PAF-antagonistic diazepines, methods of use

The invention discloses a method of treating an individual having a disorder responsive to PAF-antagonist activity by treating the individual with a compound of formula I. ##STR1## wherein A is an anellated and optionally substituted benzene or 5- or 6- membered heterocyclic ring; R.sub.5 and Z are each independently hydrogen or an optionally substituted C.sub.1 -C.sub.8 alkyl, alkenyl or alkynyl; R.sub.6 is an optionally substituted phenyl, or is thienyl or .alpha.-pyridyl; Y is ##STR2## and nontoxic, pharmaceutically acceptable acid addition salts thereof.

The invention relates to pharmaceutical compositions containing diazepines 
and having a PAF-antagonistic activity. Many of the diazepines are already 
known, while others are new. 
PAF (platelet activating factor) is acetylglyceryl-ester-phosphorylcholine 
(AGEPC). This compound is known as a powerful lipid mediator which is 
released by animal and human pro-inflammatory cells. Among these cells are 
found mainly basophilic and neutrophilic granulocytes, macrophages (from 
the blood or tissues) and thrombocytes which participate in inflammatory 
reactions. 
In pharmacological experiments, PAF shows the following activities: 
(a) bronchoconstriction which is about a hundred times more powerful than 
that of histamine; 
(b) a lowering of blood pressure, which is presumably due to direct 
peripheral vasodilation; 
(c) triggering of thrombocyte aggregation (demonstrated in vitro and in 
vivo); 
(d) pro-inflammatory activity by adhesion and aggregation of neutrophils, 
followed by the release of lysosomal enzymes and activation of arachidonic 
acid metabolism (tested in vitro). 
These experimentally demonstrable activities of PAF indicate, directly or 
indirectly, possible functions of this mediator in anaphylaxis, in the 
pathophysiology of bronchial asthma and in inflammation. 
PAF antagonists are needed to clarify further pathophysiological functions 
of this mediator in humans and animals and for treating pathological 
conditions and diseases in which PAF is involved. Examples of indications 
of a PAF antagonist include inflammatory processes of the tracheobronchial 
tree, acute and chronic bronchitis, bronchial asthma, anaphylactic 
conditions, allergies and inflammation in the mucous membranes and skin. 
Substances with a PAF-antagonistic activity are already known, for example 
substances whose structures resemble that of PAF (European Patent 
Application No. 94586; U.S. Pat. No. 4,329,302; Japanese Patent 
Application Nos. 57165394, 58035116, 58154512) and 
11-oxopyridoquinazolines (European Patent Application No. 94080). 
Moreover, compounds from the following ranges of indications have been 
investigated for a PAF-antagonistic activity: calcium antagonists, 
anti-allergic agents, anti-inflammatory agents and .alpha.-adrenergic 
agents 
Surprisingly, it has now been found that numerous substances from a group 
of compounds which has hitherto not been taken into consideration, namely 
the diazepines, have a powerful PAF-antagonistic activity. 
In the last twenty years, thousands of molecular variants of diazepines 
have been synthesized and tested pharmacologically, biochemically and in 
some cases clinically as well. The majority of diazepines, particularly 
those of the 1,4 series, have anticonvulsive, anxiolytic, muscle-relaxant 
and, to a greater or lesser extent, sedative activities (S. Garratini et 
al. "The Benzodiazepines", Raven Press, New York, 1973). Of the variety of 
structures, there are a few exceptions whose activity profile shows a 
different picture. Thus, diazepines which are effective against bilharzia 
(Drugs of the future 5, 43 (1980)) and against high blood pressure 
(European Patent Application No. 87850) are known. Other diazepines were 
found to have analgesic properties (D. Romer et al. Nature 298, 759 
(1982)) and an affinity with the dopamine receptor (Ruhland and Liepmann, 
C.I.N.P. Congress, Nuremberg (1983)). The PAF-antagonistic activity of 
diazepines was never considered. 
The invention therefore relates to a method of treating an individual 
having a disorder responsive to PAF-antagonist activity, which method 
comprises treating said individual with one or more compounds of formula: 
##STR3## 
In this formula: A is an anellated ring of the following formula 
##STR4## 
R.sub.1 and R.sub.2, are each independently hydrogen, a C.sub.1 -C.sub.8 
straight-chained or branched alkyl, alkenyl or alkynyl optionally mono- or 
polysubstituted by halogen, hydroxy, alkoxy, alkylmercapto, amino, 
alkylamino, dialkylamino, alkylcarbonyl, alkyloxycarbonyl or an acid amide 
group; a saturated carbocyclic or heterocyclic ring condensed on, which 
may contain oxygen, sulphur or nitrogen as a heteroatom while the 
nitrogen-containing ring may carry an alkyl group at the nitrogen atom; 
halogen, trifluoromethyl, nitro, cyano, optionally substituted amino, 
alkylmercapto, alkylcarbonyl, alkoxy, alkyloxycarbonyl or an acid amide 
group; 
R.sub.3 and R.sub.4, are each independently hydrogen, a C.sub.1 -C.sub.8 
straight-chained or branched alkyl, alkenyl or alkynyl optionally mono- or 
polysubstituted by halogen, hydroxy, alkoxy, alkylmercapto, amino, 
alkylamino, dialkylamino, alkylcarbonyl, alkyloxycarbonyl, cyano or an 
acid amide group; 
R.sub.5 and Z are each independently hydrogen, a C.sub.1 -C.sub.8 
straight-chained or branched alkyl, alkenyl or alkynyl optionally mono- or 
polysubstituted by halogen, hydroxy, alkoxy, alkylmercapto, amino, 
alkylamino, dialkylamino, alkylcarbonyl, alkyloxycarbonyl or an acid amide 
group; 
R.sub.6 is phenyl, which can optionally be substituted, preferably in the 2 
position, by methyl, methoxy, halogen, nitro or trifluoromethyl, or 
R.sub.6 can be thienyl or .alpha.-pyridyl; 
Y is 
##STR5## 
and non-toxic, pharmaceutically acceptable acid addition salts thereof. 
A subgeneric aspect of compounds of formula I relates to those wherein A is 
an anellated ring of the following formula 
##STR6## 
R.sub.1 and R.sub.2 are each independently hydrogen, a C.sub.1 -C.sub.8 
straight-chained or branched alkyl; halogen such as fluorine, chlorine or 
bromine, a nitro or alkoxy group or alkyloxycarbonyl, or together are a 
heterocyclic ring with at least 1 oxygen or sulphur heteroatom; 
R.sub.3 and R.sub.4 are each independently hydrogen, a C.sub.1 -C.sub.8 
straight-chained or branched alkyl which can optionally be further 
substituted by a cyano group; 
R.sub.5 is hydrogen or a C.sub.1 -C.sub.8 straight-chained or branched 
alkyl; 
R.sub.6 is a phenyl or an .alpha.-pyridyl group; 
Y is 
##STR7## 
and Z is hydrogen or a C.sub.1 -C.sub.8 straight-chained or branched 
alkyl optionally substituted by a dialkylamino group; and non-toxic, 
pharmaceutically acceptable acid addition salts thereof. 
Unless otherwise stated Hal is fluorine, chlorine, bromine or iodine; and 
alkyl is a C.sub.1 -C.sub.8 straight-chained or branched alkyl. The term 
"acid amide group" means an aminocarbonyl group which is mono- or 
disubstituted by alkyl at the nitrogen; this definition also covers an 
aminocarbonyl group which is closed, incorporating the nitrogen atom to 
form a five or six membered ring, while the hetero ring can optionally 
contain as a further heteroatom an oxygen, nitrogen or sulphur atom, and 
any nitrogen atom additionally present in the ring can carry an alkyl 
group as substituent. 
The above-mentioned compounds of formula I are known or may be obtained by 
known methods such as those described, for example, in Jeffrey W. H. 
Watthey et al "Heterocyclic Compounds" Volume 43 (1984), "Azepines" Part 
2, published by John Wiley & Sons Inc. and L. H. Sternbach "Progress in 
Drug Research" Volume 22 (1978) page 229-263, Birkhauser Verlag Basel. 
Compounds of formula I which contain a carboxylic acid function may be 
obtained as water-soluble alkali or alkaline earth metal salts. 
PHARMACOLOGICAL TEST RESULTS 
The PAF-antagonistic activity of compounds of formula I was investigated in 
terms of the inhibition of blood platelet aggregation in vitro and the 
antagonizing of PAF-induced bronchoconstriction in anesthetized guinea 
pigs. 
1. Inhibition of blood platelet aggregation in vitro 
The PAF-induced aggregation of human thrombocytes in vitro is used to 
determine PAF-antagonistic activity. To obtain thrombocyte-rich plasma 
(TRP) blood is taken from an uncongested vein using a plastic syringe 
containing 3.8% sodium citrate solution. The ratio of sodium citrate 
solution to blood is 1:9. After careful mixing the citrate blood is 
centrifuged for 20 minutes at 150 g (1200 rpm). The thrombocyte 
aggregation is measured using the method devised by Born and Cross (G. V. 
R. Born and M. J. Cross, J. Physiol. 168, 178 (1963)), by adding an 
aggregation initiator (PAF) to the TRP with constant stirring. 
The test substance is added 2 to 3 minutes before aggregation is initiated 
in a volume of 10 .mu.l. Either distilled water, ethanol and/or 
dimethylsulphoxide (each in suitable concentrations) can be used as 
solvent. 
Control mixtures contain corresponding volumes of these solvents. After the 
initial absorption has been recorded (2 to 3 minutes) aggregation is 
induced with PAF (5.times.10.sup.-8 M). 
The maximum of the first aggregation wave is used to assess the effects of 
the substance. The PAF-induced maximum absorption rate (=maximum 
aggregation.times.100%) is tested at the same time in a parallel mixture 
(=control mixture in one of the channels of the two-channel aggregometer) 
as each test mixture (second channel) and is used as a 100% value. 
The aggregation value attained under the effect of the test substance is 
given as a percentage of the control value, i.e. less than 100% in the 
case of an inhibition of aggregation and over 100% in the case of an 
increase. 
Each test substance is investigated at concentrations of from 10.sup.-3 to 
10.sup.-8 M with a random sampling of n=4 with regard to an inhibitory 
effect on the PAF-induced thrombocyte aggregation. Then a 
concentration-activity curve is drawn up using 3 concentrations and the 
IC.sub.50 is determined (concentration giving a 50% inhibition of 
aggregation). 
2. Antagonism of PAF-induced bronchoconstriction in anesthetized guinea 
pigs 
Spontaneously breathing male guinea pigs weighing from 300 to 450 g are 
pretreated orally with the test substance or a control vehicle 1 hour 
before the intravenous infusion with PAF (30 mg/kg.times.min). The test 
animals are then anesthetized by intraperitoneal route with 2 mg/kg of 
urethane, after which the jugular vein, carotid artery and trachea are 
cannulated. In the control animals the infusion of PAF induces a powerful 
and long-lasting bronchoconstriction which is measured in terms of the 
volume of the respiratory tract, compliance and resistance, and also a 
lowering of blood pressure. After about 7 to 8 minutes death occurs. These 
effects on respiration and blood pressure and the onset of death are 
prevented by the PAF antagonists described. Suitable dosages include from 
about 1 to about 50 mg/kg p.o. and 0.1 to 1.0 mg/kg i.v. 
The following Table gives the IC.sub.50 of a number of compounds of formula 
I: 
__________________________________________________________________________ 
##STR8## 
PAF-Antag. 
No. A Z R.sub.5 
Y R.sub.6 IC.sub.50 ; 
Mp. 
__________________________________________________________________________ 
.degree.C. 
1 Diazepam 
##STR9## CH.sub.3 
H 
##STR10## 
##STR11## 
250 128-130 
2 Nitrazepam 
##STR12## H H 
##STR13## 
##STR14## 
220 228-230 
3 Clonazepam 
##STR15## H H 
##STR16## 
##STR17## 
80 236-238 
4 Flunitrazepam 
##STR18## CH.sub.3 
H 
##STR19## 
##STR20## 
46 169-170 
5 Flurazepam Dihydrochlorid 
##STR21## 
##STR22## 
H 
##STR23## 
##STR24## 
80 218-220 
6 Bromazepam 
##STR25## CH.sub.3 
H 
##STR26## 
##STR27## 
1000 234-238 
7 
##STR28## CH.sub.3 
H 
##STR29## 
##STR30## 
380 221-222 
8 
##STR31## H H 
##STR32## 
##STR33## 
250 224 
9 
##STR34## H H 
##STR35## 
##STR36## 
760 231-233 
10 
##STR37## H H 
##STR38## 
##STR39## 
325 210-212 
11 
##STR40## CH.sub.3 
H 
##STR41## 
##STR42## 
170 112-114 
12 
##STR43## H H 
##STR44## 
##STR45## 
280 253-254 
13 
##STR46## CH.sub.3 
H 
##STR47## 
##STR48## 
200 160-161 
14 
##STR49## H H 
##STR50## 
##STR51## 
234 225-227 
15 
##STR52## H H 
##STR53## 
##STR54## 
870 255-256 
16 
##STR55## H H 
##STR56## 
##STR57## 
500 222-224 
17 
##STR58## H H 
##STR59## 
##STR60## 
1000 231-235 
18 
##STR61## CH.sub.3 
H 
##STR62## 
##STR63## 
110 205-208 
19 
##STR64## H H 
##STR65## 
##STR66## 
620 240-245 
20 
##STR67## H H 
##STR68## 
##STR69## 
250 280-281 
21 
##STR70## CH.sub.3 
H 
##STR71## 
##STR72## 
360 190-192 
22 
##STR73## H H 
##STR74## 
##STR75## 
580 226-228 
23 
##STR76## H C.sub.2 H.sub.5 
##STR77## 
##STR78## 
810 126-128 
24 
##STR79## H C.sub.2 H.sub.5 
##STR80## 
##STR81## 
530 234-236 
25 
##STR82## H H 
##STR83## 
##STR84## 
&gt;100 220-223 
26 
##STR85## H H 
##STR86## 
##STR87## 
79 225-227 
27 
##STR88## H H 
##STR89## 
##STR90## 
356 167 
28 
##STR91## H H 
##STR92## 
##STR93## 
&gt;100 197-198 
29 
##STR94## H H 
##STR95## 
##STR96## 
120 255-256 
30 
##STR97## H H 
##STR98## 
##STR99## 
&gt;50 334-335 
__________________________________________________________________________ 
The compounds of formula I can be administered to warm-blooded animals 
topically, orally, parenterally or by inhalation. The compounds are 
administered as active ingredients in conventional pharmaceutical 
preparations, e.g. in compositions comprising an inert pharmaceutical 
vehicle and an effective dose of the active substance, such as tablets, 
coated tablets, capsules, lozenges, powders, solutions, suspensions, 
aerosols for inhalation, ointments, emulsions, syrups, suppositories, etc. 
The effective dose range of the compounds according to the invention 
includes at least 10 to 500, preferably between 25 and 100 mg per dose for 
oral administration, and between 0.01 and 100, preferably between 0.1 and 
50 mg per dose for intravenous or intramuscular application. Solutions 
containing 0.01 to 1.0, preferably 0.1 to 0.5% of active substance can be 
used for inhalation.

Some examples of pharmaceutical compositions will now be given in which 
compounds of formula I are used as the active ingredient. 
Unless otherwise specifically stated, the parts given are parts by weight. 
1. Tablets 
The tablets include the following ingredients: 
______________________________________ 
Active substance of formula I 
0.050 parts 
Stearic acid 0.010 parts 
Dextrose 1.890 parts 
Total 1.950 parts 
______________________________________ 
PREATION 
The substances are mixed together in known manner and the mixture is 
compressed to form tablets, each of which weighs 1.95 g and contains 10 to 
50 mg of active substance. 
2. Ointment 
The ointment includes the following ingredients: 
______________________________________ 
Active substance of formula I 
2.000 parts 
Sodium pyrosulphite 0.050 parts 
Mixture (1:1) of cetyl alcohol 
20.000 parts 
and stearyl alcohol 
White Vaseline 5.000 parts 
Synthetic bergamot oil 
0.075 parts 
Distilled water ad 100.000 
parts 
______________________________________ 
PREATION 
The ingredients are intimately mixed in known manner to form an ointment of 
which 100 g contain 2.0 g of the active substance. 
3. Inhalation aerosol 
The aerosol includes of the following ingredients: 
______________________________________ 
Active substance of formula I 
1.00 parts 
Soya lecithin 0.20 parts 
Propellant gas mixture (Frigen .RTM.) 
ad 100.00 
parts 
11, 12 and 14 
______________________________________ 
PREATION 
The ingredients are mixed together in a manner known per se and the mixture 
is transferred into aerosol containers which contain a metering valve 
releasing between 1 and 20 mg of active substances on each actuation. 
4. Solution for ampoules 
The solution includes of the following ingredients: 
______________________________________ 
Active substance of formula I 
5.0 parts 
Polyethylene glycol 400.0 parts 
Benzyl alcohol 15.0 parts 
Ethyl alcohol (95%) 100.0 parts 
Sodium benzoate 50.0 parts 
Benzoic acid 1.2 parts 
Doubly distilled water 
ad 1000.0 
parts 
______________________________________ 
PREATION 
The active substance is dissolved in benzyl alcohol and then polyethylene 
glycol and alcohol are added. The sodium benzoate and benzoic acid are 
dissolved in 250 ml of water, combined with the above solution and made up 
to 1000 ml with water. The resulting solution is filtered free from 
pyrogens and the filtrate is transferred under aseptic conditions into 1 
ml ampoules which are then sterilized and sealed by fusion. Each ampoule 
contains 1 to 5 mg of the active substance. 
5. Suppositories 
Each suppository includes: 
______________________________________ 
Active substance of formula I 
1.0 parts 
Cocoa butter (m.p. 36-37.degree. C.) 
1200.0 " 
Carnauba wax 5.0 " 
______________________________________ 
PREATION 
The cocoa butter and carnauba wax are melted together. At 45.degree. C. the 
active substance is added and the mixture is stirred until a complete 
dispersion is formed. The mixture is poured into moulds of a suitable size 
and the suppositories ae suitably packaged.