Aminobenzophenones as inhibitors of interleukin and TNF

Details Aminobenzophenones as inhibitors of interleukin and TNF Aminobenzophenones as inhibitors of interleukin and TNF

1999-07-21

2001-11-06

Killos, Paul J. (Department: 1623)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Nitrogen containing other than solely as a nitrogen in an...

C564S319000

Reexamination Certificate

active

06313174

ABSTRACT:

This application is the national phase of international application PCT/DK98/00008 filed Jan. 8, 1998 which designated the U.S.
This invention relates to a hitherto unknown class of compounds which shows anti-inflammatory effects, to pharmaceutical preparations containing these compounds, to dosage units of such preparations, and to their use in the treatment and prophylaxis of asthma, allergy, rheumatoid arthritis, spondyloarthritis, gout, atherosclerosis, chronic inflammatory bowel disease, proliferative and inflammatory skin disorders, such as psoriasis, and atopic dermatitis.
The compounds of the present invention are represented by the general formula I
in which formula R
1
and R
2
stands independently for one or more, similar or different substituents selected from the group consisting of hydrogen, halogen, hydroxy, mercapto, trifluoromethyl, amino, alkyl, alkoxy, alkylthio, alkylamino, and alkoxycarbonyl, the C-content of which can be from 1 to 5, cyano, carboxy, carbamoyl, phenyl, or nitro; R
3
stand for hydrogen, halogen, hydroxy, mercapto, trifluoromethyl, amino, alkyl, alkoxy, alkylthio, alkylamino, or alkoxycarbonyl, the C-content of which can be from 1 to 5, phenyl, cyano, carboxy, or carbamoyl; R
4
, R
5
and R
6
stands independently for hydrogen, trifluoromethyl, alkyl, carbamoyl, alkoxycarbonyl, or alkyloxo, the C-content of which can be from 1 to 5; X stands for oxygen, N—OH, N—O—alkyl, dialkoxy, cyclic dialkoxy, dialkylthio, or cyclic dialkylthio, the C-content of which can be from 1 to 5.
The compounds can be used in the form of their salts which are formed with pharmaceutically acceptable inorganic or organic acids, such as hydrochloric, hydrobromic and hydroiodic acid, phosphoric acid, sulphuric acid, nitric acid , p-toluenesulphonic acid, methanesulphonic acid, formic acid, acetic acid propionic acid, citric acid, tartaric acid, succinic acid, benzoic acid, maleic acid, these examples being considered as non-limiting for the invention.
Previously, a series of closely related aminobenzophenones (e.g. 4-(2-amnino4-nitrophenylamino)benzophenone) have been described (Hussein, F. A. et al, Iraqi J. Sci., 22, 54-66 (1981)). However, there is no description of their uses.
Now we have surprisingly found that novel aminobenzophenones according to general formula I are potent inhibitors of interleukin 1&bgr; (IL-1&bgr;) and tumour necrosis factor &agr; (TNF-&agr;) secretion in vitro, making them potentially useful for treatment of inflammatory diseases, in which the production of cytokines is involved in the pathogenesis, e.g. asthma, rheumatoid arthritis, psoriasis, contact dermatitis, and atopic dermatitis.
To study the effect of the compound of the present invention, in vitro, the inhibition of the IL-1&bgr; and TNF-&agr; secretion was measured using the following procedure:
Cytokine production was measured in the media from lipopolysaccharide (LPS) stimulated peripheral blood mononuclear cells. The mononuclear cells were isolated from human peripheral blood by Lymphoprep® (Nycomed, Norway) fractionation and suspended in RPMI 1640 (growth medium) with foetal calv serum (FCS, 2%), at a concentration of 5×10
5
cells/ml. The cells were incubated in 24-well tissue culture plates in 1 ml aliquots. Test compounds were dissolved in dimethylsulfoxide (DMSO, 10 mM) and were diluted with the medium. Compounds were added to the cells for 30 minutes, then LPS (1 &mgr;g/ml final concentration) was added. The plates were incubated for 18 hours, and the concentration of IL-1&bgr; and TNF-&agr; in the medium was determined by enzyme-linked immunosorbent assays. The median inhibitory concentrations (IC
50
) of the compounds were calculated. The results are shown in table 1.
The compounds of the present invention also show similar activities in the ability to inhibit PMN (polymorphonuclear) superoxide secretion which is also indicative of potentially useful anti-inflammatory drugs. The compounds were tested using the following procedure:
Human polymorphonuclear (PMN) granulocytes were isolated from human blood by dextran sedimentation, Lymphoprep® fractionation and hypotonic lysis of contaminating erythrocytes.
Superoxide anion generation was measured as the superoxide dismutase inhibitable reduction of ferricytochrome C (Madhu, S. B. et al, Inflammation, 16, 241, (1992)).
The cells were suspended in Hanks' balanced salt solution, and incubated for 10 minutes at 37° C. with test compounds. The cells were primed by the addition of TNF-&agr; (3 ng/ml final concentration) for 10 minutes, and then ferricytochrome C, (final concentration 750 &mgr;g/ml), bovine serum albumin (BSA, final concentration 1 mg/ml) and formyl-methionyl-leucyl-phenylalanine (fMLP, final concentration 10
−7
M) were added for 3 minutes.
The cells were chilled on ice, and were spun down. The optical densities in the cell-free supernatant was measured in a spectrophotometer.
The median inhibitory concentration (IC
50
) of the compounds was calculated. The results are shown in Table 1.
TABLE 1
Inhibition of cytokines and PMN-superoxide production
in vitro by compounds of the following examples of the
present invention.
The median inhibition concentration (IC
50
, nM) of
Compound from
IL-1&bgr;
TNF-&agr;
PMN-superoxide
Example no. 1
250
790
160
Example no. 13
160
200
40
Example no. 32
100
130
>10000
Example no. 56
13
7.1
5.0
Example no. 73
32
5.0
5.0
These results show that the compounds of the present invention are able to inhibit the production of IL-1, TNF-&agr; and PMN-superoxide, thus making them potentially useful in the treatment of inflammatory diseases.
To study the compounds of the present invention in vivo the 12—O—tetradecanoylphorbol-13-acetate (TPA) induced murine chronic skin inflammation model were used (De Young, L. M. et al, Agents Actions, 26, 335-341 (1989); Carlson, R. P. et al, Agents Actions, 17, 197-204 (1985); Alford, J. G. et al, Agents Action, 37, (1992); Stanley, P. L. et al, Skin Pharmacol, 4, 262-271 (1991)). The compounds were tested using the following procedure:
In groups of 6 female mice weighing 18-25 grams, ear skin inflammation was induced by multiple topical applications of TPA on alternate days during a 10 day period. The resulting inflammation was treated topically with compounds in acetone (20 &mgr;l/ear) twice daily on day 8, 9 and 10 and once on day 11. The increased ear thickness (ET, right ear thickness minus left ear thickness) was determined approximately 6 hours after the treatment, the mice were sacrificed and the myeloperoxidase (MPO)-activity was determined in ear biopsies. The results are shown in Table 2.
TABLE 2
Effect in the TPA induced murine skin inflammation model by
compounds of the following examples of the present invention.
% inhibition of
Compound from
Dose (mg/ear)
% inhibition of ET
MPO
Example no. 1
0.1
50
65
Example no. 2
0.1
40.
76
Example no. 27
0.1
44
48
Hydrocortisone
0.1
a
58
69
0.03
36
51
a
Reduction of spleen and thymus weight.
These results shows that the compounds of the present invention are of the same potency compared to known reference compounds, e.g. hydrocortisone with its known side effects, whereas the compounds of the present invention are well tolerated and are non-toxic. Some members of the present class of compounds show a very low absorption, thus making them especially useful in the treatment of various dermatological diseases. In general, they may be administered by oral, intravenous, interperitoneal, intranasal, topically or transdermal routes.
The present invention also relates to methods for preparing the desired compounds of the general formula I. The compounds of the formula I may conveniently be prepared by standard procedures detailed in the art. The routes are outlined in the following reaction scheme.
Scheme 1: Synthesis of the compounds of the general formula I



Notes to scheme 1
a Potassium tert-butoxide/dimethylsulfoxide/20° C./24-60 hours
b Hydrazine hydrate/10% Pd/C/ethanol/20° C./24 hours or Stannous chloride dihydrate/ethanol/70° C./1-4 hours
c Alkyl- or aryl-sulfonyl chlor

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