Pyrrolopyrrolone derivatives as inhibitors of neutrophil...

Details Pyrrolopyrrolone derivatives as inhibitors of neutrophil... Pyrrolopyrrolone derivatives as inhibitors of neutrophil...

2000-06-02

2001-05-08

Lambkin, Deborah C. (Department: 1626)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Heterocyclic carbon compounds containing a hetero ring...

C514S322000, C514S414000, C546S199000, C548S453000

Reexamination Certificate

active

06228853

ABSTRACT:

The present invention relates to therapeutically active bicyclic compounds, processes for their manufacture, pharmaceutical formulations containing them and their use in chemotherapy. In particular, we have found a group of novel bicyclic compounds which are effective in treating inflammatory diseases.
Inflammation is a primary response to tissue injury or microbial invasion and is characterised by circulating leukocytes binding to and extravasation through vascular endothelium. Circulating leukocytes include neutrophils, eosinophils, basophils, monocytes and lymphocytes. Different forms of inflammation involve different types of infiltrating leukocytes.
The inflammatory process can be triggered in a number of ways, including by infection, tissue damage and autoimmune reactions. As part of the inflammatory process, neutrophils move from the bloodstream into the tissue at the site of tissue lesion. The neutrophils contain large numbers of different intracellular granules and when activated at the site of inflammation the contents of these granules are secreted into the tissue. The different granules contain a variety of enzymes and other proteins, many of which have antibacterial properties.
One of the enzymes found in the azurophilic granules is neutrophil elastase. Neutrophil elastase has a wide spectrum of activities in the body. For example, within the lung the enzyme increases mucus production and changes the cellular composition of the epithelium The enzyme also causes vascular permeability changes within the microcirculation of many tissues and it is a potent destructive agent against a number of connective tissue components.
Although there are within the body endogenous inhibitors of elastase, including the anti-trypsin and the leukocyte protease inhibitor, elastase activity has been implicated in the pathogenesis of a number of disease states including inflammatory diseases of the airways, the joints and the skin. The enzyme is also responsible for some or most of the symptoms of acute respiratory distress syndrome (ARDS) and other acute inflammatory states brought about by trauma and/or sepsis.
We have now found a group of novel compounds which inhibit neutrophil elastase. These compounds are therefore of potential therapeutic benefit in the treatment and amelioration of symptoms of diseases where elastase activity is implicated.
Thus, according to one aspect of this invention, we provide a compound of the formula (I)
(relative stereochemistry indicated)
wherein:
R
1
represents C
1-6
alkyl;
R
2
represents C
2-4
alkyl or C
2-4
alkenyl;
a represents 1 or 2;
R
3
represents C
1-8
alkyl or (CH
2
)
n
Ar;
n represents 1 or 2;
Ar represents optionally substituted phenyl;
and salts and solvates thereof (hereinafter “compounds of the invention”).
Formula (I) shows the relative stereochemistry of the chiral centres. The invention embraces compounds of the invention in racemic form as well as in a form in which one enantiomer predominates or is present exclusively. Generally, we prefer to provide a compound of formula (I) in enantiomerically pure form, most particularly the enantiomer having the absolute stereochemistry illustrated in formula (I).
The present invention also covers the physiologically acceptable salts of the compounds of formula (I). Suitable physiologically acceptable salts of the compounds of formula (I) include inorganic and organic acid salts such as hydrochloride and tartrate.
Examples of groups by with the phenyl of Ar may be substituted include halogen, C
1-4
alkyl, C
1-4
alkoxy, nitro, hydroxy, amine (optionally substituted by one or two C
1-4
alkyl groups), CF
3
, COOH, COOC
1-4
alkyl and CONH
2
(optionally substituted by one or two C
1-4
alkyl groups).
When used herein “alkyl” includes branched as well as straight chain alkyl and may also include cycloalkyl when 3 or more carbon atoms are present.
Suitable R
1
alkyl groups include methyl, ethyl and propyl.
Suitable R
3
C
1-8
alkyl groups include n-butyl, cyclopropyl and t-butyl.
We prefer R
1
to represent methyl or ethyl, especially methyl.
We prefer R
2
to represent isopropyl or propyl, especially isopropyl.
We prefer a to represent 1.
We prefer R
3
to represent C
1-8
alkyl or CH
2
Ar. Preferred Ar includes phenyl and phenyl substituted by one or more halogen groups.
The potential for compounds of the invention to inhibit neutrophil elastase activity may be demonstrated, for example, using the following in vitro and in vivo assays:
In vitro assays of human neutrophil elastase
Assay contents:
50 mM Tris/HCl (pH 8.6)
150 mM NaCl
11.8 nM purified human neutrophil elastase
Suitable concentrations of compound under test diluted with water from a 10 mM stock solution in dimethylsulphoxide. Values above are final concentrations after the addition of substrate solution (see below).
The mixture above is incubated for fifteen minutes at 30° C. at which time the remaining elastase activity is measured for 10 minutes in a BioTek 340i plate-reader, after the addition of 0.6 mM MeO-succinyl-alanyl-alanyl-prolyl-valyl-p-nitroanilide. The rate of increase in absorbance at 405 nm is proportional to elastase activity. Enzyme activity is plotted against concentration of inhibitor and an IC
50
determined using curve fitting software.
In vivo activity of inhibitors of human neutrophil elastase:
An oral in vivo model using IL-8 induced lung infiltrates for the assessment of intracellular elastase inhibition
Adult hamsters (100-150 g) are randomised into groups (n=4) and fasted overnight. Under gaseous anaesthetic (3% isofluorane) animals are dosed orally with 1 mL/100 g water as vehicle or containing predissolved compounds. Either at the same time, or subsequently under anaesthetic, animals are dosed intratracheally with 1 ug recombinant human IL-8 in 100 uL sterile saline. Six hours after IL-8 dosing animals are sacrificed using intraperitoneal pentobarbitone. The lungs are lavaged with 2×2.5 mL sterile saline and femurs are removed by dissection.
Intracellular elastase is prepared from neutrophils collected by lavage and from femoral bone marrow. This is achieved by sonication of the neutrophils and centrifugation to yield intracellular granules. These are disrupted by freeze/thawing and sonication. Elastase and myeloperoxidase assays are then performed on these samples to assess the efficacy of the compounds and to normalise for neutrophil recovery.
Human whole blood elastase inhibition assay
Triplicate aliquots of fresh, heparinised human whole blood (200 &mgr;l) are added to appropriately diluted samples (10 &mgr;l) of compounds under test. Control samples (6 replicates) contain water in place of compound. Samples are mixed thoroughly by pipette, and are then incubated for 30 minutes at 37° C. Cold red cell lysis buffer (750 &mgr;l of 155 mM ammonium chloride, 0.12 mM EDTA, 10 mM potassium bicarbonate, pH 7.4) is then added. Tubes are capped, inverted several times, and maintained at 4° C. for 15 minutes, inverting every 5 minutes. After centrifugation at 250 g for 10 minutes, at 4° C., the resulting pelleted cells are washed. The wash is with saline (300 &mgr;l), followed by centrifugation at 100 g for 10 minutes at 4° C. Pellets are washed twice more, before resuspension of the final cell pellet in buffer (200 &mgr;l of 100 mM Tris, 300 mM NaCl, 1% (w/v) HTAB, pH 8.6). Samples are stored at −20° C. After freeze-thawing of the samples four times, elastase activity is determined by a calorimetric assay in 50 mM Tris, 150 mM NaCl, 0.6 mM MeO-Succ-Ala-Ala-Ala-Pro-Val-pNA at pH 8.6, measuring the rate of increase in absorbance at 405 nm.
Accordingly, the compounds of the invention are of potential therapeutic benefit in the treatment and amelioration of symptoms of diseases where elastase activity is implicated. Such diseases particularly include bronchitis, including chronic bronchitis. Also any chronic obstructive pulmonary disease (COPD).
Examples of disease states in which the compounds of the invention have potentially beneficial effects include inflammatory diseases of the respiratory

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