Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Heterocyclic carbon compounds containing a hetero ring...
Reexamination Certificate
2001-08-16
2003-07-22
Shah, Mukund (Department: 1624)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Heterocyclic carbon compounds containing a hetero ring...
C514S269000, C514S274000, C544S122000, C544S296000, C544S319000, C544S320000, C544S321000
Reexamination Certificate
active
06596719
ABSTRACT:
The present invention relates to novel bis-sulfonamides of the general formula I and their use as active ingredients in the preparation of pharmaceutical compositions. The invention also concerns related aspects including processes for the preparation of the compounds, pharmaceutical compositions containing one or more compounds of the general formula I and especially their use as endothelin antagonists.
Endothelins (ET-1, ET-2, and ET-3) are 21-amino acid peptides produced and active in almost all tissues (Yanagisawa M et al.: Nature (1988) 332:411). Endothelins are potent vasoconstrictors and important mediators of cardiac, renal, endocrine and immune functions (McMillen M A et al.: J Am Coll Surg (1995) 180:621). They participate in bronchoconstriction and regulate neurotransmitter release, activation of inflammatory cells, fibrosis, cell proliferation and cell differentiation (Rubanyi G M et al.: Pharmacol Rev (1994) 46:328).
Two endothelin receptors have been cloned and characterized in mammals (ET
A
, ET
B
) (Arai H et al.: Nature (1990) 348:730; Sakurai T et al.: Nature (1990) 348:732). The ET
A
receptor is characterized by higher affinity for ET-1 and ET-2 than for ET-3. It is predominant in vascular smooth muscle cells and mediates vasoconstricting and proliferative responses (Ohlstein E H et al.: Drug Dev Res (1993) 29:108). In contrast, the ET
B
receptor has equivalent affinity for the 3 endothelin isopeptides and binds the linear form of endothelin, tetra-ala-endothelin, and sarafotoxin S6C (Ogawa Y et al.: BBRC (1991) 178:248). This receptor is located in the vascular endothelium and smooth muscles, and is also particularly abundant in lung and brain. The ET
B
receptor from endothelial cells mediates transient vasodilator responses to ET-1 and ET-3 through the release of nitric oxide and/or prostacyclin whereas the ET
B
receptor from smooth muscle cells exerts vasoconstricting actions (Sumner M J et al.: Brit J Pharmacol (1992) 107:858). ET
A
and ET
B
receptors are highly similar in structure and belong to the superfamily of G-protein coupled receptors.
A pathophysiological role has been suggested for ET-1 in view of its increased plasma and tissue levels in several disease states such as hypertension, sepsis, atherosclerosis, acute myocardial infarction, congestive heart failure, renal failure, migraine and asthma. As a consequence, endothelin receptor antagonists have been studied extensively as potential therapeutic agents. Endothelin receptor antagonists have demonstrated preclinical and/or clinical efficacy in various diseases such as cerebral vasospasm following subarachnoid hemorrhage, heart failure, pulmonary and systemic hypertension, neurogenic inflammation, renal failure and myocardial infarction.
Today, no endothelin receptor antagonist is marketed yet, several are in clinical trials. However, these molecules possess a number of weaknesses such as complex synthesis, low solubility, high molecular weight, poor pharmacokinetics, or safety problems (e.g. liver enzyme increases). Furthermore, the contribution of differential ET
A
/ET
B
receptor blockade to the clinical outcome is not known. Thus, tailoring of the physicochemical and pharmacokinetic properties as well as the selectivity profile of each antagonist for a given clinical indication is mandatory. We have discovered a new class of bis-sulfonamide compounds of the structure below and found that they allow the specific tailoring described above.
The inhibitory activity of the compounds of formula I on endothelin receptors can be demonstrated using the test procedures described hereinafter:
For the Evaluation of the Potency and Efficacy of the Compounds of the General Formula I the Following Tests Were Used
1) Inhibition of Endothelin Binding to Membranes from CHO Cells Carrying Human ET Receptors:
For competition binding studies, membranes of CHO cells expressing human recombinant ET
A
or ET
B
receptors were used. Microsomal membranes from recombinant CHO cells were prepared and the binding assay made as previously described (Breu V., et al, FEBS Lett 1993; 334:210).
The assay was performed in 200 uL 50 mM Tris/HCl buffer, pH 7.4, including 25 mM MnCl
2
, 1 mM EDTA and 0.5% (w/v) BSA in polypropylene microtiter plates. Membranes containing 0.5 ug protein were incubated for 2 h at 20° C. with 8 pM [
125
I]ET-1 (4000 cpm) and increasing concentrations of unlabelled antagonists. Maximum and minimum binding were estimated in samples without and with 100 nM ET-1, respectively. After two h, the membranes were filtered on, filterplates containing GF/C filters (Unifilterplates from Canberra Packard S. A. Zürich, Switzerland). To each well, 50 uL of scintillation cocktail was added (MicroScint 20, Canberra Packard S. A. Zürich, Switzerland) and the filter plates counted in a microplate counter (TopCount, Canberra Packard S. A. Zürich, Switzerland).
All the test compounds were dissolved, diluted and added in DMSO. The assay was run in the presence of 2.5% DMSO which was found not to interfere significantly with the binding. IC
50
was calculated as the concentration of antagonist inhibiting 50% of the specific binding of ET-1. For reference compounds, the following IC
50
values were found: ET
A
cells: 0.075 nM (n=8) for ET-1 and 118 nM (n=8) for ET-3; ET
B
cells: 0.067 nM (n=8) for ET-1 and 0.092 nM (n=3) for ET-3.
The IC
50
values obtained with compounds of formula I are given in Table 1
TABLE 1
IC
50
[nM]
Compound of Example
ET
A
ET
B
Example 2
1960
1790
Example 5
5560
356
Example 6
8300
420
Example 7
63.6
15.8
Example 8
160
130
Example 10
67.2
193
Example 11
5110
4.3
Example 12
2120
73.3
Example 13
885
69.2
Example 14
518
451
Example 15
1320
7.3
Example 16
261
24.5
Example 17
1100
117
Example 18
209
1050
Example 21
881
21.8
Example 23
76.1
52.7
Example 27
3634
995
Example 28
3709
1043
Example 29
1253
235
Example 30
484
288
Example 31
409
735
Example 36f
478
1212
Example 36g
121
93
Example 37
5683
604
Example 38
80
84
Example 39
1048
81
Example 40
76
87
Example 41
4898
299
Example 42
587
99
Example 43
75
376
Example 44
119
323
Example 45
251
336
Example 46
140
103
Example 49
1027
274
Example 51
3450
182
Example 54
2407
603
Example 57
1625
208
Example 58
724
447
Example 59
103
189
Example 60
1442
16
Example 61
92
183
Example 62
443
163
Example 68
477
169
Example 70
282
2071
Example 71
508
231
Example 72
153
279
Example 73
233
542
Example 74
531
934
Example 77
185
5402
Example 78
627
5458
Example 79
37
>10000
Example 80
14.8
59.4
Example 81
104
1240
Example 82
311
3510
Example 87
48.1
33.1
Example 88
14.8
1.86
Example 89
1591
101
Example 90
86
4.1
Example 91
45.5
103
Example 92
82.1
15
Example 93
22.2
3.86
Example 94
77.3
18.6
2) Inhibition of Endothelin-induced Contractions on Isolated Rat Aortic Rings (ET
A
Receptors) and Rat Tracheal Rings (ET
B
Receptors)
The functional inhibitory potency of the endothelin antagonists was assessed by their inhibition of the contraction induced by endothelin-1 on rat aortic rings (ET
A
receptors) and of the contraction induced by sarafotoxin S6c on rat tracheal rings (ET
B
receptors). Adult Wistar rats were anesthetized and exsanguinated. The thoracic aorta or trachea were excised, dissected and cut in 3-5 mm rings. The endothelium/epithelium was removed by gentle rubbing of the intimal surface. Each ring was suspended in a 10 ml isolated organ bath filled with Krebs-Henseleit solution (in mM; NaCl 115, KCl 4.7, MgSO
4
1.2, KH
2
PO
4
1.5, NaHCO
3
25, CaCl
2
2.5, glucose 10) kept at 37° and gassed with 95% O
2
and 5% CO
2
. The rings were connected to force transducers and isometric tension was recorded (EMKA Technologies SA, Paris, France). The rings were stretched to a resting tension of 3 g (aorta) or 2 g (trachea). Cumulative doses of ET-1 (aorta) or sarafotoxin S6c (trachea) were added after a 10 min incubation with the test compound or its vehicle. The functional inhibitory potency of the test compound was assessed by calculating the concentration ratio, i.e. the shift to the right of the EC
5
Bolli Martin
Boss Christoph
Clozel Martine
Fischli Walter
Actelio Pharmaceuticals Ltd.
McKenzie Thomas
Pennie & Edmonds LLP
Shah Mukund
LandOfFree
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