Note: Descriptions are shown in the official language in which they were submitted.
Le A 36 571 CA 02519987 2005-09-21
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Benzofuro-1,4-diazepin-2-one derivatives
The present invention relates to novel benzofuro-1,4-diazepin-2-one
derivatives,
process for their preparation and their use as P2X4 receptor antagonists for
producing
medicaments for the treatment and/or prophylaxis of diseases, in particular of
arteriosclerosis, restenosis and other inflammatory disorders.
Arteriosclerosis is a multifactorial disorder whose development is influenced
by
many different factors. Inflammatory processes inter alia play a central part
in this,
with inflammation-inducing cytokines such as CD40L and IFN~y being involved
[P. Libby, Nature 420 (6917): 868-74 (2002)]. The purinergic receptor P2X4
belongs
to the P2X family. To date, six different P2X receptors have been described in
humans. They take the form of calcium-permeable channels which can be
activated
by ATP [F. Di Virgilio et al., Blood 97 (3): 587-600 (2001); R.A. North,
A. Surprenant, Annu. Rev. Pharmacol. Toxicol. 40: 563-80 (2000)]. It has been
possible to show that there is high-level expression of the P2X4 channel in
highly
vascularized organs and vessels [K. Yamamoto et al., Circ. Res. 87(5): 385-91
(2000)]. Surprisingly, the P2X4 receptor is also expressed on human monocytes.
A
five-fold increase in P2X4 expression was observable on incubation of human
monocytes with CD40L and IFNy. A high level of expression of the P2X4 receptor
has also been found in the vessel wall of the aorta of rabbits after damage by
balloon
angioplasty and cholesterol feeding [T.J. Pulvirenti et al., J. Neurocytol. 29
(9): 623-
31 (2000)] and in the arteriosclerotically altered vessel segments of the apoE
knockout mouse. Since activated monocytes assume a key function in the early
stage
of atherogenesis and in restenosis, and monocytes are activated by said
cytokines,
inhibition of the activation leads to reduction in atherogenesis [P. Libby,
Nature 420
(6917): 868-74 (2002)]. Since evidently monocyte activation by CD40L and IFNy
is
associated via the increase in P2X4 receptor expression and the increased
calcium
influx connected thereto, blockade of P2X4 receptors ought to reduce
inflammatory
processes [F. Di Virgilio, A. Solini, Br. J. Pharmacol. 135 (4): 831-42
(2002)]. Thus,
diseases in which inflammatory processes are involved might be treatable by
blockade of P2X4 receptors.
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Besides the indications of arteriosclerosis and restenosis, and their sequelae
(stroke,
angina pectoris, myocardial infarction, renal failure, impaired perfusion of
limbs),
treatment of other inflammatory disorders such as, for example, psoriasis and
rheumatoid arthritis might thus also be possible via the mechanism mentioned.
The synthesis of some benzofuro[3,2-eJ-1,4-diazepin-2-one derivatives is
described
in J. Heterocyclic Chem. 6 189-90 (1979) and ibid. 20 1251-1254 (1983).
Benzofuro-1,4-diazepine derivatives with an antiulcer effect are disclosed in
EP 350 131-A.
The present invention relates to compounds of the general formula (I)
R
(~~
in which
1 S R' is halogen
and
R2 is hydrogen, halogen, nitro, cyano or a group of the formula -C(O)-OR3,
-C(O)-NR4R5, -SOZ-OR3 or -SOZ-NR4R5, in which
R3, R4 and RS are independently of one another hydrogen or (C1-C6)-alkyl,
or
R' is hydrogen
WO 2004/085440 CA 02519987 2005-09-21 PCT/EP2004/002580
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and
RZ is halogen, nitro, cyano or a group of the formula -C(O)-OR3, -C(O)-NR4R5,
-S02-OR3 or -SOZ-NR4R5, in which
R3, R4 and RS are independently of one another hydrogen or (C1-C6)-alkyl.
The compounds of the invention may also exist in the form of their salts,
solvates
and solvates of their salts.
For the purposes of the present invention, the substituents generally have the
following meaning:
~C1-C6)-alkyl and (C1-C4 -alk 1 are for the purposes of the invention a
straight-chain
or branched alkyl radical having respectively 1 to 6 and 1 to 4 carbon atoms.
A
straight-chain or branched alkyl radical having 1 to 4 carbon atoms is
preferred.
Preferred examples which may be mentioned are: methyl, ethyl, n-propyl,
isopropyl
and tert-butyl.
Halogen includes for the purposes of the invention fluorine, chlorine, bromine
and
iodine. Chlorine or bromine are preferred.
The compounds of the invention may, depending on the substitution pattern;
exist in
stereoisomeric forms which either are related as image and mirror image
(enantiomers) or which are not related as image and mirror image
(diastereomers).
The invention relates both to the enantiomers or diastereomers and to
respective
mixtures thereof. The racemic forms can, just like the diastereomers, be
separated in
a known manner into the stereoisomerically pure constituents.
Certain compounds may moreover exist in tautomeric forms. This is known to the
skilled worker, and such compounds are likewise included within the scope of
the
invention.
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The compounds of the invention may also exist as salts. Physiologically
acceptable
salts are preferred for the purposes of the invention.
Physiologically acceptable salts may be salts of the compounds of the
invention with
inorganic or organic acids. Preference is given to salts with inorganic acids
such as,
for example, hydrochloric acid, hydrobromic acid, phosphoric acid or sulphuric
acid,
or salts with organic carboxylic or sulphonic acids such as, for example,
acetic acid,
propionic acid, malefic acid, fumaric acid, malic acid, citric acid, tartaric
acid, lactic
acid, benzoic acid, or methanesulphonic acid, ethanesulphonic acid,
benzenesulphonic acid, toluenesulphonic acid or naphthalenedisulphonic acid.
Physiologically acceptable salts may likewise be salts of the compounds of the
invention with bases, such as, for example, metal or ammonium salts. Preferred
examples are alkali metal salts (e.g. sodium or potassium salts), alkaline
earth metal
1 S salts (e.g. magnesium or calcium salts), and ammonium salts derived from
ammonia
or organic amines, such as, for example, ethylamine, di- or triethylamine,
ethyldiisopropylamine, monoethanolamine, di- or triethanolamine,
dicyclohexylamine, dimethylaminoethanol, dibenzylamine, N-methylmorpholine,
dehydroabietylamine, 1-ephenamine, N-methylpiperidine, arginine, lysine,
ethylenediamine or 2-phenylethylamine.
The compounds of the invention and their salts may also exist in the form
their
solvates, especially in the form of their hydrates.
Preferred compounds of the general formula (I) are those in which
R' is chlorine or bromine
and
RZ hydrogen, chlorine, bromine, vitro, cyano or a group of the formula
-C(O)-OR3 or -C(O)-NR4R5, in which
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R3, R4 and RS are independently of one another hydrogen or (C~-C4)-alkyl,
or
S R' is hydrogen
and
R2 is chlorine, bromine, nitro, cyano or a group of the formula -C(O)-OR3 or
-C(O)-NR4R5, in which
R3, R4 and RS are independently of one another hydrogen or (C1-C4)-alkyl.
Particularly preferred compounds are those of the general formula (Ia)
R
in which
R' is chlorine or bromine
and
RZ is hydrogen, chlorine, bromine, nitro or cyano,
or
R' is hydrogen
WO 2004/085440 CA 02519987 2005-09-21 PCT/EP2004/002580
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and
R2 is chlorine, bromine, nitro or cyano.
A process for preparing the compounds of the invention has also been found,
characterized in that compounds of the formula (II)
~ CN
R'
OH
in which R1 has the meanings indicated above,
are reacted in an inert solvent in the presence of a base with a compound of
the
formula (III)
. . _._. _ _.
_2
in which R2 has the meanings indicated above, and
X1 is a suitable leaving group such as, for example, chlorine, bromine or
iodine,
initially to give compounds of the formula (IV)
CN
R'
O
in which R1 and R2 have the meanings indicated above,
WO 2004/085440 CA 02519987 2005-09-21 PCT/EP2004/002580
the latter are then cyclized, with intermediate isolation or in a one-pot
reaction, in the
presence of a base to compounds of the formula (V)
R
M
in which R' and R2 have the meanings indicated above,
subsequently converted in an inert solvent in the presence of a base with a
compound
of the formula (VI)
p
x3
x
in which
X2 and X3 are identical or different and are a suitable leaving group such as,
for
example, chlorine, bromine or iodine,
into compounds of the formula (VII)
X3
R
in which R1, R2 and X3 have the meanings indicated above,
H~O
--~N
WO 2004/085440 CA 02519987 2005-09-21 PCT/EP2004/002580
_g.
finally reacted with ammonia in an inert solvent for cyclization, and the
resulting
compounds of the formula (I) are converted where appropriate with the
appropriate
solvents and/or bases or acids into their solvates, salts and/or solvates of
the salts.
Suitable solvents for the process step (II) + (III) -~ (IV) are inert organic
solvents
which are not changed under the reaction conditions. These include
halohydrocarbons such as dichloromethane, trichloromethane,
tetrachloromethane,
trichloroethane, tetrachloroethane, 1,2-dichloroethane or trichloroethylene,
ethers
such as diethyl ether, dioxane, tetrahydrofuran, glycol dimethyl ether or
diethylene
glycol dimethyl ether, hydrocarbons such as benzene, xylene, toluene, hexane,
cyclohexane or petroleum fractions, esters such as ethyl acetate, ketones such
as
acetone or 2-butanone, heteroaromatics such as pyridine, amides such as
dimethylformamide, dialkyl sulphoxides such as dimethyl sulphoxide, or
nitrites
such as acetonitrile. It is likewise possible to employ mixtures of said
solvents.
Dimethylformamide is preferred.
The usual inorganic or organic bases are suitable as base for the process step
(II) +
(III) -~ (IV). These preferably include alkali metal or alkaline earth metal
carbonates
such as sodium, potassium or calcium carbonate, alkali metal hydrides such as
sodium hydride, amides such as lithium bis(trimethylsilyl)amide or lithium
diisopropylamide, or organic amines such as pyridine, 4-N,N
dimethylaminopyridine, 4-pyrrolidinopyridine, triethylamine,
ethyldiisopropylamine,
N methylmorpholine, N methylpiperidine, 1,5-diazabicyclo[4.3.0]non-S-ene (DBN)
or 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). Triethylamine is particularly
preferred.
The base is employed in this case in an amount of from 1 to 5 mot, preferably
in an
amount of from 1 to 2 mot, based on 1 mot of the compound of the formula (II).
The reaction generally takes place in a temperature range from 0°C to
+150°C,
preferably in a temperature range from +20°C to +100°C. The
reaction can be carried
out under atmospheric, elevated or reduced pressure (e.g. from 0.5 to 5 bar).
It is
generally carried out under atmospheric pressure.
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Suitable solvents for the process step (IV) -> (V) are likewise inert organic
solvents
which are not changed under the reaction conditions. These include
halohydrocarbons such as dichloromethane, trichloromethane,
tetrachloromethane,
trichloroethane, tetrachloroethane, 1,2-dichloroethane or trichloroethylene,
ethers
such as diethyl ether, dioxane, tetrahydrofuran, glycol dimethyl ether or
diethylene
glycol dimethyl ether, alcohols such as methanol, ethanol, n-propanol,
isopropanol,
n-butanol or tent-butanol, hydrocarbons such as benzene, xylene, toluene,
hexane,
cyclohexane or petroleum fractions, ketones such as acetone or 2-butanone,
heteroaromatics such as pyridine, amides such as dimethylformamide, dialkyl
sulphoxides such as dimethyl sulphoxide, or nitriles such as acetonitrile. It
is
likewise possible to employ mixtures of said solvents. Methanol and ethanol
are
preferred.
The usual inorganic or organic bases are suitable as base for the process step
(IV) -~ (V). These preferably include alkali metal or alkaline earth metal
carbonates
such as sodium, potassium or calcium carbonate, alkali metal hydroxides such
as
lithium, sodium or potassium hydroxide, alkali metal alcoholates such as
sodium or
potassium methanolate, sodium or potassium ethanolate or potassium tert-
butoxide,
alkali metal hydrides such as sodium hydride, amides such as lithium
bis(trimethyl-
silyl)amide or lithium diisopropylamide, or organic amines such as pyridine, 4-
N,N
dimethylaminopyridine, 4-pyrrolidinopyridine, triethylamine,
ethyldiisopropylamine,
N methylmorpholine, N methylpiperidine, 1,5-diazabicyclo[4.3.OJnon-5-ene (DBN)
or 1,8-diazabicyclo[5.4.0]undec-7-ene (DBLI). Sodium methanolate and sodium
ethanolate are particularly preferred.
The base is employed in this case in an amount of from 0.5 to 5 mol,
preferably in an
amount of from 1 to 2 mol, based on 1 mol of the compound of the formula (IV).
The reaction generally takes place in a temperature range from 0°C to
+120°C,
preferably in a temperature range from +20°C to +100°C. The
reaction can be carned
out under atmospheric, elevated or reduced pressure (e.g. from 0.5 to 5 bar).
It is
generally carned out under atmospheric pressure.
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Suitable solvents for the process step (V) + (VI) -~ (VII) are all inert
organic
solvents which are not changed under the reaction conditions. These include
halohydrocarbons such as dichloromethane, trichloromethane,
tetrachloromethane,
trichloroethane,- tetrachloroethane, 1,2-dichloroethane or trichloroethylene,
ethers
such as diethyl ether, dioxane, tetrahydrofuran, glycol dimethyl ether or
diethylene
glycol dimethyl ether, hydrocarbons such as benzene, xylene, toluene, hexane,
cyclohexane or petroleum fractions, esters such as ethyl acetate, ketones such
as
acetone or 2-butanone, amides such as dimethylformamide, dialkyl sulphoxides
such
as dimethyl sulphoxide, or nitrites such as acetonitrile. It is likewise
possible to
employ mixtures of said solvents. Dichloromethane and trichloromethane are
preferred.
The usual inorganic or organic bases are suitable as base for the process step
(V) +
(VI) ~ (VII). These preferably include alkali metal or alkaline earth metal
carbonates and bicarbonates such as sodium, potassium or calcium carbonate and
sodium or potassium bicarbonate, alkali metal hydrides such as sodium hydride,
amides such as lithium bis(trimethylsilyl)amide or lithium diisopropylamide,
or
organic amines such as pyridine, 4-N,N dimethylaminopyridine, 4-pyrrolidino-
pyridine, triethylamine, ethyldiisopropylamine, N methylmorpholine, N methyl-
piperidine, 1,5-diazabicyclo[4.3.0]non-5-ene (DBN) or 1,8-diazabicyclo-
[5.4.0]undec-7-ene (DBi~. Sodium bicarbonate is particularly preferred.
The base is employed in this case in an amount of from 1 to 10 mot, preferably
in an
amount of from 1 to S mot, based on 1 mot of the compound of the formula (V).
The reaction generally takes place in a temperature range from -20°C to
+50°C,
preferably in a temperature range from -20°C to +20°C. The
reaction can be carried
out under atmospheric, elevated or reduced pressure (e.g. from 0.5 to 5 bar).
It is
generally carried out under atmospheric pressure.
Suitable solvents for the process (VII) -~ (I) are all inert solvents which
are not
changed under the reaction conditions. These include halohydrocarbons such as
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CA 02519987 2005-09-21
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dichloromethane, trichloromethane, tetrachloromethane, trichloroethane,
tetrachloroethane, 1,2-dichloroethane or trichloroethylene, ethers such as
diethyl
ether, dioxane, tetrahydrofuran, glycol dimethyl ether or diethylene glycol
dimethyl
ether, or hydrocarbons such as benzene, xylene, toluene, hexane, cyclohexane
or
petroleum fractions. It is likewise possible to employ mixtures of said
solvents.
Dioxane is preferred.
The compounds of the formulae (II), (III) and (VI) are commercially available,
known from the literature or can be prepared by conventional literature
methods [cf.,
for example, J. Med. Chem. 13, 674-680 (1970)].
The process of the invention can be illustrated by the following formula
scheme:
Scheme
CN 1. ~tEt3, DPJ1F
OH 2. NaOG2H~, Ethanol
O ,Br
~ ~Br
Br " NH
3
NaHC03
The compounds of the invention show a valuable range of pharmacological
effects
which could not have been predicted and are therefore suitable for use as
medicaments for the treatment and/or prophylaxis of diseases in humans and
animals.
H~O
-~.~N
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The compounds of the invention act as antagonists of the P2X4 receptor.
Because of their pharmacological properties, the compounds of the invention
can be
employed alone or in combination with other medicaments for the treatment
and/or
prophylaxis of inflammatory disorders. They are particularly suitable for the
treatment of chronically inflammatory disorders of the vessel intima such as,
for
example, arteriosclerosis and restenosis, of inflammatory disorders of the
central
nervous system such as, for example, multiple sclerosis and pain, of
inflammatory
disorders of the connective tissue such as, for example, rheumatoid arthritis,
chronic
polyarthritis, panniculitis and tendinitis, of Bechterew's disease, of
inflammatory
disorders of the skin such as psoriasis and neurodermatitis, of chronically
inflammatory bowel disorders such as enteritis, enterocolitis, Crohn's disease
and
ulcerative colitis, of inflammatory disorders of the small airways and of
myositis and
endocarditis.
The compounds of the invention can be administered alone or, if required, in
combination with other active ingredients, preferably from the group of CETP
inhibitors, antidiabetics, antioxidants, thyroid hormones and/or thyroid
mimetics,
inhibitors of HMG-CoA reductase, inhibitors of HMG-CoA reductase gene
expression, squalene synthase inhibitors, ACAT inhibitors, cholesterol
absorption
inhibitors, fibrates, MTP inhibitors, triglyceride-lowering agents, nicotinic
acid and
derivatives thereof, platelet aggregation inhibitors, anticoagulants, calcium
antagonists, ACE inhibitors, angiotensin II receptor antagonists, beta Mockers
and
steroidal and nonsteroidal antiinflammatory drugs.
The activity of the compounds of the invention can be tested for example by
the tests
described in the example section.
The present invention further relates to medicaments which comprise at least
one
compound of the invention, preferably together with one or more
pharmacologically
acceptable excipients or Garners, and to the use thereof for the
aforementioned
purposes.
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The compounds of the invention may have systemic and/or local effects. They
can
for this purpose be administered in a suitable way, such as, for example, by
the oral,
parenteral, pulmonary, nasal, sublingual, lingual, buccal, rectal, dermal,
transdermal,
conjunctival or otic route or as implant or stmt. Oral administration is
preferred.
S
For these administration routes it is possible to administer the active
ingredients in
suitable administration forms. Suitable for oral administration are
administration
forms which function according to the prior art and deliver the active
ingredient
rapidly and/or in modified fashion, such as, for example, tablets (uncoated
and
coated tablets, e.g. with coatings which are resistant to gastric juice),
capsules, sugar-
coated tablets, granules, pellets, powders, emulsions, suspensions and
solutions.
Parenteral administration can take place with avoidance of an absorption step
(e.g.
intravenous, intraarterial, intracardiac, intraspinal or intralumbar) or with
inclusion of
an absorption (e.g. intramuscular, subcutaneous, intracutaneous or
intraperitoneal).
Administration forms suitable for parenteral administration are, inter alia,
preparations for injection and infusion in the form of solutions, suspensions,
emulsions, lyophilisates and sterile powders.
Examples suitable for the other administration routes are pharmaceutical forms
for
inhalation (inter alia powder inhalers, nebulizers), nasal drops/solutions,
sprays,
tablets or capsules for lingual, sublingual or buccal administration,
suppositories,
preparations for the ears and eyes, vaginal capsules, aqueous suspensions
(lotions,
shaking mixtures), lipophilic suspensions, ointments, creams, milk, pastes,
dusting
powder, implants or stems.
The active ingredients can be converted in a manner known per se into the
stated
administration forms. This takes place with use of inert nontoxic,
pharmaceutically
suitable excipients. These include inter alia carriers (e.g. microcrystalline
cellulose),
solvents (e.g. liquid polyethylene glycols), emulsifiers (e.g. sodium dodecyl
sulphate), dispersants (e.g. polyvinylpyrrolidone), synthetic and/or natural
biopolymers (e.g. albumin), stabilizers (e.g. antioxidants such as, for
example,
ascorbic acid), colours (e.g. inorganic pigments such as iron oxides) or taste-
and/or
odour-masking agents.
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It has generally proven advantageous to administer on parenteral
administration
amounts of about 0.001 to 10 mg/kg, preferably about 0.005 to 3 mg/kg, of body
weight to achieve effective results. The amount of oral administration is
about 0.001
to 100 mg/kg, preferably about 0.005 to 30 mg/kg, of body weight.
It may nevertheless be necessary where appropriate to deviate from the amounts
mentioned, specifically as a function of the body weight, administration
route,
individual response to the active ingredient, type of preparation and time or
interval
over which administration takes place. Thus, in some cases it may be
sufficient to
make do with less than the aforementioned minimum amount, whereas in other
cases
the stated upper limit must be exceeded. It may in the event of administration
of
larger amounts be advisable to divide these into a plurality of individual
doses over
the day.
The percentage data in the following tests and examples are, unless indicated
otherwise, percentages by weight; parts are parts by weight. Solvent ratios,
dilution
ratios and concentration data for liquid-liquid solutions are in each case
based on
volume.
The following exemplary embodiments illustrate the invention. The invention is
not
confined to the examples.
Abbreviations:
ICI chemical ionization (in MS)
DCI direct chemical ionization (in MS)
DMF N,N dimethylformamide
DMSO dimethyl sulphoxide
ESI electrospray ionization (in MS)
LC/MS coupled liquid chromatography-mass spectroscopy
MS mass spectroscopy
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NMR nuclear magnetic resonance spectroscopy
Rf retention index (in TLC)
Rt retention time (in LC/MS)
TLC thin layer chromatography
LC/MS methods:
Method l:
MS instrument type: Micromass ZQ; HPLC instrument type: Waters Alliance 2790;
column: Uptisphere C 18, 50 mm x 2.0 mm, 3.0 ~,m; eluent B: acetonitrile +
0.05%
formic acid, eluent A: water + 0.05% formic acid; gradient: 0.0 min 5% B --~
2.0 min
40% B -~ 4.5 min 90% B -~ 5.5 min 90% B; oven: 45°C; flow rate: 0.0 min
0.75 ml/min ~ 4.5 min 0.75 ml/min -~ 5.5 min 1.25 ml/min; UV detection: 210
nm.
Method 2:
Instrument: Micromass Platform LCZ with HPLC Agilent series 1100; column:
Grom-SIL 120 ODS-4 HE, 50 mm x 2.0 mm, 3 pm; eluent A: 1 1 of water + 1 ml of
50% formic acid, eluent B: 1 1 of acetonitrile + 1 ml of 50% formic acid;
gradient:
0.0 min 100% A -~ 0.2 min 100% A ~ 2.9 min 30% A ~ 3.1 min 10% A ~
4.5 min 10% A; oven: 55°C; flow rate: 0.8 ml/min; UV detection: 208-
400 nm.
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A. Starting compounds:
Example I
(3-Aminobenzofuran-2-yl)-(3-bromophenyl)methanone
~!H_
2.00 g (I6.8 mmol) of 2-hydroxybenzonitrile, 4.67 g (16.8 mmol) of 3-bromo-
phenacyl bromide and 1.87 g (18.5 mmol) of triethylamine are stirred in 20 ml
of
dimethylformamide at 70°C for 2 h. After addition of 100 ml of ethyl
acetate, the
reaction mixture is washed with water (3 x 100 ml) and saturated sodium
chloride
solution (2 x 100 ml). The organic phase is dried over magnesium sulphate, and
the
solvent is removed under reduced pressure. 'The residue is dissolved in 30 ml
of
ethanol. After addition of 5.98 g (18.5 mmol) of sodium ethanolate, the
mixture is
heated under reflux for 2 h. 50 ml of ethyl acetate are added, and the mixture
is
washed with water (3 x 100 ml) and saturated sodium chloride solution (2 x 100
ml).
The organic phase is dried over magnesium sulphate. After removal of the
solvent
under reduced pressure, 5.08 g (92% of theory) of the desired product are
obtained.
MS (DCI): m/z = 315.9 [M+H]+
'H-NMR (200 MHz, DMSO-d6): 8 = 7.27-7.36 (m, 1H); 7.49-7.65 (m, SH); 7.77-
7.84 (m, 1H); 8.04-8.20 (m, 3H).
Example II
2-Bromo-N {2-[(3-bromophenyl)carbonyl]benzofuran-3-yl}acetamide
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3.53 g (17.5 mmol) of bromoacetyl bromide are added to a mixture of 5.03 g
(15.9 mmol) of the compound from Example I and 5.35 g (63.6 mmol) of sodium
bicarbonate in 250 ml of chloroform at 0°C. The mixture is stirred at
0°C for 1 h.
After removal of the solvent under reduced pressure, 200 ml of ethyl acetate
are
added, and the mixture is washed with saturated sodium bicarbonate solution
(100
ml) and saturated sodium chloride solution (100 ml). The organic phase is
dried over
magnesium sulphate. After removal of the solvent under reduced pressure, 5.81
g
(83% of theory) of the desired product are obtained.
MS (CI): m/z = 436 [M+H]+
1H-NMR (200 MHz, DMSO-d6): 8 = 4.20 (s, 2H); 7.37-8.14 (m, 8H); 10.98 (s, 1H).
B. Exemplary embodiments
Example 1
5-(3-Bromophenyl)-1,3-dihydro-2H benzofuro[3,2-a]-1,4-diazepin-2-one
O
~l I
I
Br
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245 ml (123 mmol) of a 0.5 M solution of ammonia in dioxane are added to 5.37
g
(12.3 mmol) of the compound from Example II in 100 ml of diethyl ether. The
mixture is stirred at room temperature for 2 days. 100 ml of ethyl acetate are
added,
and the mixture is washed with water (3 x 250 ml) and saturated sodium
chloride
solution. The organic phase is dried over magnesium sulphate, and the solvent
is
removed under reduced pressure. 1.81 g (42% of theory) of the desired product
are
obtained.
Rf= 0.24 (dichloromethane/methanol 100:2)
MS (ESn: m/z = 355 [M+H]+
LC/MS (method 1): Rt = 3.47 min, m/z = 354 [M]+
1H-NMR (200 MHz, DMSO-d6): 8 = 4.45 (s, 2H); 7.37-7.80 (m, 6H); 7.88-7.93 (m,
1H); 7.96-8.03 (m, 1H); 11.59 (s, 1H).
The following are obtained in an analogous manner:
Example 2
1,3-Dihydro-5-(3-nitrophenyl)-2H benzofuro[3,2-a]-1,4-diazepin-2-one
O
%w
2
LC/MS (method 2): Rt = 3.5 min, m/z = 321 [M+H]+.
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Example 3
9-Bromo-1,3-dihydro-5-phenyl-2H benzofuro[3,2-a]-I,4-diazepin-2-one
Br
LC/MS (method 2): Rt = 3.5 min, m/z = 355 [M+H]+.
C. Description of the biological tests:
a) Cellular assay
The P2X4 receptor is a ligand-activated ion channel. Binding of the agonist
ATP
leads to activation of the P2X4 receptor, opening of the ion channel and
influx of 1~
extracellular calcium into the cell. This calcium influx is measured with the
aid of the
calcium-sensitive photoprotein acquorin. For this purpose, a recombinant CHO
cell
line (Chinese hamster ovary cells) with constitutive expression of the human
P2X4
receptor and of apoaequorin was prepared.
The experiment is carried out by seeding the CHO cells I-2 days beforehand
onto
microtitre plates in the 96-, 384- or 1536-well format, and specifically, in
accordance
with the microtitre plate format used, with 5000 (96 format), 2000 (384
format) or
500 (1536 format) cells per well. On the day of the experiment, the cell
culture
medium is removed and the cells are incubated with 5 ~g/ml coelenterazine in
physiological saline (Tyrode buffer) for 4 hours. Test substances are added 5
minutes
before the actual experiment. The P2X4 receptor is then activated by adding
ATP in a
concentration of 1-2 Vim, and the ATP-induced calcium signal is measured as
aequorin luminescence in a luminometer.
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Substances with antagonistic activity on the P2X4 receptor can inhibit the ATP-
induced calcium signal either by interference with the binding of ATP to the
P2X4
receptor, by preventing channel opening or by blocking calcium influx through
the
opened channel.
Exemplary embodiments 1-3 show ICso values of respectively 0.5, 2 and 0.6 pm
in
this test.
b) ATP-induced oxygen free radical formation (ROS) in primary human monocytes:
The assay is carried out in Hank's balanced salt solution (HBSS) to which 10
mM
glucose is added. Monocytes are isolated for example by use of the "Becton
Dickinson Vacutainer System" as described by the manufacturer, and suspended
in
HBSS. The oxygen free radicals are detected in principle by the luminol-
enhanced
chemiluminescence method in the presence of horse radish peroxidase (HRPO)
[H. Lundqvist, C. Dahlgren, Free Radic. Biol. Med. 20 (6): 785-92 (1996)].
Firstly, the substance to be investigated, luminol (final concentration 50
Vim), HRPO
(final concentration 10 U/ml) are incubated with 5 x 105 monocytes at
37°C for
15 min. ATP is then added to the test mixture (final concentration 100 p.M).
The
final volume in the test mixture is 200 p1. Immediately after addition of ATP,
the
ROS formation is followed using a microplate luminometer over a period of
120 seconds.
c~ ATP-induced chemotaxis of primary human monocytes:
Monocytes are isolated from blood by standard methods. The chemotaxis of the
monocytes is observed in a Transwell system [C.C. Bleul et al., J. Exp. Med.
184:
1101-1109 (1996)]. The membrane used (pore size 3 Vim, polyethylene
terephthalate,
from Falcon) is initially coated with fibronectin. 105 monocytes in RPMI 1640
medium are put into the upper chamber. The lower chamber contains varying
concentrations of stimulus or constant stimulus concentration (500 pM ATP or
10 nM MCP-1) and varying concentrations of the test substance. The substances
to
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be investigated are present in both chambers. The test mixture is incubated at
37°C
with 5% C02 for 3 h [W. Falk et al., J. Immunol. Methods 38: 239-247 (1980)].
After
the incubation, the cells which have migrated into the lower chamber are
determined.
D. Exemplary embodiments of pharmaceutical comuositions:
The compounds of the invention can be converted into pharmaceutical
preparations
in the following ways:
Tablet:
Composition:
100 mg of the compound of Example 1, 50 g of lactose (monohydrate), 50 mg of
corn starch (native), 10 mg of polyvinylpyrrolidone (PVP 25) and 2 mg of
magnesium stearate.
Tablet weight 212 mg. Diameter 8 mm, radius of curvature 12 mm.
Production:
A mixture of active ingredient, lactose and starch is granulated with a 5%
strength
solution (m/m) of the PVP in water. The granules are dried and then mixed with
the
magnesium stearate for 5 min. This mixture is compressed in a conventional
tablet
press (see above for format of the tablet). A compressive force of 15 kN is
used as
guideline for the compression.
Suspension which can be administered orall,
Composition:
1000 mg of the compound of Example 1, 1000 mg of ethanol (96%), 400 mg of
Rhodigel~ (xanthan gum from FMC, Pennsylvania, USA) and 99 g of water.
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ml of oral suspension are equivalent to a single dose of 100 mg of the
compound
of the invention.
S Production:
The Rhodigel is suspended in ethanol, and the active ingredient is added to
the
suspension. The water is added while stirring. The mixture is stirred for
about 6 h
until the swelling of the Rhodigel is complete.
