Synthesis and evaluation of N1-substituted-3-propyl-1,4-benzodiazepine-2-ones as cholecystokinin (CCK2) receptor ligands

Article abstract of DOI:10.1211/0022357021779005

A novel synthetic approach towards N₁-alkylated 3-propyl-1,4-benzodiazepines was developed in five synthetic steps from 2-amino-4-chlorobenzophenone, in which the N-oxide 4 served as a key intermediate. The structure-activity relationship optimization of this 3-prophyl-1,4-benzodiazepine template was carried out on the N₁-position by selective alkylation reactions and resulted in a ligand with an improved affinity on the cholecystokinin (CCK₂) receptor. The N-allyl-3-propyl-benzodiazepine 6d displayed an affinity towards the CCK₂ (CCK-B) receptor of 170 nM in a radiolabelled receptor-binding assay. The anxiolytic activity of this allyl-3-propyl-1,4-benzodiazepine 6d was subsequently determined in in-vivo psychotropic assays. This novel ligand had ED50 values of 4.7 and 5.2 mg kg^-1 in the black and white box test and the x-maze, respectively, and no significant sedation/muscle relaxation was observed.

Full text of DOI:10.1211/0022357021779005

Journal of
JPP 2002, 54: 827–834
# 2002 The Authors
Received September 1, 2001
Accepted February 5, 2002
ISSN 0022-3573
Synthesis and evaluation of N1-substituted-3-propyl-
1,4-benzodiazepine-2-ones as cholecystokinin (CCK2)
receptor ligands
Eric Lattmann, Jintana Sattayasai, David C. Billington, David R. Poyner,
Prapawadee Puapairoj, Siriporn Tiamkao, Wanchai Airarat, Harjit Singh
and Michael Offel
Abstract
A novel synthetic approach towards N1-alkylated 3-propyl-1,4-benzodiazepineswas developed
in Ž ve synthetic steps from 2-amino-4-chlorobenzophenone, in which the N-oxide 4 served as
a key intermediate. The structure–activity relationship optimization of this 3-propyl-1,4-
benzodiazepine template was carried out on the N1-position by selective alkylation reactions
and resulted in a ligand with an improved afŽ nity on the cholecystokinin (CCK2) receptor. The
N
-allyl-3-propyl-benzodiazepine6d displayed an afŽ nity towards the CCK2 (CCK-B) receptor of
170 nM in a radiolabelled receptor-binding assay. The anxiolytic activity of this allyl-3-propyl-
1,4-benzodiazepine6d was subsequently determined in in-vivo psychotropic assays. This novel
ligand had ED50 values of 4 7 and 5 2 mg kg 1 in the black and white box test and the x-maze,
-
.
.
respectively, and no signiŽ cant sedation/muscle relaxation was observed.
Introduction
The School of Pharmacy, Aston
University, Aston Triangle,
Birmingham B4 7ET, UK
The peripheral eŒects of cholecystokinin (Trivedi & Bharat 1994) are mediated
mainly through the A receptor subtype (CCK1), while the central eŒects are
correlated with the B receptor subtype (CCK2) (Evans et al 1993). The CCK2
receptor is involved in many pathological situations. Among these, anxiety (Bain &
Candillis 2000) and panic are particularly relevant targets for therapeutic
interventions (Tullio et al 2000). Sulfated CCK-5 is widely studied as an ideal panic-
triggering agent. The best evidence that CCK is strongly related to panic attacks is
based on experiments in which CCK-4 was administered to healthy volunteers who
had panic attacks shortly after the injection (Wilson at al 1996). Those physiological
reactions were signi®cantly blocked or reduced by administrating Merck’s CCK2
selective antagonist, L-365,260, which is chemically a 3-ureido-1,4-benzodiazepine
(Evans et al 1986) (Figure 1).
Eric Lattmann, David C.
Billington, David R. Poyner,
Harjit Singh, Michael Offel
Department of Pharmacology,
Faculty of Medicine, Khon Kaen
University, 40002 Khon Kaen,
Thailand
Jintana Sattayasai, Prapawadee
Puapairoj, Siriporn Tiamkao,
Wanchai Airarat
Correspondence: E. Lattmann,
The School of Pharmacy, Aston
University, Aston Triangle,
Birmingham B4 7ET, UK. E-mail:
e.lattmann!aston.ac.uk
We have previously identi®ed the n-propyl side chain in the 3-position of 1,4-
benzodiazepines as the best substituent for CCK ligands by a solid phase
combinatorial approach and included structure±activity relationship studies on the
ketone moiety of this molecule (Lattmann et al 2001). Here, we report a synthetic
approach towards the synthesis of this template 5 and the in¯ uence of the N1-
alkylation of the 3-propyl-1,4-benzodiazepine on the a nity at the CCK2 receptor.
Acknowledgement and funding:
We are grateful to the Peptide
Research Institute in Hannover
for providing the GABA-A
binding data on compound 6d.
This work was supported by the
EPSRC (CASE award for H.S.).

828
Eric Lattmann et al
Figure 1 Selective antagonists.
Materials and Methods
7-Chloro-2-oxo-5-phenyl-3-propyl-2,3-dihydro-1H-1,4-
benzodiazepin-4-ium-4-olate (4)
Chemistry
A solution (60 mmol) of 3 was stirred for 3±4 h at 40°C
with a mixture of 400 mL ethanol and 125 mL 2 m
sodium hydroxide. The solid that separated out was
removed by ®ltration. The crude material was collected
and recrystallized from a mixture of acetone and pet-
roleum ether. A higher amount of the title compound
was obtained by adding salt to the mixture. Recrystal-
Atmospheric pressure chemical ionization mass spec-
trometry (APCI-MS) was carried out on a Hewlett-
Packard 5989B quadrupole instrument connected to a
59987A unit with an APCI accessory. IR spectra were
recorded as KBr discs on a Mattson 3000 FTIR spectro-
photometer. Proton NMR spectra were obtained on a
Bruker AC 250 instrument operating at 250 MHz, with
tetramethylsilane as internal standard.
}
lization of the crude material with ethanol petrolether
gave the target molecule in 60% yield.
}
}
APCI-m s: m z ¯ 329 (80% ), 313 (20% ); TLC
(ether): Rf ¯ 0.45; IR (KBr) max ¯ IR (KBr disc)
max
2-Bromo-N-²4-chloro-2-[(hydroxyimino)(phenyl)-
methyl] phenyl´pentanamide (3)
¯ 3430, 3189, 3070, 2935, 1694, 1486, 1440, 1291, 1208
1
1
-
(N-oxide), 825, 688 cm ; H NMR (CDCl3) 1.0 (tr;
3H, J ¯ 7 Hz, C3:-(CH2)2CH3), 1.3±1.6 (m, 2H, C3:
-CH2CH2CH3), 2.1-2.5 (two br s, 2H, C3:
-CH2CH2CH3), 4.15 (tr, 1H, J¯ 5 Hz, C3:-H), 7.0±7.6
(m, 8 arom. H), 10.1 (s, 1H, N1:-H); 13C NMR (CDCl3)
14.0 (C3:-(CH2)2CH3), 19.4 (C3:-CH2CH2CH3), 36.9
(C3:-CH2CH2CH3), 69.6 (C3:-(CH2)2CH3), 123.2, 128.1,
129.9, 130.1, 130.5, 130.8, 131.5, 135.0, 136.4, 165.4,
170.1 ppm.
A solution of 0.10 mol (2-amino-5-chlorophenyl)-
(phenyl)methanone oxime 2 and 0.12 mol 2-bromo-1-
chloropentan-1-one in 200 mL 1,2-dichloroethane was
re¯ uxed for 3±4 h, cooled and washed with brine. The
organic layer was separated, dried with anhydrous so-
dium sulfate, ®ltered and concentrated in-vacuo. The
crystalline residue was recrystallized to give 70% of the
1
-
title product: 409.7 g mol .
}
}
APCI-m s: m z¯ 393 (50% , fragmentation, minus
OH), 329 (30% ), 313 (5% ), 283 (15% ); TLC (ether):
Rf ¯ 0.7; IR (KBr) max ¯ 3378, 2962, 2857, 1721, 1513, Preparation of 7-chloro-5-phenyl-3-propyl-1,3-dihydro-
1
1
-
1384, 1285, 1120, 1071, 695 cm ; H NMR (CDCl3)
2H-1,4-benzodiazepin-2-one (5)
1.0 (m, 3H, -CHBr(CH2)2CH3), 1.25±2.0 (m, 4H, To a suspension of 0.06 mol 3-propyl-benzodiazepine
-CHBr(CH2)2CH3), 4.25 (m, 1H, -CHBr(CH2)2CH3), N-oxide 4 in 300 mL chloroform, 17.2 mL (0.2 mol) of
7.0±8.0 ppm. (m, 8 arom. H and -NHCO).
phosphorus trichloride was added. The stirred mixture

829
Benzodiazepines as CCK ligands
m
Table 1 Chemical yields and IC50 ( ) receptor binding data on the cholecystokinin (CCK2) receptor
m
of the 1,4-benzodiazepines (standard: 10 n L-365,260, Merck).
Compound Reagent R1
Yield
CCK2
aGABA-A binding > 50 m. RT indicates reaction carried out at room temperature.
was re¯ uxed for 1 h and the solvent was removed under (5% ); mp: 197±198 °C; TLC (ether): Rf ¯ 0.72; IR
reduced pressure. To this residue, methylene chloride (KBr disc) max ¯ 3218, 3123, 2923, 2851, 1687, 1604,
1
1
and a large excess of 50% potassium hydroxide were 1476, 1320, 1220, 826, 699 cm ; H NMR (CDCl3) 1H
added. The aqueous layer was extracted three times with NMR (CDCl3) 0.99 (t; 3H, J ¯ 7 Hz, C3:-(CH2)2CH3),
methylene chloride. The organic layers were combined, 1.3±1.5 (m, 2H, C3:-CH2CH2CH3), 2.23 (m, 2H, C3:
dried, ®ltered to remove a ®ne amorphous impurity and -CH2CH2CH3), 3.53 (t, 1H, J ¯ 5 Hz, C3:-H), 7.15±7.7
concentrated in-vacuo. The residue was recrystallized (m, 8 arom. H), 10.3 (s, 1H, N1:-H); 13C NMR (CDCl3)
-
with ether yielding 80% of the desired molecule.
}
14.5 (C3:-(CH2)2CH3), 19.3 (C3:-CH2CH2CH3), 33.1
}
APCI-m s: m z¯ 313 (80% ), 268 (15% ), 264 (C3:-CH2CH2CH3), 63.2 (C3:-(CH2)2CH3), 122.8, 124.9,

830
129.1, 130.2, 130.8, 131.6, 135.7, 137.4, 168.0 (C-5) 7-Chloro-5-phenyl-3-propyl-1-prop-2-ynyl-1,3-dihydro-
Eric Lattmann et al
172.5 ppm. (C?O).
2H-1,4-benzodiazepin-2-one (6f )
}
}
APCI-m s: m z ¯ 351; TLC (ether): Rf ¯ 0.87; IR
(KBr) max ¯ 3295, 2954, 2867, 1691, 1608, 1478, 1407,
General experimental for the N-alkylation of 3-propyl-
benzodiazepines
1
1
-
1324, 1193, 1133, 825, 685 cm ; H NMR (CDCl3)
1.01 (t; 3H, J ¯ 7 Hz, C3:-(CH2)2CH3), 1.2±1.8 (m, 4H,
C3:-(CH2CH2CH3), 2.35 (m, 1H, N1:-CH2C@CH), 3.5
(m, 1H, C3:-H), 4.6 (m, 2H, N1:-CH2C@CH), 7.25±8.0
}
The N1-alkyl aryl derivatives (Table 1) were obtained
by deprotonating the parent amide 5 with 1.1 equiv.
sodium hydride in N,N-dimethylformamide, followed
by the reaction of the in-situ-formed anion with 1.2
equiv. of the electrophile. The alkylation towards the
methyl, ethyl, propyl and butyl derivatives 6a±6d was
carried out at20°C. The alkylation with the electrophiles
giving the phthalimide, morpholino, piperidine deriva-
tives 6o±6q was carried out at 50°C (Table 1).
The N1-hydroxylmethyl-benzodiazepine 6l was ob-
tained from formaldehyde, generated by thermal de-
composition of paraformaldehyde. After 30 min, the
reactions were quenched with 10% aqueous HCl. The
mixture was extracted three times with methylen-
chloride, concentrated in-vacuo to give the N1-alkylated
benzodiazepines. The crude product was puri®ed further
by solid extraction with ether.
(m, 8 arom. H); 13C NMR (CDCl3)
10.1 (C3:
-(CH2)2CH3), 19.3 (C3:-(CH2CH2CH3), 33.5, 36.8, 63.3
(C-3)), 72.1, 122.9, 128.3, 129.5, 129.8, 130.5, 131.1,
138.2, 140.5, 167.3 (C?O), 169.6 ppm.
2-(7-Chloro-2-oxo-5-phenyl-3-propyl-2,3-dihydro-1H-
1,4-benzodiazepin-1-yl)acetonitrile (6g)
}
}
APCI-m s: m z¯ 352 (90% ), 268 (10% ); TLC
(ether): Rf ¯ 0.81; IR (KBr) max ¯ 2930, 2861, 2280
(N1:-CH2-C@N), 1723, 1687, 1598, 1324, 1265, 825,
1
1
-
744, 695 cm ; H NMR (CDCl3) 1.01 (t, 3H, J ¯
7 Hz, C3:-CH2CH2CH3), 1.1±1.9 (m, 4H, C3:
-CH2CH2CH3), 3.45-3.6 (two m, 2H, N1:-CH2-C?N),
4.5 (q, 1H, J ¯ 5Hz, C3:-H), 7.1±7.8 7.1±7.9 (m, 8 arom.
H); 13C NMR (CDCl3) 14.1 (C3:-CH2CH2CH3), 19.3
(C3:-CH2CH2CH3), 33.4 (C3:-CH2CH2CH3), 38.9 (N1:
-CH2-C@N), 62.9 (C3:-H), 115.4 (N1:-CH2-C@N),
122.8, 128.4, 128.9, 129.4, 130., 130.9, 131.5, 137.4,
7-Chloro-1-methyl-5-phenyl-3-propyl-1,3-dihydro-2H-
1,4-benzodiazepin-2-one (6a)
}
}
APCI-m s: m z ¯ 327; TLC (ether): Rf ¯ 0.75; IR 139.2, 167.4, 169.3 ppm.
(KBr) max ¯ 2971, 2861, 1715, 1669, 1461, 1253, 1114,
1
1
-
1073, 738, 705 cm ; H NMR (CDCl3) 0.99 (t; 3H,
J ¯ 7 Hz, C3:-(CH2)2CH3), 1.35±1.52 (m, 2H, C3:
-(CH2)2CH3), 1.99 (m, 2H, C3:-CH2CH2CH3), 3.48 (s,
3H, N1:-CH3); 4.17 (t, 1H, J ¯ 5 Hz, C3:-H), 7.2±7.87
1-(sec-Butyl)-7-chloro-5-phenyl-3-propyl-1,3-dihydro-
2H-1,4-benzodiazepin-2-one (6i)
}
}
APCI-m s: m z¯ 369 (90% ), 313 (10% ); TLC
(ether): Rf ¯ 0.91; IR (KBr) max ¯ 2955, 2857, 1737,
(m, 8 arom. H), 13C NMR (CDCl3)
14.1 (C3:
1
-
1683, 1461, 1378, 1272, 1116, 1069, 960, 740, 695 cm
1H NMR (CDCl3)
;
0.85±1.9 (m, 15H, C3:
-(CH2)2CH3), 19.1 (C3:-CH2CH2CH3), 28.9 (N1:-CH3),
35.0 (C3:-CH2CH2CH3), 68.0 (C-3), 122.6, 128.5, 129.5,
130.8, 131.3, 138.1, 141.2, 167.7, 171.7 ppm.
-(CH2CH2CH3-N1:-CH(CH3)CH2CH3), 4.2 (m, 2H,
C3:-H-N1:-CH(CH3)CH2CH3), 7.3±7.7 (m, 8 arom.
H); 13C NMR (CDCl3) 11.5, 14.1, 18.7, 19.4, 28.8,
30.3, 38.6, 67.9 (C-3), 128.3, 128.7, 129.1, 130.4, 130.8,
132.4, 138.1, 140.9, 167.6, 169.1 ppm.
1-Allyl-7-chloro-5-phenyl-3-propyl-1,3-dihydro-2H-1,4-
benzodiazepin-2-one (6d)
}
}
APCI-m s: m z ¯ 353; TLC (ether): Rf ¯ 0.85; IR
(KBr) max ¯ 3062, 2954, 2867, 1671, 1602, 1557, 1482, 7-Chloro-1-phenethyl-5-phenyl-3-propyl-1,3-dihydro-
1
1
-
1403, 14, 1197, 1131, 921, 825, 699 cm ; H NMR 2H-1,4-benzodiazepin-2-one (6k)
}
}
(CDCl3) 0.73 (t, 3H, N1:-CH2CH2CH3), 1.01 (t; 3H, APCI-m s: m z¯ 417 (85% ), 313 (5% ), 279
J ¯ 7 Hz, C3:-(CH2)2CH3), 1.25-1.9 (m, 4H, C3: (10% ); TLC (ether): Rf ¯ 0.91; IR (KBr) max ¯ 2957,
-(CH2CH2CH3), 3.52 (m, 2H, N1:-CH2CH ? CH2), 4.56 2924, 2857, 1717, 1683, 1461, 1283, 1121, 1069, 740,
1
(m, 1H, C3:-H), 5.06±5.13 (m, 2H, N1:-CH2CH? 695 cm ; 1H NMR (CDCl3)
0.98 (t, 3H, J ¯
-
CH2), 5.8±5.85 (m, 1H, N1:-CH2CH?CH2), 7.25±7.6 7 Hz, C3:-CH2CH2CH3); 1.45±1.75 (m, 4H, C3:
(m, 8 arom. H); 13C NMR (CDCl3)
-(CH2CH2CH3), 19.3, 33.6, 50.1, 63.5 (C-3), 117.1 (N1: Ph), 6.8±7.75 (m, 13 arom. H); 13C NMR (CDCl3) 4.1
-CH2CH?CH2), 123.3, 129.4, 129.8, 130.4, 131.3, 131.8, (C3:-CH2CH2CH3), 22.9 (C3:-CH2CH2CH3), 30.1 (C3:-
14.2 (C3: -(CH2CH2CH3), 4.25 (m, 3H, C3:-H - N1:-CH2-CH2-
132.9, 138.2, 141.2, 167.1, 169.4 ppm.
CH2CH2CH3), 38.6, 68.1 (C3:-H), 123.9, 126.4, 126.9,

831
128.5, 128.7, 130.8, 132.3, 136.6, 139.3, 141.3, 167.2 (s, 2H, N1:-CH2-CO-NH2), 4.55 (m, 1H, C3:-H),
Benzodiazepines as CCK ligands
(C?O), 168.8 ppm.
5.82-6.45 (2 s, 2H, N1:-CH2-CONH2), 7.25±7.7 (m, 8
arom. H); 13C NMR (CDCl3) 13.9 (C3:-CH2CH2CH3),
22.9 (C3:-CH2CH2CH3), 28.9 (C3:-CH2CH2CH3), 57.1
(N1:-CH2-CO-NH2), 65.3 (C3:-H), 126.4, 128.2, 128.6,
7-Chloro-1-(2-hydroxyethyl)-5-phenyl-3-propyl-1,3-
dihydro-2H-1,4-benzodiazepin-2-one (6m)
}
}
APCI - m s: m z ¯ 357 (65% ), 313 (35% ); TLC 129.5, 130.4, 130.8, 131.2, 131.6, 135.0, 139.4, 141.2,
(ether): Rf ¯ 0.59; IR (KBr) max ¯ 3428, 2954, 2867, 167.7 (C?O), 170.6, 192.3 (N1:-CH2-CO-NH2) ppm.
1
1
-
1675, 1579, 1405, 1326, 1076, 823, 685 cm ; H NMR
(CDCl3) 0.97 (t, 3H, J ¯ 7 Hz, C3:-(CH2CH2CH3);
1.1±2.3 (m, 4H, C3:-(CH2CH2CH3), 3.53 (t, 2H, J ¯
5 Hz, N1:-CH2-CH2-OH), 3.8±4.0 (m, 3H, N1:-CH2-
Pharmacology
"$"
I-CCK-8 receptor binding assay
CH2-OH), 4.2 (m, 1H, C3:-H), 7.2±7.6 (m, 8 arom. H); The CCK1 and CCK2 receptor binding assays were
13C NMR (CDCl3) 14.5 (C3:-CH2CH2CH3), 19.3, 33.4 performed by using guinea-pig pancreas or guinea-pig
(C3:-CH2CH2CH3), 51.5, 61.2 (C-3), 102.4, 124.3, 128.4, cerebral cortex, respectively. For the CCK2 assay, mem-
129.4, 130.5, 131.4, 136.8, 141.8, 168.2, 170.1 ppm.
branes from male guinea-pig brain tissues were prepared
according to the modi®cation described by Saita et al
(1994). For the CCK1 binding assay, pancreatic mem-
branes were obtained as described by Charpentier et al
7-Chloro-1-(2-morpholin-4-ylethyl)-5-phenyl-3-propyl-
1,3-dihydro-2H-1,4-benzodiazepin-2-one (6p)
}
}
APCI-m s: m z ¯ 426; TLC (ether): Rf ¯ 0.82; IR (1988). All the binding assays were carried out in dupli-
(KBr) max ¯ 3432, 2954, 2853, 1675, 1608, 1476, 1320, cate with L-365,260 and devazepide as internal
1
1
-
1118, 817, 695 cm ; H NMR (CDCl3) 0.97 (t, 3H, standards.
J ¯ 7 Hz, C3:-CH2CH2CH3), 1.2±1.65 (m, 4H, C3:
-CH2CH2CH3), 2.1±2.55 (m, 8H, N1:-CH2-CH2-), 3.45 weighedafter dissection and then homogenized in 25 mL
In order to prepare the tissue, the cerebral cortex was
1
-
(m, 4H, N1:-CH2-CH2-), 4.58 (m, 1H, C3:-H), 7.25±7.65 ice-cold 0.32 m sucrose for 15 strokes at 500 rev min .
1
(m, 8 arom. H); 13C NMR (CDCl3)
-CH2CH2CH3), 19.3 (C3:-CH2CH2CH3), 33.5, 33.9 (C3: 10 min. The supernatant was centrifuged at 20000 g
14.1 (C3: It was then centrifuged at 1000 g (3000 rev min ) for
-
1
-
-CH2CH2CH3), 43.2, 53.9, 55.2, 63.3, (C-3), 66.8, 123.4, (13000 rev min ) for 20 min. This pellet was
128.2, 129.6, 130.4, 131.2, 136.5, 140.8, 166.3, redispensed in the required volume of assay buŒer as
de®ned below with 5 strokes of homogenizer at
169.3 ppm.
1
-
500 rev min . The ®nal tissue concentration was 1 g
}
7-Chloro-1-(3,3-dimethyl-2-oxobutyl)-5-phenyl-3-
propyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one (6s)
original weight 120 mL buŒer. The tissue was stored in
aliquots at ®70°C.
}
}
APCI-m s: m z¯ 411 (90% ), 313 (5% ), 268 (5% );
For the receptor binding assay, the radio ligand (125I-
TLC (ether): Rf ¯ 0.72; IR (KBr) max ¯ 2957, 2875, Bolton Hunter labelled CCK; NEN) and the drugs
1
-
1717, 1675, 1598, 1482, 1283, 1121, 1069, 736, 690 cm
1H NMR (CDCl3)
;
0.8±1.05 (m, 12H, C3: (0.1 mg mL ) in assay buŒer containing 20 mm Hepes,
(25 pm) to be tested were incubated with membranes
1
-
-CH2CH2CH3-N1:-CH2-COC(CH3)3, 1.4±1.9 (m, 4H, 1 mm EGTA, 5 mm MgCl2, 150 mm NaCl at pH 6.5 for
C3:-CH2CH2CH3), 4.25 (s, 2H, N1:-CH2-COC(CH3)3), 2 h at room temperature. The incubations were termin-
4.7 (tr, 1H, J ¯ 5 Hz, C-3), 7.0±7.75 (m, 8 arom. H); 13
NMR (CDCl3) 13.9 (C3:-CH2CH2CH3), 19.6 (C3: washed twice with water and bound radioactivity was
C
ated by centrifugation. The membrane pellets were
-CH2CH2CH3), 26.4 (N1:-CH2-COC(CH3)3), 33.5 (C3: measured in a -counter.
-CH2CH2CH3), 53.3 (N1:-CH2-COC(CH3)3), 63.8 (C3:
The GABA-A binding assay was performed as de-
3
-H), 122.3, 128.2, 128.9, 129.6, 130.5, 131.2, 136.8, 141.7, scribed by Speth et al (1979) using H-diazepam.
167.2, 169.3, 209.4 (N1:-CH2-COC(CH3)3) ppm.
In-vivo evaluation: black and white box test, elevated
2-(7-Chloro-2-oxo-5-phenyl-3-propyl-2,3-dihydro-1H-
1,4-benzodiazepin-1-yl)acetamide (6u)
x-maze and motor activity
Male OF1 mice (20±25 g) were kept in conventional
}
}
APCI-m s: m z¯ 370 (90% ), 313 (5% ), 279 (5% ); plastic cages in groups of 10 and had free access to food
TLC (ether): Rf ¯ 0.52; IR (KBr) max ¯ 3405, 2955, and water. They were housed in air-conditioned facilities
1
-
2869, 1723, 1663, 1463, 1278, 1128, 1069, 740, 690 cm
1H NMR (CDCl3)
-CH2CH2CH3), 1.3±1.8 (m, 4H, C3:-CH2CH2CH3), 4.2 University Faculty of Medicine (HO 2434-76).
;
at 27±28°C on a 12-h light±dark cycle. The animal work
0.99 (t, 3H, J¯ 7 Hz, C3: was approved by the Bioethics committee of Khon Kaen

832
Eric Lattmann et al
Figure 2 Synthesis of N-alkylated 3-propyl-1,4-benzodiazepines. Reagents: i. Bromovaleryl-bromide, re¯ ux, dichloroethane; ii. KOH,
+
.
pH 11, RT; iii. PCl3, CHCl3, re¯ ux; iv. NaH, THF, RT or 50°C, 1 2 eq E (Table 1).
The test compounds and diazepam (as standard) were amide 3 in high yield. Under basic conditions, the
dissolved in propylene glycol. The solution was admin- cyclization furnished the N-oxide 4, unlike the known
}
istered intraperitoneally at a volume of 0.05 mL 10 g at Polonowski rearrangement (Bell & Childress 1962).
30 min before the experiments ; the group size was 7 for Compared with the hydroxy group, the more nucleo-
each dose. The time the mice spent in the open arm was philic nitrogen reacted in the 7-exo-tet reaction only
recorded for an observation period of 5 min in the into the desired N-oxide 4. No formation of the 3-
elevated x-maze test. The time spent in the light was hydroxy-3-propyl-1,4-benzodiazepine (oxazepam ana-
recorded for an observation period of 10 min in the logue) via an 8-membered ring was observed. The
black and white box test. The equipment was purchased N-oxide 4 was subsequently reduced to the target 5
from San Diego Instruments (7758 San Diego, CA by using an excess of phosphorous trichloride.
92126). The motor activity of the mice was recorded as
The N-alkylated benzodiazepines 6a±6r were syn-
the time that the mice were able to hold on the sieve at thesized from the 1,4-benzodiazepine template 5 with
an angle of 180 °. The data were tested by using one-way sodium hydride in tetrahydrofuran at ambient tempera-
analysis of variance (Tukey test) and the full experi- ture or 50°C (Evanset al1987). No dialkylation products
mental details of each assay are available from Vogel & were obtained and no column chromatography was
Vogel (1997).
required for further puri®cation.
Pharmacology and structure±activity relationship
studies
Results and Discussion
The ligand containing a C3 unit in the 3-position of the
1,4-benzodiazepine was previously found to bind best to
A synthetic approach towards the synthesis of the 3- a postulated lipophilic pocket at the CCK2 receptor
Chemistry
propyl-1,4-benzodiazepine 5 and its alkylation in the (Lattmann et al 2001). In order to optimize the a nity
N1-position are outlined in Figure 2.
of the 3-propyl-1,4-benzodiazepines, additional sub-
2-Amino-4-chloro-amino-benzophenone (Sternbach stituents had to be introduced in the N1-position, as
et al 1962) was converted into the corresponding oxime are outlined in Table 1.
2 with hydroxylamine, which was subsequently reacted More complex electrophiles forming piperidino-,
with bromo-butyric acid chloride giving the desired morpholino- and phthalimido-benzodiazepines 6o±6q

833
Benzodiazepines as CCK ligands
The benzodiazepines 6d±6 g, containing a cyano-
methyl-, propargyl- and allyl-group, displayed an in-
creased a nity. Obviously, large and bulky substituents
reduce the binding a nity, and small, lipophilic elec-
tron-rich groups, such as the allyl group, are ideal for an
enhancement of the bioactivity. A substituent in the N1
position is desirable, ®rstly to enhance the a nity and
secondly to diŒerentiate the binding pro®le between the
CCK and the benzodiazepine receptor. It is known that
most of the substituents in the N1 position block the
a nity on the GABA-A receptor (Martin & Lattmann
1999) (Figure 3).
In-vivo studies
The neuropharmacological eŒects of the 3-propyl ben-
zodiazepine template 5 and the best 1,3-dialkylated
benzodiazepine 6d were evaluated in comparison with
diazepam in four diŒerent in-vivo assays in mice. The
cyanomethyl benzodiazepine 6 g, with a similar a nity
to 6d, was excluded from further evaluation owing to
insolubility in water and organic solvents. Compounds
5 and 6d have been found to be inactive in the evaluation
of pain in the hot-plate and the tail-¯ ick assays (O’Neill
Figure 3 Overview of selected 1,4-benzodiazepines.
did not show an enhanced binding a nity on the CCK et al 1989).
receptor. As previously reported for the 3-ureido-benzo-
The anxiolytic eŒect was evaluated by using the black
diazepines, N1-alkylation (Semple et al 1996) should and white box test (Matto et al 1997) and the elevated
have shown enhanced binding on the CCK2 receptor, x-maze (Johnson & Rodgers 1996) as two standard
but with the majority of the electrophiles used here the anxiolytic assays. For diazepam, the anxiolytic activity
}
a nity was signi®cantly lower than for the N1-unalkyl- correlated with sedation muscle relaxation. The 3-
propyl-benzodiazepine 5 displayed a week anxiolytic
ated 1,4-benzodiazepines.
1
-
Functionalized hydrophilic substituents such as the eŒect, having an ED50 of approximately 20 mg kg . In
hydroxy-alkyl series 6l±6m resulted in the loss of contrast to 5, the dialkylated 1,4-benzodiazepine 6d dis-
binding.
played an unexpectedly strong anxiolytic eŒect, with a
In order to gain a high chemical diversity, an amide magnitude comparable with diazepam (Table 2). Based
1
-
6u, an ester 6r and two ketones 6s and 6t were attached on doses of 1, 5, 20, 100 and 200 mg kg , the ED50
to the N1-position. Unlike the a nity observed for the values for the black and white box and x-maze were 4.7
1
-
N1 alkylated propyl-1,4-benzodiazepines 6s and 6t, and 5.2 mg kg , respectively, with no reduced motor
}
Merck’s 3-ureido-1,4-benzodiazepines have shown the activity observed. A signi®cant sedation muscle re-
best binding with the N1-substituents, analogues to the laxation compared with the control was observed at
90 mg kg 1 (ED50), a dose that is 18-times greater than
-
compounds 6s and 6t (Semple et al 1997).
Table 2 Results of receptor binding data compared with in-vivo studies (elevated x-maze, black and white box model and motor activity).
1
-
Receptor binding
ED50 (mg kg
)
Elevated x-maze
Black and white box
Motor activity
m
.
.
.
.
.
.
Diazepam
GABA-A: 70³5 n
1 1³0 2
1 3³0 3
1 3³0 2
"
200³15
"
m
CCK2 : 100³11
"
m
.
.
. .
4 7³0 8
6d
GABA-A: 50³7
5 2³0 9
m
CCK2 :170³23 n

834
Eric Lattmann et al
Johnson, N. J., Rodgers, R. J. (1996) Ethological analysis of
cholecystokinin (CCK-A and CCK-B) receptor ligands in the
Psychopharmacology Berl
.) 124: 355±
the eŒective anxiolytic dose. In a literature search, a
similar anxiolytic compound, dihydrohonokiol, with
a propyl and an allyl group attached to an aromatic
centre, was found to act on the steroid-binding site
of the GABA receptor (Stavinoha 1999).
elevated plus maze in mice.
364
(
Lattmann, E., Billington, D. C., Poyner, D. R., Howitt, S. B., OŒel,
O. (2001) Solid phase synthesis of 3-alkylated-1,4-benzodiaze-
Pharm
pines as non-peptidal cholecystokinin-(CCK)-agonists.
Pharmacol Lett
.
.
. 11: 18±21
Martin, I. L., Lattmann, E. (1999) Benzodiazepine recognition site
Conclusion
Expert Opin Ther Patents
. . 9:
ligands and GABA-A receptors.
1347±1358
A novel ®ve-step chemical approach from 2-amino-
benzophenone towards the synthesis of 3-n-propyl-1,4-
benzodiazepines was developed. Introducing substitu-
ents in the N1-position further enhanced the a nity of
the 1,4-benzodiazepine template. Based on the results of
the receptor-binding assay, the best compounds were
evaluated in animal experiments. The 1-allyl-3-propyl-
1,4-benzodiazepine 6d, having anIC50of approximately
0.17 m, was found to be active as a novel anxiolytic
Matto, V., Harro, J., Allikmet, L. (1997)The eŒects of drugs acting on
J
the CCK receptors and rat exploration in the exploration box.
Physiol Pharmacol
.
.
. 48: 239±251
O’Neill, M. F., Dourish, C. T., Iversen, S. D. (1989) Morphine in-
duced analgesia in the rat paw pressure test is blocked by CCK and
Neuropharmacology
enhanced by CCK antagonist MK-329.
243±247
28:
Saita, Y., Yazawa, H., Honma, Y., Nishida, A., Miyata, K., Honda,
}
K. (1994) Characterization of YM022: its CCKB gastrin receptor
binding pro®le and antagonism to CCK-8-induced Ca mobiliz-
2
+
with an ED50 of approximately 5 mg kg 1 in the x-maze
-
Eur J Pharmacol
. . . 269: 249±254
ation.
Semple, G., Ryder, H., Kendrick, D. A., Szelke, M., Ohta, M., Satoh,
M., Nisida, A., Akuzawa, S., Miyata, K. (1996) Synthesis and
biological activity of 1-alkylcarbonylmethyl analogues of YM022.
and the black and white box assays. There is a current
development programme to optimize this lead structure
and to study the binding pro®le of these novel
anxiolytics.
Bioorg Med Chem Lett
. . . . 6: 51±54
Semple, G., Ryder, H., Kendrick, D. A., Szelke, M., Ohta, M., Satoh,
M., Nisida, A., Akuzawa, S., Miyata, K., Rooker, D. P., Batt,
A. R. (1997) (3R)-N(tert-Butyl-carbonylmethyl)-2,3-dihydro-2-
oxo-5-(2-pyridyl)-1H-1,4-benzodiazepin-3-yl)-N«-(3-(methyl-
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