Thiadiazolodiazepine analogues as a new class of neuromuscular blocking agents: Synthesis, biological evaluation and molecular modeling study

Article abstract of DOI:10.1016/j.ejmech.2016.09.096

The synthesis, biological evaluation and molecular modeling study of 6,7-dihydro-[1,3,4] thiadiazolo[3,2-a][1,3]diazepine analogues as new class of neuromuscular blocking agents are described. The new compounds act via competitive mechanism with ACh which

Full text of DOI:10.1016/j.ejmech.2016.09.096

Accepted Manuscript
Thiadiazolodiazepine analogues as a new class of neuromuscular blocking agents:
Synthesis, biological evaluation and molecular modeling study
Hussein I. El-Subbagh, Adel S. El-Azab, Ghada S. Hassan, Shahenda M. El-Messery,
Alaa A.-M. Abdel-Aziz, Kamal E.H. El-Taher
PII:
S0223-5234(16)30859-5
10.1016/j.ejmech.2016.09.096
EJMECH 8962
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 13 September 2016
Revised Date: 28 September 2016
Accepted Date: 29 September 2016
Please cite this article as: H.I. El-Subbagh, A.S. El-Azab, G.S. Hassan, S.M. El-Messery, A.A.-M. Abdel-
Aziz, K.E.H. El-Taher, Thiadiazolodiazepine analogues as a new class of neuromuscular blocking
agents: Synthesis, biological evaluation and molecular modeling study, European Journal of Medicinal
Chemistry (2016), doi: 10.1016/j.ejmech.2016.09.096.
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1
Thiadiazolodiazepine Analogues as a New Class of
Neuromuscular Blocking Agents: Synthesis,
Leave this area blank for abstract info.
Biological Evaluation and Molecular Modeling Study.
Hussein I. El-Subbagh1,2*, Adel S. El-Azab3,4, Ghada S. Hassan1, Shahenda M. El-Messery5, Alaa A.-M. Abdel-
Aziz1,3, Kamal E. H. El-Taher6
Structures of the most active neuromuscular blocking compounds GS-53 (30), and AAH1 (33)

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Thiadiazolodiazepine Analogues as a New Class of Neuromuscular Blocking Agents: Synthesis,
Biological Evaluation and Molecular Modeling Study.#
Hussein I. El-Subbagh1*, Adel S. El-Azab2,3, Ghada S. Hassan1, Shahenda M. El-Messery4, Alaa A.-
M. Abdel-Aziz1,2, Kamal E. H. El-Taher5
1Department of Medicinal Chemistry, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt
2Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P.O.Box 2457,
Riyadh 11451, Saudi Arabia
3Department of Organic Chemistry, Faculty of Pharmacy, Al-Azhar University, Cairo 11884, Egypt
4Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Mansoura University, Mansoura
35516, Egypt
5Department of Pharmacology, College of Pharmacy, King Saud University, P.O.Box 2457, Riyadh 11451,
Saudi Arabia
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
The synthesis, biological evaluation and molecular modeling study of 6,7-dihydro-[1,3,4]
thiadiazolo[3,2-a][1,3]diazepine analogues as new class of neuromuscular blocking agents are
described. The new compounds act via competitive mechanism with ACh which could be reversed
by the anticholinesterase - Physostigmine. Compounds GS-53 (30) and AAH1 (33) induced dose-
dependent neuromuscular blockade with onset time of 3 and 10 min, ED50 0.15 and 0.36 mmoles/kg
i.p., respectively, in rats. Compound 30 proved to be as twice as potent as 33 with rapid onset and
shorter duration (P < 0.05). Docking profile of 30 and 33 closely resembles HIE-124 (3), in α7β2
nAChR receptor. Molecular modeling analysis indicated that hydrogen bonding to Thr120 and
Thr124 beside hydrophobic interactions play effective role incorporating the active ligands to
nAChR. The obtained model could be useful for further development of new skeletal muscle
relaxants.
Keywords: Synthesis, Thiadiazolodiazepine Analogues, Neuromuscular Blocking Agents, Molecular
Modeling Study
____________
# Part of the Patents EP 2 514 754 B1 and US 8,846,665 B2, ref. [16, 17]
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1.
Introduction
Neuromuscular blockade and anesthesia are essential for surgical procedures. The use of
neuromuscular blockers or skeletal muscle relaxants will decrease the doses of the general
anesthetics. Neuromuscular blockers are used to ease tracheal intubation and to suppress the patient’s
spontaneous breathing. Other uses of skeletal muscle relaxants include prevention of fractures during
electroshock therapy, suppression of titanic convulsions and diagnosis of myasthenia gravis. The
history of these drugs goes back to the use of curare, the alkaloid extract of Chardrodendron
tomentosum which followed by the introduction of Atracurium besylate (1) and Succinylcholine
chloride (2), Figure 1 [1-7]. Neuromuscular blockers can be classified according to their mechanism
of action into competitive and depolarizing blockers. Most of the drugs currently in use are of the
competitive type. Competitive neuromuscular blockers block the nicotinic acetylcholine receptor
(nAChR) located postsynaptic on skeletal muscle membranes. They compete with acetylcholine and
prevent its action on evoking muscular contraction and thus muscle relaxation ensues gradually. The
depolarizing blockers mimic ACh in their ability to activate the postsynaptic nicotinic receptors on
the skeletal muscles but differ from ACh in their ability to induce persistent depolarization of the
skeletal muscles rendering them insensitive to any released ACh. The only drug of this group that is
still in use is Succinylcholine (2) due to its rapid action and short duration (up to 5 minutes following
bolus injections at 0.5-2 mg/kg i.v.), [2-4]. The competitive neuromuscular blockers are mostly used
in medicine due to their antagonism by anticholinesterases e.g. Neostigmine, Pyridostigmine or
Edrophonium. However, those drugs suffer from various side effects e.g. apneas, non-neuromuscular
blockade, release of histamine with consequent hypotension, broncho-constriction and excessive
mucus secretions, and headache as observed with 1 [5-7], stimulation of sympathetic autonomic
ganglia, induction of tachycardia, elevation of the arterial pressure, increase in the intraocular
pressure and induction of hyperkalemia as observed with 2 [2,3]. An ideal neuromuscular blocker
should possess a rapid onset of action, reasonable duration, and rapid reversibility after ending its use
together with freedom from non-neuromuscular blockade side effects. The majority of selective
nicotinic acetylcholine receptor (nAChR) antagonists are natural products with marked complexity in
size which makes them not suitable as lead compounds for drug discovery. To date, 17 nAChR
subunits have been cloned, and among them five are of muscle-type. Development of selective
agonists or antagonists may therefore result in new and potentially useful therapeutic agents [8, 9].
Recently, 6,7-dihydro-[1,3,4]thiadiazolo[3,2-a][1,3]diazepine analogues were synthesized as
structure modification derivatives of the patented “HIE 124, 3” [10-14]. The obtained compounds
proved to possess short acting hypnotic activity in addition to in vivo potentiating effect toward the
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known ultra-short acting hypnotic "Thiopental sodium" [15]. Some of the studied derivatives, at
doses around 0.4 mmole/kg, showed respiratory depression to the tested animals as a sign of sever
muscle relaxation. The use of lower doses of those derivatives led to the discovery of a new class of
neuromuscular blocking compounds discussed in the present study. New structure modified
derivatives were synthesized which showed competitive neuromuscular blocking activity, devoid of
the disadvantages of the previous generations of muscle relaxants, with reasonable onset and
intermediate duration of action which can be rapidly reversed by anticholinesterases. The obtained
results contributed in part to the issuance of European and US patents [16, 17].
CH3
O
H3C
O
O
O
H3C
O
CH3
O
O
CH3
O
H3C
O
O
H3C
CH3
N
N
CH3
CH3
O
O
Besylate
Atracurium, 1
EtOOC
Cl
N
H3C
CH3
O
O
H3C
H3C
O
N
N
CH3
S
O
N
CH3
Cl
O
Succinylcholine, 2
HIE-124, 3
Figure 1: Structures of literature neuromuscular blocking agents (1, 2),
and the ultra-short acting hypnotic 3.
2.
Results and Discussion
Chemistry
2.1
1,3,4-Thiadiazolo[3,2-a][1,3]diazepine as a heterocyclic system, usually obtained according to
literature procedures [18,19]. Compounds of the present study (30-37) were synthesized according to
the inventive method showed in Schemes 1 and 2. The proper aldehyde and thio-semicarbazide were
mixed in alcohol with stirring to get the required thiosemicarbazones 6-13 which were suspended in
FeCl3 solution to get the required 2-amino-5-substituted-1,3,4-thiadiazole 14-21 [20]. Compounds
14-21 were 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 22-29, which were purified by
silica gel and neutral alumina chromatography. The butanamide analogues 22-29 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 30-37, (Scheme 1). Structure elucidation of the new
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1
13
synthesized intermediates and final products was attained by the aid of elemental analysis, H, C
NMR, and Mass spectrometry.
Scheme 1: Synthesis of the target compounds 30-37
2.2
Evaluation of the Neuromuscular Blocking Activity
The neuromuscular blocking activity of the test compounds 30-37 was performed by measuring its
effect on rat tibialis muscle and the sciatic nerve as well as its effect in chicks using standard
reported procedures [21, 22].
2.2.1. The neuromuscular blocking activity of test compounds on rat tibialis muscle electrically-
induced twitches and the sciatic nerve
Each of the test compounds (30-37), Atracurium besylate (1), and Succinylcholine (2); as positive
controls, or vehicles were administered (i.p.) at various doses. The effect of each dose on the rat
tibialis muscle twitches response was measured as a percentage of the pre-drug amplitude. The ED50
and the dose that produced 90% of twitches depression were calculated for each drug. The response
of the muscle to titanic stimulation was tested before administration of any drug and following 90%
inhibition of the twitches. To investigate the reversibility of any neuromuscular block, the
anticholinesterase Physostigmine was injected in a single bolus injection of 100 µg/kg. The onset
time of reversal and the percentage of reversal were monitored and calculated, respectively. The
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duration of each block was monitored. Only compounds GS-53 (30) and AAH1 (33) showed
remarkable activity, the rest of the test compounds proved to be devoid of neuromuscular blocking
activity. Administration of compounds 30 and 33 in various doses into the anaesthetized rats induced
dose-dependent inhibitions of the twitches i.e. neuromuscular blockade on skeletal muscle relaxation.
The ED50 were 0.15 and 0.36 mmoles/kg i.p., respectively. The corresponding values for the
competitive neuromuscular blocker Atracurium besylate (1) and the depolarizing neuromuscular
blocker Succinylcholine chloride (2) were 0.016 and 0.03 mmoles, respectively. The onset times of
30 and 33 were 3 and 10 min, with duration of 70 and 110 min; whereas those of 1 and 2 were 5 and
2 min, with duration of 30 and 20 min, respectively. Compound GS-53 (30) seemed to be twice as
potent as AAH1 (33) (P < 0.05) with rapid onset and shorter duration (P < 0.05). GS-53 (30)
blockade was completely reversed following administration of Physostigmine while that of AAH1
(33) was not. The potency of 30 seemed to be 1/10th that of Atracurium (1) and 20% that of
Succinylcholine (2). The duration of action of 30 seemed to be 2.3 times, whereas the duration of 33
was more than 3 times that of Atracurium (1), Table 1.
Table 1: Effects of GS-53 (30), AAH1 (33), Atracurium (1) and Succinylcholine (2) on the rat
tibialis electrically-induced twitches together with the effects of tetanus and
Physostigmine at a dose of 100 mg/kg i.p. (N = 4-8 animals).
GS-53 (30)
AAH1 (33)
*Parameter
Atracurium
Succinyl choline
0.15
0.36
0.016
0.03
ED50 (mmole/kg i.p.)
Time of onset of Block (min)
Time of 50% Block (min)
Time of 90% Block (min)
Effect of Tetanus (30 Hz)
Effect of Physostigmine
3 ± 0.9
7 ± 0.6
10 ± 3
10 ± 2
18 ± 3
28 ± 2.7
5 ± 0.8
8 ± 0.2
12 ± 1.3
2 ± 0.1
3 ± 0.1
5 ± 0.2
Non-Maintained Non-Maintained Non-Maintained Well-Maintained
100% Reversal
1.0
No Reversal
100% Reversal
2.0
No Reversal
-
-
-
-
Time of onset of Reversal (min)
Time for 100% Reversal (min)
Duration of the Block (min)
6 ± 1.2
3 ± 0.4
70 ± 12
110 ± 17
30 ± 6
20 ± 1.8
*The results reported were the mean±S.E., N = number of animals used. Significant differences between the
various treatments were performed using paired or un-paired t-test. P values < 0.05 were considered significant.
2.2.2. The neuromuscular blocking activity of test compounds in chicks
The effects of the test compounds GS-53 (30) and AAH1 (33) together with those of the standard
drugs Atracurium (1) and Succinylcholine (2) were administered to 4-day old chicks to confirm the
mode of the neuromuscular blockade [22, 23]. For this purpose submaximal dose of each of the test
compounds and the standards, as revealed in the rats tibialis muscle twitches studies, were
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administered (i.p) to the chicks and the type of paralysis was noted. Compounds GS-53 (30, 0.15
mmoles/kg) and AAH1 (33, 0.3 mmoles/kg) induced flaccid paralysis. The paralysis started with
head movement, head drop, and then complete flaccid paralysis. The onset of head drops were 15 sec
and 11 min, whereas the flaccid paralysis was 1 and 15 min, respectively following administration.
Atracurcium (1, 0.016 mmole/kg) induced similar head drop after 5 min and flaccid paralysis of the
chicks after 10 min. However, Succinylcholine (2, 0.03 mmole/kg) induced rapid spastic paralysis
characterized by neck and limbs extension with rigidity of the muscles within 30 seconds. These
experiments revealed similar mechanisms for both of 30 and 33 with the clear difference of the
rapidity of onset in case of 30 (P < 0.05). The mechanism of action of both drugs seemed to be
similar to that of 1. The onset of action of 30 was more rapid than that of 1 and almost similar to that
of 2 (P < 0.05), Table 2.
Table 2: Effects of GS-53 (30), AAH1 (33), Atracurium (1) and Succinylcholine (2)
in chicks (N = 4-8 animals).
GS-53 (30)
AAH1 (33)
*Parameter
Atracurium Succinyl choline
0.15
0.3
0.016
0.03
-
ED50 (mmole/kg i.p.)
Onset of head drop (min)
Onset of muscle relaxation (min)
Type of neuromuscular paralysis
Mechanism of action
0.25 ± 0.1
1 ± 0.08
Flaccid
11 ± 2
15 ± 1.9
Flaccid
5 ± 0.8
10 ± 0.8
Flaccid
0.5 ± 0.02
Spastic
Competitive Competitive Competitive Depolarizing
*The results reported were the mean ± S.E., N = number of animals used. Significant differences
between the various treatments were performed using paired or un-paired t-test. P values < 0.05 were
considered significant.
2.3
Mode of Action of the Test Compounds Induced Neuromuscular Blocking Activity
The experiments performed in this study both in rats and chicks clearly pointed to the neuromuscular
blocking action of the two test compounds GS-53 (30) and AAH1 (33). The results obtained in rats
following tetanus application suggest that both compounds (30 and 33) acted via competitive
mechanism with acetyl choline released following electrical stimulation of the sciatic nerve to the
postsynaptic nicotinic receptors on the tibialis muscle membrane. In presence of competitive
blockers, tetanus is not well maintained. Such a competitive mechanism is supported by the studies
in chicks in which both compounds induced head drop and flaccid paralysis of the limbs [22, 24].
The complete reversal of 30 induced blocks by the anticholinesterase-Physostigmine confirms the
competitive mechanisms of action of this compound; while the effect of 33 was not reversed by the
anticholinesterase which gives the impression that 33 induced neuromuscular blockade was not
competitive in nature [21, 25, 26]. The LD50 value of compound GS-53 (30) was performed adopting
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known procedures [27]. The calculated LD50 was found to be 195.0 mg/kg with 95% confidence
limits of 185.25-204.75 mg/kg.
Structure activity correlation of the obtained results showed that the type of substituents on the
phenyl ring attached to position 2- of the 1,3,4-thiadiazolo[3,2-a][1,3]diazepine nucleus affected the
onset and duration times of the neuromuscular blocking activity. Introduction of 4-bromo or 4-fluoro
function to the phenyl ring produced GS-53 (30) and AAH1 (33) with remarkable activity. The
presence of 4-nitro (35) or 4-methyl (36) groups, moving those functions to position 2- (32, 34), or
the isosteric replacement of the phenyl ring with 4-pyridyl function (37) led to the complete loss of
the neuromuscular blocking activity.
3.
Molecular Modeling Study
3.1. Docking Study
Molecular modeling provides important data in the rational drug design; it can predict the bonding
affinity, spatial orientation and total binding energy of the small molecule drug candidates to the
active site of their target enzymes. AChE’s active site is located on the bottom of a long and narrow
gorge; acetylcholine and substrate guidance down the gorge is facilitated by cation–pinteractions
with aromatic side-chains residues [28, 29]. In a valuable addition to the present research work, it
was thought worthwhile to search for the binding mode of the active compounds into the nicotinic
acetylcholine receptors (nAChRs) and hence rationalize the results of the in vivo experiments and
compare them against the inactive compounds.
Figure 2: Lowest energy conformers of the active compounds (a) 30 and (b) 33 and (c) HIE-124
(3) with balls and cylinders rendering
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Figure 3: Lowest energy conformers of the inactive compounds (a) 32 and (b) 35 with balls and
cylinders rendering.
Figure 4: 2D Binding mode of Compounds (a) 32 and (b) 35 docked to the model of α7β2 nAChR
receptor.
Docking of the most active muscle relaxant derivatives 30 and 33, as well as the least active
derivatives 32 and 35 in comparison to HIE-124 (3) into the α7β2 nAChR (PDB ID: 2MAW) have
been performed [30]. Conformational analysis of the selected compounds by AM1 calculation
produced the most stable; and least energetic structures which are favorable to start the docking
experiment (Figures 2 and 3). The best calculated pose for 3 was assessed, docked and resulted in the
recognition of two amino acid residues, Thr120 and Thr124 which bind via hydrogen bonds
through interaction of its carbonyl oxygen by 41% and 36% respectively, showing pronounced high
affinity (binding energy -11.32 kcal/mol), supplementary Figure a. Compound 30 binds to nAChR
site via Thr124 by hydrogen bonding interaction, the same as 3, via carbonyl oxygen (binding energy
-10.25 kcal/mol), supplementary Figure b, while 33 binds with Thr124 residue by hydrogen bonding
through its diazepine ring carbonyl oxygen by 22 % (binding energy -10.34 kcal/mol),
supplementary Figure c. On that basis, it could be stated that 30 and 33 displayed their muscle
relaxant action by nicotinic binding site antagonism at AchE receptor. When comparing 3 and the
inactive compounds 32 and 35, it is clearly seen that the compounds did not display any binding
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residues at the used model (Figure 4 a,b); and expected to be deprived of any activity. Therefore, the
docking study results, could explain the variance in the activity of the tested compounds although of
their similar chemical structure.
3.2. Flexible Alignment
Ligands alignment at active site is a famous most used technique for the structural interpretation of
protein–ligand interactions. A successful alignment is stated if the strain energy for every molecule is
small, with similar shape and overlap of their aromatic moieties [31]. The aim of the alignment is to
maximize the similarity between those ligands, after using their best found conformations. The
similarity testing of the 3D structures among the most active compounds 30 and 33, then the inactive
32 and 35, followed by comparing the active 30 against the inactive 35 were done using
MOE/MMFF94 flexible alignment which smoothly produced superposition of the compounds under
study in minimal user bias [32]. 200 conformers of each compound were given then minimized by a
distance-dependant dielectric model. The lower energy set of 100 was used for further results
analysis. It was quite remarkable to note the similarity in flipping mode attitude and direction of the
active candidates and the inactive analogues in Figure 5a,b respectively, while there was a strong
deviation in alignment angle between compound 30 and 35 and therefore they could not have the
same activity pattern (Figure 5c).
Figure 5: Flexible alignments of (a) the most active compounds 30 (violet) and 33 (yellow), (b) the
least active compounds 32 (red) and 35 (green), (c) the most active compound 30 (violet))
against least active 35 (green).
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Figure 6: Surface map for compounds (a) 30, (b) 33, (upper), (c) 32 and (d) 35 (lower). Pink:
hydrogen bond, blue: mild polar, green: hydrophobic regions.
3.3. Surface Mapping
It could be noted that the difference in the aromatic substituent seems to have an impact on the
interactions with the nAChRs, so in a valuable extension to this work, surface mapping comparing
study between the most active 30 and 33, against least active counterparts 32 and 35 was performed.
Compounds 30 and 33 showed some hydrogen bond acceptor–donor region on the 4-carbonyl
(hydrophilic region in red), N-1, non-polar area located at the thiazole ring (in green), in addition to
the mild non polar center on the aryl moiety (Figure 6a,b). It can be recognized that higher lipophilic
distribution at the 4- position of the active candidates (Figure 7a,b) than the inactive analogues which
have less lipophilic at position 4- in comparison to position 2- (Figure 6c,d). There is a clear
difference in the distribution of surface mapping among the most and the least active derivatives
especially at 4- position of the aromatic substitution. These differences could play some role in the
binding affinities of the compounds at the nAchR receptor site and hence the biological activity.
4.
Conclusion
The biological evaluation of the new compounds revealed that compounds GS-53 (29) and AAH1
(32) are neuromuscular blockers (Figure 7). The obtained results clearly point to the discovery of a
new group of muscle relaxants acting via competitive mechanism with ACh which could be reversed
by the anticholinesterase Physostigmine. Compounds 30 and 33 induced dose-dependent
neuromuscular blockade with onset time of 3 and 10 min, ED50 0.15 and 0.36 mmoles/kg i.p.,
respectively, in rats. Compound 30 seemed to be as twice as potent as 33 (P < 0.05) with rapid onset
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and shorter duration (P < 0.05). Thus, the discovery of the neuromuscular blocking action of the 2-
(4-bromophenyl)-6,7-dihydro-[1,3,4]thiadiazolo[3,2-a][1,3]diazepin-8(5H)-one (GS-53, 30) and 2-
(4-fluorophenyl)-6,7-dihydro-[1,3,4]thiadiazolo[3,2-a][1,3]diazepin-8(5H)-one (AAH1, 33) mark the
era of introduction of a new group of intermediate acting skeletal muscle relaxants. Molecular
docking of 30 and 33 into nAChR receptor provided the binding mode to be through hydrogen
bonding interaction with Thr120 and Thr124. The obtained model could be useful for further
development of new neuromuscular blocking agents.
Figure 7: Structures of the most active neuromuscular
blocking compounds GS-53 (30) and AAH1 (33).
5.
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) was employed for routine column chromatography
separations. Compounds 6-13, 14-21 [20], 22, 27, 28, 30, 34, and 36 [15] were previously reported.
Adult male Wistar rats (250 g) and 4-day old chicks (50 g) were used to conduct the neuromuscular
blocking activity 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. The animals were provided with Purina
rodent’s chow pellets supplied by Grain silos and Flour Mills Organization, Riyadh, Saudi Arabia
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and had both food and water ad libitum. The chicks were bought from the local market in Riyadh.
Male Wistar rats (250 g) were randomly divided into various groups (N = 4-8 animals). The animals
were prepared following modifications of the reported method [21, 22]. All the procedures involving
animals were conducted in conformity with the Institutional guidelines that are in compliance with
National and International laws and policies.
5.1.
Chemistry
5.1.1. General procedure for the preparation of 4-Chloro-N-[5-(substituted phenyl)-1,3,4-
thiadiazol-2-yl]butanamide (22-29)
A mixture of 5-(substituted)-1,3,4-thiadiazol-2-amine (14-21, 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 heated
under reflux for 4 h. 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 22-29.
5.1.1.1 N-(5-(4-Bromophenyl)-1,3,4-thiadiazol-2-yl)-4-chlorobutanamide
(22).
White
solid
(Toluene), Yield 87%, m.p. 187-9oC [15].
5.1.1.2. 4-Chloro-N-[5-(4-chlorophenyl)-1,3,4-thiadiazol-2-yl]butanamide (23). White solid (EtOH),
1
Yield 75%, m.p. 125-7oC, H NMR (CDCl3): δ 2.29-2.31 (m, 2H, -CH2), 2.91 (t, J = 7.2 Hz, 2H, -
CH2), 3.72 (t, J = 7.2 Hz, 2H, -CH2), 7.47-7.90 (dd, J = 8.4, 8.4 Hz, 4H, ArH), 11.83 (br s, 1H, NH).
13C NMR: δ 27.6, 33.2, 44.0, 123.6, 128.4, 129.5, 132.3, 156.5, 161.7, 171.7. MS m/z (%): 316 (13.4,
M+), 318 (5.5, M+2). Calcd. for C12H11Cl2N3OS.
5.1.1.3. 4-Chloro-N-[5-(2-fluorophenyl)-1,3,4-thiadiazol-2-yl]butanamide (24). White gum, Yield
48%, 1H NMR (DMSO-d6): δ 1.65 (t, J = 6.5 Hz, 2H, -CH2), 3.01 (t, J = 7.5 Hz, 2H,-CH2), 3.58 (t, J
= 6.5 Hz, 2H, -CH2), 7.17-7.25 (m, 3H, ArH), 8.14-8.20 (m, 1H, ArH), 12.43 (s, 1H, NH). 13C NMR:
δ 28.9, 32.1, 45.3, 117.3, 125.1, 126.9, 131.6, 145.1, 162.3, 163.4, 172.1, 175.3. MS m/z (%): 299
(15.2, M+), 301 (5.7, M+2). Calcd. for C12H11ClFN3OS.
5.1.1.4. 4-Chloro-N-[5-(4-fluorophenyl)-1,3,4-thiadiazol-2-yl]butanamide (25). White solid (EtOH),
1
Yield 81%, m.p. 143-5oC, H NMR (CDCl3): δ 2.31-2.37 (m, 2H, -CH2), 2.75 (t, J = 8.4 Hz, 2H,-
CH2), 4.82 (t, J = 8.4 Hz, 2H, -CH2), 7.15-7.21 (m, 2H, ArH), 7.93-7.96 (m, 2H, ArH), 12.44 (s, 1H,
12

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NH). 13C NMR: δ 27.7, 33.3, 47.9, 116.4, 126.8, 129.3, 157.4, 162.9, 165.1, 173.7. MS m/z (%): 299
(18.6, M+), 301 (6.7, M+2). Calcd. for C12H11ClFN3OS
5.1.1.5. 4-Chloro-N-[5-(2-nitrophenyl)-1,3,4-thiadiazol-2-yl]butanamide (26). Yellowish white gum,
1
Yield 57%, H NMR (DMSO-d6): δ 2.15 (t, J = 6.5 Hz, 2H, -CH2), 2.32 (t, J = 7.5 Hz, 2H, -CH2),
4.17 (t, J = 6.5 Hz, 2H, -CH2), 7.83-7.73 (m, 2H, ArH), 8.09-8.07 (m, 1H, ArH), 8.88 (d, J = 8.5 Hz,
1H, ArH), 12.96 (s, 1H, NH). 13C NMR: δ 28.2, 33.6, 45.8, 124.8, 126.6, 128.9, 132.0, 142.9, 147.9,
148.6, 165.2, 172.4. MS m/z (%): 326 (25.2, M+), 328 (8.5, M+2). Calcd. for C12H11ClN4O3S.
5.1.1.6. 4-Chloro-N-[5-(4-nitrophenyl)-1,3,4-thiadiazol-2-yl]butanamide (27). White solid (EtOH),
Yield 54%, m.p. 135-7oC [15].
5.1.1.7. 4-Chloro-N-[5-(p-tolyl)-1,3,4-thiadiazol-2-yl]butanamide (28). White solid (EtOH), Yield
66%, m.p. 139-41oC [15].
5.1.1.8. 4-Chloro-N-[5-(pyridin-4-yl)-1,3,4-thiadiazol-2-yl]butanamide (29). White solid (EtOH),
1
Yield 59%, m.p. 111-4oC, H NMR (DMSO-d6): δ 2.17-2.28 (m, 2H, -CH2), 2.71 (t, J = 6.5 Hz, 2H,
-CH2), 4.16 (t, J = 6.5 Hz, 2H, -CH2), 7.95-7.96 (m, 2H, ArH), 8.74-8.75 (m, 2H, ArH), 12.10 (s, 1H,
13
NH). C NMR: δ 17.9, 30.5, 37.1, 48.7, 120.8, 137.8, 150.9, 160.8, 174.8. MS m/z (%): 282 (15.7,
M+), 284 (5.1, M+2). Calcd. for C11H11ClN4OS.
5.1.2. General procedure for the preparation of 2-(Substituted phenyl)-6,7-dihydro-
[1,3,4]thiadiazolo[3,2-a][1,3]diazepin-8(5H)-ones (30-37)
A mixture of 4-chloro-N-[5-(substituted)-1,3,4-thiadiazol-2-yl]butanamide (22-29, 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 30-37.
5.1.2.1. 2-(4-Bromophenyl)-6,7-dihydro-[1,3,4]thiadiazolo[3,2-a][1,3]diazepin-8(5H)-one (30).
White yellowish solid (EtOH), Yield 78%, m.p. 204-7oC [15].
5.1.2.2. 2-(4-Chlorophenyl)-6,7-dihydro-[1,3,4]thiadiazolo[3,2-a][1,3]diazepin-8(5H)-one (31).
o
1
Yellow solid (EtOH), Yield 64%, m.p. 127-4 C, H NMR (DMSO-d6): δ 2.21-2.24 (m, 2H, -CH2),
2.65 (t, J = 7.5 Hz, 2H, -CH2), 4.13 (t, J = 7.5 Hz, 2H, -CH2), 7.60-8.00 (dd, J = 8.5, 8.5 Hz, 4H,
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ArH). 13C NMR: δ 17.7, 30.7, 47.8, 128.7, 128.9, 129.4, 135.3, 157.4, 161.2, 174.2. MS m/z (%): 279
(10.4, M+), 281 (3.6, M+2). Calcd. for C12H10ClN3OS.
5.1.2.3. 2-(2-Fluorophenyl)-6,7-dihydro-[1,3,4]thiadiazolo[3,2-a][1,3]diazepin-8(5H)-one (32).
o
1
White solid (CHCl3/Hex), Yield 91.2%, m.p. 88-90 C, H NMR (DMSO-d6): δ 2.39 (t, J = 6.5 Hz,
2H, -CH2), 2.45 (t, J=6.5 Hz, 2H, -CH2), 4.02 (t, J=8.5 Hz, 2H, -CH2), 7.37-7.46 (m, 3H, ArH), 8.34
(d, J = 8.5 Hz, 1H, ArH). 13C NMR: δ 17.4, 31.3, 44.8, 116.2, 124.9, 127.8, 131.7, 144.4, 160.2,
162.2, 162.9, 172.5. MS m/z (%): 263 (18.0, M+). Calcd. for C12H10FN3OS.
5.1.2.4. 2-(4-Fluorophenyl)-6,7-dihydro-[1,3,4]thiadiazolo[3,2-a][1,3]diazepin-8(5H)-one (33).
White solid (EtOH), Yield 51%, m.p. 180-2 oC, 1H NMR (CDCl3): δ 2.21-2.24 (m, 2H, -CH2), 2.67-
2.70 (m, 2H, -CH2), 4.11-4.14 (m, 2H, -CH2), 7.37-7.41 (m, 2H, ArH), 8.02-8.05 (m, 2H, ArH).
13C NMR: δ 17.7, 30.7, 47.7, 116.5, 129.4, 157.2, 161.3, 162.4, 164.4, 174.1. MS m/z (%): 263 (22.0,
M+). Calcd. for C12H10FN3OS.
5.1.2.5.
2-(2-Nitrophenyl)-6,7-dihydro-[1,3,4]thiadiazolo[3,2-a][1,3]diazepin-8(5H)-one
(34).
White yellowish solid (CHCl3/Hex), Yield 72%, m.p. 108-7oC [15].
5.1.2.6.
2-(4-Nitrophenyl)-6,7-dihydro-[1,3,4]thiadiazolo[3,2-a][1,3]diazepin-8(5H)-one
(35).
Yellowish browen solid (EtOH), Yield 70%, m.p. 154-6 oC, 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, 129.9,
141.3, 163.7, 174.1. MS m/z (%): 290 (25.2, M+). Calcd. for C12H10N4O3S.
5.1.2.7. 2-(p-Tolyl)-6,7-dihydro-[1,3,4]thiadiazolo[3,2-a][1,3]diazepin-8(5H)-one (36). White
yellowish solid (EtOH), Yield 73%, m.p. 166-8oC [15].
5.1.2.8.
2-(pyridin-4-yl)-6,7-dihydro-[1,3,4]thiadiazolo[3,2-a][1,3]diazepin-8(5H)-ones
(37).
Yellowish white solid (EtOH), Yield 85%, m.p. 145-7 oC. 1H NMR (DMSO-d6): δ 2.19-2.28 (m, 2H,
-CH2), 2.71 (t, J = 6.5 Hz, 2H, -CH2), 4.16 (t, J = 6.5 Hz, 2H, -CH2), 7.95-7.96 (m, 2H, ArH), 8.74-
13
8.76 (m, 2H, ArH). C NMR: δ 17.7, 30.7, 47.8, 62.9, 120.9, 136.9, 150.8, 160.3, 174.5. MS m/z
(%): 246 (10.6, M+). Calcd. for C11H10N4OS.
5.2.
Measurement of the Neuromuscular Blocking Activity in Rats
Male Wistar rats were prepared following modifications of the reported methods [21, 22]. The
animals were anaesthetized using 25% urethane in water (w/v, 1.25 g/kg i.p). Each animal was laid
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ACCEPTED MANUSCRIPT
on its back and fixed to a surgical board and body temperature was maintained by an over-head
lamp. An incision was made on the neck, the trachea was located freed and a cannula was inserted to
supply artificial ventilation with room air delivered by Parvalux Electric Motors Ltd rodents’
respirator (Wallisdown, Bournermouth, England) at a frequency of 90 breaths per minute and a tidal
volume of 20 c.c./kg. Furthermore, an incision was made to remove the skin covering the tibialis and
its neighbor muscles on the right leg. The membrane covering the tibialis muscle was removed and
its tendon freed from the Knob in the middle of the foot. The tendon was tied with a strong thread,
passed through a bully, attached to a force displacement transducer (10-100 g, Narco-Biosystems,
Myograph F 2000; USA) which was connected to a Narco Physiograph via Universal Coupler Type
7173. An incision was made on the lateral right side of the animal just above the site of the sciatic
nerve supplying the tibialis muscle. The sciatic nerve was freed and the nerve was secured between a
platinum electrodes. A strong tie was made on the portion of the nerve nearer to the spinal cord to
prevent a generalized electrical stimulation. The electrodes were attached to an electric stimulator
(Science and Research Instruments, Ltd, Kent, U.K.). Both of the muscle and the nerve were covered
with paraffin oil at 37°C to prevent dryness of the tissues. The Tibialis muscle twitches were induced
using the following parameters: supramaximal voltage of 40 volts, at a frequency of 0.3 Hz and 0.5
msec duration. When tetanus was performed the frequency was increased to 30 Hz and the speed of
recording was increased from 0.25 cm/sec to 1 cm/sec and stimulation was performed for 5-10 sec.
The calibration system built in the transducer was used to measure the tension of the muscle.
Changes in the muscle tension (twitches) were expressed as percentage change from the pre-drug
values. Test compounds (30-37), standards or vehicles were administered intraperitoneally dissolved
in dimethyl sulfoxide, or water as appropriate in various doses 1-100 mg/kg. The effect of each dose
on the twitch response was calculated as a percentage of the pre-drug amplitude. The ED50 was
calculated for each compound. Also the dose that produced 90% of twitches depression was
calculated. The response of the muscle to titanic stimulation was tested before administration of test
compounds and following 90% inhibition of the twitches. To investigate the reversibility of any
block, the anticholinesterase-Physostigmine was injected in a single bolus injection of 100 µg/kg.
The onset time of reversal and the percentage of reversal were monitored and calculated,
respectively. The duration of each block was monitored. In all animals rectal temperature was
maintained at 37±1°C, (Table 1).
5.3.
Measurement of the Neuromuscular Blocking Activity in Chicks
The test compounds (30-37), the standard drugs Atracurium (1) and Succinylcholine (2) were
administered to 4-day old chicks to confirm the mode of the neuromuscular blockade [22, 23]. Sub-
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maximal dose of test compounds and the standards as revealed in the rat's studies were administered
(i.p) to the chicks and the type of paralysis was noted. Intraperitoneal administration of test
compounds in single dose of 0.15 mmoles/kg, induced flaccid paralysis of the chicks within one
minute. The paralysis started with head movement and drop; then complete flaccid paralysis
occurred. The onset of head drop was 15 sec whereas the flaccid paralysis was one minute following
administration. 1 and 2 were used as positive controls (Table 2).
5.4.
Determination of the Lethal Dose (LD50) of GS-53 (30).
Male mice were divided into various groups. Compound 30 was administered in various doses
ranging from 0.1-5 mmole/kg, intraperitoneal. 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 outlined
[27].
5.5. Molecular Modeling Methodology
Conformational analysis of the selected compounds classified as active muscle relaxant compounds
30 and 33 in addition to the least active counterparts 32 and 35 , together with HIE124 (3) as a
standard were performed using MMX force field method preceded by AM1 calculation as implied in
MOE 2009.10. The model of the nicotinic AchE receptor was used for docking onto the active site of
nAChR derived from three-dimensional structure of the enzyme complex (PDB ID: 2MAW), [30].
Three-dimensional structures of the representatives selected thiadiazolo-diazepine derivatives, in
their neutral forms were built using the MOE of Chemical Computing Group Inc software. The
lowest energy conformers in their ‘global-minima’ were docked.’ The energy-minimized structures
were 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 every
ligand, energy minimizations were done by 1000 steps of steepest descent, followed by conjugate
gradient minimization to an RMSD energy gradient of 0.01 kcal/mol_A. The alpha triangle
placement method and the London dG scoring method were selected for docking experiments. 300
results for each ligand were generated, excluding the results having RMSD value >3 [32]. The best
scored result of the remaining conformations for each ligand was further analyzed. The studied
compounds were subjected to flexible alignment. Their geometry was optimized using the MMFF94
forcefield followed by a flexible alignment using systematic conformational search [32, 33]. Surface
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mapping calculations were performed using ‘Molecular Operating Environment’ software (MOE of
Chemical Computing Group Inc., on a Core i7, 2.7 GHz workstation).
Acknowledgements
Thanks are due to King Saud University patency authority for financing the patency process.
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Captions
Table 1: Effects of GS-53 (30), AAH1 (33), Atracurium (1) and Succinylcholine (2) on the rat
tibialis electrically-induced twitches together with the effects of tetanus and Physostigmine
at a dose of 100 mg/kg i.p. (N = 4-8 animals).
Table 2: Effects of GS-53 (30), AAH1 (33), Atracurium (1) and Succinylcholine (2) in chicks (N =
4-8 animals).
Figure 1: Structures of literature neuromuscular blocking agents (1, 2), and the ultra-short acting
hypnotic 3.
Figure 2: Lowest energy conformers of the active compounds (a) 30 and (b) 33 and (c) HIE-124 (3)
with balls and cylinders rendering
Figure 3: Lowest energy conformers of the inactive compounds (a) 32 and (b) 35 with balls and
cylinders rendering.
Figure 4: 2D Binding mode of Compounds (a) 32 and (b) 35 docked to the model of α7β2 nAChR
receptor.
Figure 5: Flexible alignments of (a) the most active compounds 30 (violet) and 33 (yellow), (b) the
least active compounds 32 (red) and 35 (green), (c) the most active compound 30 (violet))
against least active 35 (green).
Figure 6: Surface map for compounds (a) 30, (b) 33, (upper), (c) 32 and (d) 35 (lower). Pink:
hydrogen bond, blue: mild polar, green: hydrophobic regions.
Figure 7: Structures of the most active neuromuscular blocking compounds GS-53 (30) and AAH1
(33).
Scheme 1: Synthesis of the target compounds 30-37
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Research Highlights
•
•
•
Synthesis of 6,7-dihydro-[1,3,4]thiadiazolo[3,2-a][1,3]diazepines
Compounds GS-53 (30) and AAH1 (33) induced neuromuscular blockade
Compound 30 proved to be as twice as potent as 33 with rapid onset and shorter
duration
•
Recognition with Thr120 and Thr124 at α7β2 nAChR receptor is essential for activity