Benzimidazoles as NMDA glycine-site antagonists: Study on the structural requirements in 2-position of the ligand

Article abstract of DOI:10.1002/(SICI)1521-4184(20005)333:5<123::AID-ARDP123>3.0.CO;2-5

A series of different substituted benzimidazole derivatives has been synthesized and evaluated for the ability to displace [³H]MDL-105,519 to rat cortical membranes. Two benzimidazole-2-carboxylic acids 9 b and 9 c, in this substitution pattern not yet described as glycine antagonists, showed IC₅₀ values of 0.89 μM (9 b) and 38.0 μM (9 c). Replacement of the carboxylate function in 2-position by a sulfonic acid moiety appreciably increased solubility, but decreased the affinity giving evidence for the strong need of the carboxylate group within the ligand. Further structure-activity studies using benzimidazol-2-one derivatives with an acetic acid moiety adjacent to a ring nitrogen revealed new insights into the importance of amide functionalities within the heterocycle for the affinity of antagonist glycine-site ligands.

Full text of DOI:10.1002/(SICI)1521-4184(20005)333:5<123::AID-ARDP123>3.0.CO;2-5

Benzimidazoles as NMDA Glycine-Site Antagonists
123
Benzimidazoles as NMDA Glycine-Site Antagonists:
Study on the Structural Requirements in 2-Position of the Ligand
Gerd Dannhardt* and Beate K. Kohl
Department of Pharmacy, Johannes Gutenberg University, D-55099 Mainz, Germany
Key Words: NMDA-receptor; glycine antagonists; benzimidazoles; binding studies; structure-activity relationships
Summary
A series of different substituted benzimidazole derivatives has
been synthesized and evaluated for the ability to displace
3
[
H]MDL-105,519 to rat cortical membranes. Two benzimid-
azole-2-carboxylic acids 9 b and 9 c, in this substitution pattern not
yet described as glycine antagonists, showed IC50 values of
0
.89 µM (9 b) and 38.0 µM (9 c). Replacement of the carboxylate
function in 2-position by a sulfonic acid moiety appreciably in-
creased solubility, but decreased the affinity giving evidence for
the strong need of the carboxylate group within the ligand. Further
structure-activity studies using benzimidazol-2-one derivatives
with an acetic acid moiety adjacent to a ring nitrogen revealed new
insights into the importance of amide functionalities within the
heterocycle for the affinity of antagonist glycine-site ligands.
Introduction
Antagonists acting on the strychnine-insensitive glycine
site (glycine ) of the NMDA (N-methyl-D-aspartate) recep-
B
tor have long been considered as promising therapeutic
agents for a huge number of acute and chronic neurodegen-
erative disorders [ . Recent studies
1]
[2]
on glycine antago-
Figure 1
B
nists as potential therapeutics, however, indicate that the wide
range of putative applications for this class of drugs might
have to be restricted to a core of disorders including epilepsy
CO H
2
CO H
2
(
status epilepticus) and central ischaemic processes, which
N
O
O
are of a special pharmaceutical interest considering the high
morbidity and invalidity associated with these emergencies.
A further field of interest, in which glycine-site antagonists
were proved to be active is hyperalgesia suggesting that
N
O
N
H
Cl
N
H
Cl
[3]
glycine antagonists could present a new analgesic class
.
6
15 c
The advantages of glycine antagonists over other NMDA
receptor antagonists include the lack of psychotomimetic side
effects as well as the absence of vacuolisation in the cingulate
Figure 2
cortex. However, glycine antagonists are not totally free of
B
side effects. The side effect profile previously observed in-
Generally, the carboxylate function plays a role of interest
concerning the molecular architecture of the glycine binding
cludes sedation, ataxia, and myorelaxation [
2]
.
A difficulty still facing medicinal chemists designing gly- site since it could be demonstrated for diverse heterocycles,
cine antagonists is to attain the triad: affinity in vitro, potency i.e. indoles, tetrahydroquinolines, and quinoxaline-2,3-dio-
[1]
in vivo, and satisfactory solubility in physiological media
.
nes (Figure 1), that a carboxylate group introduced into the
The glycine unit incorporated in many antagonistic ligands northern region of the ligand causes an increase in affinity
for the glycine site gives rise to an often decreased in vivo giving evidence for a second acidic binding pocket in the
activity and unfavourable properties concerning the physico- receptor protein.
chemical profile, i.e. the hydrophilic-lipophilic balance and
We therefore decided to evaluate a series of benzimidazoles
solubility. Replacement of the carboxylate function in 2-po- bearing a sulfonic acid function instead of a carboxylate
sition by other acidic but solubility enhancing groups could group in 2-position of the heterocycle in order to increase the
therefore present a possibility to afford new compounds with solubility and to refine structure-activity relationships ac-
altered bioavailability.
cording to the 2-position.
Arch. Pharm. Pharm. Med. Chem.
© WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2000
0365-6233/00/0505/0123 $17.50 +.50/0

124
Dannhardt and Kohl
Table 1. Structures of benzimidazole-2-carboxylic acids and benzimidazole-
-sulfonic acids.
2
NH
3
Cl C C
NH
2
O CH
3
a)
N
R
+
R
CCl
3
N
H
NH
2
—
—————————————————————————————
8
7
1
2
1
2
3
Cpd.
R
R
Cpd.
R
R
R
—
—————————————————————————————
N
9
9
9
a
H
Cl
11 a
11 b
11 c
H
H
Cl
H
H
Br
H
H
H
F
b)
R
COOH
b
c
Cl
H
Cl
H
Cl
N
H
NO2
H
Cl
1
1
1
1
1
1 d
1 e
1 f
1g
Cl
H
Cl
9 a-c
Br
H
Br
Scheme 1. a) CH3COOH, 1 h, room temp. b) 1 N NaOH.
H
CH3
CH3
potassium ethyl xanthate in ethanol 95% (route A) [11]. As an
alternative synthetic pathway to afford 10 under milder con-
ditions suitable for readily oxidizing diamines, compound 7
was reacted with 1,1′-thiocarbonyldiimidazole in dry tetrahy-
drofuran as exemplified for the 5,6-dichloro-benzimidazole-
1 h
CH3
——————————————————————————————
Table 2. Structures of benzimidazolones.
2
-thiol (route B). The thiol group of compound 10 was easily
transferred into the sulfonic acid moiety by an oxidation
procedure using H O 10% and sodium hydroxide under
2
2
[
12]
reflux conditions
. Purification from hot water yielded
compounds 11 a–h.
S
+
C
2
H
5
O
SK
NH
2
route A
route B
N
R
a)
R
SH
NH
2
N
H
7
10
A further study based on a small series of benzimidazolones
with the favourable carboxylate function in the northern
region of the molecule should clarify the importance of an
amide structure in 2-position of the heterocycle (compound
S
+
N
N
N
N
1
5 c) relative to the bis-amide structure (compound 6) as
N
O
depicted in Figure 2.
b)
R
S
O
OH
N
H
1 a-h
Chemistry
1
The compounds, which were synthesized and evaluated for
their ability to displace [ H]MDL-105,519, are summarized
in Tables 1 and 2. The physical data of compounds 9 a–c,
3
Scheme 2. a) route A: EtOH 95%, reflux, 3 h; route B: THF, 3 h, 0 °C and
12 h room temp. b) H2O2 10%, NaOH, reflux.
1
1 a–h, 15 a–c and 18 a, b are given in Table 3.
Synthesis of the benzimidazole-2-carboxylic acid deriva-
Compounds 15 a–c were synthesized as shown in
tives 9 a–c was carried out as depicted in Scheme 1. Accord- Scheme 3. The isopropenyl intermediate 12 was prepared by
[
10]
ing to a procedure described by Louvet et al.
the reacting the o-phenylenediamine derivative 7 with ethyl ace-
[13]
o-phenylenediamine derivative 7 was reacted with trichlo-
romethyl acetimidate in acetic acid to give the corresponding
trichloromethylbenzimidazole derivative 8, which was iso-
lated and treated with a solution of sodium hydroxide to
afford the carboxylic acids 9 a–c.
The preparation of the benzimidazole-2-sulfonic acid de-
rivatives 11 a–h was performed according to Scheme 2. For
the routine synthesis of the benzimidazole-2-thiol derivative the carboxylic acids 15 a–c were precipitated by acidification
toacetate according to Rossi et al.
and Gómez-Parra et
[14].
al.
3 following a standard procedure
Compound 12 was converted into the ester derivative
[15]
1
using freshly prepared
sodium ethoxide and ethyl bromoacetate. After acidic cleav-
age of the isopropenyl moiety with diluted sulfuric acid and
saponification of the ethyl ester using 2 N sodium hydroxide,
10 the appropriate o-phenylenediamine 7 was refluxed with of the reaction mixture with concentrated hydrochloric acid.
Arch. Pharm. Pharm. Med. Chem. 333, 123–129 (2000)

Benzimidazoles as NMDA Glycine-Site Antagonists
125
Table 3. Physical data of the final compounds.
—
————————————————————————————————————————————————————————————–
Cpd.
Mp. (°C)
IR (KBr)
1H-NMR (200 MHz), δ (ppm), J (Hz)/
MS (EI, 70 eV): m/z
—
————————————————————————————————————————————————————————————–
9
a
162
3500 s (NH), 2500 w (-COOH),
680–1650 s (-C=N-; –C=O), 1600 s (arom.)
7.22 (d, 1H, J = 1.46, Ar-H), 7.34 (d, 1 H, J = 8.78, Ar-H), 7.66
(d, 1 H, J = 8.79, Ar-H), 8.28 (s, 1 H, NH)
1
1
98 (M+• , 0.1%), 154 (32%), 125 (13%)
9
9
b
c
220
192
3480 s (NH), 2500 w (-COOH), 1680–1650 s
-C=N-; C=O), 1600 s (arom.)
7.85 (s, 2 H, Ar-H)
190 [(M – CO2) , 9%], 188 (56%), 186 (88%), 159 (10%)
+
(
3460 s (NH), 2500 w (-COOH), 1680-1650 s
-C=N-; C=O), 1600 s (arom.), 1530 s (N=O)
7.78 (d, 1 H, J = 2.93, Ar-H), 8.11 (d, 1 H, J = 9.20, Ar-H), 8.22
(s, 1 H, NH), 8.53 (d, 1 H, J = 9.20, Ar-H)
(
2
07 (M+• , 0,34%), 164 (9%), 163 (100%), 44 (43%)
11 a
11 b
11 c
>300
>300
>250
3530 s (-OH), 3480 s (NH), 1640 s (-C=N-),
7.39 (d, 1 H, J = 1.96, Ar-H), 7.46 (d, 1 H, J = 8.3, Ar-H),
7.50 (d, 1 H, J = 8.3, Ar-H)
1
1
610 m (arom.), 1260 m (SO2-OH),
060 s (-SO2-OH)
216 (M+• , 7%), 136 (100%), 109 (44%)
3560 s (-OH), 3480 s (NH), 1640 s (-C=N-),
250 s (SO2-OH), 1080 s (-SO2-OH)
7.54 (d, 1 H, J = 1.47, Ar-H), 7.63 (dd, 1 H, J = 7.32, Ar-H),
7.70 (d, 1 H, J = 7.32, Ar-H)
1
a)
+•
MS (FD) : 234 (M , 5%), 232 (4%)
3590 s (-OH), 3460 s (NH), 1640 s (-C=N-),
610 s (arom.), 1240 s (SO2-OH), 1070 s
-SO2-OH)
6.70 (s, 1 H, NH), 7.49 (m, 2 H, Ar-H)
1
267 (M+• , 62%), 234 (56%), 196 (62%), 174 (65%)
(
1
1 d
1 e
>300
>250
3440 s (NH), 1670 s (-C=N-), 1610 s (arom.),
270 s (SO2-OH), 1070 s (-SO2-OH)
7.9 (s, 2 H, Ar-H)
MS (FD) : 268 (M , 3%)
a)
+•
1
1
3540 s (-OH), 3460 s (NH), 1640 s (-C=N-),
610 s (arom.), 1270 s (SO2-OH), 1060 s
-SO2-OH)
7.54 – 7.73 (m, 3 H, Ar-H))
+
1
186 [(M – 91) , 3%], 118 (100%), 93 (63%), 80 (13%), 79 (11%)
(
1
1 f
1g
>300
>300
3540 s (-OH), 3460 s (NH), 1640 s (-C=N-),
600 s (arom.), 1270 s (SO2-OH), 1070 s (C-Br)
7.63 (d, 1 H, J = 1.47, Ar-H), 7.69 (d, 1 H, J = 1.47, Ar-H)
1
355 (M+• , 2%), 353 (1%), 275 (20%), 77 (100%)
1
3460 s (NH), 2960 s (-CH3), 1640 s (-C=N-),
2.45 (s, 3 H, -CH3), 7.48 (m, 3 H, Ar-H)
1
1
610 s (arom.), 1470 s (CH3), 1260 s (-SO2-OH),
060 s (-SO2-OH)
212 (M+• , 2%), 132 (100%), 131 (75%), 77 (14%)
11 h
>300
3460 s (NH), 2960 s (-CH3), 1640 s (-C=N-),
1
2.34 (s, 6 H, -CH3), 7.46 (s, 2 H, Ar-H)
610 s (arom.), 1470 s (CH3), 1260 s (-SO2-OH), 226 (M+• , 1%), 146 (100%), 131 (65%), 118 (8%)
060 s (-SO2-OH)
1
15 a
15 b
15 c
18 a
18 b
>250
>250
>250
>250
>240
3400 s (NH), 3300 s (-COOH), 1700 s (-CO),
680 s (NHCO), 1470 s (-CH2-)
4.26 (s, 2 H, -CH2), 6.97 (m, 4 H, Ar-H), 10.80 (s, 1 H, NH)
195 (M+• , 15%), 192 (100%), 148 (68%), 147 (79%), 119 (88%)
1
3460 s (NH), 3000 m (-COOH), 1710–1690 s
CO), 1600 s (arom.), 1430 m (-CH2-)
4.02 (s, 2 H, -CH2), 6.89 (m, 3 H, Ar-H), 11.01 (s, 1 H, NH)
(
226 (M+• , 2%), 182 (3%), 167 (72%), 166 (24%), 92 (100%), 78 (50%)
3400 s (NH), 3000 m (-COOH), 1700 s (-CO),
680 (NHCO), 1480 s (-CH2-)
4.02 (s, 2 H, -CH2), 6.90 (m, 3 H, Ar-H), 11.01 (s, 1 H, NH)
1
229 (M+• , 3%), 228 (30%), 226 (88%), 182 (87%), 181 (100%), 153 (60%)
3350 s (-COOH), 2980 m (-CH2-), 1700 s (-CO),
600 s (arom.), 1480 s (-CH2-)
4.57 (s, 4 H, -CH2), 7.06 (m, 4 H, Ar-H)
1
252 (M+• , 2%), 250 (100%), 205 (44%), 162 (39%), 133 (35%)
3000 m (-COOH), 1700 s (-CO), 1600 m (arom.)
4.63 (s, 4 H, -CH2), 7.09 (d, 1 H, J = 8.30, Ar-H), 7.20
(
d, 1 H, J = 8.28, Ar-H), 7.28 (s, 1 H, Ar-H), 11.08 (s, 2 H, -COOH)
86 (M+• , 32%), 284 (100%), 239 (44%), 228 (30%), 181 (97%), 153 (50%)
————————————————————————————————————————————————————————————–
2
—
a)
For FD-mass spectra see Experimental Part
Arch. Pharm. Pharm. Med. Chem. 333, 123–129 (2000)

126
Dannhardt and Kohl
Table 4. Binding studies of benzimidazole-2-carboxylic acids 9 a–c.
NH
2
N
a)
R
R
O
O
NH
2
N
H
7
12
O
2 5
C H
N
N
b)
c)
R
O
O
1
3
O
O
Table 5. Binding studies of benzimidazole-2-sulfonic acids 11 a–h.
O
C
2
H
5
OH
N
N
d)
R
O
R
N
H
N
H
1
4
15 a-c
——————————————————————————————
Scheme 3. a) ethyl acetoacetate, xylene, KOH b) NaOEt, ethyl bromoacetate
c) diluted H2SO4 d) 2 N NaOH, room. temp.
Cpd.
IC50 [µM]
Cpd.
IC50 [µM]
3
3
vs. [ H]MDL-105,519
vs. [ H]MDL-105,519
—
—————————————————————————————
The N,N′-disubstituted derivatives 18 a and 18 b were
yielded as outlined in Scheme 4. For this purpose the o-
phenylenediamine derivative 7 was treated with N,N′-car-
bonyldiimidazole to afford compound 16, which was reacted
with potassium hydroxide in acetone followed by slow addi-
1
1
1
1
1 a
399
445
304
223
11 e
11 f
11 g
11 h
347
319
401
328
1 b
1 c
1 d
[
16]
tion of ethyl bromoacetate to obtain compound 17
.
——————————————————————————————
Saponification of 17 with 2 N sodium hydroxide followed by
acidification with concentrated hydrochloric acid gave the
crude bis-carboxylic acids (18 a, b), which were recrystal-
lized from hot ethanol.
Table 6. Binding studies of benzimidazolones 15 a–c.
O
—
—————————————————————————————
N
N
N
N
H
N
Cpd.
IC50 [µM]
vs. [ H]MDL-105,519
Cpd.
IC50 [µM]
vs. [ H]MDL-105,519
NH
2
3
3
a)
b)
R
O
R
——————————————————————————————
N
H
NH
2
15 a
15 b
>1000
>1000
>1000
18 a
18 b
no displacement
no displacement
7
16
O
O
15 c
C
2
2
H
5
O
O
OH
N
N
N
N
——————————————————————————————
c)
R
O
R
O
cording to Foster and Wong [18] using the brains of male
Sprague-Dawley rats (250–260 g body weight). The
photometric protein determination method by Hartfree [
was used to quantify the final amount of protein (250–
OH
C
H
5
19]
O
O
1
7
18 a, b
5
00 µg/ml) in the membrane preparation. The IC value of
50
[6]
Scheme 4. a) THF, 72 h, room. temp. b) KOH, acetone, 30 min reflux, ethyl
bromoactate, 1 h reflux c) 2 N NaOH, 72 h room. temp.
compound 9 a is included for purpose of comparison.
Results and Discussion
Pharmacology
All the final compounds synthesized were evaluated for
The binding affinities of compounds 9a–c, 11 a–h, 15 a–c their ability to displace [ H]MDL-105,519 to rat cortical
3
and 18a, b were determined by measuring the ability to membranes. The results are summarized in Tables 4–6.
3
displace [ H]-MDL-105,519 to rat cortical membranes ac-
The benzimidazole-2-carboxylic acids 9 a–c generally
cording to the method of Siegel et al. [ . Each value is an show considerable binding affinities in the range of 0.89 to
average of triplicates. Tissue preparation was performed ac- 38.0 (IC50 values [µM] vs. [ H]MDL-105,519). Compared to
17]
3
Arch. Pharm. Pharm. Med. Chem. 333, 123–129 (2000)

Benzimidazoles as NMDA Glycine-Site Antagonists
127
receptor
protein
receptor
protein
N
O
O
N
O
S
O
N
H
N
H
Cl
Cl
O
9
a
11 b
-
-
-
increase in bulk
affinity
solubility
positive modulatory
effect on affinity
weak interaction:
no limitation of
the affinity loss
O
O
O
O
O
O
N
N
N
H
Cl
N
H
O
Cl
6
15 c
drastically reduced
interaction with the
receptor protein
Figure 3
the standard compound 9 a, the dichlorosubstituted analogue contrast to other classes of glycine antagonists, no apprecia-
b might be considered as equipotent, whereas the nitro ble modifications in binding affinity as a result of changes in
compound 9 c leads to a 20-fold loss in receptor affinity, a substitution could be observed.
9
fact also observed in other antagonists for the glycine site.
The strong effect of minor changes in the eastern region of
The displacement studies for the benzimidazole-2-sulfonic the ligand on the binding affinity could be further demon-
acids 11 a–h indicate in a very drastic manner that carboxyl- strated by the benzimidazolone derivatives 15 a–c. Com-
replacement by the solubility enhancing sulfonic acid moiety pound 15 c showed an extremely poor affinity (IC
>
0
5
3
is not tolerated to gain affine agents in this class of com- 1000 µM vs. [ H]MDL-105,519) compared to the quinox-
3
pounds. Compounds 11 a–h were of excellent solubility even aline-2,3-dione derivative 6 (IC = 0.52 µM vs. [ H]Gly)
50
[9]
under aqueous conditions but the 240–250 fold loss in affinity evaluated by Novonordisk . This result indicates the impor-
as demonstrated for compounds 11 b and 11 d compared to tance of a second amide moiety within the heterocycle, which
their carboxylic acid analogues 9 a and 9 b makes them tools dramatically enhances the binding affinity. The glycine unit
for structure-activity studies which clearly show the tightrope in the northern region of 15 c (Figure 3) had obviously not
walk between in vitro potency and lack of potency. A possible the ability to compensate the lack of the additional amide
reason for the decreased affinity in the benzimidazole-2-sul- moiety within the heterocycle. This finding accumulates the
fonic acid series might be the increased bulk of the tetrahedral evidence already suggested in Figure 1, that the glycine unit
sulfonic acid group relative to the carboxylic group predomi- in the northern region of the quinoxalinedione derivative 6
nantly orientated into the plane. Thus an optimal fitting of the positively modulates the affinity to the receptor protein how-
sulfonic acid function into the acidic binding pocket might be ever contributing to the interaction with the glycine binding
impeded, leading to a markedly reduced physical contact site to an all in all smaller extent.
(
coulombic interactions) of the ligand with the receptor pro-
The inability of compounds 18 a and 18 b to displace
[6]
3
tein. Findings by former groups , indicating that glycine- [ H]MDL-105,519 is in line with the common pharmaco-
antagonist potency might be altered by varying substitution phore model for glycine antagonists suggesting a H-bond
patterns of the benzene ring fused to the heterocycle, led us donorgroupin the southernpositionofthe ligand. A summary
to investigate the effects of halogen and methyl substituents of the structure-activity relationships outlined above is illus-
to the 4-, 5-, and 6-positions in the sulfonic acid series. In trated in Figure 3.
Arch. Pharm. Pharm. Med. Chem. 333, 123–129 (2000)

128
Dannhardt and Kohl
evaporated under reduced pressure and the resulting residue recrystallized
from ethanol 95% using charcoal (yield 43%).
Conclusions
The binding studies indicated in a very clear manner, that
structural elements often giving rise to unfavourable physical
properties of the whole molecule as for example the carboxyl-
ate group or the bis-amide moiety, are in the case of glycineB
antagonists exactly the molecular parts, which strongly inter-
act with the receptor protein and mainly contribute to the
binding affinity. The research interest leading to a further
o-Phenylenediamine derivatives
o-Phenylenediamine derivatives, which were not commercially available
comprise the following compounds:
– 3,5-dichloro-o-phenylenediamine
–
–
3,5-dibromo-o-phenylenediamine
4-bromo-o-phenylenediamine
development of glycine antagonists concerns both the eval-
B
uation of structures suitable as diagnostic tools and the design
of therapeutically interesting compounds. The discrepancy
between solubility, affinity, and of course bioavailability to
the CNS forces medicinal chemists to search for completely
new leads and further strategies to optimize drug delivery in
a specific manner (i.e. Brain Specific Chemical Delivery
Systems (CDS) [ ).
The first two o-phenylenediamine derivatives were synthesized from the
commercially available mono-nitro compounds using the following proce-
dure: The appropriate mono-nitro compound (5 g) and powdered tin (15 g)
were added to 80 ml hydrochloric acid (18%) and boiled under reflux for 1 h.
After this time, the colour of the mixture had changed from dark yellow to
pale yellow, indicating a successful reduction. Undissolved tin was filtered
off, the resulting clear solution was diluted with 50 ml water and extracted
with diethylether (2 × 50 ml). The aqueous layer was now poured into an
excess of diluted sodium hydroxide and the resulting primary amine extracted
with diethylether (3 × 100 ml). The organic layers were combined, dried over
magnesium sulfate, filtered, and evaporated under reduced pressure. After a
short cooling procedure, the oily residue turned into a white solid (yield
60%).
20]
Acknowledgements
We wish to thank Dr. G. Quack and Dr. C. G. Parsons, Department of
Pharmacology, Merz & Co., D-60318 Frankfurt am Main, for providing the
3
binding data of the sulfonic acid series. [ H]-MDL-105,519 was a generous
4-Bromo-o-phenylenediamine was synthesized from 4-bromo-2-nitroanil-
ine, which had to be prepared according to Gardner [ and Philipps
21]
[22]
.
gift from Hoechst Marion Roussel. Financial support by Fonds der Chemi-
schen Industrie is gratefully acknowledged.
4-Bromo-2-nitro-aniline was reduced according to the above described pro-
cedure.
Experimental Part
Benzimidazole-2-sulfonic Acids 11 a–h
1
1 mmol of the corresponding benzimidazole-2-thiol was dissolved in
Chemistry
diluted sodium hydroxide, an excess of H2O2 10% added and the mixture
stirred under reflux conditions [ . After the onset of a very vigorous reac-
tion, reflux was continued for another 10 min. Cooling of the resulting
solution and acidification with concentrated hydrochloric acid to pH 1–2
caused precipitation of the product. Further purification of the crude product
was achieved by recrystallization from hot water. The crystals obtained in
the last step were dried in vacuo (yield 90%).
12]
Melting points were measured on a Büchi apparatus (Dr. Tottoli) and are
uncorrected. IR spectra (KBr) were recorded on a Beckman IR Model 4220
spectrophotometer. H NMR spectra were obtained on a Bruker AC-200 (200
1
MHz) and were consistent with proposed structures. Chemical shifts are
described in parts per million. Tetramethylsilane was used as internal stand-
ard. Coupling constants (J) are reported in hertz. Mass spectra (electron
impact) were obtained on a Varian MAT 311 A spectrometer. FD-mass
spectra were performed on a Finnigan MAT 95 FD-spectrometer (5 kV
ionisation energy). For the compounds recrystallized from water, elemental
analyses were not be obtained due to the strong tendency of these compounds
to incorporate the solvent. Thin-layer chromatography (TLC) was carried out
with E. Merck silica gel 60 F254 plates. Yields are reported in per cent from
their theoretically calculated value.
Benzimidazolones 15 a–c
Compounds15a–c werepreparedfrom theircorresponding1-isopropenyl-
,3-dihydro-1H-benzo[d]imidazol-2-one derivatives. These were synthe-
2
sized from the o-phenylenediamine derivatives with ethyl acetoacetate
according to Rossi et al. [ and Gómez-Parra et al.
13]
[14]
. The resulting
compounds were reacted with freshly prepared sodium ethoxide and ethyl
bromoacetate following a standard procedure [ . Cleavage of the iso-
propenyl moiety with diluted H2SO4 and saponification of the ethyl ester
with 2 N sodium hydroxide afforded 15 a–c (yields 45–50%).
1,1′-Thiocarbonyldiimidazole and 1,1′-carbonyldiimidazole were pur-
15]
chased from Fluka. All other chemicals were obtained from Aldrich Chemi-
cal Co. (Steinheim, Germany). For column chromatography silica gel
(
230–240 mesh ASTM) from Merck was used.
1
,3-Bis-Substituted Benzimidazole-2-ones 18 a,b
Compound 18 a was prepared according to Clark [23] and Kloetzel [16]. For
Benzimidazole-2-carboxylic Acids 9 a–c
The synthesis of compounds 9 a–c was performed as described by Lou-
[10]
the synthesis of 18 b 5-chloro-2,3-dihydro-1H-benzo[d]imidazole-2-one was
synthesized as follows:
vet
for 4,6-dichlorobenzimidazole-2-carboxylic acid.
8
0 mmol 4-chloro-o-phenylenediamine and 80 mmol 1,1′-carbonyldiimid-
Benzimidazole-2-sulfonic Acids 11 a–h
azole were dissolved in 400 ml dry tetrahydrofuran each and added dropwise
to 150 ml dry tetrahydrofuran. After the addition of both educts the reaction
mixture was stirred for 72 h at room temperature. The formation of a grey
precipitate indicated a successful reaction. Finally the precipitate was col-
lected by filtration and purified by chromatography on silica gel (eluent:
CH2Cl2/MeOH 9:1). 5-Chloro-2,3-dihydro-1H-benzo[d]imidazole-2-one
was reacted to yield 18 b as described for 18 a (yield 60%).
Route A: 75 mmol of the corresponding o-phenylenediamine derivative
and 82 mmol potassium ethyl xanthate were dissolved in a mixture of 75 ml
ethanol 95% and 11 ml water and heated under reflux conditions for 3 h [
.
11]
After adding 3 g of active charcoal the mixture was heated for another 10 min,
filtered, and diluted with 75 ml of tap water (60–70 °C). The product was
precipitated by the addition of diluted acetic acid (6.25 ml glacial acetic acid
in 12.5 ml water). Purification of the crude product was achieved by recrys-
tallization from ethanol 95% (yields 40–60%).
Pharmacology
Route B: 14 mmol 1,1′-Thiocarbonyldiimidazole and 14 mmol 4,5-di-
chloro-o-phenylenediamine were dissolved in 500 ml dry THF (freshly
All centrifugation steps for the tissue preparation were performed on a
Beckman Optima LE-80 K centrifuge using a Ti 60 rotor. D (+)-Sucrose
0
TM
distilled over K /benzophenone) each and added dropwise into 100 ml of
cooled and dry THF under an atmosphere of nitrogen. The reaction mixture
was stirred for 3 h at 0 °C followed by a 12 h stirring procedure at room temp.,
and tris(hydroxymethyl)aminomethane were purchased from Fluka. Stock
solutions were made using DMSO (Aldrich) and Tris-buffer. Serial dilutions
Arch. Pharm. Pharm. Med. Chem. 333, 123–129 (2000)

Benzimidazoles as NMDA Glycine-Site Antagonists
129
were made with Ampuwa® (Fresenius). [ H]-MDL-105,519 (74.0 Ci/mmol)
(
3
References
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®
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were obtained from Schleicher & Schuell. Scintillation liquid (Ultima
[2] W. Danysz, C. G. Parsons, M. Karcz-Kubicha, A. Schwaier, P. Popik,
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TM
Gold ) was purchased from Packard. Radioactivity was determined using
a Wallac 1409 scintillation counter and MultiCalc Routine (Laboratory
[
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[
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Tissue Preparation
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Tissue preparation was performed according to the method described by
[5] P. D. Leeson, R. W. Carling, K. W. Moore, A. M. Moseley, J. D. Smith,
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Foster and Wong . Male Sprague-Dawley rats (200–260 g) were decapi-
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homogeniser (potter-Elvehjem). The homogenate was centrifuged at
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[6] P. D. Leeson, R. Baker, R. W. Carling, N. R. Curtis, K. W. Moore, B.
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4
200 rpm for 8 min. The pellet was discarded and the supernatant centrifuged
at 16000 rpm for 30 min. The resulting pellet was resuspended in 15 ml of
ice-cold Ampuwa® and centrifuged at 10000 rpm for 30 min. Then the
supernatant and the isolated buffy coat were centrifuged at 30000 rpm for
0 min. The pellet was now resuspended in 15 ml 5 mM Tris-buffer (pH 7.4;
°C) and centrifuged at 30000 rpm for 30 min. The last step was repeated
times. Resuspension of the pellet in 5 ml 5 mM Tris-buffer (pH 7.4)
resulted the final preparation, which was frozen rapidly at –80 °C. On the
day of assay the membranes were thawed and washed with 15 ml 5 mM
Tris-buffer and centrifuged at 30000 rpm for 30 min. The membrane prepa-
ration used for the displacement studies was suspended in assay buffer
[
[
7] P. D. Leeson, L. L. Iversen, J. Med. Chem. 1994, 37, 4053–4067.
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3
4
6
[
9] A. S. Jørgensen, C. E. Stidsen, P. Faarup, F. C. Grønvald (Novo
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[
[
[
11] J. A. van Allan, B. D. Deacon, Org. Synth. 1950, 30, 56–57.
(
50 mM Tris-buffer; pH 8.0; 4 °C).
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Radioligand Binding-Studies
[
14] V. Gómez-Parra, M. Jiménez, F. Sánchez, T. Torres, Liebigs Ann.
Chem. 1989, 539–544.
Stock solutions (10 mM) were made in Tris-buffer/dimethyl sulfoxide
1:1). To improve solubility of the compounds bearing carboxylic acid
(
[15] J. Davoll, D. H. Laney, J. Chem. Soc. 1960, 314–318.
moieties, an equimolar quantity of diluted sodium hydroxide was added.
®
Serial dilutions of the stock solutions were made in Ampuwa .
[16] I. J. Pachter, M. C. Kloetzel, J. Amer. Chem Soc. 1952, 74, 1321–1322.
Incubations were performed according to the methods modified from
[
17] B. W. Siegel, B. M. Baron, B. L. Harrison, R. S. Gross, C. Hawes, P.
[17]
Siegel et al.
. Membranes were suspended and incubated in 50 mM
Towers, J. Pharmacol. Exp. Ther. 1996, 279, 62–68.
3
Tris-buffer (pH 8.0; 4 °C) for 60 min at 4 °C with a fixed [ H]-MDL-105,519
concentration of 2 nM. Non-specific binding was defined by the addition of
unlabeled glycine at 1 mM. Incubations were terminated by a filtration
procedure. The samples, all in duplicate for each concentration, were rinsed
two times with 2.5 ml ice-cold assay buffer over glass fibre filters (GF 52)
from Schleicher & Schuell under constant vacuum. Following rapid separa-
[
[
18] A. C. Foster, E. H. F. Wong, Br. J. Pharmacol. 1987, 91, 403–409.
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[22] M. A. Philipps, Soc. 1930, 2400–2401.
[
TM
tion, the filters were placed in 5 ml Ultima Gold scintillation liquid, each.
[
23] R. L. Clark, A. A. Pessolano, J. Amer. Chem. Soc. 1958, 80, 1657–1667.
Radioactivity retained on the filters was determined with a scintillation
counter.
Received: November 24, 1999 [FP439]
Arch. Pharm. Pharm. Med. Chem. 333, 123–129 (2000)