; barbiturates showed respiratory, renal and cardiovascular depression whereas
benzodiazepines caused anterograde amnesia and menstrual disorders. The common disadvantages
of those two generations included impaired day psychomotor performance, appearance of depressant
residual actions; the morning-after effects e.g. headache, drowsiness; the precipitation of tolerance,
addiction and rebound insomnia [3-6]. It was thought that ideal hypnotic was probably reached upon
the discovery of Gaboxadol (1, Figure 1) which was introduced as a new non-benzodiazepine
hypnotic [7]. This is followed by the introduction of the cyclopyrrolone “Zopiclone” (2), the
imidazopyridine “Zolpidem” (3) and the pyrazolopyrimidine “Zaleplon” (4) [8]. This third
generation (2-4) was initially thought to be devoid of rebound insomnia yet later experience revealed
their inherent capabilities to exert tolerance, dependence and rebound insomnia [3-5,9,10]. However,
Gaboxadol (1) possessed a serious side effect by increasing the duration of spontaneous Petit-mal
seizures [13].
CH3
O
N
H3C
N CH3
O
N
N
O
O
OH
N
N
N
H3C
N
N
N
N
N
Cl
CH3
HN
O
N
N
O
CN
Zolpidem, 3
Zaleplon, 4
Gaboxadol,1
Zopiclone, 2
EtOOC
O
N
N
S
N
O
N
S- Na+
O
H
Thiopental sodium, 6
HIE-124, 5
Figure 1: Structures of some literature hypnotic lead compounds.
GABAA receptors regulate behavioral functions, including memory, and anxiety, and considered to
be therapeutic targets for the treatment of insomnia, mood disorders, memory dysfunction, and pain.
These receptors are responsive to sedative, hypnotic and general anesthetic drugs [14]. Several drugs
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are currently in the pipeline for treating insomnia, high development costs limit the further
development of new drugs [15-17]. The ultra-short acting hypnotic activity of ethyl 8-oxo-5,6,7,8-
tetrahydro-thiazolo[3,2-a][1,3] diazepin-3-carboxylate (HIE-124, 5, Figure 1), overcame many of the
disadvantages and problems that usually associated with the use of thiopental sodium (6) as
intravenous anesthetic agents. Compound 5 proved to be a very short acting hypnotic with onset of
action less than 60 seconds, and its duration of action is significantly shorter than that of 6.
Compound 5 did not show any sign of acute tolerance [18-20].
In the present investigation, 6,7-dihydro-[1,3,4]thiadiazolo[3,2-a][1,3]diazepine analogues were
synthesized as a new structure modification derivatives of the previously patented agent “HIE 124,
5”. The new compounds proved to possess short acting hypnotic activity in addition to in vivo
potentiating effect toward the known ultra-short acting hypnotics 6; this combination allowed the use
of lower doses of both to avoid the undesirable side effects. The obtained results contributed in part
to the issuance of European and US patents [21,22].
Scheme 1: Synthesis of the target compounds 30-36
S
S
O
H
R
N
EtOH
-H2O
+
N
NH2
N
H
NH2
R
H
NH2
8
7
9-15
FeCl3
N N
R
NH2
R = CH3, CF3, CH3CH2, C6H5, 4-BrC6H4,
4-CH3C6H4, 4-NO2C6H4
S
16-22
Cl
Cl
K2CO3,
Toluene,
r.t.
O
O
Cl
N N
N N
piperidine
Toluene, reflux
N
R
N
O
R
S
S
H
30-36
23-29
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2.
Results and Discussion
2.1 Chemistry
1,3,4-Thiadiazolo[3,2-a][1,3]diazepine analogues usually obtained by the use of reported literature
procedures [23,24]. Compounds of the present study (30-36) were synthesized according to an
inventive method showed in Scheme 1. The proper aldehyde and thiosemicarbazide in alcohol were
mixed with stirring to get the required thiosemicarbazones (9-15). Compounds 9-15 were stirred in
FeCl3 solution to get the required 2-amino-5-substituted-1,3,4-thiadiazoles (16-22) [25]. Compounds
16-22 were then acylated with 4-chlorobutyryl chloride in toluene at room temperature to afford 4-
Chloro-N-(5-substituted-1,3,4-thiadiazol-2-yl)butanamide analogues (23-29), which were purified by
silica gel and neutral alumina chromatography. The butanamide analogues 23-29 were then heated
under reflux with piperidine in toluene to give the cyclized derivatives 2-substituted-6,7-dihydro-
thiazolo[3,2-a][1,3]diazepin-8(5H)-ones (30-36), Scheme 1, Table 1. Structure elucidation of the
1
new synthesized intermediates and final products was attained by the aid of elemental analysis, H,
13C NMR, and Mass spectrometry.
Table 1: Physicochemical properties of the synthesized compounds 23-36
Yield
%
M.p.
oC
Compound
R
Solvent
Molecular Formulaea
CH3
EtOH
82
79
85
85
87
89
67
77
61
68
90
78
70
77
112-5
C7H10ClN3OS
C7H7ClF3N3OS
C8H12ClN3OS
C12H12ClN3OS
C12H11BrClN3OS
C13H14ClN3OS
C12H11ClN4O3S
C7H9N3OS
23
24
25
26
27
28
29
30
31
32
33
34
35
36
CF3
CH3CH2
C6H5
4-BrC6H4
4-CH3C6H4
4-NO2C6H4 EtOH
EtOH
EtOH
108-10
68-70
159-62
187-9
122-4
130-2
138-40
152-4
120-2
189-92
204-7
175-7
159-62
Toluene
Toluene
EtOH
CH3
CF3
EtOH
EtOH
EtOH
EtOH
EtOH
EtOH
C7H6F3N3OS
C8H11N3OS
C12H11N3OS
CH3CH2
C6H5
4-BrC6H4
4-CH3C6H4
C12H10BrN3OS
C13H13N3OS
4-NO2C6H4 EtOH
C12H10N4O3S
aAnalysed for C,H,N; results were within ± 0.4 % of the theoretical values for the given
formulae.
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2.2 Evaluation of the Hypnotic Activity of Compounds 30-36
The hypnotic activity of test compounds 30-36 in mice was measured using the standard righting
reflex method [26-29]. Intraperitoneal administration of compounds 30, 31, 33 (GS-62), 34 (GS-53),
35 and thiopental sodium (6) but not 32 or 36 at doses of 0.2–2 mmoles/kg into mice induced
hypnosis and the loss of the righting reflux albeit with wide variations. The obtained minimal
effective dose, onset time and duration of sleep for each compound are shown in Table 2. The onset
(6.4 ± 0.2 min) and duration of sleep (94.8 ± 5.3 min) for compound 33 (GS-62) were significantly
greater than the values obtained for the reference drug 6 (2 ± 0.1 min, 45 ± 3.6 min, 6.1 respectively
;
P<0.05; n=6). Compounds 32 and 36 showed no hypnotic activity; while 34 and 35 showed
respiratory depression and thus the duration was not calculated. The onset and duration times of
sleep induced by 30 and 31 were significantly different compared with those of GS-62 (33) and 6;
with delayed onsets and shorter durations. Compound GS-62 (33) seemed to possess a delay in
regard to the induction of the onset of sleep but twice as potent regarding the duration (P<0.05,
N=6). The LD50 values and the therapeutic indices of the tested compounds were calculated.
Compounds were given intraperitoneally in doses ranging from 0.1-5 mmole/kg [30]. The percentage
of death at each dose level was then calculated, and the LD50 values were obtained, Table 2. It can be
clearly observed, that the Therapeutic Index of 33 (G5-62) is similar to that of 6. The Therapeutic
Indices of compounds 30 and 31 were 53-57% of 33. Compounds GS-53 (34) and 35 seemed to be
toxic causing respiratory failure to the tested animals. Thus, compound GS-62 (33) emerged as the
most effective and the safest among the tested compounds. Its safety is comparable to that of
thiopental sodium (6), but its duration is significantly longer. Compounds 33 and 34 did not show
any sign of acute tolerance reported with the second (maintenance) dose of 6.
Table 2: Influence of the test compounds 30-36 and thiopental sodium on sleep in mice (N = 6),
LD50 and the Therapeutic Indices values.
Effective Dose
mmole/kg (i.p)
Onset of sleep Duration of sleep LD50
Compound
Therapeutic Index
(minutes)
(minutes)
mmole/kg (i.p)
1.0
3.55
2.74
3.55
3.32
30
25 ± 4.1**
14 ± 2.6**
None
6 ± 1.3**
14 ± 2.9**
None
0.8
31
None
0.4
32
-
-
2.65
0.6
1.0
6.62
1.5
1.25
33 (G5-62)
34 (G5-53)
35
6.4 ± 0.2**
43.4 ± 8.3
20 ± 2.8
None
94.8 ± 5.3**
N.A.
0.4
1.6
N.A.
None
0.2
None
36
-
-
6.1
1.22
Thiopental Na
2 ± 0.1*
45 ± 3.6*
*: Significantly longer compared with that of thiopental Na (P < 0.05, N = 6).
N.A.: Values are not available due to Respiratory depression.
**: Significantly different compared with 33 (G5-62) and Thiopental Na.
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2.3 Mode of Action of the Test Compounds Induced Hypnosis
In order to study the mode(s) of action of the hypnotic activity of compounds GS-62 (33) or GS-53
(34), animals were injected with the minimal hypnotic doses (0.4 mmole/kg, i.p). Following
induction of sleep, each animal was injected with caffeine as adenosine R1 receptor blocker at doses
up to 800 mg/kg [31]; ketanserin as serotonin S2 (5-HT2) receptor blocker at 3 mg/kg [32]; picrotoxin
as GABAA receptor blocker at doses up to 40 mg/kg [33]; and flumazenil as non-selective but
specific benzodiazepine receptor blocker at 3 mg/kg [34-36]; or a combination of flumazenil and
picrotoxin in the above indicated doses. The animals were then carefully observed for arousal from
sleep and regaining of the righting reflex. Regaining of the righting reflex following injection of any
of the above treatments was considered as antagonism of the mechanism(s) involved in the induced
sleep. The onset time for reversal was noted. The results of the experiments revealed the failures of
caffeine, ketanserin, and flumazenil to reverse compounds GS-62 (33) and GS-53 (34) induced
hypnosis. This proves the dis-involvement of the brain adenosinergic, serotoninergic and
benzodiazepinergic systems or receptors in the induced sleep. However, the tendency of reversal of
the induced sleep following the administration of picrotoxin alone or the full reversal of sleep and
regaining of the righting reflex following the administration of combination of picrotoxin and
flumazenil clearly proved the involvement of the combined activation of GABAA and
benzodiazepine ω1 (Bnz-1) types of receptors either directly or indirectly.
Table 3: Synergism between compound GS-62 (33) or GS-53 (34)
and thiopental in induction of sleep in mice
Duration of sleep
(Minutes)
Dose
Onset time of sleep
Treatment
33 +
(mmole/kg, i.p) (Minutes)
0.05
15 ± 1.5
0.06
5 ± 0.8*
Thiopental
33 +
Thiopental
0.06
0.06
7.5 ± 1.3*
62.5 ± 5.9*
34 +
Thiopental
0.03
0.06
3.1 ± 1.1
75 ± 4.3*
45 ± 3.6
Thiopental
0.2
2 ± 0.3
*P < 0.05, compared with thiopental alone (N = 6).
2.4 Evaluation of the Test Compounds Synergistic Effect on Thiopental Sodium Sleep
Induction
Combined Intraperitoneal administration of one tenth of the hypnotic dose of 33 and 34 (0.03-0.06
mmole/kg) and one third of the hypnotic dose of 6 (0.06 mmole/kg, P<0.05, N=6) synergized each
other and produced a stable sleep of rapid onset and significantly longer duration than that induced
by 6 alone. Combination of an equimolar amounts (0.06 mmol) of 33 and 6 produced an onset of 7.5
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± 1.3 min and duration of 62.5 ± 5.9 min; combination of 34 (0.03 mol) and 6 (0.06 mol) produced
an onset of 3.1 ± 1.1 min and duration of 75 ± 4.3 min; while thiopental sodium alone (6, 0.2 mol)
showed an onset of 2 ± 0.3 min and duration of 45 ± 3.6 min. (Table 3).
3.
Structure Activity Correlations
The type of substituents at position 2- of the 1,3,4-thiadiazolo[3,2-a][1,3]diazepine analogues
affected the onset and duration times of the hypnotic activity. Aliphatic or aromatic substituents at 2-
position produced compounds with variable onsets (6.4-43.4 min). The presence of 2-methyl
function produced compound 30 with onset time and duration of 25±4.1, 6±1.3 min; respectively.
Replacement of the 2-methyl group by 2-triflouromethyl moiety produced 31 with shorter onset and
longer duration. The introduction of 2-ethyl group with one carbon atom longer afforded 32 with the
loss of hypnotic activity. The presence of phenyl ring at position 2- produced the most active
compound in the present study (33) with onset and duration of 6.4±0.2, 94.8±5.3 min; respectively.
Introduction of 4-bromo or 4-methyl function to the phenyl ring of 33 produced 34 and 35 with
respiratory depression, while the presence of 4-nitro group produced 36 with complete loss of
hypnotic activity.
4.
Molecular Modeling Study
4.1 Docking study
In order to further explore whether the test compounds could fit into the benzodiazepine (BZD)-
binding site of the GABAA receptor and hence possibly rationalize the results of the in vivo
experiments, the most active short acting hypnotic derivative 33, the least active derivatives 32, 36 as
well as 34 which caused respiratory depression were docked against the appropriate standard
GABAA agonist "diazepam", into the extracellular domain of the receptor. Although its crystal
structure has not been available yet, there are many homology models which are in a suitable
matching with the experimental findings [37,38]. A recently reported unified model of GABAA
receptor complex based on the glutamate-gated chloride ion channel was selected. The BDZ binding
site is located at the interface of GABAA receptors, taking into account that the most reliable docking
parameters for the evaluation of the whole extracellular domain of the receptor was explored [39].
Conformational analysis of the selected hypnotic compounds by AM1 calculation led to the best
conformers suited for the docking study (Figures 2 and 3). The favorable calculated pose for
diazepam (binding energy 10.55 kcal/mol) was assessed.
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Figure 2: Lowest energy conformers of compounds 33 (a) and 34 (b).
Figure 3: Lowest energy conformers of the inactive compounds 32 (a) and 36 (b).
Docking of diazepam identified a triad of amino acid residues, Arg87 and Arg149 which bind with
diazepam via cationic-arene bonding through its aromatic ring substituent, in addition to Thr150 with
a hydrogen bonding interaction of its carbonyl oxygen with a percentage of 15% which showed
pronounced influence to higher diazepam affinity (Figure 4a). Providing a considerable attention to
the tested biologically active hypnotic derivatives of this study, compound 33 binds to the BDZ site
via Arg87 and Arg149 by cationic-arene interaction; the same as diazepam but both amino acids
binds with its aromatic ring substituent (Figure 4b) while compound 34 binds with Arg87 residue by
cationic-arene and with Thr151 by hydrogen bonding through its diazepine ring carbonyl oxygen by
59%, which probably explain its different behavior and the respiratory depression effect (Figure 4c).
Thus compound 33 could exert its action via BDZ binding site agonism at GABAA receptor. When
compared to diazepam, it is clearly seen that 32 and 36 did not occupy any regions of the model that
could lead to any binding (Figure 5a,b), therefore, not expected to act as full agonists like diazepam,
as they do not have the necessary required interactions into the model under study for positive
allosteric modulation of the BDZ receptor.
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Figure 4: 2D Binding mode interaction and residues involved in the recognition of (a)
diazepam, (b) 33 and (c) 34 into BZD-binding site of GABAA receptor.
Figure 5: 2D Binding mode interaction and residues involved in the recognition
of the inactive compounds 32 and 36 into BDZ-binding pocket of GABAA
receptor.
According to the docking study results, the difference in the biological activity of the tested
compounds despite of their similar chemical structure could be explained. It could be noted that the
presence of aromatic substituent directly attached to the thiazole core without a spacer, seems to be
crucial for the interaction with the BZD-binding pocket. One of the explanations (within
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the“aromatic” series) could be illustrated in conformational freedom property to the molecule. Thus,
important structural characteristics of the active molecules may have favorable, diazepam like spatial
arrangements. The inactive molecules such as 32 and 36 were much more constrained and possibly
the absence of aromatic core in 32 or having certain structural attributes (such as highly electron
withdrawing group) attached to the aromatic ring as in 36 cannot avoid the unfavorable interactions
with “forbidden” regions of the receptor.
4.2 Flexible alignment and Superposition
Ligand-based active site alignment is a well-known technique for structural analysis of ligand
complexes. In the present study, flexible alignment comparative modeling experiment among the
most active 33 and the least active 32 against diazepam was performed. An alignment is considered
to be successful when the molecule strain energy is small, possessing similar shapes and their
aromatic atoms overlap [40]. The goal of computing a multiple ligand alignment of a set of input
ligands is to maximize the similarity between these ligands, while placed in sound conformations. In
order to test the similarity between the 3D structures of the most active 33 and diazepam, flexible
alignment was performed using MOE/MMFF94 to generate superposition with minimal user bias
[41]. It was interesting to note the similarity in the direction of flipping mode of both 33 and
diazepam (Figure 6a) while 32 seems to have different alignment angle which prevent its proper far
reach into the receptor active site (Figure 6b).
Figure 6: (a) Flexible alignment of the most active compound 33 (pink) and diazepam
(blue). (b) Flexible alignment of the least active compound 32 (yellow) and diazepam (blue).
Superposition of 3D putative ligands could help to deduce specific structural requirements for
biological activity [42,43]. Structure superposition of 33 and 34 (Figure 7a) with mark points such
as, the π-systems represented by aryl groups of the thiadiazolodizepine ring system core, phenyl ring
substituent, and both N and S center atoms, have a fairly well alignment. This superposition implies
the possible existence of region(s) on the receptor suitable for the specific binding and recognition of
the previously mentioned features. This assumption is in accordance with the obtained experimental
10

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data. Moreover, these selected mark points could be used as template model for further optimization.
In addition, upon conducting the flexible alignment experiments it was noticed that there is a
similarity in the alignment profiles among the inactive derivatives 32 and 36 (Figure 7b). On the
other hand, applying the same conditions on the active 33 versus the inactive 36 showed a different
pattern with the minimal alignment profile (Figure 7c) which explains their different biological
activity behavior.
Figure 7: (a) Superposition of the most active compounds 33 (pink) and 34 (cyan). (b) Flexible
alignment of the least active compounds 32 (yellow) and 36 (green). (c) Flexible alignment of
the most active compound 33 (pink), against least 36 (yellow).
Figure 8: Surface map for (a) diazepam, (b) the most active compound 33, (c) the least
active compound 32. Pink: hydrogen bond, blue: mild polar, green: hydrophobic regions.
11

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4.3 Surface mapping
In a valuable extension, surface mapping comparing study among the most active 33, and the least
active counterpart 32 against diazepam was performed. Compound 33 showed hydrophilic region (in
red), hydrogen bond acceptor–donor region located on the 4-carbonyl, N-1, non-polar area located on
the thiazole ring (in green), in addition to the mildly non polar center on the aryl moiety which are
generally having a similar structural features with the surface mapping distribution of diazepam
(Figure 8a,b). On the contrary there is a clear difference in surface mapping distribution between 32
and diazepam (Figure 8a,c). These results provided structure mapping similarity and hence explained
the biological resemblance of 33 and diazepam. These findings supported the hypothesis that the
tested compounds might exert their hypnotic action via GABAA agonism.
Table 4: Pharmacokinetic parameters of compounds 33 and 34
Compound b_rotN Lip_acc Lip_don Lip_druglike Lip_viol LogP TPSA
Mwt
1
1
4
4
0
0
1
1
0
0
3.77
4.57
45
48
245.30
324.20
33
34
b_rotN: Number of rotatable bonds, Lip.acc: Number of hydrogen bond acceptors, Lip.don: Number of
hydrogen bond donors, Lip_druglike: Drug calculated ability, Lip.V: Number of violations of Lipinski rule,
Log P: Calculated lipophilicity, Mwt: Molecular weight, TPSA: Polar surface area
.
4.4 Molecular properties and drug-likeness
Molecular properties are mixture of structural balanced features which determine the feasibility of
comparing particular molecule to known drugs. Acceptable drug bioavailability could be achieved
upon reaching an appropriate balance between solubility and partitioning properties. The most active
compounds in this study were subjected to solubility prediction analysis, their conformity with
Lipinski's rule of five, in addition to other properties shown in Tables 4. The Lipinski's rule of five
depends on the statistical calculations stating that the most biologically active drugs have number of
hydrogen bond donor not higher than 5, hydrogen bond acceptor sites not higher than 10, molecular
weight of not more than 500, and logP values not higher than 5 [44]. Topological polar surface area
(TPSA) and number of rotatable bonds are linked to drug bioavailability. Molecules violating more
than one of Lipinski's rule of five, and molecules with more than 10 rotatable bonds may have
problems with bioavailability [45-47]. TPSA is a feasible descriptor for drug absorption, including
intestinal bioavailability and blood–brain barrier penetration. Molecules with TPSA values around
140 Å or less are expected to exhibit high intestinal absorption [45]. All descriptors were calculated
by the MOE package for the active compounds. Results shown in Table 4 indicated that compounds
33 and 34 have TPSA values <140 Å, and all compounds have zero violation of Lipinski's rule;
12

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therefore, they are not expected to have problems with bioavailability. Based on these data, it could
be suggested that 33 and 34 have a lipiniski drug likeness of 1 which make them suitable drug
candidates in addition to their expected orally absorbed tendency as short acting hypnotics.
5.
Conclusion
The obtained results clearly point to the discovery of compounds GS-62 (33) and GS-53 (34) as a
new group of short acting hypnotics that induce their actions via interaction with GABAA and
benzodiazepine ω1 receptors as proven experimentally. Thus, a new means for treatment of insomnia
with all of its sleep disorders seemed to be at hand. The safety of the new compounds is comparable
to that of thiopental sodium (6) and its duration is significantly longer. In addition, 33 and 34 did not
show any sign of acute tolerance reported with the second (maintenance) dose of 6. Therefore, the
new compounds have the potential use as a preanesthetic medication, induction of anesthesia, and
treatment of insomnia. Combined administration of one tenth of the hypnotic dose of 33 and 34
together with one third of 6, both in doses lower than the effective dose, attained the same hypnotic
potency avoiding the drawbacks and side effects associated with 6 full dose administrations. The
durations were significantly longer than that induced by the hypnotic dose of 6 alone. Figure 9
showed the structures of the most active short acting hypnotics GS-62 (33) and GS-53 (34).
Molecular docking interactions between the active ligands 33, 34 and diazepam at interface residues
in GABAA receptor extracellular domain homology model have been calculated showing two types
of interactions. A cationic-arene interaction with Arg 87and Arg149 in addition to hydrogen bonding
interaction with Thr150 and Thr151 as being essential for high-affinity at BDZ binding site. The
incorporation of the thiadiazolzepine core and aromatic ring were judged to be crucial structural
requirements for the observed hypnotic activity. Additional modeling studies including flexible
alignment, surface mapping and drug likeness have shed more light on the explanation of the activity
patterns. Thus, the herein presented data could be considered as a starting point for a new chapter in
the design of new thiadaizolodiaepine based CNS active agents.
Figure 9: Structures of the most active short acting hypnotic
compounds GS-62 (33) and GS-53 (34).
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6.
Experimental Part
Melting points (°C) were determined on Mettler FP80 melting point apparatus and are uncorrected.
Microanalyses were performed on a Perkin-Elmer 240 elemental analyzer at the Central Research
Laboratory, College of Pharmacy, King Saud University. All of the new compounds were analyzed
for C, H and N and agreed with the proposed structures within ± 0.4% of the theoretical values. 1H,
13C-NMR spectra were recorded on a on Bruker 500 MHz FT spectrometer (the Central Research
Laboratory, College of Pharmacy, King Saud University); chemical shifts are expressed in δ ppm
with reference to TMS. Mass spectral (MS) data were obtained on a Perkin Elmer, Clarus 600
GC/MS and Joel JMS-AX 500 mass spectrometers. Thin layer chromatography was performed on
pre-coated (0.25 mm) silica gel GF254 plates (E. Merck, Germany), compounds were detected with
254 nm UV lamp. Silica gel (60−230 mesh) and neutral alumina were employed for routine column
chromatography separations. Compounds 9-15 and 16-22 were previously reported [23-25]. Adult
male Swiss albino mice (22-28 g), approximately 10-week old, were used to conduct the short acting
hypnotic evaluation. They were housed in cages and kept at a temperature of 20±2°C and a relative
humidity of 55±5% with a light-dark cycle of 12 h. and fed with standard diet and water ad libitum.
Compounds have been dissolved in dimethylsulfoxide and administered in a maximum volume of 1
ml/kg intraperitoneal whereas larger doses were suspended in 0.25% aqueous sodium carboxy-
methylcellulose and thoroughly homogenized and administered in volumes up to 10 ml/kg
intraperitoneal. In all experiments a control group of mice was included and received intraperitoneal
injections of the test compounds’ vehicle. All the procedures involving animals were conducted in
conformity with the Institutional guidelines that are in compliance with National and International
laws and policies.
6.1.
Chemistry
6.1.1. General procedure for the preparation of 4-Chloro-N-(5-substituted-1,3,4-thiadiazol-2-
yl)butanamide (23-29)
A mixture of 5-substituted-1,3,4-thiadiazol-2-amine (16-22, 0.04 mol), 4-chloro-butyryl chloride
(11.3 g, 9.0 ml, 0.08 mol) and potassium carbonate (5.5 g, 0.04 mol) in toluene (100 ml) was stirred
at room temperature for 4 hr. The toluene was then evaporated under reduced pressure. The residue
was then quenched with water, stirred, and filtered then purified by silica gel and neutral alumina
chromatography. The obtained solid was washed, dried and recrystallized to give the required
products 23-29 (Table 1).
1
6.1.1.1. 4-Chloro-N-(5-methyl-1,3,4-thiadiazol-2-yl)butanamide (23). H NMR (DMSO-d6): δ 1.88-
14

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2.04 (m, 2H, -CH2), 2.43-2.72 (m, 2H, -CH2), 3.69 (s, 3H, -CH3), 3.69-3.72 (m, 2H, -CH2), 10.74 (br
s, 1H, NH). 13C NMR: δ 20.3, 26.3, 33.1, 44.6, 142.3, 153.1, 171.7. MS m/z (%): 219 (25.7, M+).
6.1.1.2. 4-Chloro-N-[5-(trifluoromethyl)-1,3,4-thiadiazol-2-yl]butanamide (24).1H NMR (DMSO-
d6): δ 1.91-2.02 (m, 2H, -CH2), 2.47 (t, J = 7.0 Hz, 2H, -CH2), 3.69 (t, J = 7.0 Hz, 2H,-CH2), 11.65
(br s, 1H, NH). 13C NMR: δ 27.6, 32.9, 43.1, 120.6, 154.4, 158.5, 171.2. MS m/z (%): 273 (14, M+).
1
6.1.1.3. 4-Chloro-N-(5-ethyl-1,3,4-thiadiazol-2-yl)butanamide (25). H NMR (DMSO-d6): δ 1.21 (t,
J = 7.5 Hz, 3H, CH3), 1.89-2.06 (m, 2H, -CH2), 2.41 (t, J = 7.0 Hz, 2H, -CH2), 3.02-3.31 (q, J = 7.5
13
Hz, 2H, -CH2), 3.64 (t, J = 7.0 Hz, 2H, -CH2), 10.35 (br s, 1H, NH). C NMR: δ 14.3, 25.2, 26.9,
33.3, 43.4, 153.4, 164.5, 171.8. MS m/z (%): 273 (14.0, M+).
1
6.1.1.4. 4-Chloro-N-(5-phenyl-1,3,4-thiadiazol-2-yl)butanamide (26). H NMR (DMSO-d6): δ 2.07-
2.10 (m, 2H, -CH2), 2.67-2.70 (m, 2H, -CH2), 3.70-3.72 (m, 2H, -CH2), 7.48-7.54 (m, 3H, ArH),
7.93-7.94(m, 2H, ArH), 12.65 (br s, 1H, NH). 13C NMR: δ 27.9, 32.7, 45.1, 127.4, 129.8, 130.7,
131.0, 158.5, 162.4, 171.2. MS m/z (%): 281 (15.7, M+).
1
6.1.1.5. N-[5-(4-Bromophenyl)-1,3,4-thiadiazol-2-yl]-4-chlorobutanamide (27). H NMR (DMSO-
d6): δ 1.96-2.22 (m, 2H, -CH2), 2.69-2.81 (m, 2H, -CH2), 3.71-3.72 (m, 2H, -CH2), 7.49-7.88 (dd, J =
13
7.0 Hz, 4H, ArH), 12.76 (br s, 1H, NH). C NMR: δ 27.3, 32.1, 44.6, 123.8, 128.7, 129.4, 132.3,
158.5, 160.7, 170.7. MS m/z (%): 360 (15.7, M+).
1
6.1.1.6. 4-Chloro-N-(5-(4-methylphenyl)-1,3,4-thiadiazol-2-yl)butanamide (28). H NMR (DMSO-
d6): δ 2.07-2.09 (m, 2H, CH2), 2.38 (s, 3H, CH3), 2.68 (t, 2H, J = 14.5 Hz, CH2), 3.71 (t, 2H, J = 14.5
Hz, CH2), 7.34 (d, 2H, J = 7.5 Hz, Ar-H), 7.83 (d, 2H, J = 7.5 Hz, Ar-H), 12.68 (s, 1H, NH).
13C NMR: δ 20.9, 27.3, 32.1, 44.7, 126.8, 127.5, 129.9, 140.5, 157.9, 161.8, 170.6. MS m/z (%): 295
(3.5, M+).
1
6.1.1.7. 4-Chloro-N-(5-(4-nitrophenyl)-1,3,4-thiadiazol-2-yl)butanamide (29). H NMR (DMSO-d6):
δ 2.35-2.40 (m, 2H, CH2), 2.78 (t, 2H, J = 12.0 Hz, CH2), 4.31 (t, 2H, J = 12.0 Hz, CH2), 7.26 (s, 1H,
13
NH), 8.15 (d, 2H, J = 8.5 Hz, Ar-H), 8.35 (d, 2H, J = 8.5 Hz, Ar-H). C NMR: δ 18.3, 31.2, 47.9,
124.5, 128.0, 136.3, 148.8, 158.4, 161.4, 173.8. MS m/z (%): 326 (12.9, M+).
6.1.2. General procedure for the preparation of 2-substituted-6,7-dihydro-[1,3,4]thiadiazolo[3,2-
a][1,3]diazepin-8(5H)-ones (30-36)
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A mixture of 4-chloro-N-(5-substituted-1,3,4-thiadiazol-2-yl)butanamide (23-29, 0.004 mol) and
piperidine (0.7 g, 0.8 ml, 0.008 mol) in toluene (50 ml) was heated under reflux for 3 h. The reaction
mixture was cooled, poured into water and stirred. Toluene was separated dried and evaporated to
give a crude product which was purified by repeated silica gel and neutral alumina column
chromatography eluting with EtOAc/hexane (50:50 v/v) and CHCl3/hexane (80:20 v/v). The solid
obtained was washed, dried and recrystallized to give the required products 30-36 (Table 1).
6.1.2.1. 2-Methyl-6,7-dihydro-[1,3,4]thiadiazolo[3,2-a][1,3]diazepin-8(5H)-one (30). 1H NMR
(CDCl3): δ 1.31 (s, 3H, CH3), 1.98 (m, 2H, -CH2), 2.36 (t, J = 7.5 Hz, 2H, -CH2), 2.75 (t, J = 7.5 Hz,
2H, -CH2). 13C NMR: δ 20.3, 24.1, 31.3, 47.9, 156.4, 162.9, 173.6. MS m/z (%): 183 (15.0, M+).
6.1.2.2. 2-(Trifluoromethyl)-6,7-dihydro-[1,3,4]thiadiazolo[3,2-a][1,3]diazepin-8(5H)-one (31).
1H NMR (CDCl3): δ 2.08 (m, 2H, -CH2), 2.42 (t, J = 7.5 Hz, 2H, -CH2), 2.68 (t, J = 7.5 Hz, 2H, -
CH2). 13C NMR: δ 24.3, 31.7, 48.9, 120.0, 136.4, 162.1, 173.3. MS m/z (%): 237 (22.0, M+).
6.1.2.3. 2-Ethyl-6,7-dihydro-[1,3,4]thiadiazolo[3,2-a][1,3]diazepin-8(5H)-one (32). 1H NMR
(CDCl3): δ 1.13 (t, J = 7.0 Hz, 3H, -CH3), 2.08 (m, 2H, -CH2), 2.42 (t, J = 7.5 Hz, 2H, -CH2), 2.61
13
(q, J = 7.0 Hz, 2H, -CH2), 2.75 (t, J = 7.5 Hz, 2H, -CH2). C NMR: δ 17.3, 25.6, 29.3, 31.7, 48.7,
156.4, 161.1, 172.9. MS m/z (%): 197 (17.0, M+).
6.1.2.4. 2-Phenyl-6,7-dihydro-[1,3,4]thiadiazolo[3,2-a][1,3]diazepin-8(5H)-one (33). 1H NMR
(CDCl3): δ 2.33-2.36 (m, 2H, -CH2), 2.75 (t, J = 7.5 Hz, 2H, -CH2), 4.28-4.31 (t, J = 7.5 Hz, 2H, -
13
CH2), 7.46-7.52 (m, 3H, ArH), 7.94-8.10 (m, 2H, ArH). C NMR: δ 18.3, 31.3, 47.9, 127.4, 129.1,
130.5, 130.6, 157.4, 163.9, 173.6. MS m/z (%): 245 (9.0, M+).
6.1.2.5. 2-(4-Bromophenyl)-6,7-dihydro-[1,3,4]thiadiazolo[3,2-a][1,3]diazepin-8(5H)-one (34).
1H NMR (CDCl3): δ 2.32-2.40 (m, 2H, -CH2), 2.76 (t, J = 7.5 Hz, 2H, -CH2), 4.29 (t, J = 7.5 Hz, 2H,
-CH2), 7.62-7.84 (dd, J = 8.5 Hz, 4H, ArH). 13C NMR: δ 18.3, 31.2, 47.9, 125.0, 128.7, 129.5, 132.3,
157.5, 162.9, 173.7. MS m/z (%): 324 (7.0, M+).
6.1.2.6. 2-(4-Methylphenyll)-6,7-dihydro-[1,3,4]thiadiazolo[3,2-a][1,3]diazepin-8(5H)-one (35).
1H NMR (DMSO-d6): δ 2.22 (t, 2H, J = 14.5 Hz, CH2), 2.38 (s, 3H, CH3), 2.68 (t, 2H, J = 16.0 Hz,
CH2), 4.12 (t, 2H, J = 14.0 Hz, CH2), 7.35 (d, 2H, J = 7.5 Hz, Ar-H), 7.87 (d, 2H, J = 8.0 Hz, Ar-H).
13C NMR: δ 17.7, 20.9, 30.7, 47.7, 123.5, 125.2, 126.9, 127.1, 129.9, 140.7, 156.8, 166.8, 174.1. MS
m/z (%): 259 (29.1, M+).
16

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6.1.2.7.
2-(4-Nitrophenyl)-6,7-dihydro-[1,3,4]thiadiazolo[3,2-a][1,3]diazepin-8(5H)-one
(36).
1H NMR (DMSO-d6): δ 2.28 (t, 2H, J = 15.5 Hz, CH2), 2.72 (t, 2H, J = 14.5 Hz, CH2), 4.16 (t, 2H, J
= 15.0 Hz, CH2), 8.28 (d, 2H, J = 7.0 Hz, Ar-H), 8.36 (d, 2H, J = 7.5 Hz, Ar-H). 13C NMR: δ 21.3,
30.7, 47.8, 124.5, 125.0, 126.9, 128.2, 129.9, 141.3, 154.0, 163.7, 174.1. MS m/z (%): 290 (25.2,
M+).
6.2.
Measurement of the hypnotic effect of compounds 30-36
The hypnotic activity of the test compounds in mice was measured using the standard righting reflex
method as described [26-29,48,49] with some modifications. Mice were initially tested for the
presence of the righting reflex by placing each mouse on its back and observing the rapid correction
to the normal position i.e. the righting reflex. Then groups of mice were injected (i.p) with various
doses of the test compound and placed each separately under a 30-cm glass funnel. The animals were
then observed carefully for any change in behavior such as unsteady movements, drowsiness, ataxia
and loss of the righting reflex. Failure of any treated mouse to correct its posture to the normal
condition of standing on its feet within one minute was considered as loss of the righting reflex and
hence onset of sleep. The onset of sleep was carefully noted and recorded and the mice were
continuously monitored visually and by videotaping and the duration of the sleep was noted. The end
of the duration of sleep was noted by the regaining of the righting reflex 3 times within one minute
albeit some drowsiness is still observed. Intraperitoneal administration of compounds 30-36 and
thiopental sodium in doses of 0.2–2 mmol/kg were performed and the minimal effective doses, the
onset times and the durations of sleep were recorded, Table 2.
6.3.
Study of the hypnotic effect mode(s) of action of compounds 30-36
Male mice were injected (i.p) with the minimal hypnotic doses. Following induction of sleep, the
animals were allowed to enjoy their sleep for 5 minutes. Then each animal was injected with caffeine
at doses up to 800 mg/kg, ketanserin (3 mg/kg), picrotoxin up to (40 mg/kg), flumazenil (1.2 or 3
mg/kg) (i.p) or a combination of flumazenil and picrotoxin in the above indicated doses. The animals
were then carefully observed visually and by the aid of a video camera for arousal from sleep and
regaining of the righting reflex. Regaining of the righting reflex following injection of any of the
above treatments was considered as antagonism of the mechanism(s) involved in the induced sleep
[31]. The onset time for reversal was noted.
6.4. Potentiating effect of compounds 33 (GS-62) or 34 (G-53) to the non-hypnotic dose of
thiopental sodium in mice
17

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Combined intraperitoneal administration of GS-62 (33) or G-53 (34) and thiopental sodium in doses
of 0.03–0.06 mmol/kg into mice induced hypnosis. The minimal effective doses, the onset times and
the durations of sleep were recorded (Table 3).
6.5.
Determination of the Lethal Dose (LD50) and the Therapeutic Indices of compounds 30-36
Male mice were divided into various groups and test compounds were administered in various doses
ranging from 0.1-5 mmole/kg, (i.p.). Following treatments, the animals were observed for up to 6
hours continuously and were then kept under observation for 72 hours. All behavioral changes and
death during the observation periods were recorded. The percentage of death at each dose level was
then calculated, converted to probits and the LD50 values were calculated as reported [30]. The
Therapeutic Index of test compounds were calculated following the determination of the minimal
effective hypnotic and the LD50 values (Table 2) by the formula:
.
6.6.
Docking and Molecular modeling study
Conformational analysis of some selected compounds represent the active short hypnotic derivatives
(33 and 34), and the least active counterparts 32 and 36, along with diazepam as a standard were
performed. Basically through MMX force field method followed by AM1 calculation implemented
in MOE 2009.10. The model of the GABAA receptor was used for docking. [39]. Three-dimensional
structures of the selected thiadiazolodiazepine representatives, in their neutral forms were built using
the MOE of Chemical Computing Group Inc software. Lowest energy conformer of every new
analogue ‘global-minima’ was docked into the homology model used. The energy-minimized
structure was used for molecular modeling studies. Ligand structures were built with MOE and
minimized using the MMFF94x force field until an RMSD gradient of 0.05 kcal/mol_A was reached.
For each ligand, energy minimizations (EM) were performed using 1000 steps of steepest descent,
followed by conjugate gradient minimization to an RMSD energy gradient of 0.01 kcal/mol_A. The
docking experiments were done using the alpha triangle placement method and the London dG
scoring method. 300 results for each ligand were generated, excluding the results having RMSD
value >3 [36]. The best scored result of the remaining conformations for each ligand was further
analyzed. The investigated compounds were subjected to flexible alignment and superposition. Their
geometry was optimized using the MMFF94 forcefield followed by a flexible alignment using
systematic conformational search [40,50]. Surface mapping and molecular properties calculations
18

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using ‘Molecular Operating Environment’ software (MOE of Chemical Computing Group Inc., on a
Core i7, 2.7 GHz workstation) were performed.
Acknowledgements
Thanks are due to King Saud University patency authority for financing the patency process.
7.
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Captions
Table 1: Physicochemical properties of the synthesized compounds 23-36
Table 2: Influence of the test compounds 30-36 and thiopental sodium on sleep in mice (N = 6),
LD50 and the Therapeutic Indices values.
Table 3: Synergism between compound GS-62 (33) or GS-53 (34) and thiopental in
induction of sleep in mice
Table 4: Pharmacokinetic parameters of compounds 33 and 34
Figure 1: Structures of some literature hypnotic lead compounds.
Figure 2: Lowest energy conformers of compounds 33 (a) and 34 (b).
Figure 3: Lowest energy conformers of the inactive compounds 32 (a) and 36 (b).
Figure 4: 2D Binding mode interaction and residues involved in the recognition of (a) diazepam,
(b) 33 and (c) 34 into BZD-binding site of GABAA receptor.
Figure 5: 2D Binding mode interaction and residues involved in the recognition of the
inactive compounds 32 and 36 into BDZ-binding pocket of GABAA receptor
Figure 6: (a) Flexible alignment of the most active compound 33 (pink) and diazepam (blue). (b)
Flexible alignment of the least active compound 32 (yellow) and diazepam (blue).
Figure 7: (a) Superposition of the most active compounds 33 (pink) and 34 (cyan). (b) Flexible
alignment of the least active compounds 32 (yellow) and 36 (green). (c) Flexible
alignment of the most active compound 33 (pink), against least 36 (yellow).
Figure 8: Surface map for (a) diazepam, (b) the most active compound 33, (c) the least active
compound 32. Pink: hydrogen bond, blue: mild polar, green: hydrophobic regions.
Figure 9: Structures of the most active short acting hypnotic compounds GS-62
(33) and GS-53 (34).
Scheme 1: Synthesis of the target compounds 30-36
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Research Highlights
•
•
•
•
•
Synthesis of (E)-6,7-dihydro-[1,3,4]thiadiazolo[3,2-a][1,3]diazepines.
Compound GS-62 (33) is a short acting hypnotic.
Compound 33 potentiate the in vivo hypnotic effect of thiopental sodium
Recognition with Arg87, Arg149 at BDZ binding site essential for activity
The new compounds exert their action via GABAA agonism.