New ultra-short acting hypnotic: Synthesis, biological evaluation, and metabolic profile of ethyl 8-oxo-5,6,7,8-tetrahydro-thiazolo[3,2-a][1,3]diazepin-3-carboxylate (HIE-124)

Article abstract of DOI:10.1016/j.bmcl.2007.11.011

Ethyl 8-oxo-5,6,7,8-tetrahydro-thiazolo[3,2-a][1,3]diazepin-3-carboxylate (HIE-124, 4) is a member of a new generation of ultra-short acting hypnotics. HIE-124 (4) exhibited potent in vivo activity with a rapid onset of action and a short duration of action, with no acute tolerance or noticeable side effects. The metabolic profile of 4 is also performed. HIE-124 (4) has the potential use as a preanesthetic medication, anesthesia inducer, and could be used with thiopental sodium to maintain anesthesia for longer duration.

Full text of DOI:10.1016/j.bmcl.2007.11.011

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 72–77
New ultra-short acting hypnotic: Synthesis, biological
evaluation, and metabolic profile of ethyl
8
-oxo-5,6,7,8-tetrahydro-thiazolo[3,2-a][1,3]diazepin-
q
3
-carboxylate (HIE-124)
a,
b
a
*
Hussein I. El-Subbagh, Hassan A. El-Kashef, Adnan A. Kadi,
Alaa A.-M. Abdel-Aziz, Ghada S. Hassan, Justice Tettey and Jochen Lehmann
a
c
d
e
a
Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia
b
Department of Pharmacology, College of Pharmacy, Mansoura University, Mansoura 35516, Egypt
Department of Medicinal Chemistry, College of Pharmacy, Mansoura University, Mansoura 35516, Egypt
c
d
Institute of Pharmaceutical and Biomedical Sciences, University of Strathclyde, Glasgow, United Kingdom
e
Institut of Pharmacy, Friedrich-Schiller-University Jena, Philosophenweg 14, D-07743 Jena, Germany
Received 4 July 2007; revised 1 November 2007; accepted 5 November 2007
Available online 7 November 2007
Abstract—Ethyl 8-oxo-5,6,7,8-tetrahydro-thiazolo[3,2-a][1,3]diazepin-3-carboxylate (HIE-124, 4) is a member of a new generation
of ultra-short acting hypnotics. HIE-124 (4) exhibited potent in vivo activity with a rapid onset of action and a short duration of
action, with no acute tolerance or noticeable side effects. The metabolic profile of 4 is also performed. HIE-124 (4) has the potential
use as a preanesthetic medication, anesthesia inducer, and could be used with thiopental sodium to maintain anesthesia for longer
duration.
Ó 2007 Elsevier Ltd. All rights reserved.
Surgical procedures require the administration of sev-
eral intravenous drugs to ensure hypnosis, analgesia,
relaxation, and control of visceral reflex responses.
The use of intravenous drugs adds flexibility and permits
the administration of lower doses of inhalational anes-
thetic agents. General anesthesia most often is initiated
by an injection of thiopental, an ultra-short acting bar-
biturate, to induce sleep prior to administration of the
agents that are necessary for maintaining anesthesia
during the surgical procedure. Ultra-short acting barbi-
turates and benzodiazepines have an important place in
the practice of anesthesiology. Thiopental sodium re-
mains the standard for comparison with new agents.
anesthesia may increase for up to 40 s and then de-
creases progressively until consciousness returns in 20–
30 min. However, recovery may require many hours if
2
a large dose of thiopental sodium is administered. Thio-
pental is metabolized slowly in the liver, which together
with other factors may influence the depth of anesthesia,
3
time of recovery, and duration of action. The concen-
tration employed of thiopental should not exceed 2.5%
in aqueous solution. When greater concentration is in-
jected extravascularly, severe pain and tissue necrosis
may occur. Intra-arterial injection of a concentrated
solution of thiopental may result in damage to the arte-
rial endothelium, followed by endarteritis, often with
thrombosis exacerbated by subsequent arteriolar spasm.
4
Single intravenous anesthetic dose of thiopental pro-
duces unconsciousness within 10–20 s. The depth of
Vascular ischemia and even gangrene may result. Thio-
5
pental produces a long lasting hangover. Patients using
a large initial dose of thiopental will awake despite plas-
ma concentrations that normally would cause sleep. The
nature of this acute tolerance is not known. For this rea-
son, thiopental cannot be used to maintain surgical
anesthesia, but only as an induction agent. Thiopental
produces a dose-related depression of respiration that
Keywords: Synthesis; Thiazolo[3,2-a][1,3]diazepine; Ultra-short acting
hypnotic; Metabolic profile.
q
Ref. 1.
Corresponding author. Tel.: +966 1 467 7366; fax: +966 1 467
383; e-mail: subbagh@yahoo.com
*
6
0
960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.11.011

H. I. El-Subbagh et al. / Bioorg. Med. Chem. Lett. 18 (2008) 72–77
73
6
–8
can be profound. Larger doses of thiopental sodium
cause more profound changes and respiration is main-
tained only by movements of the diaphragm.
(HIE-124, 4) was synthesized according to an inventive
method (Scheme 1). Ethyl 2-amino-thiazole-4-carboxyl-
1
1
ate (1) was prepared using reported procedure, and
then alkylated with 4-chloro-butyryl chloride (2),
in presence of anhydrous potassium carbonate to
afford ethyl 2-(4-chloro-butanamido)thiazole-4-carbox-
ylate (3). Compound 3 was cyclized using piperidine
as a base to produce HIE-124 (4), which was purified
using silica gel column chromatography. Structure elu-
cidation of compounds 3 and 4 was obtained based on
Benzodiazepines were first introduced for the treatment
of anxiety, then later on, they have been used as seda-
tives, anticonvulsants, and muscle relaxants. Hypnosis
and unconsciousness may be produced with large doses.
Benzodiazepines are not analgesics, nor can they pro-
duce a state of surgical anesthesia when used alone. It
is necessary to combine several drugs to achieve surgical
levels of anesthesia with a balance of sedation, analge-
sia, amnesia, relaxation, and freedom from reflex stimu-
lation. Benzodiazepines are useful as the sole agent for
procedures that do not require analgesia, such as endos-
copy, cardiodepression, cardiac catheterization and a
spectrum of radiodiagnostic procedures. For induction
of anesthesia, the benzodiazepines are given intrave-
nously, to minimize the burning sensation and avoid
venous thrombosis that might accompany the iv
1
13
analysis of the H, C, DEPT, COSY, HMBC, and
HMQC NMR spectra, in addition to mass spectrometry
for each compound.
Intravenous administration of ethyl 8-oxo-5,6,7,8-tetra-
hydro-thiazolo[3,2-a][1,3]diazepin-3-carboxylate (HIE-
124, 4), at dose levels of 50 and 100 mg/kg, induced hyp-
nosis, in all mice within few seconds measured by using
the loss of righting reflex method (Table 1). Loss of
righting reflex was defined as the inability of a mouse
to right itself within 30 s. Return of the righting response
was defined as the ability of a mouse to right itself twice
in 1 min. Upon loss of righting reflex, mice were placed
on their backs, and the time to regain righting reflex was
measured. Sleeping time or duration of loss of righting
reflex was defined as the time between loss and return
of the righting response, while onset of action is the time
between injection of the compound and loss of righting
9
administration.
In the present investigation, we would like to report the
ultra-short acting activity of ethyl 8-oxo-5,6,7,8-tetrahy-
dro-thiazolo[3,2-a][1,3]diazepin-3-carboxylate
1
(HIE-
24, 4), a member of a novel class which might over-
come many of the disadvantages and problems, such
as hangover and tolerance, that are usually associated
with the use of thiopental or benzodiazepines as intrave-
nous anesthetic agents. HIE-124 (4) has been shown to
not only induce anesthesia but also maintain the anes-
thetic state during the surgical procedure. HIE-124 (4)
exhibited a very rapid onset of action and a shorter
duration of action with no acute tolerance or noticeable
side effects such as salivation, dizziness, muscle twitches,
and convulsions, when compared with thiopental so-
dium. This new finding granted the issuance of a Ger-
1
2a
reflex. Tachypnea was observed in three mice out of
10 that received 100 mg/kg and dyspnea was also
observed in one mouse out of 10 mice that received
100 mg/kg. Both doses of HIE-124 (4) potently potenti-
ated the hypnotic effect produced by 50 mg/kg iv
thiopental sodium (Table 1). Intraperitoneal administra-
tion of HIE-124 (4) at dose levels of 100 and 200 mg/kg
induced hypnosis in all mice with onset of action less
than 1 min (Table 2). Tachypnea was observed in two
mice out of 10 that received 100 mg/kg, and ataxia and
tachypnea were also observed in three mice out of 10
mice that received 200 mg/kg. The tachypnea, observed
in 30% of animals received the high dose of HIE-124
(4) compound, may indicate that high doses of the com-
1
man patent.
The thiazolo[3,2-a][1,3]diazepine nucleus can be obtained
0
1
by the use of published methods. Ethyl 8-oxo-5,6,7,
8
-tetrahydro-thiazolo[3,2-a][1,3]-diazepin-3-carboxylate
EtOOC
Cl
EtOOC
N
K2CO3
Cl
+
N
Cl
S
NH2
N
S
O
H
O
1
2
3
piperidine
EtOOC
N
S
N
O
HIE-124 (4)
Scheme 1.

74
H. I. El-Subbagh et al. / Bioorg. Med. Chem. Lett. 18 (2008) 72–77
Table 1. Hypnotic effect of ethyl 8-oxo-5,6,7,8-tetrahydro-thiazolo[3,2-
a][1,3]diazepin-3-carboxylate (HIE-124, 4), 50, and 100 mg/kg iv, and
its effect on the thiopental sodium, 50 mg/kg iv,-induced hypnosis in
mice
pound could have undesirable effects on respiratory
system. Both doses of the compound, 100 and 200 mg/
kg, potently potentiated the hypnotic effect produced
12b
by 65 mg/kg ip thiopental sodium
(Table 2).
Treatment
Dose (mg/kg iv) Sleeping time (min)
Acute toxicity test was performed according to the
13
method described by Litchfield and Wilcoxon. The
Thiopental sodium
HIE-124 (4)
HIE-124 (4)
50
50
4.90 ± 1.13
0.75 ± 0.01
1.48 ± 0.21
19.90 ± 5.21
25.70 ± 5.11
a
a
a
a
100
calculated LD50 HIE-124 (4) was found to be
681.4 mg/kg with 95% confidence limits of 482.8–876.4.
Acute tolerance test was conducted following the ip
administration of 200 mg/kg of HIE-124 (4), three
times/day for three consecutive days. There was no sig-
nificant difference among the sleeping times after the
first and the last administration of the compound. The
sleeping time after the first administration of 4 was
HIE-124 (4) + Thiopental 50 + 50
HIE-124 (4) + Thiopental 100 + 50
Data are expressed as means ± SE. n = 10.
a
Significantly different as compared to thiopental, using Student’s t-
test, P < 0.05.
10.3 ± 1.4 min, while after the ninth administration,
the sleeping time was 8.7 ± 2.1 min.
Table 2. Hypnotic effect of ethyl 8-oxo-5,6,7,8-tetrahydro-thiazol-
o[3,2-a][1,3]diazepin-3-carboxylate (HIE-124, 4),100 and 200 mg/kg ip,
and its effect on the thiopental sodium, 65 mg/kg ip, induced hypnosis
in mice
Initial ESI LC–MS analysis of HIE-124 (4) (Fig. 1) dis-
solved in 60:40 CH CN:H O showed the compound
3
2
Treatment
Dose (mg/kg ip) Sleeping time (min)
eluting at ꢀ17 min with a protonated molecular ion
peak [M+1]+ at m/z 241 in the positive ion mode
(Fig. 2). Gradient elution was tailored such that HIE-
124 (4) elutes at a relatively late analysis time to allow
for sufficient time window for the elution of other
metabolites, which are expected to be polar and hence
elute at an earlier time. Screening for potential metabo-
lites was performed by searching for ions with molecular
weights corresponding to metabolites generated through
one of the most common phase I and phase II metabolic
transformations. For HIE-124 (4), these transforma-
tions were expected to be ester hydrolysis, aromatic
hydroxylation, and glucuronic acid conjugation. The
primary strategy for metabolite identification was thor-
ough analysis of total ion current (TIC) chromatograms
obtained from various LC–MS analyses. Blank TIC and
UV chromatograms were subtracted from those con-
taining compound 4 in either a urine sample or an
in vitro incubation.
Thiopental sodium
HIE-124 (4)
HIE-124 (4)
65
15.2 ± 1.40
2.4 ± 0.83
9.1 ± 1.23
49.2 ± 3.71
a
a
a
100
200
HIE-124 (4) + Thiopental 100 + 65
HIE-124 (4) + Thiopental 200 + 65
a
155.7 ± 16.35
Data are expressed as means ± SE. n = 10.
a
Significantly different as compared to thiopental, using Student’s t-
test, P < 0.05.
HOOC
N
S
N
O
5
Figure 1. Structure of the major metabolite 5.
3 2
Figure 2. ESI mass spectrum of HIE-124 (4) dissolved in 60:40 CH CN:H O.

H. I. El-Subbagh et al. / Bioorg. Med. Chem. Lett. 18 (2008) 72–77
75
Figure 3. TIC chromatogram of blank hepatocyte incubation.
Screening of TIC chromatograms of pooled mice urine
samples showed a prominent peak eluting at ꢀ11 min,
with a protonated molecular ion peak at m/z 213, with
the absence of the peak at m/z 241 corresponding to
the protonated 4 (Fig. 2). The peak was proposed to
results were consistent with those obtained from
in vivo experiments. The various incubation conditions
were designed to obtain more details about the possible
enzymatic processes involved in the in vitro metabolism
of 4. Incubations with NADPH alone with no cells were
performed to determine the presence of non-enzymatic
hydrolysis of the ester. Incubation with cells but no
NADPH was designed to determine involvement of
non-CYP450 isoforms in the metabolism of 4 since
CYP450 isoforms need NADPH for the catalysis. Incu-
bation with glutathione (GSH) was designed to deter-
mine the presence of GSH adducts, frequently formed
+
be the de-ethylated metabolite 5 [MÀ28] , resulting
from hydrolysis of the ethyl ester of 4. It is a well-docu-
mented route of biotransformation.
1
4–17
Careful screen-
ing of the TIC chromatogram showed the absence of
any other peak corresponding to one of the expected
metabolites resulting from either phase I reactions (e.g.
m/z 229 resulting from hydroxylation) or phase II reac-
tions (e.g. m/z 389 resulting from glucuronidation).
2
0,21
with carboxylic acids. Troglitazone (TGZ), known
to form GSH adducts, was used as a positive control.
2
2
In vitro drug metabolism studies are widely regarded as
reliable and accurate models for the prediction of drug
metabolism in a variety of mammalian species. Among
the different techniques to study in vitro metabolism
are hepatic microsomal models and hepatocyte prepara-
LC–MS analysis of thein vitro studies gave similar and
consistent results to the in vivo experiments. In RLM
studies, the only metabolite detected for HIE-124 (4)
+
was the de-ethylated product 5 [MÀ28] at m/z 213
1
8,19
tions.
This study attempted to utilize such tech-
(Fig. 3). The chromatogram showed no evidence of the
parent compound after the conclusion of the incubation
time (Fig. 4). No other drug-related peaks were detected
in the run compared to a blank TIC chromatogram.
niques to gain more information about the metabolic
profile of HIE-124 (4). Incubation of compound 4 with
rat liver microsomes (RLMs) was straightforward and
Figure 4. TIC and SIM (m/z 213) chromatograms of 30-min RLM incubation with HIE-124 (4).

76
H. I. El-Subbagh et al. / Bioorg. Med. Chem. Lett. 18 (2008) 72–77
Figure 5. Chromatograms of hepatocyte incubation with HIE-124 (4) after 120 min. (a) TIC; (b) UV; (c) SIM (m/z 213); and (d) ESI mass spectrum
of 5.
Compound 5 was also the only metabolic product ob-
served for 4 after hepatocyte incubations (Fig. 5). The
compound appears to be the product of enzymatic
hydrolysis by the action of liver esterases rather than a
simple chemical hydrolysis as confirmed by the absence
of any significant amounts of the hydrolyzed product, as
in the case of RLM and hepatocyte studies. Further-
more, the involvement of esterases in the hydrolysis is
supported in part by the presence of the hydrolyzed
product in RLM incubations, which does not contain
NADPH, the necessary co-factor for cytochrome
P450-mediated metabolism but not for esterase-medi-
ated hydrolysis. The presence of carboxyesterase activity
in freshly thawed hepatocytes was confirmed by the car-
boxyfluorescein diacetate assay, frequently used to easily
HIE-
23–27
determine cell-derived enzymatic esterification.
124 (4) appeared to undergo rapid hydrolysis to the corre-
sponding carboxylic acid derivative, with undetected lev-
els of the parent compound remaining in aliquots
2
8–30
untreated with CH CN taken at 0 min hepatocyte
3
incubation time. RLM incubations of 4 in the presence
of GSH did not result in the formation of GSH adducts
based on positive ion ESI LC–MS results.
The biological evaluation of HIE-124 (4) revealed that
the compound is a very short acting hypnotic. The onset
of action of this compound is very short compared to
thiopental sodium. In all animals used, the onset of ac-

H. I. El-Subbagh et al. / Bioorg. Med. Chem. Lett. 18 (2008) 72–77
77
tion was less than 60 s. The duration of action when gi-
ven iv or ip is significantly shorter than that of thiopen-
tal sodium. In addition, the investigated compound did
not show any sign of acute tolerance reported with the
second (maintenance) dose of thiopental sodium. There-
fore, compound HIE-124 (4) has the potential use not
only as a preanesthetic medication and induction of
anesthesia but also has the potential to be used with
thiopental sodium to maintain anesthesia for longer
duration than using thiopental sodium alone. Com-
pound 4 appears to undergo enzymatic hydrolysis by
the action of liver esterases. Further metabolic studies
are needed to characterize the metabolism of this com-
pound from other mammalian systems.
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