Synthesis and chemiluminescent high throughput screening for inhibition of acetylcholinesterase activity by imidazo[2,1-b]thiazole derivatives

Article abstract of DOI:10.1016/j.ejmech.2005.05.010

The synthesis of a new series of imidazo[2,1-b]thiazole derivatives is described. They were tested as acetylcholinesterase inhibitors by means of a chemiluminescent method suitable for high throughput screening. The compounds without quaternization had no appreciable inhibitory potency probably because they are poorly soluble in water. The corresponding quaternized compounds were good inhibitors with activity related to the spacer employed.

Full text of DOI:10.1016/j.ejmech.2005.05.010

European Journal of Medicinal Chemistry 40 (2005) 1331–1334
www.elsevier.com/locate/ejmech
Short Communication
Synthesis and chemiluminescent high throughput screening for inhibition
of acetylcholinesterase activity by imidazo[2,1-b]thiazole derivatives
Aldo Andreani *, Massimiliano Granaiola, Massimo Guardigli, Alberto Leoni,
Alessandra Locatelli, Rita Morigi, Mirella Rambaldi, Aldo Roda
Dipartimento di Scienze Farmaceutiche, Università di Bologna, Via Belmeloro 6, 40126 Bologna, Italy
Received 23 March 2005; received in revised form 16 May 2005; accepted 25 May 2005
Available online 29 August 2005
Abstract
The synthesis of a new series of imidazo[2,1-b]thiazole derivatives is described. They were tested as acetylcholinesterase inhibitors by
means of a chemiluminescent method suitable for high throughput screening. The compounds without quaternization had no appreciable
inhibitory potency probably because they are poorly soluble in water. The corresponding quaternized compounds were good inhibitors with
activity related to the spacer employed.
© 2005 Elsevier SAS. All rights reserved.
Keywords: Imidazo[2,1-b]thiazole; Bisammonium salts; Chemiluminescence; Antiacetylcholinesterase activity; Neuromuscular transmission; Alzheimer’s
disease; Miastenia gravis; Glaucoma
1. Introduction
6-methylimidazo[2,1-b]thiazole-5-carboxylic acid that con-
tained a four-carbon spacer (the amide 5a and the ester 6a)
showed a unique activity.At low doses (0.50–1 mg kg–1) they
releasedACh at the presynaptic level, with a stimulating effect
(37–50% increase in contractile force) that was not related to
activity on the muscle. By contrast, at high doses (16–
40 mg kg–1) both compounds produced a blockade (50–
100%) by acting at the postsynaptic level. Based on the data
obtained, the effect at low doses could be due to ACh release
or AChE inhibition. The biochemical evaluation for 3a–7a
was performed using a chemiluminescent (CL) method suit-
able for high throughput screening ofAChE inhibitors [11,12].
To further optimize the activity observed, a new series of
imidazo[2,1-b]thiazole derivatives with different spacers was
prepared (8–15, 8a–15a) (see Scheme 1) to investigate the
effect of different distances between the two portions of the
imidazothiazoles. These compounds were tested with the same
CL method. Quaternary compounds are not considered use-
ful drugs for the treatment of the Alzheimer’s disease since
they do not cross the blood–brain barrier but the authors of
the already mentioned review [8] underline that as early as
1992 Rosenberry asserted that compounds such as am-
benonium chloride are important tools for exploring AChE
catalytic mechanism and supposed that simple analogs could
penetrate the blood-brain barrier [13].
Acetylcholinesterase (AChE) inhibitors have been widely
used as therapeutic agents in disease states such as Alzhe-
imer’s, miastenia gravis and glaucoma [1–4]. They are widely
used in anesthesia to reverse the skeletal muscle relaxation
induced by non-depolarizing neuromuscular blocking agents
[5]. By inhibiting the hydrolysis of acetylcholine (ACh),
AChE inhibitors increase the levels of ACh in the neuromus-
cular junction facilitating cholinergic neurotransmission and
recovery of muscle function. Despite the widespread use of
neostigmine, a reversible inhibitor ofAChE (IC50 = 11.3 nM)
that is used as a reversal agent for neuromuscular block in
surgical anesthesia [5], there is a need for an equally effec-
tive reversal agent with reduced cardiovascular side effects
[6,7]. Also, the use of bis-quaternary acetylcholinesterase
inhibitors may be a strategy for development of effective drugs
for treatment of Alzeimer’s disease [8,9].
In a prior report the 6-methylimidazo[2,1-b]thiazole deriva-
tives (3a–7a) were known to act on neuromuscular transmis-
sion [10]. Two symmetrical bisammonium salts arising from
* Corresponding author. Tel.: +39 051 209 9714.
E-mail address: aldo.andreani@unibo.it (A. Andreani).
0223-5234/$ - see front matter © 2005 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2005.05.010

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A. Andreani et al. / European Journal of Medicinal Chemistry 40 (2005) 1331–1334
Scheme 1. (R see Table 1).
2. Chemistry
3. Biological results and conclusion
The new compounds were prepared from 6-methyl and
6-phenyl-imidazo[2,1-b]thiazole-5-carboxylic acids [14]
using the procedure previously reported [10]. The correspond-
ing 1-acylimidazole, prepared by reacting the acid with 1,1′-
carbonyldiimidazole (DCI), gave the alkyl carboxylate in
fairly good yields (see Table 1) after reaction with 1,6-
hexanediol (10), 1,8-octanediol (8, 11), 1,10-decanediol (9,
12), diethylene glycol (13), triethylene glycol (14), or tetra-
ethylene glycol (15) in the presence of 1,8-diaza-
bicyclo[5.4.0]undec-7-ene (DBU) as activator of the alcohol.
The quaternary salts 8a–15a were prepared by quaterniza-
tion of the tertiary bases 8–15 with a large excess of refluxing
iodomethane [10] (see Scheme 1).
The biological activity of the compounds was evaluated
by means of a high throughput CL assay based on a series of
coupled enzymatic reactions involving AChE, choline oxi-
dase and horseradish peroxidase, with luminol as the CL sub-
strate [11,12]. Assay validation was performed using tacrine,
a knownAChE inhibitor [15]. The IC50 values are reported in
Table 3 and represent the mean S.D. of at least three inde-
pendent measures. Most of the compounds without quater-
nization (4, 7–9, 11–15) were poorly soluble in water or
water/DMSO and were not assayed for the AChE activity,
however compounds 3, 5, 6, 10 were soluble and showed weak
activity (IC50 = 20–70 µM). Most of the quaternized com-
pounds were acetylcholinesterase inhibitors with significant
Table 1
Compounds 5–15
Compound
Z-n
A
R
Formula
MW
M.p. (°C) or References
[10]
5
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
CH3
CH3
CH3
CH3
CH3
CH3
C18H20N6O2S2
C20H26I2N6O2S2
C18H18N4O4S2
C20H24I2N4O4S2
C20H22N4O4S2
C22H28I2N4O4S2
C22H26N4O4S2
C24H32I2N4O4S2
C24H30N4O4S2
C26H36I2N4O4S2
C30H26N4O4S2
C32H32I2N4O4S2
C32H30N4O4S2
C34H36I2N4O4S2
C34H34N4O4S2
C36H40I2N4O4S2
C18H18N4O5S2
C20H24I2N4O5S2
C20H22N4O6S2
C22H28I2N4O6S2
C22H26N4O7S2
C24H32I2N4O7S2
416.5
700.4
418.5
702.4
446.5
730.4
474.6
758.5
502.7
786.5
570.7
854.6
598.7
882.6
626.79
910.7
434.5
718.4
478.6
762.4
522.6
806.5
5a
6
6a
7
7a
8
8a
9
9a
10
10a
11
11a
12
12a
13
13a
14
14a
15
15a
A
[10]
B-4
B-4
B-6
B-6
B-8
B-8
B-10
B-10
B-6
B-6
B-8
B-8
B-10
B-10
C-2
C-2
C-3
C-3
C-4
C-4
[10]
[10]
[10]
[10]
124–126
217–218 Dec.
93–95
216–217 Dec.
148–150
200–201 Dec.
112–114
165–166 Dec.
Oil
174–175 Dec.
159–161
216–217 Dec.
145–147
200–202 Dec.
91–92
152–153 Dec.

A. Andreani et al. / European Journal of Medicinal Chemistry 40 (2005) 1331–1334
1333
Table 2
IR and 1H-NMR of compounds 8–15 (th = thiazole, ar = aromatic)
Compound
IR: mmax (cm−1
)
1H-NMR:a d (ppm) in DMSO-d6
8
1670, 1320, 1230, 1140, 1100
1705, 1595, 1370, 1230, 1120
1670, 1320, 1230, 1135, 1095
1700, 1580, 1230, 1150, 825
1685, 1120, 750, 685, 655
1690, 1120, 950, 820, 655
1695, 1315 1225, 1110, 680
1720, 1220, 1130, 835, 730
1675, 1225, 1130, 750, 685
1710, 1205, 1120, 825, 690
1685, 1235, 1148, 717, 666
1725, 1230, 1122, 835, 738
1715, 1250, 1153, 764, 682
1696, 1127, 835, 748, 661
1700, 1291, 1148, 840, 687
1700, 1245, 825, 738, 666
1.36 (8H, m, 4CH2), 1.71 (4H, qui, 2CH2, J = 6.5), 2.48 (6H, s, 2CH3), 4.27 (4H, t, 2COOCH2, J = 6.5)
7.43 (2H, d, th, J = 4.4), 8.06 (2H, d, th, J = 4.4)
8a
1.37 (8H, m, 4CH2), 1.77 (4H, qui, 2CH2, J = 6.3), 2.70 (6H, s, 2CH3), 3.95 (6H, s, 2CH3I), 4.40 (4H,
t, 2COOCH2, J = 6.3) 7.89 (2H, d, th, J = 4.1), 8.42 (2H, d, th, J = 4.1)
9
1.32 (12H, m, 6CH2), 1.69 (4H, qui, 2CH2, J = 6.6), 2.49 (6H, s, 2CH3), 4.25 (4H, t, 2COOCH2,
J = 6.6), 7.43 (2H, d, th, J = 4.4), 8.05 (2H, d, th, J = 4.4)
9a
1.33 (12H, m, 6CH2), 1.76 (4H, qui, 2CH2, J = 6.7), 2.71 (6H, s, 2CH3), 3.95 (6H, s, 2CH3I), 4.40 (4H,
t, 2COOCH2, J = 6.7) 7.89 (2H, d, th, J = 4.2), 8.43 (2H, d, th, J = 4.2)
10
1.17 (4H, m, 2CH2), 1.55 (4H, qui, 2CH2, J = 6.2), 4.19 (4H, t, 2COOCH2, J = 6.2), 7.39 (6H, m, ar),
7.52 (2H, d, th, J = 4.4), 7.77 (4H, m, ar), 8.20 (2H, d, th, J = 4.4)
10a
11
0.78 (4H, s, 2CH2), 1.30 (4H, qui, 2CH2, J = 6), 3.75 (6H, s, 2CH3I), 4.14 (4H, t, 2COOCH2, J = 6),
7.64 (10H, s, ar), 8.00 (2H, d, th, J = 4.2), 8.59 (2H, d, th, J = 4.2)
1.16 (8H, s, 4CH2), 1.57 (4H, qui, 2CH2, J = 6.3), 4.20 (4H, t, 2COOCH2, J = 6.3), 7.39 (6H, m, ar),
7.52 (2H, d, th, J = 4.4), 7.77 (4H, m, ar), 8.20 (2H, d, th, J = 4.4)
11a
12
0.88 (4H, s, 2CH2), 1.00 (4H, s, 2CH2), 1.38 (4H, qui, 2CH2, J = 5.3), 3.74 (6H, s, 2CH3I), 4.16 (4H, t,
2COOCH2, J = 5.3), 7.63 (10H, s, ar), 7.98 (2H, d, th, J = 3.5), 8.58 (2H, d, th, J = 3.5)
1.18 (12H, s, 6CH2), 1.57 (4H, qui, 2CH2, J = 6.3), 4.21 (4H, t, 2COOCH2, J = 6.3), 7.40 (6H, m, ar),
7.53 (2H, d, th, J = 4.4), 7.79 (4H, m, ar), 8.20 (2H, d, th, J = 4.4)
12a
13
0.93 (4H, m, 2CH2), 1.11 (8H, s, 4CH2), 1.40 (4H, qui, 2CH2, J = 6.4), 3.74 (6H, s, 2CH3I), 4.17 (4H,
t, 2COOCH2, J = 6.4), 7.64 (10H, s, ar), 7.99 (2H, d, th, J = 4.1), 8.58 (2H, d, th, J = 4.1)
2.40 (6H, s, 2CH3), 3.83 (4H, t, 2CH2, J = 4.6), 4.41 (4H, t, 2CH2, J = 4.6), 7.33 (2H, d, th, J = 4.4),
7.97 (2H, d, th, J = 4.4)
13a
14
2.69 (6H, s, 2CH3), 3.94 (6H, s, 2CH3I), 4.54 (8H, m, 4CH2), 7.84 (2H, d, th, J = 4.2), 8.35 (2H, d, th,
J = 4.2)
2.44 (6H, s, 2CH3), 3.63 (4H, s, 2CH2), 3.76 (4H, t, 2CH2, J = 4.6), 4.34 (4H, t, 2CH2, J = 4.6), 7.39
(2H, d, th, J = 4.4), 8.00 (2H, d, th, J = 4.4)
14a
15
2.70 (6H, s, 2CH3), 3.64 (4H, s, 2CH2), 3.80 (4H, t, 2CH2, J = 4.7), 3.94 (6H, s, 2CH3I), 4.50 (4H, t,
2CH2, J = 4.7), 7.88 (2H, d, th, J = 4), 8.39 (2H, d, th, J = 4)
2.48 (6H, s, 2CH3), 3.55 (8H, s, 4CH2), 3.73 (4H, m, 2CH2), 4.35 (4H, m, 2CH2), 7.41 (2H, d, th,
J = 4.4), 8.03 (2H, d, th, J = 4.4)
15a
2.70 (6H, s, 2CH3), 3.57 (8H, m, 4CH2), 3.79 (4H, m, 2CH2), 3.94 (6H, s, 2CH3I), 4.49 (4H, m, 2CH2),
7.88 (2H, d, th, J = 4.2), 8.40 (2H, d, th, J = 4.2)
Table 3
The effect of the spacers was comparable for all the quat-
ernized compounds with the C type (13a–15a) giving good
Inhibition of acetylcholinestearase activity a
Compound
IC50 (µM)
70 15
inhibition (IC50 = 1.8–2.8 µM) and the most active com-
pounds being the B-type (8a, 10a, IC50 = 0.8, 1.4 µM, respec-
tively).Among this limited number of compounds, the nature
of the spacer influenced biological activity more than the
length of the spacer itself or the nature of the substituent at
the 6 position.
3 [10]
3a [10]
4a [10]
5 [10]
5a [10]
6 [10]
6a [10]
8a
10
3
3.5 0.7
40
13
6
4
45 15
30
0.8 0.1
20
6
4. Experimental section
10
4
10a
13a
14a
15a
1.4 0.5
1.8 0.4
1.8 0.4
2.8 0.4
0.20 0.03 b
4.1. Chemistry
The melting points are uncorrected. Elemental analyses
(C, H, N) were within 0.4% of the theoretical values. Bak-
erflex plates (silica gel IB2-F) were used for TLC: a mixture
of chloroform/methanol in various proportions was used as
the eluent and exposure to a UV lamp or iodine vapor was
used as the visualization method. The IR spectra were
recorded in Nujol on a Nicolet Avatar 320E.S.P.; mmax is
expressed in cm−1 (see Table 2). The 1H-NMR spectra were
recorded in (CD3)2SO on a Varian Gemini (300 MHz); the
chemical shift (referenced to solvent signal) is expressed in d
(ppm) and J in Hz. (see Table 2).
Tacrine
a Mean S.D. of at least three independent measures.
b Used as the reference compound [15].
activity. The improvement in activity shown by the quater-
nized compounds were most likely dependant on an interac-
tion of the positive charged imidazo[2,1-b]thiazoles with an
active site of the enzyme. The prior activity seen for 5a and
6a (IC50 = 13, 30 µM, respectively) suggests that the increase
in contractile force at low doses [10] was due more to ACh
release than AChE inhibition.

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A. Andreani et al. / European Journal of Medicinal Chemistry 40 (2005) 1331–1334
4.1.1. Alkyl carboxylates 8–15
DCI (22 mmol) was added to a suspension of
6-methylimidazo[2,1-b]thiazole-5-carboxylic
ing 10 µl of a 10 mM water solution ofACh to each well, then
the CL signal from the whole 384-well microtiter plate was
measured at 1-min time intervals by using a low-light imag-
ing luminograph (NightOWL, PerkinElmer Berthold, Bad
Wildbad, Germany). Upon evaluation of the CL intensity val-
ues for each well at different times, the slopes of the lumines-
cence kinetic profiles within 5 min after the start of the CL
reaction were calculated. The AChE inhibition in each well
was then determined by using a calibration curve obtained in
the same analytical session in wells with different amounts
of AChE. Finally, the IC50 values were evaluated by fitting
the experimental inhibition data with a four-point dose–
response curve.
acid
(1,
20 mmol) or 6-phenylimidazo[2,1-b]thiazole-5-carboxylic
acid (2, 20 mmol) in anhydrous CHCl3 (50 ml) and held at
reflux under stirring until effervescence ceased. After cool-
ing 1-acylimidazole was treated with the appropriate alcohol
(20 mmol) or diol (10 mmol) and DBU (22 mmol). The reac-
tion mixture was stirred under reflux for 4 h, cooled, and par-
titioned between CHCl3 and water. The organic layer was
washed with water to neutrality, dried and concentrated under
reduced pressure. The residue was crystallized from ethanol
(8, 10, 11), methanol (13, 15) and ethyl acetate (14) or puri-
fied by column chromatography (9 SiO2/ethyl acetate; 12
Al2O3/CHCl3) with a yield of 35% (8, 9, 11) and 60% (10,
12–15).
Acknowledgments
4.1.2. Quaternary salts 8a–15a
This work was supported by University of Bologna (Funds
for selected research topics).
Tertiary bases 8–15 (7–10 mmol) and CH3I (60 ml) were
stirred under reflux using a coil condenser cooled at –2 °C.
The reactions were allowed to proceed until TLC analysis of
the crude mixture (methanol solution) indicated both the lack
of the tertiary base and the presence of a single adduct. The
required time ranged from 120 (8a, 9a) to 240 h (10a–15a).
The reaction mixtures were then cooled and the suspended
solid filtered and washed with Et2O in order to remove the
excess CH3I. Additional purification from traces of unre-
acted tertiary base or monoquaternary salt was achieved by
treating a saturated ethanol solution with Et2O (8a, 9a) or by
recrystallization with H2O (10a–12a) or by washing with
acetone (13a–15a) with a yield of 80–90%.
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