Butyne diol derivatives

Details Butyne diol derivatives Butyne diol derivatives

2002-06-24

2004-04-13

Ramsuer, Robert W. (Department: 1626)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Heterocyclic carbon compounds containing a hetero ring...

C544S123000, C544S296000, C544S298000, C544S319000

Reexamination Certificate

active

06720322

ABSTRACT:

This is a national stage application of International Application PCT/EP00/12743, filed Dec. 14, 2000, which was published under PCT Article 21(2) as PCT Publication No. WO 01/46156 in English, and which claims the benefit of International Application PCT/EP99/10276 filed Dec. 22, 1999. Both International Applications PCT/EP00/12743 and PCT/EP99/10276 are hereby incorporated by reference in their entireties.
The present invention relates to novel butyne diol derivatives 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 receptor 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, pharmacokinetic properties and the selectivity profile of each antagonist for a given clinical indication is mandatory. We have discovered a new class of butyne-diol derivatives 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 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
ET
A
ET
B
Example 1g
26
77
Example 2c
126
44
Example 3e
22
1520
Example 4d
53
2030
Example 5b
38
635
Example 9d
16
49
Example 11d
49
97
Example 18
79
36
Example 19
112
45
Example 21
230
44
Example 58
7
123
Example 70
94
375
Example 71
13
28
Example 72
1
42
Example 81
1
197
Example 84
2
241
Example 89
13
1140
Example 94
13
107
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 (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) keep at 37° C. 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
50
induced by different concentrations of test compound. EC
50
is the concentration of endothelin needed to get a half-maximal contraction, pA
2
is the negative logarithm of the antagonist concentration which induces a two-fold shift in the EC
50
value.
The pA
2
values obtained with compounds of formula I are given in Table 2.
TABLE 2
pA
2
Compound of
aortic rings
trachea
Example 4d
7.15
5.89
Example 9d
7.11
6.47
Example 11d
7.05
7.03
Example 19
<5
7.62
Example 58
7.57
Example 59
7.70
Example

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