New non competitive AMPA antagonists

Bioorganic & Medicinal Chemistry 8 (2000) 2127±2143

New Non Competitive AMPA Antagonists

Gizella Abraham, Sandor Solyom,* Emese Csuzdi, Pal Berzsenyi, Istvan Ling,^y

Istvan Tarnawa,^{Tamas Hamori, Istvan Pallagi, Katalin Horvath,^x

Ferenc Andrasi, Gabor Kapus, Laszlo G. Harsing Jr., Istvan Kiraly,

Miklos Patthy and Gyula Horvath

Institute for Drug Research, Ltd, Berlini-u. 47-49, H-1045 Budapest, Hungary^k

Received 13 April 2000; accepted 24 May 2000

AbstractÐNew halogen atom substituted 2,3-benzodiazepine derivatives condensed with an azole ring on the seven membered part

of the ring system of type 3 and 4 as well as 5 and 6 were synthesized. It was found that chloro-, dichloro- and bromo-substitutions

in the benzene ring and additionally imidazole ring condensation on the diazepine ring can successfully substitute the methylene-

dioxy group in the well known molecules GYKI 52466 (1) and GYKI 53773 (2) and the 3-acetyl-4-methyl structural feature in 2,

respectively, preserving the highly active AMPA antagonist characteristic of the original molecules. From the most active com-

pounds (3b,i) 3b (GYKI 47261) was chosen for detailed investigations. 3b revealed an excellent, broad spectrum anticonvulsant

activity against seizures evoked by electroshock and dierent chemoconvulsive agents indicating a possible antiepileptic ecacy. 3b

was found to be highly active in a transient model of focal ischemia predictive of a therapeutic value in human stroke. 3b also

reversed the dopamine depleting eect of MPTP and antagonized the oxotremorine induced tremor in mice indicating a potential

Introduction

The 2,3-benzodiazepine derivative GYKI 52466 (1) was

the prototype of the noncompetitive AMPA antagonists⁴

(Fig. 1). From our structure±activity relationship study

GYKI 53773 (2, LY300164, talampanel), a highly active

AMPA antagonist emerged from the 3-acylated 3,4-dihy-

dro analogues of 1,⁵which is now one of the clinically

most advanced agents among the non competive AMPA

antagonists.⁶Further structure±activity relationship stu-

dies revealed several structural features which are

important to maintain the potent AMPA antagonistic

character of the original molecules 1 and 2.⁷We have

found that halogen atoms in the benzene ring can success-

fully substitute the dioxolane ring in 1 and 2^7dand the

biological activity was also retained when the 3-acyl-4-

methyl substitution pattern in 2 was replaced by some

nitrogen containing heterocycles attached to the 3,4-

positions of the 2,3-benzodiazepine ring system.^7bIn this

paper we report the synthesis and preliminary pharma-

cological studies of compounds 3±6, where halogen

atom(s) and condensed nitrogen containing ®ve membered

heterocycles were simultaneously applied to the 2,3-ben-

zodiazepine ring system.^7e

Excessive activation of the glutamate receptors may be a

major factor in the pathologenesis of several acute and

chronic neurological disorders.¹Attention has been

focused to identify selective antagonists according to the

types of the ionotropic glutamate receptors classi®ed

according to their exogenous ligands N-methyl-d-aspartic

acid (NMDA), 2-amino-3-(3-hydroxy-methylisoxazol-4-

yl)propionic acid (AMPA) and kainic acid.^1cSelective

AMPA antagonists have gained special importance

recently.²Among them the noncompetitive antagonists

exerting their eect at an allosteric site of the receptor are

of particular interest because of the theoretical possibility

that they are eective even at extraordinarily high levels

of the natural transmitter glutamate, as well.²Some of

the potential therapeutic targets of these antagonists

may be epilepsy, spasticity, pain, and neurodegenerative

disorders.³

*Corresponding author. Tel.:+36-1-399-3471; fax: +36-1-399-3356;

e-mail: h13759sol@ella.hu

^yRecent address: EGIS, Kereszutuvi ut 30-38, Budapest, Hungary.

^{Gedeon Richter Ltd, Gyomroi-u. 19-21, H-1103 Budapest, Hungary.

Chemistry Results

^xPharmaceutical Control and Development Laboratory, Mexikoi u

Â t 9,

H-1149 Budapest, Hungary.

One of the key steps in the synthesis of new 2,3-benzo-

diazepines with halogen atoms in the benzene ring is the

^kA subsidiary of IVAX Corporation, Miami, Florida, USA.

PII: S0968-0896(00)00133-4

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G. Abraham et al. / Bioorg. Med. Chem. 8 (2000) 2127±2143

2128

Figure 1.

formation of isochromanes 9a±d from the correspond-

ing alcohols and an aldehyde (Scheme 1). While the

same reaction can be easily performed in the case of alco-

hols with electron donating groups, like, e.g. with 2-(3,4-

dimethoxyphenyl)- or 2-(3,4-methylenedioxyphenyl)eth-

anol or the corresponding isopropanols and 4-nitrobenz-

aldehyde using only slightly more than one equivalent of

concd hydrochloric acid,⁸the analogous reactions with

7a±c need anhydrous conditions in benzene using

freshly molten zinc chloride and dry hydrogen chloride

gas and even then the yields are moderate. But on the

other hand the enhanced electrophilicity of the reacting

benzaldehyde derivative must contribute to the success

of the reaction as well, since, e.g. under the same reaction

conditions only negligible reaction could be noticed

between 7a and unsubstituted benzaldehyde. It was also

found e.g. with the less electrophilic 8b that apart from

the unknown steric contribution of the ortho substituent

to the reaction the corresponding isochromane 9d

formed with signi®cantly lower yield than the analogous

9b in the reaction between 7b and 8a.

have chosen the corresponding thiooxo derivatives 12a±d,

which were prepared from 11a±d by phosphorous penta-

sul®de in dry pyridine with acceptable yields.⁹The latter

were reacted further with the known aminoacetals 13a±g.

This condensation step was most successful when the

reactants were heated in 2-methoxyethanol using red

mercury oxide as the sulfur binding agent. The resulting

intermediates of type 14 were generally puri®ed by column

chromatography and then further reacted with hydro-

chloric acid to give the imidazolo compounds 15a±g and

15i±n as well as 3p,q, respectively. The nitro groups in

benzodiazepines 15a±n were reduced by a standard method

using hydrazine hydrate and RaNi to give products 3a±n.¹⁰

Because of solubility considerations the reductions were

carried out generally in a mixture of methanol and di-

chloromethane. As imidazolo-benzodiazepines with

unsubstituted phenyl ring can hardly be synthesized by

the above route, because of the diculties concerning

the corresponding isochromane formation, this type of

compound was prepared by removal of the amino

group, e.g. in 3c by reaction with isoamyl nitrite and 5n

hydrochloric acid to provide 3r.¹¹In one instance the

aromatic amino group of 3b was acetylated to give 3o as

one of the potential metabolites. The prepared imidazolo

compounds are listed in Table 1.

The reaction of isochromanes 9a±d with Jones reagent in

acetone provided ketocarboxylic acids 10a±d. The latter

were reacted with excess hydrazine hydrate in ethanol to

give the intermediate hydrazones which were treated in

turn with dicyclohexylcarbodiimide to achieve ring clo-

sure. Another possibility to induce ring closing reaction

was the treatment of the intermediate hydrazone with

excess hydrochloric acid for longer time to provide the 4-

oxo-2,3-benzodiazepine derivative, e.g. 11a.

When 3b as one of the biologically most interesting

compounds had to be prepared in multigram scale we

became interested to avoid some disadvantages of the

synthetic route outlined in Scheme 1. These were the 5

step synthesis of the acetal 13b from alanine and the

optional but useful column chromatography of the

intermediates of type 14. It was attractive to make use

of a relatively scarcely used 4+1 cyclization method to

prepare the imidazole ring, where an acylamidoketone is

In order to enhance the reactivity of the 4-oxo compounds

11a±d towards condensation reactions to build up the

expected imidazolo- or triazolo-2,3-benzodiazepines, we

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G. Abraham et al. / Bioorg. Med. Chem. 8 (2000) 2127±2143

Â

2129

ꢀ

.

Scheme 1. Route I. (a) benzene, ZnCl₂, HCl(g); (b) acetone, CrO₃/H₂SO₄; (c) EtOH, H₂NNH₂H₂O, Á; (d) DCC or HCl; (e) pyridine, P₂S₅, 80_ꢀC;

ꢀ

.

(f) 2-methoxyethanol, red HgO, Á; (g) HCl, Á; (h) MeOH-CH₂CL₂, RaNi, H₂NNH₂H₂O; (i) DMF, isoamylnitrite, 65 C; (j) pyridine/AcCl, 5 C.

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G. Abraham et al. / Bioorg. Med. Chem. 8 (2000) 2127±2143

2130

yield of this condensation step to the sensitive nature of

the acetone side chain in 16 towards reactions, since

with the phenacyl derivative 17 the same condensation

could be performed with a signi®cantly better yield

(82%). (Scheme 2.)

For the formation of the triazolo ring in compounds 4a±d

acylhydrazides were used (Scheme 3).¹³Better results

could be achieved in the condensation steps when instead

of thione 12a the corresponding 4-methylthio-2,3-ben-

zodiazepine 18 was used and additionally a catalytic

amount of hydrochloric acid was applied, as well. The

nitro groups in compounds 19a±d were reduced by

the standard RaNi-hydrazine hydrate method and the

resulting compounds 4a±d are shown in Table 2.

To build up the 11H-imidazolo[3,4-c][2,3]benzodiazepine

ring system in 5 we started from 23 which was synthesized

on the analogy to well established methods (Scheme 4).¹⁴

The methyl group in 23 was oxidized by selenium diox-

ide to form the corresponding aldehyde 24, which was

then reduced to the alcohol 25 by sodium borohydride. The

Scheme 2. Route II. (a) DMF, K₂CO₃; (b) NH₄OAc, AcOH, Á.

reacted with ammonium acetate as nitrogen atom donor

in acetic acid.¹²The necessary amidoketone 16 was pre-

pared in very good yield by alkylation of 11a with chlor-

oacetone (Scheme 2). We have found that condensation

of 16 to give the corresponding imidazolo-benzodiaze-

pine 15b needs a large excess (>50 equivalents) of

ammonium acetate in acetic acid and the yield is modest

(40%). The use of other ammonium salts like formate or

carbonate as well as solvents, e.g. formamide, propionic

acid, tri¯uoroacetic acid or microwave techniques

instead of conventional heating did not give signi®cant

improvement. Despite the lower yield of the imidazole

forming step, 3b can be produced by this route without

any laborious synthetic steps. We attribute the lower

Table 1. 11H-Imidazolo[1,2-c][2,3]benzodiazepine derivatives (3a±r)

Compound X Y R¹

R²

R³

R⁴

mp

(^ꢀC)

Yield

(%)^a

3a

3b

3c

3d

3e

3f

3g

3h

3i

3j

3k

3l

3m

3n

3o

3p

3q

3r

H Cl

H Cl Me

H Cl

H Cl Me

H Cl Et

H

Me

H

NH₂

210±214 67

229±230 79

267±270 71

274±278 83

247±250 72

250±253 84

293±294^b68

223±226 81

254±255 40

284±286 71

248±251 64

263±268 77

272±275 84

295±300^b41

H

H Cl

H

4-NH₂-C₆H₄Ð H

4-pyridyl

H

Scheme 3. (a) acetone, MeJ, K₂CO₃; (b) DMF, acylhydrazine, cat.

.

HCl; (c) MeOH-CH₂Cl₂, RaNi, H₂NNH₂H₂O.

H Cl Ph

Cl Cl Me

H

Me

H

Me

4-pyridyl

H

Cl Cl

H Br Me

H Br

H Br Me

H Br

H

Table 2. 6-(4-Aminophenyl)-8-chloro-11H-1,2,4-triazolo[4,5-c][2,3]

benzodiazepine derivatives (4a±d)

H

Compound

R

mp (^ꢀC)

Yield (%)^a

H Cl Me

H Br Me

H NHCOMe 265±266 63

H

Me

Cl

H

132±140^c21

210±215^c48

166±169 39

H Br

H Cl

H

4a

4b

4c

4d

Me

4-pyridyl

4-NH₂-C₆H₄Ð

CH₃OCH₂Ð

228±231

284±288

191±193

195±197

91

92

85

83

^aYields refer to the last step of the synthesis.

^bDecomposition.

^cHydrochloride salts.

^aYields refer to the last step of the synthesis.

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G. Abraham et al. / Bioorg. Med. Chem. 8 (2000) 2127±2143

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.

Scheme 4. (a) 4-nitrobenzaldehyde, benzene, ZnCl₂, HCl(g); (b) acetone, CrO₃/H₂SO₄, then 70% HClO₄; (c) DMF, H₂NNH₂H₂O; (d) dioxane,

.

SeO₂; (e) THF-H₂O, NaBH₄; (f) THF, Ph₃P, phthalimid, DEAD; (g) MeOH, H₂NNH₂H₂O; (h) Ac₂O; (i) ClCH₂CH₂Cl, POCl₃, Á; (j) MeOH-

.

CH₂Cl₂, RaNi, H₂NNH₂H₂O.

latter was transformed under Mitsunobu conditions¹⁵with

phthalimide into 26 then hydrazinolysis and acetylation

gave 27 and 28, respectively. Cyclization of 28 with phos-

phorus oxychloride gave 29 which was reduced by RaNi-

hydrazine hydrate to give 5.

The pyrrolo derivative 6 was prepared from 23 by using a

combined alkylation-condensation procedure¹⁶with ethyl

bromopyruvate to give the nitro compound 30 which on

reduction provided 6 (Scheme 5).

Scheme 5. (a) Br-CH₂-C(O)-CO₂Et, EtOH, Á; (b) MeOH-CH₂Cl₂,

.

RaNi, H₂NNH₂H₂O.

Biological Results and Discussion

Primary pharmacological testing and structure±activity

relationships

Inhibition of AMPA-, or kainate-triggered spreading de-

pression in isolated chicken retina. Excitation of isolated

chicken retinas by glutamate receptor agonists provokes

spreading depression (SD), which is accompanied by a

characteristic change of the light scattering properties of

the preparation. The phenomenon is easily visible by

unaided eye, thus it is a convenient method for drug

Table 3 contains primary in vitro and in vivo biological

data with the newly synthesized compounds in comparison

with the standard molecules 1 (GYKI 52466) and 2

(GYKI 53773, LY300164, talampanel).

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G. Abraham et al. / Bioorg. Med. Chem. 8 (2000) 2127±2143

2132

Table 3. Screening results of the condensed 2,3-benzodiazepine derivatives 3±6^a

Compond

Behavioural changes

(100 mg/kg ip; 200 mg/kg po)

Retinal spreading depr.

IC₅₀(mM)

MES Inc. screen

ED₅₀, mg/kg po ED₅₀, mg/kg ip

1

2

Loss of righting re¯ex

Short loss of righting re¯ex

Loss of righting re¯ex

Ataxia, muscle relaxation, po: loss of righting re¯ex

Ataxia

A: 6.3 K: 9.5

A: 1.7 K: 2.6

A: 6.5 K: 3.8

37.4

8.6

61.6

24.0

47.1

13.4

47.1

36.5

100-125

>200

>150

ꢁ200

>200

47.2

3a

3b

3c

3d

3e

3f

3g

3h

3i

A: 7.3 K: 2.5

A: 4.1 K: 0.5 (¯at curve)

A: 11.8 K: 1.5

61.3

ꢁ100

Loss of righting re¯ex, ataxia, SMA#

A: >20 K: >20

A: 4.3 K: 7.1

A: 3.1 K: n.t.

A: >20 K: n.t.

A: 3.1 K: 1.9

44.6

SMA#, ataxia

>100

24.0

50±100

40.3

ꢁ100

>100

56.0

>100

ꢁ100

>100

ꢁ100

>100

SMA#, ataxia

1

Loss of righting re¯ex

po: SMA#, ataxia

Loss of righting re¯ex

SMA#, ataxia, weak muscle relaxation

SMA#, ataxia

3j

3k

3l

A: >20 (0.6±10 mM: max. 47% inhibition) K: 1.1

A: 9.3 K: 2.2

A: 4.0 K: 1.3

>200

68.4

ꢁ200

>200

ꢁ200

>200

ꢁ100

>200

ꢁ150

>200

ꢁ200

3m

3n

3o

3p

3q

3r

4a

4b

4c

4d

5

A: >20 K: 7.4

A: 3.2 K: >20

po: SMA#

SMA#, ataxia

A: >20 K: >20

A: 2.9 K: 4.1

ip: Weak SMA#

ip: SMA#

ip: Weak SMA#

Ataxia, weak muscle relaxation

1

A: >20 K: ꢁ20

1

A: >20 K: 1.0

A: 9.2 K: 5±10

SMA#, ataxia

ip: SMA#, ataxia

A: >20 K: n.t.

6

100 ip.: 1

^aSMA#: decrease of spontaneous motor activity; A: AMPA; K: kainate; n.t.: not tested.

testing. Retinal SD can be blocked by glutamate

antagonists.¹⁷In our study the majority of the tested

compounds were eective in the retinal spreading

depression (SD) test, i.e. blocked either AMPA- or kai-

nate induced SD with an IC₅₀lower than 10 mM. The

IC₅₀values of the most potent compounds were similar

to those of 1 or 2 in the AMPA test, and lower in the

kainate test. Compounds 3b,d,j,k,l,m seemed to show

some selectivity towards kainate responses, i.e. their IC₅₀

values were more than 2-fold higher against AMPA

than against kainate. The concentration-response curves

of 3b against the two agonists are shown in Fig 2.

Interestingly, some compounds such as 3e, 3m, or 4d

caused only partial inhibition of kainate-triggered SD

(maximal inhibition: 40±60% at 20mM; not shown), or had

a ¯at dose-response curve (3a, 3c, or 3i; not illustrated).

The signi®cance of the dierences between AMPA and

kainate antagonistic actions in the case of some com-

pounds is dicult to tell, as the role of the speci®c kainate

receptors in triggering SD in this preparation has not been

characterized yet. It is to be noted here that the competitive

AMPA antagonist 1,2,3,4-terahydro-6-nitro-2,3-dioxo-

benzo[f]quinoxaline-7-sulfonamide (NBQX) also had a

¯at dose-response curve against kainate in this model.¹⁷

Regarding the dose-response curves in the AMPA-

induced spreading depression test, the curves were usually

steeper than in the kainate test, except for 3j which only

partially depressed AMPA response (maximum inhibition:

45% at 2.5 mM).

Behavioral changes, eects of the compounds in the max-

imal electroshock (MES) test, and in the inclined screen

test. Most compounds elicited some gross behavioral

eects after ip (100 mg/kg) or po (200 mg/kg) applica-

tion, indicating a reasonable absorption and penetration

through the blood brain barrier. These included a

decrease in motility, ataxia, muscle weakness and even a

loss of righting re¯ex with the most eective substances.

These behavioral symptoms have also been observed

with other 2,3-benzodiazepine compounds^5aand can be

regarded as the consequence of the blockade of central

AMPA receptors function.

AMPA receptor blockade also results in an antic-

onvulsant action.^2,5aMES test has been found a suitable

method for establishing in vivo structure±activity rela-

tionships among another group of 2,3-benzodiazepine

Fig. 2. Inhibition of kainate- and AMPA induced spreading depres-

sion in isolated chick retina by 3b. & 5 mm AMPA; ^ 5 mm kainate.

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G. Abraham et al. / Bioorg. Med. Chem. 8 (2000) 2127±2143

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2133

AMPA antagonists.^5aThe two most eective compounds

of the present series were 3b and 3i, both with an ED₅₀

of 24 mg/kg po, which is between the ED₅₀values of 1

and 2.

pretreatment was applied. Similar metabolic activation

was described among other 2,3-benzodiazepine AMPA

antagonists.^5a

Similarly to earlier 2,3-benzodiazepine type AMPA

antagonists^5a,7the ecacy of the present compounds is

also generally restricted to the 4-aminophenyl derivatives

(see e.g. the dezamino compounds 3p,q,r), and even an

acetylation of the amino group (e.g. in 3o) decreases

biological activity.

The ataxia inducing or muscle relaxant action was

quanti®ed in the inclined screen assay. The ED₅₀values

of the compounds in this test (30 min ip pretreatment)

were in general slightly higher than in the MES test (60

min oral pretreatment). A bigger gap between the

anticonvulsant and motor impairment inducing doses

would be expected if identical treatment schedules were

used. This MES preference does not hold true in respect

of 3a, which was more potent in the muscle relaxant

assay. A possible explanation for this discrepancy is that

the compound seems to have an extremely short duration

of action (probably due to a rapid metabolism in mice),

and 60 min after the treatment its blood concentration

decreases to a level that can not protect the animal from

MES. Another possible explanation is thatÐin contrast

to most of the 2,3-benzodiazepines³Ð3a has a poor

absorption from the intestines.

On the basis of the screening results 3b and in certain

cases additionally 3i were chosen for more detailed and

comparative investigations.

Inhibition of AMPA-, or kainate-induced whole-cell cur-

rents by 3b in isolated neurons. The AMPA receptor

antagonistic eect of 3b in freshly isolated cerebellar Pur-

kinje cells is shown in Fig. 3. In this preparation 3b is

nearly equiactive with 2 and more potent than 1. Its IC₅₀

against AMPA (2.3 mM) was somewhat lower than

against kainate (4.5mM, not shown). However, under our

experimental conditions most current elicited by kainate

was probably mediated by AMPA receptors. Thus these

data are not suitable for analyzing a possible subtype

selectivity of the compound. The current blocking

action of 3b (10 mM) against 5 mM AMPA developed

slowly and the recovery from the blockade took several

seconds (t_onand t_oof the current trajectories were

4.2Æ0.8 and 5.2Æ0.7 sec, respectively), suggesting con-

siderably slower binding and unbinding kinetics than

those of 1 and the racemate of 2 (t_onand t_ovalues

below 1 sec).¹⁸To assess the biological signi®cance of

this feature of 3b warrants further studies. Just as 1 or

2,^18,193b acted in a non-competitive way, as its current

blocking action was not dependent on the agonist con-

centration (not shown).

Correlating the in vitro and in vivo pharmacological

data with the structural features of the new compounds

the following relationships can be established. Applying

chloro-, dichloro- or bromo-substitution instead of the

methylendioxy group of molecules 1 and 2 and substituting

simultaneously the hydrogen bond acceptor type acetyl

group of 2 by a condensed nitrogen containing ®ve mem-

bered heterocycle preserve the high AMPA antagonist

character of the original molecules. Even among the

bromo compounds we found a compound (3k) with sig-

ni®cant activities.

Concerning the condensed heterocycles optimum ecacy

was found among the imidazolo[1,2-c][2,3]benzodiaze-

pines of type 3. Although most of the triazolo-derivatives

4a±d showed acceptable in vitro ecacy, the additional

hydrogen bond acceptor atom in the condensed ring may

have prevented a proper absorption resulting in an in

vivo inactivity. With pyrrolo-derivative 6, where no

hydrogen bond possibility exists in a similar way as it

may occur for molecules 3, 4 and 2 or where the acceptor

nitrogen atom is probably at a wrong place (e.g. imida-

zolo[3,4-c][3,4]benzodiazepine derivative 5) neither in

vitro nor in vivo eects were found.

Anticonvulsant pro®le of selected 2,3-benzodiazepines.

The broad-spectrum anticonvulsant activity of 1 and 2

has already been described.^3,5aTable 4 illustrates that

the anticonvulsant pro®le of 3b and 3i is similar to the

former compounds. Their potencies were usually

between those of 1 and 2. It is notable, however, that

both newly synthesized compounds protected mice from

Within the imidazolo-derivatives 3 the optimum ecacy

was found among the compounds substituted with little

aliphatic groups (3b,c,e,i,k). Bigger aryl substitutions

resulted in inactive or nearly inactive compounds, e.g.

3f,g,h,n, whereas methyl substitution in position 3 seems

to enhance kainate selectivity, e.g. 3c,d,j,l. But double

methyl substitution, e.g. in 3d and 3m, resulted in loss of

in vivo activity, which may be explained by a worsened

ADME pro®le of these derivatives. It is worth mentioning

that 3e, a homologue of one of the most active sub-

stances (3b), is inactive in vitro but still has a reasonable

anticonvulsant activity. Formation of biologically active

metabolites may underlie this discrepancy, which is

supported by the observation that the compound was

ineective in the inclined screen assay, where a shorter ip

Fig. 3. Inhibition of AMPA induced whole cell current in freshly iso-

lated cerebellar Purkinje cells by 3b.

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G. Abraham et al. / Bioorg. Med. Chem. 8 (2000) 2127±2143

2134

Table 4. Anticonvulsive eects of selected compounds (3b and 3i) against MES and various chemical convulsants

ED₅₀mg/kg po

1

2

3b

3i

MES

Metrazole

Strychnine

Bemegride

Bicuculline

Nicotine

4-aminopyridine

3-mercapto-propionic acid

37.4 (29.2±47.5)

119.8 (108.5±132.3)

86.7 (71.7±104.9)

71.9 (62.0±83.4)

35.0 (28.5±43.1)

71.8 (49.8±103.7)

43.0 (39.9±59.8)

47.0 (32.9±67.2)

8.6 (7.0±10.6)

16.8 (10.2±27.6)

17.4 (10.6±28.4)

23.9 (16.0±35.7)

14.6 (5.8±36.7)

22.7 (16.2±31.9)

8.4 (5.6±12.5)

24.0 (17.9±32.1)

53.8 (43.7±66.2)

67.4 (43.7±66.2)

40.7(21.9±56.9)

22.8 (16.7±31.2)

13.1 (8.1±21.2)

55.5 (47.1±65.4)

38.1 (28.7±50.5)

24.0 (17.9±32.1)

62.2 (44.1±87.6)

44.2 (38.5±50.7)

39.9 (26.5±60.1)

22.5 (13.4±37.7)

18.3 (14.2±23.8)

6.7 (3.5±12.7)

17.1 (9.7±30.1)

66.8 (43.0±103.1)

nicotine-induced seizures and death more potently than

2 did, which may indicate a potential antiparkinsonian

ecacy. Further, 3i was the most eective of the four

compounds in preventing 4-aminopiridine (4-AP)-induced

seizures.

Table 6. Eect of 3b on the size of ischemic damage in rats after

transient occlusion of the medial cerebral artery

Comp.

Doses

(mg/kg iv)

Infarct size scores

(mean Æ S.E.)

Decrease

(%)

Veh.^a

1

48920Æ4012

32218Æ6325*

40518Æ5928

13229Æ2313*

42795Æ7513

16122Æ4368*

6Â5

6Â2

6Â1

34.1

67.4

62.3

Muscle relaxant eect of selected 2,3-benzodiazepines. A

common feature among 2,3-benzodiazepine AMPA

antagonists is the inhibition of motor functions with a

primarily spinal site of action.^3,20The ataxia/muscle

relaxation inducing ED₅₀values of 3b and 3i are listed

and compared to those of 1 and 2 in the inclined screen

and rotarod tests (Table 5).

Veh.^a

3b

Veh.^a

3b

^aVehicle. * P<0.01 Signi®cance was calculated by one-way ANOVA

followed by the Duncan test. Treatment schedule: ®rst injection 30

min after occlusion and repeated 5 times in every 30 min.

Antiischemic eect of 3b in a transient focal ischemia

model, in rats. The antiischemic property of AMPA

antagonists renders them promising drug candidates in

various acute and chronic neurodegenerative disorders

such as stroke.^{1 3}Compound 1 has been shown to pro-

tect animals from ischemic damage in various models.²¹

The transient focal ischemia model is a relevant stroke

model.^22,23Table 6 shows that 3b, applied at 2.5±5-fold

lower doses, had a considerably more potent eect on

infarct size than 1.

Table 7. Antagonism of oxotremorine induced tremor in mice

Oxotremorine antagonism ED₅₀mg/kg po

1

2

3b

20.5 (14.9±28.3)

5.6 (3.6±8.5)

16.8 (12.0±23.6)

.

N-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine HCl

(MPTP) neurotoxicity. Degeneration of dopaminergic

neurons in Parkinsons disease is well known.²⁴Recent

®ndings indicate that impairment of striatal dopami-

nergic neurotransmission results in an overactivity of

glutamatergic neurons in the subthalamic nucleus.²⁵

This glutamatergic overactivity is thought to play an

important role in the expression of some parkinsonian

symptoms, such as hypokinesia and rigidity as well as in

development of neuronal damage.²⁶Therefore we tested

the eects of 3b against MPTP-induced neurotoxicity in

mice, a relevant rodent model of Parkinsons disease, in

comparison with those of 1 and of the competitive

AMPA antagonist NBQX.

The antiparkinsonian eects of 3b

Oxotremorine induced tremor. The cholinergic agonist

oxotremorine, applied systemically, provokes tremor

with a central action. Table 7 shows that the 2,3-benzo-

diazepine compounds 1, 2 and 3b potently mitigated the

tremor induced by oxotremorine. Salivation, a peripheral

eect of oxotremorine, was not aected by these drugs.

Since Parkinson's disease is characterized by a disturbed

balance between cholinergic and dopaminergic neuro-

transmission in the basal ganglia, an indirect central

anticholinergic eect of compounds 1, 2 and 3b may be

indicative for a therapeutically suitable antiparkinsonian

eect.

Table 5. Muscle relaxant eects of selected compounds (3b and 3i) in the inclined screen and rotarod tests, in mice

ED₅₀mg/kg ip

Method

1

2

3b

3i

Inclined screen

Rotarod

47.1 (44.2±50.2)

24.0 (22.0±26.2)

13.4 (11.2±16.0)

2.3 (1.6±3.4)

36.5 (29.4±45.2)

15.8 (13.0±19.3)

47.2 (41.9±53.3)

13.7 (12.4±15.1)

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G. Abraham et al. / Bioorg. Med. Chem. 8 (2000) 2127±2143

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2135

Table 8. Eects of 3b on MPTP induced depletion of dopamine and its metabolites in mouse striatum^a

Treatment

Concentration (mg/g) of

1

0 min.

2

30 min.

3

150 min.

4

270 min.

5

390 min.

DA

DOPAC

HVA

Vehicle

MPTP

Vehicle

3b

1

NBQX

Vehicle

3b

1

NBQX

Vehicle

1

Vehicle

3b

Vehicle

11.14Æ0.43

1.07Æ0.17^b

4.38Æ0.89^c

3.60Æ0.37^c

2.74Æ0.52^d

0.54Æ0.03

0.10Æ0.04^b

0.21Æ0.05

0.27Æ0.03^d

0.16Æ0.04

0.87Æ0.05

0.33Æ0.06^b

0.45Æ0.06

0.36Æ0.03

0.37Æ0.03

NBQX

^aSigni®cance was calculated by one-way ANOVA followed by the Duncan's test.

^b<0.01 versus saline treated control group.

^cP<0.01 versus MPTP treated group.

^dP<0.05 versus MPTP treated group.

Table 9. Determination of acute toxicity in mice

spectrum anticonvulsant activity against seizures evoked

by electroshock and dierent chemoconvulsive agents

indicating a possible antiepileptic ecacy. 3b (GYKI

47261) was also found to be highly active in a transient

model of focal ischemia predictive of a therapeutic value

in human stroke. A signi®cant eect antagonizing the

MCAO induced infarct size in rat brain was found in a

®ve times lower dose than for GYKI 52466 (1), the repre-

sentative of the non competitive AMPA antagonists. In

addition 3b reversed the dopamine depleting eect of

MPTP and antagonized the oxotremorine induced tremor

in mice. These ®ndings indicate that the non-competitive

AMPA antagonist 3b may have therapeutical potential

not only in acute but chronic neurodegenerative disorders

as well.²⁷

LD₅₀mg/kg

Compound

ip

po

1

3b

302.1 (273.9±333.3)

259.4 (205.8±327.1)

296.9 (258.6±340.8)

372.9 (322.5±431.1)

The eects of 3Â20 mg/kg (ip dose) of 3b, 1 and NBQX

on MPTP-induced dopamine (DA) depletion in mouse

striatum are shown in Table 8. MPTP injection reduced

DA concentration in the striatum by 90% and the levels of

DA metabolites, dihydroxyphenylacetic acid (DOPAC)

and homovanillic acid, (HVA) were also decreased sig-

ni®cantly (Table 8). MPTP administration did not aect

the concentrations of serotonin and 5-hydroxyindoleacetic

acid (5-HIAA) in the striatum indicating the speci®c toxic

eect of MPTP on dopaminergic neurons (data not

shown). 3b and 1, when they were injected repeatedly,

reversed the MPTP-induced decrease in striatal con-

centrations and NBQX exhibited similar eect (Table 8).

The neuroprotective eects of 2,3-benzodiazepines against

MPTP neurotoxicity suggest that non-competitive

AMPA receptor antagonists may have a bene®cial role

in the treatment of Parkinson's disease.^25,26

Experimental

Chemistry

Melting points were measured on a Boetius hotstage

microscope and are uncorrected. IR (KBr): Bruker IFS-85

1

spectrophotometer. HNMR (CDCl₃, internal standard

TMS,T=298^ꢀK): Bruker AC 250, other solvents are

indicated. Mass spectra: Finnigan MAT 8430 mass

spectrometer. Operating conditions: electron ionization,

E_el=70eV, I_el=0.5 mA, U_acc=3 kV, R=1250. FAB:

ION-TECH atom gun with Xe, in m-nitrobenzyl alcohol

matrix. Column chromatography: silica gel, Kieselgel 60,

Merck. Satisfactory elemental analyses (Æ0.4 %) for C, H,

N and S were obtained for all new described compounds

except for 25±29 which were not measured this way. The

following compounds were prepared according to the lit-

erature: 7a,²⁸7b,²⁸7c,²⁹13a,³⁰13b,³¹13c,³⁰13d,³⁰13e,³¹

13f,³²13g.³²

Acute toxicity. Compound 3b caused lethality in mice

only at doses several fold higher than its ED₅₀values in

the pharmacological assays. Its toxicity is similar to that

of 1. The LD₅₀values of 3b and 1 were almost the same

after ip and oral administrations suggesting a good

gastro-intestinal absorption (Table 9).

Conclusion

Among the newly synthesized, with heterocycle condensed

and halogen substituted 2,3-benzodiazepine derivatives

several non-competitive AMPA antagonists were identi-

®ed with potencies that approached or exceeded those of

the reference compounds 1 or 2. Some of them also had

potent in vivo activity. 3b, the compound selected for

detailed studies, displayed a pharmacological pro®le

largely similar to that of the formerly described 2,3-

benzodiazepine AMPA antagonists,³but a potent anti-

parkinsonian activity was also revealed as an additional

feature. The compound showed an excellent, broad

General procedure for the synthesis of isochromanes 9a±d.

To a stirred solution of the phenylethanol derivative

(7a±c) (0.1 mol) and the corresponding benzaldehyde

8a,b (0.1 mol) in dry benzene (300 mL) freshly molten

and ground zinc chloride (13.6 g, 0.1 mol) was given.

Dry hydrogen chloride gas was introduced into the sus-

pension over 4 h and the mixture was stirred overnight.

The resulting mixture was then washed with water and a

5% sodium-hydrogensul®t solution. The organic phase

was dried and after ®ltration the solvent evaporated.

The residue was recrystallized to give the title products.

Â

G. Abraham et al. / Bioorg. Med. Chem. 8 (2000) 2127±2143

2136

7-Chloro-1-(4-nitrophenyl)isochromane (9a). Yield: 56%.

Mp 98±101^ꢀC (ethanol). C₁₅H₁₂ClNO₃(289.7). ¹H

NMR: d 2.79 (ddd, J₁=16.5 Hz, J₂=J₃=3.6 Hz, 4-H)

and 3.12 (ddd, J₁=16.5 Hz, J₂=9.7 Hz, J₃=5.6 Hz, 4-H),

3.92 (ddd, J₁=11.5 Hz, J₂=9.7 Hz, J₃=3.6Hz, 3-H) and

4.21 (ddd, J₁=16.5 Hz, J₂=5.6 Hz, J₃=3.6Hz, 3-H), 5.75

(s, 1H, 1-H), 6.65 (d, J=1.9Hz, 1H, 8-H), 7.13 (d,

J=8.3Hz, 1H, 5-H), 7.17 (dd, J₁=8.3 Hz, J₂=1.9Hz,

1H, 6-H), 7.50 (d, J=8.8 Hz, 2H) and 8.25 (d, J=8.8Hz,

2H, nitrophenyl).

J₂=2.0 Hz, 1H, 5-H), 7.95 (d, J=8.9 Hz, 2H) and 8.38

(d, J=8.9 Hz, 2H, nitrobenzoyl), 12.1±12.7 (1H).

4,5-Dichloro-2-(4-nitrobenzoyl)phenylacetic acid (10c).

The crude product prepared by the evaporation of its

solution in acetone was recrystallized from 96% acetic

acid and further puri®ed by column chromatography.

Eluent: chloroform:methanol (9:1). Yield: 54 %. Mp 187±

190 ^ꢀC. C₁₅H₉Cl₂NO₅(354.1). H NMR: (DMSO-d₆): d

1

3.90 (s, 2H, CH₂), 7.68 (s, 1H), 7.85 (s, 1H), 7.97 (d,

J=8.7Hz, 2H) and 8.38 (d, J=8.7 Hz, 2H, nitrobenzoyl).

7-Bromo-1-(4-nitrophenyl)isochromane (9b). Yield: 62%.

Mp 104±107 ^ꢀC (ethyl acetate). C₁₅H₁₂BrNO₃(334.2). ¹H

NMR: d 2.78 (ddd, J₁=16.2 Hz, J₂=J₃=3.6 Hz, 4-H)

and 3.12 (ddd, J₁=16.2 Hz, J₂=9.9 Hz, J₃=5.8 Hz, 4-H),

3.93 (ddd, J₁=13.5 Hz, J₂=9.9 Hz, J₃=3.6Hz, 3-H) and

4.21 (ddd, J₁=13.5 Hz, J₂=5.8 Hz, J₃=3.6Hz, 3-H), 5.78

(s, 1-H), 6.82 (d, J=1.9 Hz, 8-H), 7.10 (d, J=8.2 Hz, 5-H),

7.35 (dd, J₁=8.2Hz, J₂=1.9Hz, 6-H), 7.55 (d, J=8.8Hz,

2H) and 8.22 (d, J=8.8 Hz, 2H, nitrophenyl).

4-Bromo-2-(2-chlorobenzoyl)phenylacetic acid (10d).

Yield: 47%. Mp 135±138 ^ꢀC. C₁₅H₁₀BrClO₃(353.6). H

NMR: (DMSO-d₆): 3.90 (s, 2H, CH₂), 7.37 (d, J=2.2Hz,

1H, 3-H), 7.43 (d, J=8.1Hz, 1H, 6-H), 7.78 (dd,

J₁=8.1 Hz, J₂=2.2 Hz, 1H, 5-H), 7.47 (m, 2H) and 7.60

(m, 2H, chlorophenyl), 12.40 (br. s).

1

8-Chloro-1-(4-nitrophenyl)-3,5-dihydro-4H-2,3-benzodiaz-

epin-4-one (11a). Method A. A solution of 10a (17.6 g,

55.0 mmol) and 85% hydrazine hydrate (8 mL) in etha-

nol (340 mL) was heated at re¯ux for 4 h. The mixture

was then chilled, treated with 1N hydrochloric acid

(115 mL) and the solvent was evaporated. The residue was

treated with water (50 mL) and the precipitate ®ltered and

dried. This intermediate hydrazone was dissolved in di-

chloromethane (300mL) and treated with a solution of

dicyclohexylcarbodiimide (13.4 g 65.0 mmol) in dichloro-

methane (210mL). The mixture was stirred overnight at rt

and the resulting precipitate was ®ltered and washed

with dichloromethane to give 11a (12.5 g). Yield: 72 %.

Mp 275±278 ^ꢀC.

7,8-Dichloro-1-(4-nitrophenyl)isochromane (9c). Yield:

30%. Mp 130±132^ꢀC (ethanol). C₁₅H₁₁Cl₂NO₃(324.1). ¹H

NMR: d 2.80 (ddd, J₁=16.9 Hz, J₂=J₃=3.9 Hz, 4-H) and

3.13 (ddd, J₁=16.9 Hz, J₂=10.0 Hz, J₃=5.9 Hz, 4-H),

3.91 (ddd, J₁=11.5 Hz, J₂=10.0 Hz, J₃=3.9 Hz, 3-H) and

4.20 (ddd, J₁=11.5 Hz, J₂=5.9 Hz, J₃=3.9Hz, 3-H), 5.72

(s, 1H, 1-H), 6.76 (s, 1H, 8-H), 7.30 (s, 1H, 5-H), 7.48 (d,

J=8.7 Hz, 1H) and 8.23 (d, J=8.7 Hz, 1H, nitrophenyl).

7-Bromo-1-(2-chlorophenyl)isochromane (9d). Yield:

40%. Mp 62±65 ^ꢀC (ethanol). C₁₅H₁₂BrClO (323.6). H

1

NMR: d 2.75 (ddd, J₁=16.4 Hz, J₂=J₃=3.6 Hz, 4-H)

and 3.09 (ddd, J₁=16.4 Hz, J₂=9.7 Hz, J₃=5.7 Hz, 4-H),

3.93 (ddd, J₁=13.6 Hz, J₂=9.7 Hz, J₃=3.6Hz, 3-H) and

4.21 (ddd, J₁=13.6 Hz, J₂=5.7 Hz, J₃=3.6Hz, 3-H), 6.88

(d, J=1.7Hz, 1H, 8-H), 7.05 (d, J=8.0 Hz, 1H, 5-H),

7.14±7.34 (m, 4H, 2-clorophenyl), 7.45 (dd, J₁=8.0Hz,

J₂=1.7Hz, 1H, 5-H).

Method B. The reaction was performed as in Method A,

but isopropanol was used as solvent and after re¯ux it was

treated with 1N hydrochloric acid (125mL) and stirred

overnight at rt The resulting precipitate was ®ltered and

the crude product was recrystallized from 2-methox-

yethanol. Yield: 88%. Mp 275±277 ^ꢀC. C₁₅H₁₀ClN₃O₃

1

General procedure for the synthesis of phenylacetic acid

derivatives 10a±d. To a solution of the corresponding

isochromane (9a±d) (90 mmol) in acetone (360 mL)

Jones reagent (260 mL) was gradually given and the

mixture was stirred for 16 h. The separated chromium

sulfate was ®ltered and the ®ltrate evaporated to dry-

ness. The residue was treated with excess 10% sodium

carbonate solution and dichloromethane. After separa-

tion the aqueous layer was acidi®ed with concd hydro-

chloric acid and the separated crystals were collected by

®ltration.

(315.7). H NMR: (DMSO-d₆): d 3.62 (s, 2H, 5-H), 7.20

(d, J=2.0 Hz, 1H, 9-H), 7.58 (d, J=8.2Hz, 1H, 6-H), 7.70

(dd, J₁=8.2Hz, J₂=2.0Hz, 1H, 7-H), 7.80 (d, J=8.7Hz,

2H) and 8.30 (d, J=8.7 Hz, 2H, nitrophenyl).

8-Bromo-1-(4-nitrophenyl)-3,5-dihydro-4H-2,3-benzodiaz-

epin-4-one (11b). Starting from 10b (12.7 g, 35.0 mmol)

Method A was essentially followed as described for 11a.

Yield: 65% (8.19 g). Mp 264±267 ^ꢀC. C₁₅H₁₀BrN₃O₃

1

(360.2). H NMR: (DMSO-d₆): d 3.60 (br. s, 2H, 5-H),

7.29 (d, J=1.8 Hz, 1H, 9-H), 7.52 (d, J=8.3 Hz, 1H, 6-

H), 7.77 (overlapping, 7-H), 7.78 (d, J=8.7 Hz) and 8.32

(d, J=8.7 Hz, 2H, nitrophenyl), 11.41 (s, 1H, N-H).

4-Chloro-2-(4-nitrobenzoyl)phenylacetic acid (10a). Yield:

78%. Mp 150±152 ^ꢀC (ethyl acetate). C₁₅H₁₀ClNO₅

(319.7). ¹H NMR: (DMSO-d₆): d 3.82 (s, 2H, CH₂), 7.42

(d, J=1.8 Hz, 1H, 3-H), 7.48 (d, J=8.2Hz, 1H, 6-H), 7.62

(dd, J₁=8.2Hz, J₂=1.8 Hz, 1H, 5-H), 7.90 (d, J=8.7Hz,

2H) and 8.32 (d, J=8.7 Hz, 2H, nitrobenzoyl).

7,8-Dichloro-1-(4-nitrophenyl)-3,5-dihydro-4H-2,3-benzo-

diazepin-4-one (11c). A solution of 10c (6.1 g, 17.2 mmol)

and 85% hydrazine hydrate (6 mL) in isopropanol

(300 mL) was treated at re¯ux for 6 h. The solvent was

evaporated and the residue was dissolved in a mixture of

40% acetic acid (40 mL) and dichloromethane (400 mL).

After separation the organic layer was washed with

water, dried and treated with dicyclohexylcarbodiimide

(3.60 g 17.5 mmol). The reaction mixture was stirred

4-Bromo-2-(4-nitrobenzoyl)phenylacetic acid (10b). Yield:

62 %. Mp 128±130 ^ꢀC. C₁₅H₁₀BrNO₅(364.2). ¹H NMR:

(DMSO-d₆): d 3.80 (s, 2H, CH₂), 7.46 (d, J=8.2 Hz, 1H,

6-H), 7.57 (d, J=2.0 Hz, 1H, 3-H), 7.81 (dd, J₁=8.2 Hz,

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G. Abraham et al. / Bioorg. Med. Chem. 8 (2000) 2127±2143

Â

2137

overnight and the precipitate ®ltered. The ®ltrate was

evaporated to dryness and the residue was boiled with

methanol (120mL) and ®ltered while hot to give the pro-

duct (4.40 g). Yield: 73%. Mp 268±270 ^ꢀC. C₁₅H₉Cl₂N₃O₃

(350.1). ¹H NMR: (DMSO-d₆): d 3.76 (s, 2H, 5-H), 7.48 (s,

1H, 6-H), 7.90 (d, J=8.8 Hz, 2H) and 8.39 (d, J=8.8 Hz,

2H, nitrophenyl), 8.01 (s, 1H, 9-H).

removed and the residue was puri®ed by chromatography,

eluent was chloroform:methanol (98:2). The fractions

containing the condensation product of type 14 were eva-

porated to dryness and the residue was treated with a 1:1

mixture of concd hydrochloric acid and ethanol and

heated at re¯ux for 1±2 h. Evaporation gave the title pro-

ducts as hydrochloride salts. In a few cases (15f,g,n) the

intermediate condensation product was reacted with

methanesulfonic acid at rt for 1±2 h to induce the ring

closure reaction. In these cases the reaction mixture was

diluted with water and made alkaline with 5N sodium

hydroxide. The products were collected by ®ltration. The

yields, physical and spectroscopic date are collected in

Table 10.

8-Bromo-1-(2-chlorophenyl)-3,5-dihydro-4H-2,3-benzodiaz-

epin-4-one (11d). A solution of 10d (4.28 g, 12.1 mmol)

and 98% hydrazine hydrate (1.8 mL) in ethanol (45 mL)

was re¯uxed for 5 h. The mixture was evaporated to

dryness and the residue was dissolved in dichhorometane

(120 mL) and washed with water (3Â20 mL). The organic

layer was dried and evaporated. The crude product was

recystallized from ethanol to give the product (2.26 g).

Yield: 53%. Mp 255±258 ^ꢀC. C₁₅H₁₀BrClN₂O (349.6).

¹H NMR: (DMSO-d₆): d 3.62 (s, 2H, 5-H), 7.02 (d,

J=1.8 Hz, 1H, 9-H), 7.52 (d, J=8.1 Hz, 1H, 6-H), 7.79

(dd, J₁=8.1 Hz, J₂=1.8 Hz, 1H, 7-H), 7.55±7.75 (m, 4H,

2-chlorophenyl), 11.35 (br. s, 1H, 3-H).

8-Chloro-1-(4-nitrophenyl)-3-(2-oxopropyl)-3,5-dihydro-

4H-2,3-benzodiazepin-4-one (16). To a solution of 11a

(36.3 g, 115 mmol) in DMF (250 mL) potassium carbon-

ate (19.9 g, 144 mmol) was given and the mixture was

heated at 80 ^ꢀC for 10 min. Then chloroacetone (11 mL,

138 mmol) was added and the mixture was heated at

80 ^ꢀC for 2 h. The reaction mixture was then cooled to rt

and poured onto ice (1.2 kg). The precipitate was collected

by ®ltration and washed with water. The crude product

was recrystallized from acetic acid to give 16 (39.3 g).

General method for the synthesis of 3,5-dihydro-4H-2,3-

benzodiazepine-4-thione derivatives 12a±d. To a solution

of the corresponding 4-oxo-2,3-benzodiazepine derivative

(11a±d) (38.0 mmol) in dry pyridine (150 mL) phosphorous

pentasul®de (13.3 g, 60.0 mmol) was given and the mixture

was kept at 80 ^ꢀC for 2±3 h. The reaction mixture was then

cooled to rt and poured onto ice (1 kg). The precipitate was

collected by ®ltration and washed with water. The crude

products were recrystallized from 2-methoxyethanol.

ꢀ

1

Yield: 92%. Mp 222±224 C. C₁₈H₁₄ClN₃O₄(371.8). H

NMR: (DMSO-d₆): d 2.12 (s, 3H, CH₃), 3.75 (br. s, 2H, 5-

H), 4.72 (s, 2H, CH₂), 7.23 (d, J=2.1 Hz, 1H, 9-H), 7.63

(d, J=8.3 Hz, 1H, 6-H), 7.74 (dd, J₁=8.3 Hz, J₂=2.1Hz,

1H, 7-H), 7.83 (d, J=8.8 Hz, 2H) and 8.33 (d, J=8.8Hz,

2H, nitrophenyl).

8-Chloro-1-(4-nitrophenyl)-3,5-dihydro-4H-2,3-benzodiaz-

epine-4-thione (12a). Yield: 71%. Mp 231±234 ^ꢀC.

C₁₅H₁₀ClN₃O₂S (331.8). H NMR: (DMSO-d₆): d 4.00

(br. s, 2H, 5-H), 7.48 (d, J=8.2 Hz, 1H, 6-H), 7.15 (d,

J=1.9Hz, 1H, 9-H), 7.72 (dd, J₁=8.2 Hz, J₂=1.9Hz,

1H, 7-H), 7.80 (d, J=8.7 Hz, 2H) and 8.28 (d, J=8.7Hz,

2H, nitrophenyl).

8-Chloro-1-(4-nitrophenyl)-3-phenacyl-3,5-dihydro-4H-

2,3-benzodiazepin-4-one (17). The compound was syn-

thesized from 11a according to the procedure as given

above by using 2-chloroacetophenone. Yield: 83%. Mp

223±225 ^ꢀC. C₂₃H₁₆ClN₃O₄(433.8). ¹H NMR: (DMSO-

d₆): d 3.8 (br. s, 2H, 5-H), 5.4 (br. s, 2H, phenacyl), 7.22

(d, J=1.9 Hz, 1H, 9-H), 7.64 (d, J=8.3 Hz, 1H, 6-H),

7.74 (dd, J₁=8,3 Hz, J₂=1.9 Hz, 1H, 7-H), 7.54 (dd,

J₁=J₂=7.5 Hz, 2H, phenyl), 7.66 (dd, J₁=J₂=7.5 Hz,

1H, phenyl), 7.98 (d, J=7.5 Hz, 2H, phenyl), 7.83 (d,

J=8.8 Hz, 2H) and 8.32 (d, J=8.8 Hz, 2H, nitrophenyl).

1

8-Bromo-1-(4-nitrophenyl)-3,5-dihydro-4H-2,3-benzodiaz-

epine-4-thione (12b). Yield: 67%. Mp 220±223 ^ꢀC. C₁₅

H₁₀BrN₃O₂S (376.8). ¹H NMR: (DMSO-d₆): d 4.05 (br. s,

2H, 5-H), 7.35 (d, J=1.8 Hz, 1H, 9-H), 7.48 (d, J=8.3Hz,

1H, 6-H), 7.88 (dd, J₁=8.3Hz, J₂=1.8 Hz, 1H, 7-H), 7.84

(d, J=8.8Hz, 2H) and 8.36 (d, J=8.8Hz, 2H, nitrophe-

nyl), 13.1 (s, 1H, N-H).

8-Chloro-2-methyl-6-(4-nitrophenyl)-11H-imidazolo[1,2-c]

[2,3]benzodiazepine hydrochloride (15b). (Alternative

route.) To a solution of ammonium acetate (180 g) in

acetic acid (180mL) 16 (9.0g, 24.2mmol) was added and

the mixture was heated at 140 ^ꢀC for 3 h. The reaction

mixture was then cooled to rt and poured onto ice

(0.9 kg). The precipitate was collected by ®ltration and

washed with water. The crude product was recrystallized

twice from DMF to give the product as base (3.42 g).

Yield: 40%. Mp 246±250 ^ꢀC. A sample was converted to

the hydrochloride with mp 230±231 ^ꢀC which was iden-

tical with the substance prepared by the general method

described earlier, for further data see Table 10.

7,8-Dichloro-1-(4-nitrophenyl)-3,5-dihydro-4H-2,3-benzo-

diazepine-4-thione (12c). Yield: 61%. Mp 210±213 ^ꢀC.

C₁₅H₉Cl₂N₃O₂S (366.2). MS(EI): m/z: M: 366/368.

8-Bromo-1-(2-chlorophenyl)-3,5-dihydro-4H-2,3-benzodiaz-

epine-4-thione (12d). Yield: 79%. Mp 198±20 ^ꢀC. C₁₅H₁₀

BrClN₂S (365.7). MS(EI): m/z: M: 365/367.

General procedure for the synthesis of 11H-imidazolo

[1,2-c][2,3]-benzodiazepine derivatives (15a±g, 15i±n, 3p,

q). A mixture of the appropriate 2,3-benzodiazepine-4-

thione derivative (12a±d) (10.0 mmol), the correspond-

ing aminoacetal (13a±g) (20.0 mmol) and red mercury

oxide (10.0 mmol) in 2-methoxyethanol was stirred and

heated at re¯ux for 1±10 h. After ®ltration the solvent was

8-Chloro-6-(4-nitrophenyl)-2-phenyl-11H-imidazolo[1,2-c]

[2,3]benzodiazepine (15h). The compound was prepared

from 17 according to the procedure as described above

for 15b. (For yield, physical and spectroscopic data see

Table 10.)

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G. Abraham et al. / Bioorg. Med. Chem. 8 (2000) 2127±2143

2138

Table 10. Physical and spectroscopic data of the 11H-imidazolo[1,2-c][2,3]benzodiazepine derivatives 15a±n, 3p±q

Compound^a

mp

(^ꢀC)

Yield

(%)

Molecular formula

(Mol. mass)

Spectroscopic data:

¹H NMR (in DMSO-d₆; d) or MS

15 a

210±215 48

229±230 79

205±208 41

207±210 46

C

₁₇H₁₂Cl₂N₄O₂(375.2)

4.55 (s, 2H, 11-H), 7.22 (d, J=1.8 Hz, 1H, 7-H), 7.65 (d, J=8.2 Hz, 1H, 10-H), 7.84

(dd, J₁=8.2 Hz, J₂=1.8 Hz, 1H, 9-H), 7.62 (overlapping) and 7.95 (2-H and 3-H), 8.00

(d, J=8.5 Hz, 2H) and 8.43 (d, J=8.5 Hz, 2H, nitrophenyl).

2.25 (d, J=1.0 Hz, 3H, 2-CH₃), 4.52 (s, 2H, 11-H), 7.23 (d, J=1.8 Hz, 1H, 7-H), 7.63

(d, J=8.2 Hz, 1H, 10-H), 7.72 (q, J=1.0 Hz, 1H, 3-H), 7.82 (dd, J₁=8.2 Hz, J₂=1.8 Hz,

1H, 9-H). 7.96 (d, J=8.6 Hz) and 8.44 d, J=8.6 Hz, nitrophenyl).

15 b

15 c

15 d

C

₁₈H₁₄Cl₂N₄O₂(389.2)

₁₈H₁₄Cl₂N₄O₂(389.2) 2.40 (d, J=1.0 Hz 3H, 3-CH₃), 7.22 (d, J=1. Hz, 1H, 7-H), 7.42 (q, J=1.0 Hz, 1H, 2-H),

7.62 (d, J=8.2 Hz, 1H, 10-H), 7.86 (dd, J₁=8.2 Hz, J₂=1.8 Hz, 1H, 9-H),

8.05 (d, J=8.6 Hz) and 8.40 (d, J=8.6 Hz, nitrophenyl).

₁₉H₁₆Cl₂N₄O₂(403.3)

2.22 (s, 3H), 2.35 (s, 3H), 4.49 (s, 2H, 11-H), 7.22 (d, J=1.9 Hz, 1H, 7-H), 7.63

(d, J=8.2 Hz, 1H, 10-H), 7.86 (dd, J₁=8.2 Hz, J₂=1.9 Hz, 1H, 9-H), 8.02 (d, J=8.7 Hz)

and 8.40 (d, J=8.7 Hz, nitrophenyl).

15 e

15 f^b

150±153 20

270±272 30

₁₉H₁₆Cl₂N₄O₂(403.3)

MS(EI): m/z: M: 403/405.

C₂₃H₁₄ClN₅O₄(459.8)

C₂₂H₁₄ClN₅O(415.8)

C₂₃H₁₅ClN₄O₂414.8

4.25 (br. s, 2H, 11-H), 7.26 (d, J=1.8 Hz, 1H, 7-H), 7.55 (s, 1H, 2-H), 7.72 (d, J=8.3 Hz,

1H, 10-H), 7.76 (dd, J₁=8.3 Hz, J₂=1.8 Hz, 1H, 9-H), 7.95 (d, J=8.9 Hz, 2H), 8.04

(d, J=8.9 Hz, 2H) and 8.36 (d, J=8.9 Hz, 2H), 8.41 (d, J=8.9 Hz, nitrophenyls).

4.10 (br. s, 2H, 11-H), 7.22 (d, J=1.9 Hz, 1H, 7-H), 7.35 (s, 1H, 2-H), 7.47 (d, J=8.3 Hz,

1H, 10-H), 7.56 (d, J=6.2 Hz, 2H, pyridyl), 7.60 (dd, J₁=8.3 Hz, J₂=1.9 Hz, 1H, 9-H), 7.92

(d, J=8.9 Hz, 2H) and 8.37 (d, J=8.9 Hz, 2H, nitrophenyl), 8.72 (d, J=6.2 Hz, 2H, pyridyl).

4.25 (br. s, 2H, 11-H), 7.21 (br. s, 1H, 7-H), 7.72 (br. s, 2H, 9-H and 10-H), 7.12±7.26

(1H, phenyl, overlapping), 7.37 (dd, J₁=1₂=7.3 Hz, 2H, phenyl), 7.78 (d, J=7.3 Hz, 2H,

phenyl), 8.00 (d, J=8.8 Hz, 2H, nitrophenyl), 8.10 (s, 1H, 3-H), 8.42

(d, J=8.8 Hz, nitrophenyl).

15 g^b

15 h^b

250±252 30

239±232 82

15 i

15 j

15 k

221±224 13 C₁₈H₁₃Cl₃N₄O₂(423.7)

240±244 41 C₁₈H₁₃Cl₃N₄O₂(423.7)

MS (EI): m/z (%): M 386/388 (100/68), 385/387 (45/30), 339/341 (16/12).

MS (EI): m/z (%): M 386/388 (73/45), 385/387 (100/68), 339/341 (20/14).

215±220 28

194±202 50

212±219 42

C

₁₈H₁₄BrClN₄O₂(433.8) 2.25 (d, J=1.0 Hz, 3H, 2-CH₃), 4.43 (br. s, 2H, 11-H), 7.35 (d, J=1.9 Hz, 1H, 7-H), 7.58

(d, J=8.3 Hz, 1H, 10-H), 7.71 (q, J=1.0 Hz, 1H, 3-H), 7.96 (dd, J₁=8.3 Hz, J₂=1.9 Hz,

1H, 9-H), 7.98 (d, J=8.82 Hz) and 8.43 (d, J=8.8 Hz, nitrophenyl).

15 l

₁₈H₁₄BrClN₄O₂(433.8)

2.42 (d, J=1.0 Hz, 3H, 3-CH₃), 4.50 (s, 2H, 11-H), 7.32 (d, J=1.8 Hz, 1H, 7-H), 7.42

(q, J=1.0 Hz, 1H, 2-H), 7.55 (d, J=8.1 Hz, 1H, 10-H), 7.97 (dd, J₁=8.1 Hz, J₂=1.8 Hz,

1H, 9-H), 8.05 (d, J=8.7 Hz) and 8.42 (d, J=8.7 Hz, nitrophenyl).

2.22 (s, 3H), 2.34 (s, 3H), 4.45 (s, 2H, 11-H), 7.42 (d, J=1.9 Hz, 1H, 7-H), 7.58

(d, J=8.1 Hz, 1H, 10-H), 7.95 (dd, J₁=8.1 Hz, J₂=1.9 Hz, 1H, 9-H), 8.01

(d, J=8.8 Hz) and 8.42 (d, J=8.8 Hz, nitrophenyl).

15 m

₁₉H₁₆BrClN₄O₂(447.8)

15 n^b

3 p^c,d

3 q^c,d

Foam

Ð

60

Ð

C₂₂H₁₄BrN₅O(460.3)

C₁₈H₁₄BrCl₂N₃(423.2)

MS(EI): m/z: M: 460/462.

^aHydrochlorides.

^bData refer to the base.

^cFor Mp and yield see Table 1.

^dFor spectroscopic data see Table 12.

8-Chloro-4-methylthio-1-(4-nitrophenyl)-5H-2,3-benzo-

diazepine (18). Thione 12a (3.32 g 10.0 mmol) was dis-

solved in acetone (200mL) and potassium carbonate

(2.76 g 20.0mmol) and methyliodide (1.87 mL, 30.0

mmol) were added. The mixture was stirred at rt for 3 h,

then the product was ®ltered, washed with water and

recrystallized from DMF _ꢀto give the title product (2.84 g).

Yield: 82%. Mp 249±252 C. C₁₆H₁₂ClN₃O₂S (345.8). ¹H

NMR: d 2.42 (s, 3H, S-CH₃), 3.35 (d, J=13.0 Hz, 5-H),

and 3.45 (d, J=13.0 Hz), 7.20 (d, J=2.0 Hz,1H, 9-H),

7.25 (d, J=8.2Hz, 1H, 6-H), 7.55 (dd, J₁=8.2Hz,

J₂=2.0Hz, 1H, 7-H), 7.87 (d, J=8.8Hz) and 8.38 (d,

J=8.8Hz, nitrophenyl).

were recrystallized. The yields, physical and analytical

data are collected in Table 11.

7-Chloro-3-methyl-1-(4-nitrophenyl)isochromane (21).

Prepared from 1-(4-chlorophenyl)-2-propanol³³accord-

ing to the general method as described for 9a±d. Yield:

32%. Mp 120±123 ^ꢀC. C₁₆H₁₄ClNO₃(303.8). ¹H NMR: d

1.42 (d, J=6.4 Hz, 3H, 3-CH₃), 2.7±2.95 (m, 2H, 4-H),

4.02 (m, 1H, 3-H), 5.80 (s, 1H, 1-H), 6.60 (d, J=2.0Hz,

1H, 8-H), 7.10 (d, J=8.2Hz, 1H, 5-H), 7.16 (dd,

J₁=8.2 Hz, J₂=2.0 Hz, 1H, 6-H), 7.53 (d, J=8.8 Hz, 2H)

and 8.24 (d, J=8.8 Hz, 2H, nitrophenyl).

7-Chloro-3-methyl-1-(4-nitrophenyl)-2-benzopyrilium per-

chlorate (22). To an ice cold solution of 21 (6.8 g,

22.4 mmol) in acetone (70 mL) Jones reagent (29 mL,

78 mmol) was added dropwise during 1h and the mixture

was then stirred at rt for 4 h. The separated chromium

salt was ®ltered and the solution evaporated to dryness.

The residue was suspended with water (25 mL) and ®l-

tered. The precipitate was then dissolved in hot acetic

acid (76 mL) and 70% perchloric acid (1.48 mL) was

General procedure for the synthesis of 3-substitued 8-

chloro-6-(4-nitrophenyl)-11H-1,2,4-triazolo[4,5-c][2,3]ben-

zodiazepine derivatives (19a±d). 18 (3.45 g 10.0 mmol)

was dissolved in DMF (120 mL) then the corresponding

acylhydrazide (25.0 mmol) and concd HCl (0.5 mL)

were added and the mixture was stirred and heated at

120±130 ^ꢀC for 9±15 h. The reaction mixture was poured

onto ice and the product ®ltered. The crude products

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G. Abraham et al. / Bioorg. Med. Chem. 8 (2000) 2127±2143

Â

2139

Table 11. Physical and spectroscopic data of the 3-substitued 8-chloro-6-(4-nitrophenyl)-11H-1,2,4-triazolo[4,5-c][2,3]benzodiazepine derivatives

19a±d

Compound

mp

(^ꢀC)

Yield

(%)

Molecular formula

(Mol. mass)

Spectroscopic data:

¹H NMR or MS

19 a

19 b

271±274

287±289

72

86

C₁₇H₁₂ClN₅O₂(353.8)

2.58 (s,3H) 4.16 (s, 2H), 7.12 (d, J=1.9 Hz, 1H), 7.45 (d, J=8.3 Hz, 1H), 7.55

(dd, J₁=8.3 Hz, J₂=1.9 Hz, 1H), 7.90 (d, J=8.7 Hz) and 8.35 (d, J=8.7 Hz, nitrophenyl).

C₂₁H₁₃ClN₆O₂(416.8) (in DMSO-d₆) 4.46 (br. s, 2H), 7.24 (br. s, 1H), 7.78 (br. s, 2H), 7.98 (d, J=8.8 Hz, 2H)

and 8.42 (d, J=8.8 Hz, 2H, nitrophenyl), 8.18 (d, J=5.5 Hz, 2H) and 8.83

(d, J=5.5 Hz, 2H, pyridyl).

19 c

19 d

287±290

266±268

68

88

C₂₂H₁₃ClN₆O₄(460.8)

₁₈H₁₄ClN₅O₃(383.8)

MS(EI):m/z: M:460/462.

MS(EI): m/z: M: 383/385.

C

added. A product separated which was ®ltered after

cooling and washed with acetic acid to give the title

product. Yield: 42% (3.73 g). Mp 247±255 ^ꢀC. C₁₆H₁₁

azodicarboxylate (1.52 mL, 9.7 mmol) in THF (11 mL)

was added dropwise. After stirring for 3 h at rt the sol-

vent was removed and the residue recrystallized from

ethanol to give 26 (1.34 g). Mp 254±256 ^ꢀC (dec.).

(MS(FAB): M: 458/460.)

1

Cl₂NO₇(400.2). IR: 1096 cm (ClO₄).

8-Chloro-4-methyl-1-(4-nitrophenyl)-5H-2,3-benzodiaze-

pine (23). Compound 22 (4.1g, 10.2 mmol) was added to

a solution of 98% hydrazine hydrate (1.5 mL, 70.7 mmol)

in DMF (20 mL) at 10±15 ^ꢀC. The reaction mixture was

then stirred for 1.5 h at rt Water (25 mL) was added and

the separated precipitate was collected by ®ltration and

washed with water. The crude product was recrystallized

from isopropanol. Yield: 87% (2.82 g). Mp 199±203 ^ꢀC.

The solution of the intermediate 26 (1.34 g, 2.9 mmol) and

98% hydrazine hydrate (1.09 mL, 21.7 mmol) in methanol

(134mL) was heated at boiling for 4 h. After evaporation

of the solvent the residue was treated with methanol

(50 mL) and the resulting precipitate was ®ltered. The ®l-

trate was then evaporated and the residue suspended with

water to give 27 (0.97 g, mp 105±107 ^ꢀC (dec.)). This crude

intermediate was taken up with acetic anhydride (8 mL)

and stirred for 2 h. Dilution with water (40 mL) gave a

solid substance which was collected by ®ltration. Column

chromatography of this product with the eluent ethyl

acetate:benzene (4:1) gave the acetylamino derivative 28

(0.60 g). Mp 216±218 ^ꢀC. (MS(FAB): M: 370/372.)

1

C₁₆H₁₂ClN₃O₂(313.7). H NMR: (DMSO-d₆): d 2.11 (s,

3H, CH₃), 2.93 and 3.65 (d, J=12.2 Hz, 2H, 5-H), 7.33 (d,

J=2.0Hz, 1H, 9-H), 7.61 (d, J=8.2 Hz, 1H, 6-H), 7.73

(dd, J₁=8.2Hz, J₂=2.0 Hz, 1H, 7-H), 7.82 (d, J=8.7Hz,

2H), 8.32 (d, J=8.7Hz, 2H).

8-Chloro-4-formyl-1-(4-nitrophenyl)-5H-2,3-benzodiaze-

pine (24). To a solution of 23 (9.17 g, 29.0 mmol) in diox-

ane (120 mL) powdered selenium dioxide (2.27 g,

20.5 mmol) was given and the mixture was stirred at 90^ꢀC

for 40min. Charcoal was added to the mixture and it was

®ltered. The clear solution was poured into water (1.5 L)

and the separated crystalls were ®ltered and washed with

water. Column chromatography with benzene as eluent

gave the aldehyde. Yield: 29% (2.8g). Mp 208±210 ^ꢀC.

A solution of 28 (0.59 g, 1.6 mmol) and POCl₃(0.73 mL,

7.95 mmol) in 1,2-dichloroethane (30 mL) was heated at

re¯ux for 3 h. The solution was then chilled with ice and

treated with sodium hydrogencarbonate solution. After

separation the organic phase was washed with water,

dried and evaporated. The residual oil was chromato-

graphed with an eluent of ethyl acetate:benzene (4:1) to

give 8-chloro-3-methyl-6-(4-nitrophenyl)-11H-imidazolo

[3,4-c][2,3]benzodiazepine (29), which was then reduced

according to the general procedure as described for

compounds 3a±n. The crude product was boiled with

ethanol to remove impurities. By the above sequence

0.12 g of pure 5 could be prepared. Mp 256±258 ^ꢀC

(dec.). C₁₈H₁₅ClN₄(322.8). MS(EI): m/z (%): M: 322/

324 (100/33), 321/323 (26/9), 108.5 (22), 65 (11), 219

(10), 106 (10), 218 (8), 217 (8).

1

C₁₆H₁₀ClN₃O₃(327.7). H NMR: (DMSO-d₆): 2.98 (d,

J=13.1 Hz) and 4.13 (d, J=13.1 Hz, 2H, 5-H), 7.42 (d,

J=2.2Hz, 1H, 9-H), 7.50 (d, J=8.3 Hz, 1H, 6-H), 7.73

(dd, J₁=8.3Hz, J₂=2.2 Hz, 1H, 7-H), 7.92 (d, J=8.9Hz,

2H) and 8.36 (d, J=8.9 Hz, 2H, nitrophenyl).

6-(4-Aminophenyl)-8-chloro-3-methyl-11H-imidazolo[3,4-c]

[2,3]benzodiazepine (5). The solution of 24 (2.15 g,

6.60mmol) in a 1:1 mixture of THF:water (88 mL) was

chilled with icewater and sodium borohydride (0.12 g

3.30mmol) was given in portions. After stirring at rt for

40min the solution was diluted with water (90 mL) to give

a precipitate which was ®ltered and after drying chroma-

tographed with the eluent of benzene:ethyl acetate (1:1) to

give pure 4-(hydroxymethyl)-8-chloro-1-(4-nitrophenyl)-

5H-2,3-benzodiazepine (25; 1.62 g). Mp >163^ꢀC (decom-

position). (MS(FAB): M: 329/331.)

6-(4-Aminophenyl)-8-chloro-2-ethoxycarbonyl-11H-pyr-

rolo[1,2-c][2,3]benzodiazepine (6). The solution of 23

(0.50 g, 1.60 mmol) and ethyl bromopyruvate (0.27 mL,

2.20 mmol) in ethanol (20 mL) was heated at re¯ux for

12 h. After evaporation of the solvent the residue was

chromatographed with benzene as eluent to give 30

(0.29 g) which was reduced without further puri®cation

according to the general procedure as described for 3a±

n. It was recrystallized from ethanol (0.11 g). Mp 247±

249 ^ꢀC. C₂₁H₁₈ClN₃O₂(379.8). MS (EI): m/z (%): M:

379/381 (100/35), 378/380 (41/14), 350/352 (35/14), 306/

308 (35/12), 65 (23).

The intermediate 25 (1.62 g, 4.9 mmol), triphenylphosphine

(2.54 g, 9.7 mmol) and phthalimide (1.42 g, 9.7 mmol) were

dissolved in dry THF (72 mL) and a solution of diethyl

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G. Abraham et al. / Bioorg. Med. Chem. 8 (2000) 2127±2143

2140

General procedure for the reduction of the nitro compounds

15a±n, 19a±d, 29 and 30. (Preparation of 3a±n, 4a±d, 5

and 6). To a stirred solution of the corresponding nitro

compound (2mmol) in a 1:1 mixture of methanol and di-

chloromethane hydrazine hydrate (4±5 equiv) and RaNi

(0.1±2g) were added. After stirring at 20±40 ^ꢀC for 1±5 h

the catalyst was ®ltered and the solvents evaporated. The

residues were recrystallized from ethanol. Mp-s and yields

of the aminophenyl compounds 3a±n and 4a±d are shown

in Table 1 and 2 respectively. The composition and spec-

troscopic data of 3a±3r and 4a±d are collected in Table 12.

Electrophysiological studies. Electrophysiological experi-

ments were carried out on acutely isolated cerebellar

Purkinje cells according to Bleakman et al.¹⁹Brie¯y:

cells were isolated from the cerebellum of 6±9-day-old

rats using enzymatic treatment and mechanical tritura-

tion, then plated to poly-l-lysine coated glass coverslips.

Cells were kept alive in a tissue culture medium, in a

CO₂thermostat, and used for electrophysiological

experiments on the day of isolation. Purkinje cells

(identi®ed by their bigger size) were patched with glass

electrodes (4±6 Mꢀ). The membrane potential was ®xed

at 70 mV. Experiments were performed at room tem-

perature. The internal recording solution contained NaCl

(140mM), MgCl₂(1mM), HEPES (10mM), glucose

(10 mM), EGTA (0.1 mM), pH=7.2. The cells were

continuously perfused with a solution composed of

NaCl (138 mM), KCl (5 mM), CaCl₂(5 mM), MgCl₂

(1 mM), HEPES (10 mM), glucose (10 mM), pH=7.35.

Excitatory amino acids (kainate 100 mM or S-AMPA

5 mM) were applied alone, or in combination with various

concentrations of 1, 2 or 3b. The percentage decrease of

the plateau current was evaluated. IC₅₀ÆS.E.M. values

were calculated from 4±6 concentrations (5±6 cells for

each concentration), using the microcal origin 4.1

computer program for sigmoidal curve ®tting.

6-(4-Acetylaminophenyl)-8-chloro-2-methyl-11H-imida-

zolo[1,2-c][2,3]benzodiazepine (3o). Prepared from 3b

(0.46 g, 1.42 mmol) in pyridine (8 mL) by reacting with

acetylchloride (0.20 mL) at 5±10 ^ꢀC for 1.5 h. Dilution

with ice water gave a solid precipitate which was

recrystallized from ethanol. For yield and mp see Table

1, spectroscopic data are shown in Table 12.

8-Chloro-3-methyl-6-phenyl-11H-imidazolo[1,2-c][2,3]ben-

zodiazepine (3r). To a stirred solution of 3c (1.10 g,

3.20mmol) in DMF (12 mL) at 65 ^ꢀC isoamyl nitrite

(0.80 mL) was added droppwise over 8 min. Heating was

continued for 30min and the cooled mixture was then

treated with 5N HCl. The product was extracted with

ether and puri®ed by column chromatography. Eluent:

chloroform:methanol (98:2). For yield and mp see Table

1, spectroscopic data are shown in Table 12.

In vivo experiments

Animals were purchased from Charles River Hungary Ltd.

(Budapest, Hungary) and were housed for a minimum of

six days prior to experiments with free access to stan-

dard laboratory diet and tap water and maintained on a

12±12 h light-dark cycle (light from 6.00 am to 6.00

p.m.). All compounds under study were suspended in

saline (ip administration) or distilled water (oral

administration) containing 1±2% Tween-80. Control

animals received vehicle under the same conditions.

ED₅₀and LD₅₀values were calculated by the Litch®eld±

Wilcoxon method.³⁴The volumes of administration

were 0.1 mL/10 g and 0.5 mL/100 g body weight for mice

and rats, respectively, unless otherwise stated.

Biology, in vitro methods

Retinal spreading depression (S.D.) test. Experiments

were performed according to Sheardown.¹⁷The posterior

chamber of each eye of 1±5-day-old chickens was dis-

sected and placed in a Petri dish containing physiological

saline of the following composition: NaCl (100mM), KCl

(6mM), CaCl₂(1 mM), MgSO₄(1 mM), NaHCO₃

(30 mM), NaH₂PO₄(1mM). The solution was saturated

with 95% O₂and 5% CO₂and maintained at 26 ^ꢀC. The

eyes were initially incubated in normal saline for 30 min

and then transferred to a solution containing AMPA

(5 mM) or kainate (5 mM). These solutions triggered

S.D., which could be easily observed by eye. The latency

of S.D. was determined. The presence of a white area

(0.5 mm in diameter ) was taken as the onset of S.D. The

eye cups were then returned to normal saline. After a fur-

ther 15 min recovery period the eyes were incubated in a

solution containing the test compounds and incubated

for 15min. Thereafter the eyes were transferred to a glu-

tamate agonist containing solution which also contained

the test compound and the latency of S.D. was determined

again. 60min following this measurement, the whole pro-

cedure was repeated, with another test substance/con-

centration. Each drug concentration was tested in 6 retina

preparations. An increase in the latency of 30 s or more

was considered to be 100% inhibition of S.D. (cut-o time

was 1 min).¹⁷The drug eects therefore are expressed as

the percentage maximum inhibition obtained for a given

concentration. Dose-response curves were constructed

from 3±6 concentration points and IC₅₀ÆS.E.M. values

were calculated by sigmoidal curve ®tting using the

microcal origin 4.1 computer program.

Behavioural changes in mice. 5 male CD1 mice (21±26 g)

were starved for 16 h and treated with test compounds in

doses of 100 and 200 mg/kg ip and po, respectively. The

gross behavioural changes were evaluated continuously

for 5 h according to Irwin.³⁵

Seizure assays. 10 male CD1 mice (21±26 g) per group,

after 16 h starvation, were used. Animals were treated

orally with test compounds 60 min before maximal elec-

troshock (MES)³⁶or chemical convulsants (metrazole

130 mg/kg ip, strychnine 3 mg/kg ip, bemegride 50 mg/

kg ip, bicuculline 1 mg/kg iv, nicotine 3.5 mg/kg iv, 4-

aminopyridine 12.5 mg/kg ip and 3-mercapto-propionic

acid 110 mg/kg iv).³⁷Prevention of tonic convulsion

(MES) and/or death (convulsive agents) served as measure

of anticonvulsive potency.

Muscle relaxation in mice. (Inclined screen and rotarod

tests). For determination of muscle relaxant activity the

inclined screen test³⁸and rotarod test³⁹were used. 10

male CD1 mice (21±25 g) per group were examined.

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G. Abraham et al. / Bioorg. Med. Chem. 8 (2000) 2127±2143

Â

2141

Table 12. Composition and spectroscopic data of compounds 3a±r and 4a±d

Compound

Molecular formula

(Mol. mass)

Spectroscopic data:

¹H NMR (in DMSO-d₆; ) or MS

3a

C₁₇H₁₃ClN₄(308.8)

4.00 (br. s, 2H, 11-H), 5.85 (br. s, NH₂), 6.84 and 7.38 (d, J=1.5 Hz, 2-H and 3-H), 6.63

(d, J=8.7 Hz, 2H) and 7.36 (d, J=8.7 Hz, 2H, aminophenyl), 7.21 (d, J=2.0 Hz, 1H, 7-H), 7.58

(d, J=8.3 Hz, 1H, 10-H), 7.63 (dd, J₁=8.3 Hz, J₂=2.0 Hz, 1H, 9-H).

3b

3c

3d

3e

3f

C₁₈H₁₅ClN₄(322.8)

2.04 (d, J=1.1 Hz, 1H, 2-CH₃), 3.91 (br. s, 2H, 11-H), 5.80 (br. s, 2H, NH₂), 6.63 (d, J=8.7 Hz, 2H)

and 7.37 (d, J=8.7 Hz, 2H, aminophenyl), 7.07 (q, J=1.1 Hz, 1H, 3-H), 7.19 (d, J=2.1 Hz, 1H, 7-H),

7.56 (d, J=8.3 Hz, 2H, 10-H), 7.64 (dd, J₁=8.3 Hz, J₂=2.1 Hz, 1H, 9-H).

(in CDCl₃) 2.32 (d, J=1.0 Hz, 3H, 3-CH₃), 3.92 (br. s, 2H, 11-H), 4.08 (br. s, 2H, NH₂), 6.63

(q, J=1.0 Hz, 1H, 2-H), 6.75 (d, J=8.5 Hz, 2H) and 7.55 (d, J=8.5 Hz, 2H, aminophenyl), 7.29

(d, J=1.9 Hz, 1H, 7-H), 7.36 (d, J=8.1 Hz, 1H, 10-H), 7.44 (dd, J₁=8.1 Hz, J₂=1.9 Hz, 1H, 9-H).

2.00 and 2.18 (s, 3H, CH₃), 3.90 (br. s, 2H, 11-H), 5.82 (br. s, 2H, NH₂), 6.68 (d, J=8.8 Hz, 2H)

and 7.42 (d, J=8.8 Hz, 2H, aminophenyl), 7.20 (d, J=1.9 Hz, 1H, 7-H), 7.54 (d, J=8.3 Hz, 1H, 10-H),

7.64 (dd, J₁=8.3 Hz, J₂=1.9 Hz, 1H, 9-H).

C₁₈H₁₅ClN₄(322.8)

C₁₉H₁₇ClN₄(336.8)

C₂₃H₁₈ClN₅(399.9)

(in CDCl₃) 1.26 (t, J=7.3 Hz, 3H), 2.6 (q, J=7.3 Hz, 2H), 3.94 (br. s, 2H, 11-H), 6.72 (d, J=8.6 Hz, 2H)

and 7.52 (d, J=8.6 Hz, 2H, aminophenyl), 6.97 (s, 1H, 3-H), 7.27 (d, J=2.0 Hz, 1H, 7-H), 7.34

(d, J=8.2 Hz, 1H, 10-H), 7.45 (dd, J₁=8.2 Hz, J₂=2.0 Hz, 1H, 9-H).

3.6-4.3 (2H, 11-H), 5.25 (br. s, 2H) and 5.86 (br. s, 2H, NH₂), 6.64 (d, J=8.8 Hz, 2H), 6.66

(d, J=8.8 Hz, 2H) and 7.27 (d, J=8.8 Hz, 2H), 7.35 (d, J=8.8 Hz, 2H, aminophenyls), 7.30

(d, J=1.9 Hz, 1H, 7-H), 7.60 (d, J=8.3 Hz, 1H, 10-H), 7.65 (dd, J₁=8.3 Hz, J₂=1.9 Hz, 1H, 9-H).

3.8-4.3 (2H, 11-H), 7.30 (d, J=2.0 Hz, 1H, 7-H), 7.46 (s, 1H, 2-H), 7.62 (d, J=8.2 Hz, 1H, 10-H),

7.67 (dd, J₁=8.2 Hz, J₂=2.0 Hz, 1H, 9-H), 6.68 (d, J=8.6 Hz, 2H) and 7.40 (d, J=8.6 Hz, 2H,

aminophenyl), 7.68 (d, J=5.6 Hz, 2H) and 8.62 (d, J=5.6 Hz, 2H, pyridyl).

MS (EI): m/z (%): M: 384/386 (100/33).

(in CDCl₃) 2.22 (d, J=1.0 Hz, 3H, 2-CH₃), 3.96 (br. s, 2H, 11-H), 4.08 (br. s, 2H, NH₂), 6.98

(q, J=1.0 Hz, 1H, 3-H), 6.65 (d, J=8.0 Hz, 2H) and 7.53 (d, J=8.6 Hz, 2H,

aminophenyl), 7.30 (s, 1H), 7.39 (s, 1H).

3g

C₂₂H₁₆ClN₅(385.9)

3h

3i

C₂₃H₁₇ClN₄(384.9)

C₁₈H₁₄Cl₂N₄(357.2)

3j

C

₁₈H₁₄Cl₂N₄(357.2)

2.21 (d, J=1.0 Hz, 3H, 3-CH₃), 4.00 (br. s, 2H, 11-H), 6.60 (q, J=1.0 Hz, 1H, 2-H), 6.68

(d, J=8.6 Hz, 2H) and 7.45 (d, J=8.6 Hz, 2H, aminophenyl), 7.40 (s, 1H), 7.93 (s, 1H).

2.02 (d, J=1.2 Hz, 3H, 2-CH₃), 3.90 (br. s, 2H, 11-H), 5.90 (br. s, 2H, NH₂), 7.07 (q, J=1.2 Hz,

1H, 3-H), 6.62 (d, J=8.5 Hz, 2H) and 7.35 (d, J=8.5 Hz, 2H, aminophenyl), 7.32 (d, J=2.2 Hz,

1H, 7-H), 7.47 (d, J=8.3 Hz, 1H, 10-H), 7.75 (dd, J₁=8.2 Hz, J₂=2.2 Hz, 1H, 9-H).

2.22 (d, J=1.0 Hz, 3H, 3-CH₃), 3.92 (br. s, 2H, 11-H), 5.82 (br. s, 2H, NH₂), 6.55 (q, J=1.0 Hz,

1H, 2-H), 6.65 (d, J=8.6 Hz, 2H) and 7.42 (d, J=8.6 Hz, 2H, aminophenyl), 7.32 (d, J=1.8 Hz,

1H, 7-H), 7.50 (d, J=8.0 Hz, 1H, 10-H), 7.72 (dd, J₁=8.0 Hz, J₂=1.8 Hz, 1H, 9-H).

1.98 (s, 3H) and 2.16 (s, 3H, CH₃), 3.85 (br. s, 2H, 11-H), 5.80 (s, 2H, NH₂), 6.65 (d, J=8.6 Hz, 2H)

and 7.34 (d, J=8.6 Hz, 2H, aminophenyl), 7.31 (d, J=1.9 Hz, 1H, 7-H), 7.45 (d, J=8.2 Hz, 1H,

10-H), 7.75 (dd, J₁=8.2 Hz, J₂=1.9 Hz, 1H, 9-H).

3.7±4.3 (br, 2H, 11-H), 5.95 (br. s, 2H, NH₂), 6.67 (d, J=8.6 Hz, 2H) and 7.40 (d, J=8.6 Hz, 2H,

aminophenyl), 7.47 (s, 3H, 2-H), 7.45 (d, J=2.0 Hz, 1H, 7-H), 7.57 (d, J=8.2 Hz, 1H, 10-H), 7.78

(dd, J₁=8.2 Hz, J₂=2.0 Hz, 1H, 9-H), 7.71 (d, J=5.6 Hz, 2H) and 8.63 (d, J=5.6 Hz, 2H, pyridyl).

(in CDCl₃) 2.18 (d, J=1.0 Hz, 3H, 2-CH₃), 2.23 (s, 3H, acetyl), 3.94 (br. s, 2H, 11-H), 6.98

(q, J=1.0 Hz, 1H, 3-H), 7.18 (d, J=2.2 Hz, 1H, 7-H), 7.31 (d, J=8.2 Hz, 1H, 10-H), 7.46

(dd, J₁=8.2 Hz, J₂=1.9 Hz, 1H, 9-H), 7.63 and 7.68 (d, J=8.8 Hz,

3k

C₁₈H₁₅BrN₄(367.3)

3l

C₁₈H₁₅BrN₄(367.3)

3m

3n

3o

C₁₉H₁₇BrN₄(381.3)

C₂₂H₁₆BrN₅(430.3)

C₂₀H₁₇ClN₄O(364.8)

acetylaminophenyl), 8.13 (br. s, 1H, NH-Ac).

2.32 (d, J=1.0 Hz, 3H, 2-CH₃), 4.48 (br. s, 2H, 11-H), 7.12 (q, J=1.0 Hz, 1H, 3-H),

7.55±7.80 (m, 5H), 7.90±8.00 (m, 2H).

2.35 (d, J=1.0 Hz, 3H, 3-CH₃), 4.45 (br. s, 2H, 11-H), 7.08 (d, J=2.1 Hz, 1H, 7-H), 7.43

(q, J=1.0 Hz, 1H, 2-H), 7.58 (d, J=8.3 Hz, 1H, 10-H), 7.60±7.73 (m, 4H), 7.95

(dd, J₁=8.3 Hz, J₂=2.1 Hz, 1H, 9-H).

3p ^a

3q ^a

C₁₈H₁₄BrCl₂N₃(423.1)

3r

4a

4b

4c

C₁₈H₁₄ClN₃(307.8)

C₁₇H₁₄ClN₅(323.8)

C₂₁H₁₅ClN₆(386.8)

2.30 (br. s, 3H, CH₃), 4.05 (br. s, 2H, 11-H), 6.63 (br. s, 1H, 2-H), 7.12 (d, J=1.9 Hz,

1H, 7-H), 7.50±7.85 (m, 7H).

2.40 (br. s, 3H, 3-CH₃), 4.13 (br, 2H, 11-H), 5.94 (br, 2H, NH₂), 6.64 (d, J=8.6 Hz, 2H) and 7.43

(d, J=8.6 Hz, 2H, aminophenyl), 7.22 (br. s, 1H, 7-H), 7.65 (br. s, 2H, 9-H and 10-H).

3.95±4.6 (br, 2H, 11-H), 6.67 (d, J=8.6 Hz, 2H) and 7.42 (d, J=8.6 Hz, 2H, aminophenyl), 7.28

(br. s, 1H, 7-H), 7.68 (br. s, 2H, 9-H and 10-H), 8.02 (br. s, 2H, pyridyl), 8.82 (br. s, 2H, pyridyl).

(in CDCl₃) 4.12 (br, 2H, 11-H), 6.72 (d, J=8.6 Hz, 2H), 6.79 (d, J=8.6 Hz, 2H) and 7.54

(d, J=8.6 Hz, 2H), 7.84 (d, J=8.6 Hz, 2H, aminophenyls), 7.32 (d, J=2.1 Hz, 1H, 7-H), 7.40

(d, J=8.2 Hz, 1H, 10-H), 7.50 (dd, J₁=8.2 Hz, J₂=2.1 Hz, 1H, 9-H)

C₂₂H₁₇ClN₆(400.9)

4d

C₁₈H₁₆ClN₅O(353.8)

3.35 (s, 3H, OCH₃), 4.20 (br. s, 2H, 11-H), 4.62 (s, 2H, O-CH₂), 5.98 (br. s, 2H, NH₂), 6.63

(d, J=8.7 Hz, 2H) and 7.42 (d, J=8.7 Hz, 2H, aminophenyl), 7.22 (br. s, 1H, 7-H),

7.62 (br. s, 2H, 9-H and 10-H).

^aHydrochloride salts.

They were treated ip 30 min before testing. In the

inclined screen test the untrained mice were placed on a

screen inclining 60^ꢀ. The number of falling mice was

noted within 60 s test period and scored as positive. In

the rotarod test mice were trained to do coordinated

motor movements for 120 s on the rod (3.5 cm diameter)

rotating at 15 rpm. The impairment of coordinated

motor movements was de®ned as inability of selected

mice to remain on the rotating rod for 120 s test period.

Transient occlusion of the middle cerebral artery (MCA)

in rats. Male CD RB rats, weighing 370±410 g, were

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G. Abraham et al. / Bioorg. Med. Chem. 8 (2000) 2127±2143

2142

used. The anaesthesia was induced by 5% and maintained

by 2% halothane in a mixture of 30% N₂O and 70%

O₂. The rectal body temperature was controlled and

kept at 37 ^ꢀC throughout the experimental period using

a thermostatically controlled blanket.

HPLC mobile phase containing a-methyl-dopamine as

internal standard. The samples were centrifuged (10,000Âg

for 15min at 4 ^ꢀC) and a 5 ml aliquots were injected into the

HPLC system. Calculated values are expressed as mg/g wet

tissue.

The occlusion of the MCA was achieved as follows:^22,23

the left common carotid artery (CCA), the extracranial

external carotid artery (ECA) and internal carotid

artery (ICA) were exposed through a midline incision.

The branches of the ECA were then isolated and coa-

gulated. The pterygopalatine artery, this posteriorly

directed extracranial branch of the ICA, was occluded

by a microvascular clip. Next a silk suture was tied

loosely around the mobilised ECA stump which was cut

at its ®rst branching (leaving a stump about 2±3 mm)

and used to introduce the embolus. The CCA was

occluded by microvascular clip and a 5 cm length of 4±0

mono®lament nylon suture, its tip rounded by heating

near a ¯ame, was introduced into the ECA lumen

(22 mm distal to the carotid bifurcation). This was

secured in place by tightening the silk ligature. At this

point the intraluminal suture has blocked the origin of the

MCA occluding of blood ¯ow from the ICA, anterior and

posterior cerebral arteries.

The concentrations of dopamine, DOPAC, HVA, 5-HT

and 5-HIAA in the samples were determined by HPLC/

electrochemistry according to Patthy and Gyenge.⁴¹The

HPLC system used in this study consisted of a Knauer

64 isocratic pump with a pulse dampener, a BAS Unijet

microbore injector equipped with a 5 ml sample loop, a

BAS microbore ODS 3 mm (100Â1 mm) analytical column.

For electrochemical detection, glassy carbon electrodes

(BAS LC-4C) set at +0.75 V against an Ag/AgCl

reference electrode were used. The mobile phase com-

position was aqueous buer:acetonitrile (95:5) (^v/_v). The

aqueous buer consisted of 3 mM HFBA (hepta-

¯uorobutyric acid) as pairing ion, 100 mM sodium

hydroxide, 0.1 g/l Na₂EDTA, and the pH was adjusted

to 4.3 with 42.5% (^w/_v) phosphoric acid. All other che-

micals used were of analytical reagent grade. One-way

analysis of variance (ANOVA) followed by the Duncan's

multiple range test was used for statistical analysis.

Acute toxicity in mice. The LD₅₀values were calculated

from lethality within 14 days after ip and po adminis-

tration of the drug.⁴²

60 min after embolization a second short and light

anaesthesia was induced by face mask. The nylon suture

and the microvascular clip on the CCA were carefully

removed, so the blood-¯ow from the CCA into the ICA

was restored. 24 h after reperfusion the rats were decapi-

tated. Their brains were rapidly removed 5 mm from the

frontal pole 3 coronal sections (2 mm thick) were cut out

from the cerebrum, immersed in a 2% solution of TTC

for vital staining and placed in a 37 ^ꢀC water-bath for 10

minutes and ®xed in 10% formalin solution. TTC

stained the intact areas of brain to deep red colour, but

did not stain the infarcted tissue.

Acknowledgements

The authors wish to express their thanks to the Hun-

garian National Committee for Technological Develop-

ment (OMFB) for the generous ®nancial support of the

project.

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New non competitive AMPA antagonists

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