Arch. Pharm. Chem. Life Sci. 2008, 341, 81–89
Thiazolo[3,2-a][1,3]diazepine analog
87
Ultra-short acting hypnotic evaluation experiments were per-
formed at Pharmacology Department, College of Pharmacy,
Mansoura University, Egypt. Analytical chromatography separa-
tion and analytes' mass determination was carried out on an Agi-
lent 1100 MSDm (Agilent, Palo Alto, CA, USA) system consisting of
an Agilent 1100 series HPLC system equipped with an ultraviolet
detector and interfaced via electrospray ionization (ESI) to an
Agilent 1100 single quadrupole mass spectrometer. The chroma-
tographic separation was carried out on a Hypersil C18 25063.0
ID, 5 lm particle size column, eluting with a mobile phase con-
sisting of A: 1% formic acid and B: acetonitrile (CH3CN) in a lin-
ear gradient program running at a constant flow rate of 0.5 mL/
min. The program was started at 5% (CH3CN) which was linearly
increased to 60% over 20 minutes, kept at 20% for 2 minutes,
then returned to 5% B in one minute and kept at the composi-
tion for the final 3 minutes. UV absorbance was monitored at
254 nm. MS experiments were carried out in the positive mode,
using nitrogen as nebulizing gas at 300. Total ion current (TIC)
as well as single ion monitoring (SIM) experiments were con-
ducted to screen for the possible metabolites. TIC experiments
were conducted in the range from 100–600 amu, and SIM
experiments were set to monitor for a number of ions including
m/z 241 [M + H]+, m/z 213 [M - 28]+, m/z 229 [M + 16]+, m/z 389
[M + 176]+9 CH3CN and formic acid were all HPLC grade. NADPH,
MgCl2 were obtained from Sigma, Inc. (U.K). All buffer solutions
were prepared in-house.
Ultra-short acting hypnotic evaluation
Mice of both sexes (22–28 g) were used to conduct the ultra-
short acting hypnotic evaluation. Fifty and 100 mg/kg of com-
pound HIE-124 4 were injected intravenously into two groups of
mice, respectively; each group consists of ten mice. The sleeping
time was recorded for each mouse and compared with control
ultra-short acting hypnotic agent. The effect of the test com-
pound, administered intravenously, on thiopental sodium
(Intraval sod. May & Baker LTD, England) -induced sleeping time
was also evaluated. Three groups of mice each consists of ten
mice were used. The first group (control), injected with the sol-
vent“15% cremophore EL”, the second group, treated with
50 mg/kg, and the third group, treated with 100 mg/kg of the
test compound intravenously. All mice in the three groups were
then injected with thiopental sodium, 50 mg/kg intravenously.
The time elapsed between the loss and the recovery of righting
reflex“sleeping time” was recorded for each animal and com-
pared with control group (Table 1).
An amount of 100 and 200 mg/kg of compound HIE-124 4 was
injected intraperitoneally into two groups of mice, respectively.
Each group consists of ten mice. The sleeping time were
recorded for each mouse and compared with control ultra short-
acting hypnotic agent. The effect of the test compound, adminis-
tered intraperitoneally, on thiopental sodium-induced sleeping
time was also evaluated. Three groups of mice, each consisting
of ten mice were used. The first group (control), injected with
the solvent“15% cremophore EL”, the second group, treated
with 100 mg/kg, and the third group, treated with 200 mg/kg
compound of formula (I) intraperitoneally. All mice in the three
groups were then injected with thiopental sodium; 65 mg/kg
intraperitoneally [12]. The sleeping time was recorded for each
animal and compared with control group (Table 2).
Chemistry
Ethyl 2-(4-chlorobutanamido)-thiazole-4-carboxylate 3
A mixture of ethyl 2-aminothiazole-4-carboxylate (1, 7.0 g,
0.04 mol) and 4-chloro-butyryl chloride (2, 11.3 g, 9.0 mL,
0.08 mol) in toluene (100 mL) was heated under reflux for 4 h.
The toluene was then evaporated under reduced pressure. Then,
the residue was quenched with water, stirred, and filtered. The
solid obtained was washed, dried, and recrystallized from water
to give the required product 3 (10.2 g, 92%): m.p. 171–1728C; 1H-
NMR (CDCl3) d 1.27 (t, 3H, J = 7.0 Hz, CH3CH2), 2.02–2.08 (m, 2H,
Acute toxicity test
Five groups of mice, each consisting of ten animals were used to
conduct the acute toxicity test and to calculate the LD50. Com-
pound HIE-124 4 dissolved in 15% cremophore EL was given
intraperitoneally in doses of 100, 200, 400, 800, and 1600 mg/
kg, respectively. The final volume of injection in all groups did
not exceed 0.2 mL/mouse. Twenty-four hours later, the% mortal-
ity in each group was recorded and the LD50 was calculated using
the method described by Litchfield and Wilcoxon [13].
J = 6.5, 7.3 Hz, ClCH2CH2CH2N), 2.59 (t, 2H,
J = 7.3 Hz,
Cl(CH2)2CH2N), 3.66 (t, 2H, J = 6.5 Hz, ClCH2(CH2)2N), 4.27 (q, 2H, J
= 7.0 Hz, CH3CH2), 7.96 (s, 1H, thiazoleH), 12.5 (brs, 1H, NH). 13C-
NMR d 14.1, 27.7, 32.4, 44.9, 60.8, 122.7, 141.3, 158.3, 161.3,
171.3; MS m/e 276.0 (85). Anal. (C10H13ClN2O3S) C, H, N.
Acute tolerance test
Ten mice were used to conduct the acute tolerance experiment.
Each mouse received 200 mg/kg of compound HIE-124 4 dis-
solved in 15% cremophore intraperitoneally, three times per day
for three consecutive days. After each of the nine administra-
tions, the sleeping time induced by the test compound was
recorded for each mouse.
Ethyl 8-oxo-5,6,7,8-tetrahydro-thiazolo[3,2-a]
[1,3]diazepin-3-carboxylate 4
A mixture of ethyl 2-(4-chlorobutanamido)-thiazole-4-carboxy-
late (3, 1.0 g, 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 column chromatography elut-
ing with CHCl3/hexane (80 : 20 v/v) to give 4 (0.7 g, 80%): m.p.
210–2128C;1H-NMR (CDCl3) d 1.22 (t, 3H, J = 7.1 Hz, CH3CH2), 2.08-
2.14 (m, 2H, J = 8.0, 4.5 Hz, COCH2CH2CH2N), 2.54 (t, 2H, J =
8.0 Hz, COCH2CH2CH2N), 4.07 (t, 2H, J = 4.5 Hz, COCH2CH2CH2N),
4.19–4.23 (q, 2H, J = 7.1 Hz, CH3CH2), 7.7 (s, 1H, thiazoleH). 13C-
NMR d 14.6, 18.3, 31.9, 48.3, 61.4, 122.7, 142.1, 157.7, 161.7,
174.3; MS m/e 240.0 (65) Anal. (C10H12N2O3S) C, H, N.
In-vivo metabolic studies
Male mice (n = 4, average l25 g) were housed in special purpose
metabolism cages obtained from Nalgene1. Animals will be
acclimated in the cages for 48 hours prior to the start of the
study in a 12 hour light/dark cycle (7:00–19:00 o'clock) and the
animals were allowed free access to standard animal feed and
water ad-libitum. HIE-124 4 was formulated into a 20% ethanol
solution. Each animal received a 30 mg/kg dose of the solution
at 24, 72, and 96 hours. Urine draining into the special urine
compartments fitted to the metabolism cages were collected
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