Synthesis of substituted 3-anilino-5-phenyl-1,3-dihydro-2H-1,4- benzodiazepine-2-ones and their evaluation as cholecystokinin-ligands

Article abstract of DOI:10.1002/ardp.200500217

3-Amino-1,4-benzodiazepines as well as chemically related diverse amines were prepared from oxazepam and subsequently screened on the cholecystokinin receptor in a radiolabel binding assay. Oxazepam 2 was activated via its 3-chloro-1,4-benzodiazepine intermediate 3 and was reacted with a large series of aliphatic and aromatic amines. The substituted 3-anilino-1,4-benzodiazepine structure was identified as lead structure in a diverse series of 3-amino-1,4-benzodiazepines 4-38 and the full SAR (structure-activity relationship) optimisation provided 3-anilinobenzodiazepines 16-38 with CCK ₁ receptor selectivity to CCK₂. The compounds 18, 24, 28 and 33 have shown affinities at the CCK₁ receptor of 11, 10, 11 and 9 nM, respectively. These equipotent CCK₁ ligands were fully evaluated in behaviour pharmacological essays. An antidepressant effect was identified in the tail suspension- and the Porsolt swimming-test. The ED₅₀ values for 24 and 28 were determined in these assays as 0.46 and 0.49 mg/kg. The mixed antagonist 37 showed in addition to the antidepressant effects anxiolytic properties.

Full text of DOI:10.1002/ardp.200500217

Arch. Pharm. Chem. Life Sci. 2006, 339, 163–173
M. Offel et al.
163
Full Paper
Synthesis of Substituted 3-Anilino-5-phenyl-1,3-dihydro-
2H-1,4-benzodiazepine-2-ones and their Evaluation as
Cholecystokinin-Ligands
Michael Offel1, Pornthip Lattmann1, Harjit Singh1, D. C. Billington1, Yodchai Bunprakob2,
Jintana Sattayasai2, Eric Lattmann1
1 Aston Pharmacy School, Aston University, Birmingham, England
2 Department of Pharmacology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand
3-Amino-1,4-benzodiazepines as well as chemically related diverse amines were prepared from
oxazepam and subsequently screened on the cholecystokinin receptor in a radiolabel binding
assay. Oxazepam 2 was activated via its 3-chloro-1,4-benzodiazepine intermediate 3 and was reac-
ted with a large series of aliphatic and aromatic amines. The substituted 3-anilino-1,4-benzodia-
zepine structure was identified as lead structure in a diverse series of 3-amino-1,4-benzodiazepi-
nes 4-38 and the full SAR (structure-activity relationship) optimisation provided 3-anilinobenzo-
diazepines 16–38 with CCK1 receptor selectivity to CCK2. The compounds 18, 24, 28 and 33 have
shown affinities at the CCK1 receptor of 11, 10, 11 and 9 nM, respectively. These equipotent CCK1
ligands were fully evaluated in behaviour pharmacological essays. An antidepressant effect was
identified in the tail suspension- and the Porsolt swimming-test. The ED50 values for 24 and 28
were determined in these assays as 0.46 and 0.49 mg/kg. The mixed antagonist 37 showed in
addition to the antidepressant effects anxiolytic properties.
Keywords: 1,4-Benzodiazepines / CCK receptor / Cholecystokinin / Antidepressant /
Received: October 3, 2005; accepted: November 23, 2005
DOI 10.1002/ardp.200500217
treatment of chronic and severe pain [10]. In order to
fully understand, why it was focussed again on the class
of 1,4-benzodiazepines, a brief overview on different
classes of cholecystokinin ligands is presented. There-
fore, an early CCK antagonist, lorglumide, an example of
a peptidal antagonist, CI 988, the CCK1 selective standard
devazepide and YM 022, an optimised Merck ligand, are
outlined in Figure 1.
Lorglumide is a CCK1 selective antagonist with a low
potency and presents chemically an amide derivative of
glutamic acid [11, 12]. A research group at Parke-Davis
[13] examined the activity of CCK-30-33 fragments in
binding experiments on CCK2/gastrin receptors. This led
to the development of CI 988, which exhibited a 1600-
fold selectivity for CCK2 over CCK1 receptors with a sub-
nanomolar affinity (IC50 = 0.3 nM). However, due to the
high molecular weight and to the dipeptoid structure
these derivatives have a low bioavailability and are not
suitable for oral therapy [14].
Introduction
Cholecystokinine (CCK), which act as a neuromodulator/
gut hormone and CCK-ligands, agonists as well as antago-
nists [1], have been extensively investigated as potential
drug targets [2]. Antagonists were studied as growth inhi-
bitors in certain forms of cancer [3], as anxiolytics [4], in
the treatment of schizophrenia [5], satiety [6] and as anti-
panic agents [7]. An agonist, the shortened CCK tetrapep-
tide, was found to induce panic in patients [8]. A phase II
clinical trial of devazepide, a potent and CCK1 selective
antagonist [9], has been recently completed showing a
significant enhancement of the effect of morphine in the
Correspondence: Dr. Eric Lattmann, Aston Pharmacy School, Aston
University, Birmingham B4 7ET, England.
E-mail: e.lattmann@aston.ac.uk
Fax: + 44 121 359-0733
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164
M. Offel et al.
Arch. Pharm. Chem. Life Sci. 2006, 339, 163–173
Figure 2. Overview of selected CCK-antagonists.
work was reported using the 3-amino-1,4-benzodiazepine
template [24]. The only example of CCK ligands in the
class of substituted arylated or alkylated amines,
attached to the 3-position was a 3-amino-2-indolylmethyl-
1,4-benzodiazepine (CCK1 :IC50 = 87 nM) of in total two
examples of 3-amino-benzodiazepines with a high bind-
ing affinity [25]. This exciting structure served as our lead
structure of CCK1 selective ligands, when their relevance
was understood as agents in the treatment of depression
[26].
Here, we report the synthesis, binding affinity and in
vivo studies in mice on novel, CCK1 selective antagonists
with a much improved water solubility compared to the
ureido-1,4-benzodiazepines.
Figure 1. Overview on different classes of cholecystokinin
ligands.
Chemistry
L-364, 718 or MK-329 or devazepide revealed nanomo-
lar affinity for peripheral receptors, longer lasting effi-
cacy in vitro and in vivo, whilst orally bioavailable. devaze-
pide [15, 16] possessed potent CCK1 blocking activity in
different tissues. Pancreatic amylase secretion is antago-
nised with a potency of 2000000 times more than lorglu-
mide. Devazepide has been claimed to be a selective
antagonist of the effects of CCK-8 on food intake [17] and
it was a key tool in the autoradiographical demonstra-
tion of the presence of CCK1 receptors in the various
regions of the brain [18].
A novel potent series of 1-arylmethyl analogues of L-
365, 260 [19] was prepared in complex synthetical steps,
but the water solubility still remained a problem [20, 21].
Our early work on 1,4-benzodiazepines, based on asper-
licin, provided asperlicin- analogues [22] and other
libraries of N-alkylated 3-propyl-1,4-benzodiazepines.
With two alkyl chains, a CCK2 selectivity was achieved,
combined with a very high lipophilicity [23]. Various
Various approaches towards oxazepam were available,
such as the oxidation of the 3-position of diazepam [27].
Oxazepam was claimed to be synthesized via the known
Polonowski rearrangement of the N-oxide on an indus-
trial scale [28]. The chloroacetamide amide 1 was con-
verted into a quinazoline structure [29] in presence of a
Lewis acid, such as ZnCl2.
The formation of the desired benzodiazepine structure
2 took place from acetamide 1 under controlled alkaline
conditions enabling us to synthesise gramme quantities
of this key intermediate. The chloro-acetamide 1 was
cyclised into the desired oxazepam salt at ambient tem-
perature with an alcoholic solution of KOH at pH 11–14,
which was subsequently titrated with 3 M HCl to pH 1.8
to give the free oxazepam 2.
An activation of the 3-hydroxy group with DEAD via
phosphono-oxy derivatives was described [30], but the
most reliable route towards the 3-amino-1,4-benzodiaze-
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Arch. Pharm. Chem. Life Sci. 2006, 339, 163–173
3-Anilino-1,4-benzodiazepines as CCK Ligands
165
the receptor binding affinity towards the CCK receptors
are given in Table 1.
Following these initial findings, a large series of 3-ani-
lino-1,4-benzodiazepines 17–38 were prepared in order
to fully explore the structure activity relationships (SAR).
It was found that a meta-phenyl substituent had increas-
ed binding affinity: the m-nitro, m-methoxy and the m-
methylanilinoderivatives18,24and28showedanequipo-
tentaffinitytowardstheCCK1 receptorof10and11 nM.
Interestingly, the m-choroderivative 21 displayed only
a tenth of this binding affinity. The dimethylanilino-ana-
logue 33 was found of the same affinity, if both substitu-
ents were attached to two meta positions. N-Methylation
gave 37, a highly potent mixed CCK1/CCK2 antagonist.
The introduction of an N-ethyl group resulted in a
decreased binding affinity (entry 38).
In vivo pharmacology
The p-methoxy derivative 25 was found inactive in all in
vivo assays, as expected, in correlation with no receptor
binding activity. The CCK1 antagonists 24, 28, 31 and 33
have shown a MED (minimum effective dose) of 0.1 mg/
kg for the tail suspension test and the forced swim test in
mice. The mixed cholecystokinin antagonist 37 has
shown the same antidepressant bioactivity and in addi-
tion, an anxiolytic effect was determined in the elevated
x-maze and the light/dark box test for the same dose. No
analgesic effect and no impairment of coordination were
observed in a group of 5 mice up to a dose of 20 mg/kg for
the series outlined in Table 2.
Scheme 1. Synthesis route of compounds 4–38.
pines was found via the 3-chloro-1,4-benzodiazepine 3.
Oxazepam 2 was reacted with an excess of thionyl chlor-
ide to give a yellow 3-chloro-1,4-benzodiazepine 3, which
was reacted in situ with the corresponding amine into
the 3-aminobenzodiazepines 4–38 in presence of a cataly-
tic amount of TEA. Amines 4–16 were purified by chroma-
tography, while the anilines 17–38 precipitated out after
the addition of n-hexane to the reaction mixture.
After the first evaluation, the determination of the
MED, the two CCK1 selective ligands 24 and 28 and mixed
CCK-ligand 37 were chosen for a full data collection. The
nitro derivative 18 was not tested further due to a possi-
ble metabolism to a potentially toxic nitroso compound.
The m-toluene derivative 28 and the m-methoxy deriv-
ative 24 were evaluated at the same range using a group of
10 mice for each dose. An ED50 of 0.46, 0.46 mg/kg was
determined for the tail suspension test and an ED50 of0.49,
0.49 mg/kgwasfoundforthePorsoltswimmingtest.
The mixed CCK antagonist 37 showed in addition to
the antidepressant effects an ED50 of 0.46 and 0.42 mg/kg
for the elevated x-maze and for the light/dark box test,
respectively. Note: All three ligands antagonised CCK-8s
and CCK-4 induced contractions of the gall bladder and
the guinea pig ileum [31].
Results and discussion
SAR studies
N-alkylated derivatives 4–6 showed a binding affinity
above 20 mM; piperazines such as benzodiazepine 7 and
the substituted aminopiperidine 8, as an example,
showed a very weak binding affinity towards the CCK
receptor. Removal of substituents on the aromatic system
(entry 16) and the replacement of the aromatic phenyl
group by a cyclohexyl system (entry 13) resulted in a
reduced binding affinity. Ring closure of the aniline
structure into the quinoline derivative 9 was found less
active. A CH2-spacer between the benzodiazepine tem-
plate and the aniline structure, as for benzyl derivative
10 displayed a decreased binding affinity and the pheny-
lethyl-analogue 11 showed a loss of receptor binding. The
unsubstituted 3-anilino 1,4-benzodiazepine 16 itself dis-
played a low binding affinity, but the aniline structure
was clearly obtained as a privileged structure from this
first series (entry 4–16). An overview about the yields and
Conclusions
Chemical diversity in the relevant 3-position of the non-
toxic 1,4-benzodiazepine scaffold was used to replace the
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166
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Arch. Pharm. Chem. Life Sci. 2006, 339, 163–173
Table 1. Binding affinity of 3-amino-1,4-benzodiazepines for the CCK receptor.
Entry
Structure: R
MS (M+1) [m/z]
Yield [%]
IC50 [lM] CCK2
IC50 [lM] CCK1
4
5
6
ethyl
n-propyl
n-butyl
315
329
343
38
47
61
A20
A20
A20
A20
A20
A20
7
8
431
459
58
35
15
A20
15
A20
9
10
11
402
376
390
66
62
60
7.2
6.0
A20
A20
A20
A20
12
13
14
390
368
410
36
33
31
2.9
10.1
A15
A20
A20
A20
15
16
396
362
30
80
A15
A20
A20
4.1
17
407
49
7.5
A20
18
19
407
407
68
74
6 l 1
0.011 l 0.002
8.5
A20
20
21
396
396
41
53
9.3
6.2
A20
0.27
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Arch. Pharm. Chem. Life Sci. 2006, 339, 163–173
Table 1. Continued.
3-Anilino-1,4-benzodiazepines as CCK Ligands
167
Entry
22
Structure: R
MS (M+1) [m/z]
Yield [%]
IC50 [lM] CCK2
IC50 [lM] CCK1
396
67
2.9
20
23
392
40
1.5
A20
24
25
392
392
45
62
4.5 l 0.4
11 l 2
0.010 l 0.002
20 l 2
26
27
376
422
48
50
7.1
1.2
10
A20
28
29
30
376
376
390
85
92
22
3.8 l 0.5
4.6
0.011 l 0.002
0.29
A10
10
31
390
48
0.92 l 0.05
0.015 l 0.002
32
33
390
390
46
72
6 l 1
0.018 l 0.002
0.009 l 0.002
2.9 l 0.5
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Arch. Pharm. Chem. Life Sci. 2006, 339, 163–173
Table 1. Continued.
Entry
Structure: R
MS (M+1) [m/z]
Yield [%]
IC50 [lM] CCK2
6.1 l 0.5
IC50 [lM] CCK1
0.39 l 0.05
34
390
67
35
36
390
376
56
39
6.0 l 0.5
1.0 l 0.3
0.15 l 0.05
0.014 l 0.002
37
38
390
404
39
48
0.072 l 0.008
0.65 l 0.04
0.008 l 0.002
0.33 l 0.04
Table 2. In vivo evaluation of selected 3-anilino-5-phenyl-1,3-dihydro-2H-1,4-benzodiazepine-2-ones.
Compound
Receptor binding IC50 [nM]
CCK2
Elevated
plus-maze
Light/dark
box
Tail suspen-
sion test
Forced
swim test
Thermal tail Hot plate
Rota-rod
test
Wire mesh
grasping test
flick test
test
CCK1
24
25
28
31
33
37
10 l 2
11 000 l 2000
11 l 2
4 500 l 400
20 000 l 2000
3 800 l 500
920 l 50
2 900 l 500
72 l 8
NS
NS
NS
NS
NS
0.1
NS
NS
NS
NS
NS
0.1
0.1
NS
0.1
0.1
0.1
0.1
0.1
NS
0.1
0.1
0.1
0.1
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
15 l 2
9 l 2
8 l 2
NS = no significance could be observed at 0.05, 0.1, 0.5, 1.0, 5.0, 10.0 and 20.0 mg/kg; MED [mg/kg]: Minimum effective dose.
The mixed cholecystokinin antagonist 37 has shown anti-
depressant, as well as anxiolytic properties. Our com-
pounds occured a better water solubility than the most
recent generation of ureas containing a cationic solubi-
lising group [32]. The resolution of salts of maleic acid is
currently under investigation and will provide enantio-
merically pure pharmaceuticals.
Table 3. In vivo studies of CCK antagonist 24, 28 and 37 in
mice.
24 ED50 [mg/kg]
28 ED50 [mg/kg]
37 ED50 [mg/kg]
Elevated plus maze
Light/dark box
Tail suspension test
Forced swim test
–
–
–
–
0.46 l 0.15
0.42 l 0.20
0.50 l 0.20
0.48 l 0.21
0.46 l 0.12
0.49 l 0.13
0.46 l 0.11
0.49 l 0.11
We acknowledge Dr. David Poyner for providing pancreatic
known urea linkage by a novel 3-anilino-lead structure. and brain cortex membranes and Mr. Simon Dunn for in vitro
The CCK1 selective antagonists displayed antidepressant testing of ligand 28, 24 and 37. This work was partially sup-
properties in two standard assays carried out in mice. ported by the EPSRC and Panos Therapeutics Ltd.
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Arch. Pharm. Chem. Life Sci. 2006, 339, 163–173
3-Anilino-1,4-benzodiazepines as CCK Ligands
169
cooled in a ice bath to 0–108C over night. The precipitate was fil-
tered and dried to give the crude product (light brown solid).
Yield: 59.8% (crystallisation with ethanol or 1,4 dioxane); IR
(KBr) cm–1: 3385, 3025, 3320, 3010, 1705, 1590, 730; MF
C15H11N2O2Cl; MW 286.7; MS (APCI(+)): 287, 289 (M+1), 269, 271 (–
H2O) m/z; 1H-NMR (DMSO-d6) 300K d: 10.81 (s, N–H), 7.48 (s, phe-
nyl-H), 7.23 (s), 7.25 (d, Ar-H, J = 8.8 Hz), 7.64 (d, Ar-H, J = 8.7 Hz),
6.33 (d, OH, J = 8.7 Hz), 4.78 (d, C3-H, J = 8.7 Hz) ppm; 13C-NMR
(DMSO-d6) 300K d: 170.3 (C=N), 165.9 (C=O), 123.7, 127.1, 128.3,
128.5 (26C), 128.9, 129.7, 129.8 (26C), 131.0, 132.3, 138.5 (Ar-C),
83.3 (CH–OH) ppm.
Experimental
Chemistry
General methods
Chemicals were purchased from Aldrich UK and Lancaster Ltd
(Aldrich, Gillingham, UK; Lancaster Synthesis, Morecambe, UK).
Mass spectra were obtained by Atmospheric Pressure Chemical
Ionisation (APCI), negative or positive mode, using a Hewlett-
Packard 5989b quadrupole instrument (Hewlett-Packard, Palo
Alto, CA , USA). Samples were dissolved in HPLC grade methanol,
toluene or acetonitrile. Proton and Carbon NMR spectra were
obtained on a Bruker AC 250 instrument (Bruker, Coventry, UK)
calibrated with the solvent reference peak or TMS.
IR spectra were plotted from KBr discs on a Mattson 300 FTIR
Spectrophotometer (Mattson Instruments, Harston, UK). Melting
points were recorded from a Stuart Scientific Melting Point
(SMP1; Fisher Scientific, Loughborough, UK) and are uncor-
rected. Analytical Thin Layer Chromatography was obtained
using aluminium sheets, silica gel 60, F254 and visualised using
ultraviolet light. Preparative chromatography was performed
on 250 lm, 20620 cm silica gel TLC plates, obtained from
Aldrich. Jencons sonomatic sonicator (SO175; Fisher Scientific)
was used to prepare samples for screening. All compounds were
dissolved in DMSO for screening. Small scale solution syntheses
were carried out on a carousel reaction stations (RR 98030;
Fisher Scientific), fitted with a 12 place carousel reaction sta-
tion.
3,7-Dichloro-5-phenyl-1,3-dihydro-2H-1,4-
benzodiazepin-2-one 3
Oxazepam (0.5 g, 1.75 mmol) was treated with thionyl chloride
(4 eq, 0.4 mL) and heated to 608C for 1.5 h. The resulting inter-
mediate, a yellow solid, was washed with dry diethyl ether
(twice), to remove any excess of thionyl chloride.
Yield: 93.4%. MF: C15H10N2O2Cl2. MW: 305.2. IR (KBr-disc)
m max: 3418, 3220, 3060, 2919, 1704, 1606, 1476, 1322,
1226, 902, 823, 693 cm–1. MS (APCI(+)): 305, 306, 307
(M+1), 269, 270, 271 m/z. 1H-NMR (CDCl3) 300K d: 9.89 (s,
NH), 7.26–7.80 (m, Ar-H, 8H), 5.64 (s, C3-H) ppm.
General method for the preparation of 3-amino-
substituted 1,4-benzodiazepin-2-ones 4–38
Oxazepam (0.1 g, 3.5 mmol) was treated with thionyl chloride (4
eq, 0.1 mL) and heated to 608C for 1.5 hours. The resulting inter-
mediate, a yellow solid, was washed with dry diethyl ether
(twice) to remove any excess thionyl choride. The appropriate
amine (2.5 eq, 1.1 mmol), with TEA (drops) were refluxed for two
hours in DCM (15 mL). The organic phase was washed with
hydrochloric acid and dried over sodium sulphate. Excess hex-
ane was added and the mixture was allowed to stand overnight.
The precipitate was filtered, washed with hexane and dried.
2-Chloro-N-{4-chloro-2-
[(hydroxyimino)(phenyl)methyl]phenyl} acetamide 1
A solution of 2-amino-(5-chlorophenyl)(phenyl)methanoneox-
ime (142 mmol, 35.18 g) in ether (1000 mL) and water (300 mL)
was stirred in an ice bath at 0–58C. Chloroacetyl chloride
(160 mmol, 12.8 mL) was added dropwise, over 30 min, whilst
maintaining a slightly basic solution with the addition of 15%
aqueous sodium hydroxide. After the addition of chloroacetyl
chloride the reaction was stirred for an additional 3 h at room
temperature. The organic phase was washed with water, dried
over magnesium sulphate and concentrated to dryness to yield a
white powder.
3-(Ethylamino)-7-chloro-5-phenyl-1,3-dihydro-2H-1,4-
benzodiazepin-2-one 4
Yield: 38%, mp.: 126–1288C, Rf (ether)= 0.40. MF: C17H16ClN3O.
MW: 313.8. IR (KBr-disc) m max: 3430, 3121, 2977, 2855, 1654,
1607, 1478, 1320, 693 cm–1. MS (APCI(+)): 314, 315 (M+1), 296, 297
(M+), 269, 271 (M+) m/z. 1H-NMR (DMSO-d6) 300K d: 1.16–1.24 (t,
3H, J = 7.1 Hz), 2.70-3.0 (m, –CH2–), 4.34 (s, C3-H), 7.24–7.61 (m, Ar-
8H), 11.19 (s, NH) ppm. 13C-NMR (DMSO-d6) 300K d: 14.7 (CH3),
42.4 (–CH2–), 71.3 (C3), 123.4, 125.8 (26C), 128.3, 129.7 (26C),
130.6, 131.1, 136.3, 137.7, 166.5 (C=O), 169.0 (C=N) ppm.
Yield: 94%; IR (KBr) cm–1: 3390, 3300, 3015, 2900, 1660, 820;
MF C15H12N2O2Cl2; MW 323.2; MS (APCI(+)): 323, 325 (M+1), 305,
307 (-H20) m/z;1H-NMR (DMSO-d6) 300K d: 11.92 (s, N– OH), 9.25 (s,
NH), 7.39 (s, phenyl-H), 7.20 (s, C3-H), 7.53 (d, C6-H, J = 8.7 Hz),
7.83 (d, C5-H, J = 8.8 Hz), 4.06 (s, –CH2–) ppm; 13C-NMR (DMSO-d6)
300K d 157.2 (C=N–OH), 137.1 (C–NH–), 43.1 (–CH2–), 136.9,
136.0, 133.9, 133.1, 132.8, 132.2, 130.8, 130.1,127.9, 125.8, 124.9
(Ar-C) ppm.
3-(Propylamino)-7-chloro-5-phenyl-1,3-dihydro-2H-1,4-
benzodiazepin-2-one 5
7-Chloro-3-hydroxy-5-phenyl-1,3-dihydro-2H-1,4-
benzodiazepin-2-one 2
Yield: 47%, mp.: 128–1308C, Rf (ether)= 0.44. MF: C18H18ClN3O.
MW: 327.8. IR (KBr-disc) m max: 3433, 3108, 2980, 2851, 1764,
1471 1315, 699 cm–1. MS (APCI(+)): 326, 327 (M+1), 308, 309 (M+),
269, 271 (M+) m/z. 1H-NMR (DMSO-d6) 300K d: 1.11–1.20 (t, 3H, J =
7.0 Hz), 2.51–2.72 (m, 4H, –CH2–), 4.45 (s, C3-H), 7.25–7.83 (m, Ar-
8H), 11.12 (s, NH) ppm. 13C-NMR (DMSO-d6) 300K d: 12.9 (CH3),
34.0 & 42.4 (–CH2–), 71.1 (C3), 123.4, 125.1 (26C), 128.5, 129.9
(26C), 130.0, 131.3, 136.4, 137.7 (Ar-C), 166.2 (C=O), 169.5 (C=N)
ppm.
A
solution of 2-chloro-N-{4-chloro-2-[(hydroxyimino)(phenyl)-
methyl]phenyl} acetamide (134 mmol, 43.28 g) in ethanol
(900 mL) and sodium hydroxide (2 M, 280 mL) were stirred at
room temperature over night. The precipitate that formed was
separated by filtration and dissolved in a minium amount of
ethanol/water 60:40 mix, (undissolved oxazepam salt was col-
lected and dried). The mixture was acidified to pH 1.0–2.0 by the
addition of concentrated hydrochloric acid. The filtrate was
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Arch. Pharm. Chem. Life Sci. 2006, 339, 163–173
3-(Butylamino)-7-chloro-5-phenyl-1, 3-dihydro-2H-1, 4-
3-(Benzylamino)-7-chloro-5-phenyl-1, 3-dihydro-2H-1, 4-
benzodiazepin-2-one 6
benzodiazepin-2-one 10
Yield: 61%, mp.: 135–1378C, Rf (ethylacetate) = 0.54. MW: 341.8.
IR (KBr-disc) m max: 3425, 3100, 2930, 2850, 1700, 1640, 1615,
1480, 1330, 1080 cm–1. MS (APCI(+)): 342, 344 (M+1), 324, 326 (–
H2O), 269, 271 (M+) m/z. 1H-NMR (DMSO-d6) 300K d: 11.57 (s, NH),
9.69 (s, NH), 7.77 (dd, Ar-H, J = 8.7 Hz), 7.60–7.49 (m, phenyl-5H),
7.42 (d, Ar-H, J = 8.8 Hz), 7.30 (s, Ar-H), 5.12 (s, C3-H), 3.21 (m, –
CH–), 3.02 (m, –CH–), 1.73 (m, –CH2–), 1.39 (m, –CH2–), 0.92 (t,
CH3, J = 7.2, 7.4 Hz) ppm. 13C-NMR (DMSO-d6) 300K d: 13.7 (CH3),
21.9, 30.2, 45.2 (CH2), 70.5 (C3), 121.9, 125.1 (26C), 127.6, 128.2,
129.9 (26C), 130.8, 131.3, 132.0, 136.8, 138.2 (Ar-C), 167.5 (C=O),
168.1 (C=N) ppm.
Yield: 62.0%, mp.: 144–1468C, MF: C22H18ClN3O. MW: 375.9. IR
(KBr-disc) m max: 3430, 3335, 2975, 1705, 1580, 1330, 1095, 705
cm–1. MS (APCI(+)): 276, 278 (M+1), 358, 360 (-H2O), 269, 270 (M+)
1
m/z. H-NMR (DMSO-d6) 300K d: 11.57 (s, NH), 7.77 (dd, Ar-H, J =
8.7 Hz), 7.53–7.62 (m, phenyl-5H), 7.53 (d, Ar-H, J = 8.8 Hz), 7.40–
7.44 (m, amine phenyl-5H), 7.30 (s, Ar-H), 5.75 (s, –CH2–), 5.11 (s,
C3–H) ppm. 13C-NMR (DMSO-d6) 300K d: 52.1 (CH2), 74.2 (C3),
121.2, 125.8 (26 C), 126.5, 128.3, 128.5, 129.2 (26C), 130.1
(26C), 130.3, 130.6, 130.8 (26C), 131.0, 134.2, 136.1, 136.9 (Ar-
C), 162.1 (C=O), 167.1 (C=N) ppm.
7-Chloro-3-(methylbenzyl)-5-phenyl-1,3-dihydro-2H-1,4-
benzodiazepin-2-one 12
Yield: 78%, mp.: 151–1538C, Rf (ether)= 0.63. MW: 389.9. IR (KBr-
disc) m max: 3398, 3324, 3128, 2899, 2832, 1767, 1522, 1477,
1320, 1150, 683 cm–1. MS (APCI(+)): 390, 391 (M+1), 269, 271 (M+)
m/z. 1H-NMR (DMSO-d6) 300K d: 3.43 (s, CH3), 4.21 (m, 2H, –CH2–),
5.13 (s, C3-H), 6.65–6.70 (d, Ar-2H, J = 8.8 Hz), 7.11–7.19 (t, Ar-2H, J
= 7.9, 8.5 Hz), 7.29 (s, Ar-H), 7.34–7.40 (d, Ar-H, J = 8.9 Hz), 7.47–
7.58 (m, phenyl-5H), 7.68–6.73 (dd, Ar-H, J = 8.8 Hz), 11.59 (s, NH)
ppm. 13C-NMR (DMSO-d6) 300K d: 66.7 (–CH2–), 70.4, 71.2 (CH3 iso-
mers), 84.4 (C3), 117.83, 123.0, 123.1, 128.2 (26C), 128.5, 128.8
(26C), 129.2, 129.3 (26C), 130.2, 130.1 (26C), 131.7, 136.8,
138.8 (Ar-C), 166.2 (C=O), 169.8 (C=N) ppm.
3-(1-Phenylpiperazine)-7-chloro-5-phenyl-1,3-dihydro-2-
H-1,4-benzodiazepine-2-one 7
Yield: 58%, mp.: 168–1708C, Rf (ethylacetate) = 0.37. MW: 430.94.
IR (KBr-disc) m max: 3434, 3049, 2921, 2417, 1704, 1596, 1482,
1324, 1091, 685 cm–1. MS (APCI(+)): 431, 433 (M+1), 269, 271 (M+)
m/z. 1H-NMR (DMSO-d6) 300K d: 11.79 (s, NH), 7.76–7.82 (dd, Ar-H,
J = 9.8 Hz), 7.49–7.65 (m, phenyl-6H), 7.34 (s, Ar-H), 7.26–7.32 (d,
Ar-2H, J = 8.3 Hz), 7.02–7.05 (d, Ar-2H, J = 8.1 Hz), 6.85–6.91 (t, Ar-
H, J = 7.3, 7.2 Hz), 5.30 (s, C3-H), 3.40–3.53 (m, –CH2- 8H) ppm. 13C-
NMR (DMSO-d6) 300K d: 58.5 (26 –CH2–N–C3), 70.0 (26 –CH2-N-
Ar), 71.8 (C3), 116.4 (26C), 120.5, 124.7, 128.1, 128.3, 129.2, 129.7
(26C), 130.4 (26C), 132.2, 133.3, 134.9, 137.6, 137.8 (26C),
150.0 (Ar-C), 164.2 (C=O), 167.9 (C=N) ppm.
7-Chloro-3-(cyclohexylamino)-5-phenyl-1,3-dihydro-2H-
1,4-benzodiazepin-2-one 13
Yield: 33.0% mp.: 166–1688C, MF: C21H22ClN3O. MW: 367.9. IR
(KBr-disc) m max: 3403, 3092, 3033, 2927, 2863, 2358, 1700, 1623,
1474, 1322, 695 cm–1. MS (APCI(+)): 368, 370 (M+1), 350, 352 (-
H2O), 269, 271 (M+) m/z. 1H-NMR (DMSO-d6) 300K d: 11.57 (s, NH),
9.58 (s, NH), 7.74–7.79 (dd, Ar-H, J = 8.7 Hz), 7.43–7.63 (m, phenyl-
5H), 7.40–7.43 (d, Ar-H, J = 8.8 Hz), 7.30–7.31 (s d, Ar-H, J = 2.4 Hz),
5.15 (s, C3-H), 1.07–2.22 (m, –CH2–, 11H) ppm. 13C-NMR (DMSO-d6)
300K d: 24.7, 25.3, 28.6, 29.8, 30.8 (–CH2–), 54.6 (–CH–), 124.4
(26C), 127.9, 128.0, 129.1, 130.3, 130.7, 131.9, 133.2, 137.8
(26C), 137.9 (Ar-C), 165.7 (C=O), 167.8 (C=N) ppm.
3-Benzylpiperidin-4-anilino-7-chloro-5-phenyl-1,3-
dihydro-2H-1,4-benzodiazepin-2-one 8
Yield: 35.0%, mp.: 143–1458C, Rf (ethylacetate) = 0.44. MW: 458.9.
IR (KBr-disc) m max: 3434, 2828, 2358, 1994, 1602, 1481, 1318,
1120, 742, 699 cm–1. MS (APCI(+)): 459, 461 (M+1), 269, 271 (M +)
m/z. 1H-NMR (DMSO-d6) 300K d: 11.59 (s, NH), 7.66–7.74 (m, Ar-H,
13H), 4.30 (s, C3-H), 2.74–3.04 (m, CH), 2.66–2.83 (m, –CH2-Ar),
1.92-2.09 (m, –CH2–, 4H), 1.40–1.55 (m, –CH2–, 4H) ppm.
13C-NMR (DMSO-d6) 300K d: 32.0 (26 –CH2–), 38.9 (26 –CH2–N),
52.5 (CH), 73.1 (C3), 123.8, 127.3, 128.3 (26C), 128.6, 128.7, 128.9
(26C), 129.3 (26C), 129.7 (26C), 129.9, 131.0, 132.2, 138.3,
138.8, 139.1 (Ar-C), 164.9 (C=O), 167.2 (C=N) ppm.
3-Anilino-7-chloro-5-phenyl-1,3-dihydro-2H-1,4-
benzodiazepin-2-one 16
Yield: 80.0%, mp.: 173–1758C, MW: 361.8. IR (KBr-disc) m max:
3425, 3025, 3065, 2930, 1710, 1590, 1440, 1340, 1100, 730, 700
cm–1. MS (APCI(+)): 362, 364 (M+1), 344, 346 (–H2O), 269, 271 (M +)
1
m/z. H-NMR (DMSO-d6) 300K d: 11.09 (s, NH), 7.70 (dd, Ar-H, J =
7-Chloro-3-[3,4-dihydroquinolin-1(2H)-yl]-5-phenyl-1,3-
dihydro-2H-1,4-benzodiazepin-2-one 9
8.7 Hz), 7.42–7.50 (m, phenyl-5H), 7.36 (d, Ar-H, J = 8.8 Hz), 7.32 (s,
Ar-H), 7.23 (t, Ar-2H, J = 7.7, 7.8 Hz), 7.09 (t, Ar-H, J = 7.6, 7.3 Hz),
6.96 (d, Ar-2H, J = 7.7 Hz), 4.98 (s, C3-H) ppm. 13C-NMR (DMSO-d6)
300K d: 67.4 (C3), 114.2 (26C), 117.9, 122.8, 125.5 (26C), 127.1,
128.8, 129.9 (26C), 131.7, 136.9, 138.0, 150.2, (Ar-C), 165.1 (C=O),
167.9 (C=N) ppm.
Yield: 66.0% mp.: 160–1648C, MF: C24H20ClN3O. MW: 401.9. IR
(KBr-disc) : 3440, 3050, 2930, 2850, 2360, 1700, 1610, 1480, 830,
740 cm–1. MS (APCI(+)): 402, 404 (M+1), 384, 386 (-H2O), 269,
271(M+) m/z. 1H-NMR (DMSO-d6) 300K: 10.91 (s, NH), 7.72 (dd, Ar-
H, J = 8.7 Hz), 7.47–7.55 (m, phenyl-5H), 7.37 (d, Ar-H, J = 8.8 Hz),
7.28 (s, Ar-H), 6.95 (d, Ar-H, J = 7.2 Hz), 6.86 (d, Ar-H, J = 7.4, 7.6
Hz), 6.52 (t, Ar-H, J = 7.4, 7.2 Hz), 6.22 (d, Ar-H, J = 8.2 Hz), 5.22 (s,
C3-H), 4.09 (m, –CH–), 3.62 (m, –CH–), 2.77 (m, –CH2–), 1.98 (m, –
CH2–) ppm. 13C-NMR (DMSO-d6) 300K d: 22.0, 28.5, 45.5 (–CH2–),
78.5 (C3), 111.5, 117.5, 122.6, 122.7, 125.6 (26C), 128.1, 128.2,
128.3, 129.3, 129.9, 130.0 (26C), 130.5, 131.7, 136.9, 138.1,
148.1, (Ar-C), 165.1 (C=O), 166.3 (C=N) ppm.
7-Chloro-3-(2-nitroanilino)-5-phenyl-1,3-dihydro-2H-1,4-
benzodiazepin-2-one 17
The compound was purified further by using column chromato-
graphy with ether. Yield: 49%, mp.: A1908C, Rf (ether)= 0.30. MF:
C21H15ClN4O3. MW: 406,8. IR (KBr-disc) m max: 3349, 3279, 1702,
1590, 1527, 1469, 1316, 1297, 1114, 830, 693 cm–1. MS (APCI(+)):
i 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2006, 339, 163–173
3-Anilino-1,4-benzodiazepines as CCK Ligands
171
407 (M+1), 391, 393, 269, 271 (M+) m/z.1H-NMR (DMSO-d6) 300K d:
5.19-5.22 (d, C3-H, J = 7.0 Hz), 6.82-6.86 (d, Ar-H, J = 9.2 Hz), 7.10–
7.18 (m, Ar-2H), 7.33 (s, Ar-H), 7.44–7.55 (m, phenyl-5H), 7.70–
7.75 (dd, Ar-H, J = 8.8 Hz), 7.09–8.02 (d, Ar-H, J = 9.3 Hz), 7.04-7.07
(d, Ar-H, J = 7.1 Hz), 11.15 (s, NH) ppm. 13C-NMR (DMSO-d6) 300K d:
70.7 (C3), 113.2, 124.1, 126.3, 127.2 (26C), 128.9, 129.9, 130.3,
131.3, 132.6, 137.5, 138.2 (26C), 138.6, 153.2 (26C), 166.8 (C=O),
167.7 (C=N) ppm.
OCH3) ppm. 13C-NMR (DMSO-d6) 300K d: 55.1, 65.5 (OCH3), 67.5
(C3), 105.1, 111.7, 122.9, 125.4 (26C), 128.1, 128.6, 129.9 (26C),
130.0, 130.8, 132.1, 136.5, 137.1, 140.2 (Ar-C), 152.7, 153.1 (Ar–
O–), 165.2 (C=O), 165.9 (C=N) ppm.
7-Chloro-3-(2,4-dimethylanilino)-5-phenyl-1,3-dihydro-
2H-1,4-benzodiazepin-2-one 34
Yield: 67%, mp.: 158–1618C, Rf (ether) = 0.44. MF: C23H20ClN3O.
MW: 389.9. IR (KBr-disc) m max: 3436, 3182, 2919, 2618, 1690,
1606, 1473, 1222, 1147 cm–1. MS (APCI(+)): 390, 392, (M+1), 269,
7-Chloro-3-(3-chloroanilino)-5-phenyl-1,3-dihydro-2H-
1,4-benzodiazepin-2-one 21
1
271 (M+) m/z. H-NMR (DMSO-d6) 300K d: 2.32 (s, CH3), 2.78 (s,
Yield: 53%, mp.: 159–1618C, Rf (ether) = 0.31. MW: 396.3. IR (KBr-
disc) m max: 3438, 2919, 2856, 2362, 2338, 1653, 1594, 1318,
1014, 671 cm–1. MS (APCI(+)): 396, 397, 398 (M+1), 378, 379, 380
(–H2O), 269, 271 (M+) m/z. 1H-NMR (DMSO-d6) 300K d: 5.05 (s, C3-
H), 6.80–6.60 (d, Ar-H, J = 8.5 Hz), 6.64–6.69 (d, Ar-H, J = 8.0 Hz),
6.80 (s, Ar-H), 7.05–7.11 (t, Ar-H, J = 8.1 Hz), 7.32 (s, Ar-H), 7.33–
7.35 (d, Ar-H, J = 8.7 Hz), 7.43–7.53 (m, phenyl-5H), 7.68–7.73 (dd,
Ar-H, J = 8.8 Hz), 11.06 (s, NH) ppm. 13C-NMR (DMSO-d6) 300K d:
67.9 (C3), 117.5, 118.1, 122.6, 122.8, 125.8 (26C), 128.4, 128.7,
130.1, 130.5 (26C), 130.8, 130.9, 132.0, 133.5, 136.1, 137.2, 142.5
(Ar-C), 164.2 (C=O), 168.1 (C=N) ppm.
CH3), 4.80 (s, C3-H), 7.08–7.18 (m, Ar-2H), 7.22–7.23 (s,d Ar-H, J =
2.5 Hz), 7.28-7.31 (d, Ar-H, J = 8.5 Hz), 7.32–7.35 (d, Ar-H, J = 7.5
Hz), 7.46–7.53 (m, phenyl-5H), 7.63–7.68 (dd, Ar-H, J = 8.7, 8.8 Hz),
10.18 (s, NH), 10.84 (s, NH) ppm. 13C-NMR (DMSO-d6) 300K d: 17.5
(CH3), 21.0 (CH3), 83.2 (C3), 123.8 (26C), 125.8, 127.2, 127.8,
128.1, 128.7, 129.0 (26C), 129.4, 129.9, 131.2, 132.3, 132.4,
132.5, 138.2, 138.4 (Ar-C), 163.5 (C=O), 170.1 (C=N) ppm.
7-Chloro-3-(methylanilino)-5-phenyl-1,3-dihydro-2H-1,4-
benzodiazepin-2-one 36
Yield: 85%, mp.: 163–1658C, Rf (ether) = 0.59. MW: 375.9. IR (KBr-
disc) m max: 3430, 3216, 3129, 2923, 2851, 2358, 1708, 1596,
1496, 1318, 114, 693 cm–1. MS (APCI(+)): 376, 378 (M+1), 269, 271
7-Chloro-3-(3-methoxyanilino)-5-phenyl-1, 3-dihydro-2H-
1, 4-benzodiazepin-2-one 24
1
(M+) m/z. H-NMR (DMSO-d6) 300K d: 3.40 (s, CH3), 5.24 (s, C3-H),
Yield: 45%, mp.: 153–1558C. MF: C22H18ClN3O2. MW: 391.9. IR
(KBr-disc) m max: 3445, 3210, 3080, 2940, 1690, 1520, 1495, 1230,
1140, 1030 cm–1. MS (APCI(+)): 392, 394 (M+1), 374, 376 (-H2O),
269, 271 (M+) m/z. 1H-NMR (DMSO-d6) 300K d: 11.20 (s, NH), 7.65
(dd, Ar-H, J = 8.8 Hz), 7.43–7.51 (m, phenyl-5H), 7.31 (s, Ar-H), 7.30
(d, Ar-H, J = 8.7 Hz), 6.98 (t, Ar-H, 8.0, 8.0 Hz), 6.45 (d, Ar-H, J = 7.5
Hz), 6.27 (s, Ar-H), 6.22 (m, Ar-H), 4.89 (d, C3-H, 7.5 Hz), 3.66 (s,
OCH3) ppm. 13C-NMR (DMSO-d6) 300K d: 55.7 (OCH3), 66.9 (C3),
104.5, 107.9, 113.0, 122.7, 125.7 (26C), 128.0, 128.5, 129.8
(26C), 129.9, 130.8, 131.5, 137.1, 138.2, 142.1 (Ar-C), 160.1 (Ar-O)
164.1 (C=O), 167.9 (C=N) ppm.
6.68–6.71 (d, Ar-2H, J = 8.8 Hz), 7.12-7.18 (t, Ar-2H, J = 7.3, 8.5 Hz),
7.28 (s, Ar-H), 7.34–7.38 (d, Ar-H, J = 8.8 Hz), 7.48–7.55 (m, phenyl-
5H), 7.68–6.73 (dd, Ar-H, J = 8.7 Hz), 11.89 (s, NH) ppm. 13C-NMR
(DMSO-d6) 300K d: 70.1, 70.8 (CH3 isomers), 83.4 (C3), 117.8, 123.2,
123.3, 128.2 (26C), 128.3, 128.9 (26C), 129.4, 129.4 (26C),
130.0, 130.3 (26C), 131.7, 136.9, 138.1 (Ar-C), 165.1 (C=O), 169.4
(C=N) ppm.
7-Chloro-3-(3-dimethylanilino)-5-phenyl-1,3-dihydro-2H-
1,4-benzodiazepin-2-one 37
Yield: 39%, mp.: 163–1648C, Rf (ether) = 0.66. MF: C23H20ClN3O.
MW: 389.9. IR (KBr-disc) m max: 3420, 2925, 1700, 1600, 1481,
1320, 1121, 699 cm–1. MS (APCI(+)): 390, 392, (M+1), 269, 271 (M+)
m/z. 1H-NMR (DMSO-d6) 300K d: 2.22 (s, CH3), 3.25 (s, N-CH3), 5.61
(s, C3-H), 7.00–7.06 (t, Ar-H, J = 7.8, 7.9 Hz), 7.23 (s, Ar-H), 7.22–
7.30 (d, Ar-H, J = 7.4 Hz), 7.31 (s, Ar-H), 7.37–7.40 (d, Ar-H, J = 8.7
Hz), 7.49–7.56 (m, phenyl-5H), 7.64–7.66 (dd, Ar-H, J = 8.8 Hz),
7.69–7.74 (dd, Ar-H, J = 8.7 Hz), 10.89 (s, NH) ppm. 13C-NMR
(DMSO-d6) 300K d: 22.0 (CH3), 58.6 (N–CH3), 71.8 (C3), 122.3, 124.6
(26C), 125.2, 125.7, 127.0, 127.6, 128.6, 128.1, 128.9, 129.0,
129.3, 129.8 127.2, 127.8, 128.1, 128.7, 129.0, 129.3, 129.8 (26C),
129.9, 138.4, 138.4, 138.6, 149.5 (Ar-C), 165.1 (C=O), 169.4 (C=N)
ppm.
7-Chloro-3-(4-methoxyanilino)-5-phenyl-1,3-dihydro-2H-
1,4-benzodiazepin-2-one 25
Yield: 62%, mp.: 163–1658C, Rf (ether) = 0.38. MW: 391.9. IR (KBr-
disc) m max: 3426, 3193, 3058, 2935, 1687, 1519, 1476, 1320,
1220, 698 cm–1. MS (APCI(+)): 392, 394 (M+1), 374, 376 (-H2O), 269,
271 (M+) m/z.1H-NMR (DMSO-d6) 300K d: 3.77 (s, OCH3), 4.80 (s, C3-
H), 7.00–7.05 (d, Ar-2H, J = 7.8 Hz), 7.28-7.31 (d, Ar-H, J = 7.7 Hz),
7.32-7.36 (d, Ar-H, J = 7.9 Hz), 7.46-7.55 (m, phenyl-5H), 7.63–7.68
(dd, Ar-H, J = 8.8 Hz), 10.16 (s, NH), 10.85 (s, NH) ppm. 13C-NMR
(DMSO-d6) 300K d: 53.3 (OCH3), 68.1 (C3), 116.3 (26C), 117.2
(26C), 122.3, 124.5 (26C), 124.9, 126.6, 126.9, 129.8 (26C),
129.9, 130.6, 137.0, 137.4, 139.7, 153.6, 165.3 (C=O), 167.1 (C=N)
ppm.
Biology
7-Chloro-3-(3,4-dimethoxyanilino)-5-phenyl-1,3-dihydro-
Cholecystokinin binding assay, [125I]I-CCK-8 receptor
2H-1,4-benzodiazepin-2-one 27
binding assay
Yield: 50%, mp.: 194–1968C. MW: 421.9. IR (KBr-disc) m max: 3450,
3215, 3070, 2940, 1695, 1515, 1495, 1230, 700 cm–1. MS (APCI(+)):
422, 426 (M+1), 404, 406 (–H2O), 269, 271 (M+) m/z. 1H-NMR
(DMSO-d6) 300K d: 11.15 (s, NH), 7.70 (dd, Ar-H, J = 8.7 Hz), 7.43–
7.48 (m, phenyl-5H), 7.36 (s, Ar-H), 7.33 (d, Ar-H, J = 8.8 Hz), 6.80
(d, Ar-H, J = 8.7 Hz), 6.19 (dd, Ar-H, J = 8.7 Hz), 6.06 (d, Ar-H, J = 7.1
Hz), 5.97 (s, Ar-H), 5.03 (d, C3-H, J = 7.1 Hz), 3.82 (s, OCH3), 3.61(s,
CCK1 and CCK2 receptor binding assays were performed, by using
guinea pig cerebral cortex (CCK2) or rat pancreas (CCK1). Male
guinea pig brain tissues were prepared according to the modi-
fied method described by Saita et al. [33]. Pancreatic membranes
were prepared as described by Charpentier et al. [34].
Tissues were homogenised in cold sucrose (0.32 M, 25 mL) for
15 strokes at 500 rpm and centrifuged at 13000 rpm for 10 min.
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172
M. Offel et al.
Arch. Pharm. Chem. Life Sci. 2006, 339, 163–173
The supernatant was re-centrifuged at 13000 rpm for 20 min.
The resulting pellet was re-dispersed to the required volume of
buffer at 500 rpm and stored in aliquots. Binding was achieved
using radioligand 125I-Bolton-Hunter labeled CCK, NEN at 25 pM.
The samples were incubated with membranes (0.1 mg/mL) in
20 mM Hepes, 1mM EGTA, 5 mM MgCl2, 150 mM NaCl, at pH 6.5
for 2 h at RT and then centrifuged at 11000 rpm for 5 min. The
membrane pellets were washed twice with water and the bound
radioactivity was measured in a Packard Cobra Auto-gamma
counter (B5005; Perkin Elmer, Beaconsfield, UK). All binding
assays were carried out with L-363, 260 as an internal non-speci-
fic control.
The hot plate test
Mice were placed on a hot plate that was thermostatically main-
tained at 508C. A plexiglass box was used to confine the animal
to the hot plate. The reaction time of each animal (either paw
licking or jumping) was considered a pain response. The latency
to reaction was recorded. For prevention of heat injury, the cut-
off time of the test was 30 s.
Antidepressant tests
The tail suspension test
Mice were hung by their tail on the tail hanger using sticky tape
for tail fixation, at approximately 1 cm from the end. The
hanger was fixed in the black plastic box (20620645 cm) with
the opening at the top front. The distance between the hanger
and the floor was approximately 40 cm. The mouse was sus-
pended in the air by its tail and the immobile time was recorded
during the period of 5 min. The duration of immobility was
defined as the absence of all movement except for those
required for respiration.
Animal studies
Experiments were conducted in male IRC mice obtained from
the Animal House, Faculty of Medicine, Khon Kaen University.
Each experimental group consisted of 8 animals and the treat-
ment procedures were approved by the ethical committee,
Faculty of Medicine, Khon Kaen University. Mice were intraperi-
toneally injected with the test compound, dissolved in 5% DMSO
at the volume not more than 0.2 mL/animal. At 30 min after
treatment, animals were tested as described in the following sec-
tions.
The forced swim test
The forced swim test was carried out in a glass cylinder (20 cm
diameter, 30 cm height) filled with water to the height of 20 cm.
The water temperature was approximately 25–288C. Mice were
gently placed into the water and the immobility time was
recorded by an observer during the period of 5 min. Immobility
was defined as absence of all movement and remained floating
passively in the water with its head just above the water surface.
Anxiolytic activity tests
The light/dark box
Mice were placed in the light part of the light / dark box. The box
was a plexiglass cage, 25650620 cm, having one-third as a dark
and two-third as a light compartment. A 40-W light bulb was
used and positioned 10 cm above the centre of the light compo-
nent. The animals could walk freely between dark and light
parts through the opening. The time animals spent in light part
during the 5 min interval was recorded. The mouse was consid-
ered to be in the light part when its four legs were in the light
part.
Motor activity tests
The rota-rod test
Mouse was placed on the rotating drum with the acceleration
speed (Accelerator: Rota-rod, Jones & Roberts, for mice 7650, Ugo
Basile, Italy). The time the animal spent on the rod is recorded.
The wire mesh grasping test
Mouse was placed on a wire mesh (20630 cm). After a few sec-
onds, the mesh was turned 1808C and the time the animal hold
on the mesh was recorded.
The elevated plus-maze
The wooden elevated plus-maze consisted of two open arms
(30610 cm) without any walls, two enclosed arms of the same
size with 5 cm high side walls and end wall, and the central
arena (10610 cm) interconnecting all the arms. The maze was
elevated approximately 30 cm height from the floor. At the
beginning of the experiment the mouse was placed in the cen-
tral arena facing one of the enclosed arms. During a 5-min inter-
val, the time animals spent in the open arms of the plus-maze
was recorded. The mouse was considered to be in the open part
when it had clearly crossed the line between the central arena
and the open arm with its four legs.
Statistical methods
The data were expressed as mean + SD and one-way analysis of
variance (ANOVA) and supplementary Tukey test for pair-wise
comparison were tested to determine for any significant differ-
ence at p a0.05.
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