The application relates to the novel use of certain known hydroxamic acid derivatives for the management of diseases or conditions mediated by overproduction of, or over-responsiveness to, endothelin.

The vasoconstrictor peptide endothelin is produced by endothelial cells. It has been cloned and found to be a 21 amino-acid peptide which has two disulphide bridges between amino acids 1-15 and 3-11 (Yanagisawa et al., (1988) Nature 332 411-415). It is produced as pre-pro endothelin, a 203 amino acid precursor, which is cleaved by dibasic endopeptidases to form Big Endothelin (Big-ET). Big- ET is subsequently cleaved between Trp2i and Val22 by an endothelin converting enzyme (ECE) to the vasoactive compound, endothelin-1 (ET-1). Two additional peptides have recently been shown to be related to ET-1 ; ET-2 and ET-3 differ from ET-1 by 2 and 6 amino acids, respectively (Inoue et al., Proc. Natl. Acad. Sci USA (1989) 86 _. 2863-2867; Shinmi et al., Biochem. Biophys. Res. Comm. (1989) 164 587-593). However, ET-1 probably represents the prevalent form circulating in humans. As used herein, the term endothelin or ET means endothelin 1 and peptides having a high degree of sequence homoiogy with, and substantially similar physiological effects to, endothelin 1 , for example ET-2 and ET-3.

Endothelin has been found to be involved in a number of physiological conditions, for example vasoconstriction and bronchoconstriction. It also acts to increase the rate and force of heart beats, increase mean arterial blood pressure, decrease cardiac output, increase cardiac contractility in vitro, stimulate mitogenesis in vascular smooth muscle in vitro, contract non-vascular smooth muscle including guinea pig trachea, human urinary bladder strips and rat uterus in vitro, induce formation of gastric ulcers, stimulate release of atrial natriuretic factor, increase plasma levels of vasopressin, aidosterone and catecholamines and to decrease the blood flow to the kidney, leading to sodium retention.

Over expression of endothelin, or over-responsiveness to endothelin is therefore implicated in corresponding pathological states, for example hypertension, asthma,

congestive heart failure, or acute or chronic renal failure. An anti-endothelin antibody has been shown, upon renal infusion, to ameliorate the effects of renal ischaemia on renal vascular resistance and glomerular filtration rate (Kon etal., J. Clin. Invest. (1988) 83 1762). Treatment with anti-ET antibodies also reduced infarct size following coronary artery litigation/reperfusion in the rat (Watanabe et al., (1991), Circ Res. 69 370-377).

It would therefore be desirable to be able to provide a therapeutic agent which inhibits the conversion of Big-ET to ET, leading to a reduction in the specific pathological actions of ET. However, very few agents with such capability have been described to date. Those which have been described are the naturally occurring compound phosphoramidon and the compounds described in WO 92/13545.

This invention is based on the identification of certain compounds (known in the art as inhibitors of collagenase - a matrix metalloproteinase involved in the breakdown of connective tissue) which are able to inhibit significantly the conversion of Big-ET to ET. In its first aspect, the invention comprises the use of a compound selected from the group set forth below in the preparation of a medicament for the management (by which is meant treatment or prophylaxis) of diseases mediated by overproduction of, or over-responsiveness to, endothelin in mammals, in particular in humans. Such diseases may include, for example, hypertension, unstable angina, arrhythmia, congestive heart failure, myocardial ischemia, pulmonary hypertension, asthma, cerebral vasospasm, subarachnoid hemorrhage, pre- eclampsia, atherosclerosis, Buergers disease, Takayusu's arteritis, Raynaud's phenomenon, complications in diabetes, cancer (especially pulmonary carcinoma), gastric mucosal damage, gastrointestinal disorders, endotoxin shock, endotoxin induced multiple organ failure, septicemia, and acute or chronic renal failure.

The group of compounds which exhibit ECE inhibitory activity in accordance with the first aspect of the invention is as follows:

1 [4-(N-Hydroxyamino)-2S-isobutyl-3S-methylsuccinyl]-4-methoxy -L- phenylalanine-N-methylamide,

2 [4-(N-Hydroxyamino)-2R-isobutylsuccinyl]-4-methoxy-L-phenyla lanine-N- methylamide,

3 [4-(N-Hydroxyamino)-2R-isobutylsuccinyl]-L-phenylalanine-N-m ethylamide,

4 [4-(N-Hydroxyamino)-2R-cyclopentylmethylsuccinyl]-L-phenylal anine-N- methylamide,

5 [4-(N-Hydroxyamino)-2S-isobutylsuccinyl]-L-phenylalanine-N-m ethylamide,

6 [4-(N-Hydroxyamino)-2R-isobutylsuccinyl]-L-phenylalanine-N-( 3- pyridylmethyl)amide,

7 [4-(N-Hydroxyamino)-2R-isobutylsuccinyl]-L-phenylalanine-N-( 2- aminoethyl)amide,

8 [4-(N-Hydroxyamino)-2R-isobutylsuccinyl]-4-(oxymethylenecarb oxy)-L- phenylalanine-N-methyiamide,

9 [4-(N-Hydroxyamino)-2R-isobutylsuccinyl]-L-glutamicacid-N-be nzylamide,

10 [4-(N-Hydroxyamino)-2R-isobutylsuccinyl]-4-(oxymethylenecarb oxy-N- benzylamido)-L-phenylalanine-N-methylamide,

1 1 [4-(N-Hydroxyamino)-2R-isobutylsuccinyl]-4-(oxymethylenecarb oxyethyl)-L- phenylalanine-N-methylamide,

12 [4-(N-Hydroxyamino)-2R-isobutylsuccinyl]-4-(oxymethylenecarb onyl glycine methylester)-L-phenylalaniπe-N-methylamide,

13 [4-(N-Hydroxyamino)-2R-isobutylsuccinyl]-4-(oxymethylenecarb onylglycine)- L-phenylalanine-N-methylamide,

14 [4-(N-Hydroxyamino)-2R-isobutylsuccinyl]-4-[oxymethylene-N,N - dimethylcarboxamido)-L-phenylalanine-N-methylamide,

15 [4-(N-Hydroxyamino)-2R-isobutylsuccinyl]-4-(oxymethylenecyan o)-L- phenylalanine-N-methylamide,

16 [4-(N-Hydroxyamino)-2R-isobutylsuccinyl]-4-(oxymethyienecarb oxamido)-L- phenylalanine-N-methylamide,

17 [4-(N-Hydroxyamino)-2R-isobutylsuccinyl]-4-amino-L-phenylala nine-N- methylamide,

18 [4-(N-Hydroxyamino)-2R-isobutyl-3S-(2-thienylthiomethyl)succ inyl]-4- (oxymethylenecarboxymethyl)-L-phenyialanine-N-methylamide,

19 [(N-formyl-N-hydroxy)-2R-isobutyl-3-aminopropanoyl]-4-methox y-L- phenylaianine-N-methylamide,

20 [4-(N-Hydroxyamino)-2R-isobutylsuccinyl]-L-phenylalaninyl-L- alanine,

21 [4-(N-Hydroxyamino)-2R-isobutylsuccinyl]-L-phenylaianyl-L-ph enylalanine- N-methylamide,

22 4-(N-Hydroxyamino)-2R-isobutyl-3S-hydroxysuccinyl]-L-phenyla lanine-N- methylamide,

23 [4-(N-Hydroxyamino)-2R-isobutyl-3S-hydroxysuccinyl]-L-phenyl alanine,

24 [4-(N-Hydroxyamino)-2R-isobutyl-3S-(2-thienyithiomethyl)succ inyl]-γ-methyl-

L-glutamic acid N-methylamide,

25 [(N-formyl-N-hydroxy)-2R-isobutyl-3S-aminobutanoyl]-4-amino- L- phenylalanine-N-methylamide,

26 [4-(N-Hydroxyamino)-2R-isobutylsuccinyl]-L-phenylalanine,

27 [4-(N-Hydroxyamino)-2R-isobutylsuccinyl]-L-alanine,

28 [4-(N-Hydroxyamino)-2R-phenylmethylsuccinyl]-L-alanine,

29 [4-(N-Hydroxyamino)-2R-isopropylsuccinyl]-L-phenylalanine and salts thereof

Salts of the compounds useful in accordance with the invention include physiologically acceptable acid addition salts for example hydrochlorides, hydrobromides, sulphates, methane sulphonates, p-toluenesulphonates, phosphates, acetates, citrates, succinates, lactates, tartrates, fumarates and maleates. Salts may also be formed with bases, for example sodium, potassium, magnesium, and calcium salts.

There are several chiral centres in the compounds useful in accordance with the invention because of the presence of asymmetric carbon atoms. The presence of several asymmetric carbon atoms gives rise to a number of diastereomers with R or S stereochemistry at each chiral centre. Use of compounds as diastereomers or as diastereomeric mixtures is contemplated.

The above compounds and methods for their preparation are all known from the following sources;

Compounds 1 -5 from EP 214 639;

Compounds 6 and 7 from WO 90 05719;

Compounds 8-18 from EP 498 665; Compounds 19-23 from EP 236 872; Compound 24 from GB 9207759.3 Compound 25 from GB 9211455.2 Compounds 26-29 from US 4,618,708

Those publications disclose many additional compounds and classes of compounds which are very similar in structure, for example in terms of their molecular configurations, to the group of compounds with which this invention is concerned, and screening of such compounds using the assays described in Pharmacological Examples 1 and 2 below enables the routine identification of additional compounds disclosed therein which are also useful in inhibiting the conversion of Big-ET to ET. Since such screening and identification of active compounds is mere routine following or motivated by the primary discovery in accordance with the first aspect of the invention of inhibitory activity in the specified compounds within those earlier disclosures, this invention also includes the use of compounds disclosed in any of those publications which are active in inhibiting the conversion of Big-ET to ET, in the preparation of a medicament for the management (by which is meant treatment or prophylaxis) of diseases mediated by overproduction of, or over-responsiveness to, endothelin in mammals, in particular in humans.

Particularly preferred compounds for use in accordance with the invention are [4-(N-Hydroxyamino)-2R-isobutylsuccinyl]-L-phenylalanine; [4-(N-Hydroxyamino)-2R-isobutylsuccinyl]-4-(oxymethylenecarb oxyethyl)-L- phenylalanine-N-methylamide;

[4-(N-Hydroxyamino)-2R-isobu.ylsuccinyl]-4-(oxymethylenec arboxamido)-l_- phenylalanine-N-methyiamide; and

[4-(N-Hydroxyamino)-2R-isopropylsuccinyl]-L-phenylalanine and salts thereof.

The compounds may be prepared for administration by any convenient route. Those which are bioavailable orally may be in the form of tablets, capsules,

powders, granules, lozenges, liquid or gel preparations, such as oral, topical, or sterile parenteral solutions or suspensions. Tablets and capsules for oral administration may be in unit dose presentation form, and may contain conventional excipients such as binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, or poiyvinyl-pyrrolidine; fillers for example lactose, sugar, maize-starch, calcium phosphate, sorbitol or glycine; tabletting lubricant, for example magnesium stearate, talc, polyethylene glycol or silica; disintegrants for example potato starch, or acceptable wetting agents such as sodium lauryl sulphate. The tablets may be coated according to methods well known in normal pharmaceutical practise. Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives such as suspending agents, for example sorbitol, syrup, methyl cellulose, glucose syrup, gelatin hydrogenated edible fats; emulsifying agents, for example lecithin, sorbitan monooleate, or acacia; non-aqueous vehicles (which may include edible oils), for example almond oil, fractionated coconut oil, oily esters such as glycerine, propylene glycol, or ethyl alcohol; preservatives, for example methyl or propyl p- hydroxybenzoate or sorbic acid, and if desired conventional flavouring or colouring agents.

The compounds may also be administered parenterally in a sterile medium. Depending on the vehicle and concentration used, the compound can either be suspended or dissolved in the vehicle. Advantageously, adjuvants such as a local anaesthetic, preservative and buffering agents can be dissolved in the vehicle.

The dosage unit involved in oral administration may contain from about 1 to 250mg, preferably from about 25 to 250mg of the compound. A suitable daily dose for a mammal may vary widely depending on the condition of the patient. However, a dose of the compound of about 0.1 to 300mg/kg body weight, particularly from about 1 to 100mg/kg body weight may be appropriate.

The following examples illustrate the invention, but are not intended to limit the scope in any way.

Methods

Pharmacological Example 1 : Hypertension assay

Rats were anaesthesised with urethane (1.6g.kg- ⁱ ). The trachea was cannulated through a midiine incision in the neck, and the animals breathed spontaneously air enriched with oxygen to maintain arterial blood pθ ₂ between 100 and 140 mmHg. A carotid artery was cannulated for the measurement of blood pressure and this signal was used to trigger a rate meter to measure heart rate. A jugular vein was cannulated for the intravenous administration of test compound (1 mg/kg) and Big- ET (10μg/kg) in saline containing 50% (v/v) DMSO. The test compounds were administered 5 minutes prior to Big ET. Only one dose of endothelin was given per rat. Activity was measured by evaluating the area under the curve of the hypertensive response to Big-ET and comparing vehicle treated animals with drug treated animals (n=5-9 for each compound). Active compounds are defined as those which result in a significant (p<0.05) reduction in the area under the curve of the response to Big-ET in compound-treated animals compared to controls, and which have no significant activity against endothelin-1. The results are shown in Table 1.

Pharmacological Example 2: conversion of Big ET to ET by A10 cells

Embryonic rat, thoracic aorta smooth muscle ceils (A10) obtained from ATCC were cultured in Dulbecco's modified Eagles Medium (DMEM) with 20% foetal calf serum (FCS) in 24 well tissue culture plates until confluent. The cells were then incubated for a further 24 hr at 37°C in a humidified atmosphere of 5% C0 ₂ , 95% air in DMEM with 1 % FCS, together with human Big ET (38amino-acids) (1 μM) and the test substance at the appropriate concentration. The incubation medium was then diluted and analysed for the presence of immunoreactive ET (irET) using a commercially available radioimmunoassay kit (Amersham International Ltd. RPA 555), specific for endothelin (21 amino acids). The conversion of Big ET to irET in the presence of the inhibitor is calculated as a percentage of the conversion achieved in the presence of the vehicle alone. The results are set forth in Table 2,

and confirm that the compounds identified inhibit the conversion by A10 cells of exogenous Big ET to immunoreactive endothelin.

Table 1

Compound % inhibition

[4-(N-Hydroxyamino)-2R-isobutyisuccinyl]-L- 62 p<0.01 phenylalanine

[4-(N-Hydroxyamino)-2R-isobutylsuccinyl]-4- 63 p<0.01

(oxymethylenecarboxyethyl)-L-phenylaianine-N- methylamide

[4-(N-Hydroxyamino)-2R-isobutylsuccinyl]-4- 51 p<0.05 [oxymethylene-N,N-dimethylcarboxamido)-L- phenylalanine-N-methylamide

[4-(N-Hydroxyamino)-2R-isobutylsuccinyl]-4- 59 p<0.01

(oxymethylenecarboxamido)-L-phenylalanine-N- methylamide

[4-(N-Hydroxyamino)-2R-isobutyl-3S-(2- 46.9 p<0.05 thienylthiomethyl)succinyl]-γ-methyl-L-glutamic acid N- methylamide

[4-(N-Hydroxyamino)-2R-isobutyl-3S-(2- 28.6 p<0.05 thienylthiomethyl)succinyl]-4- (oxymethylenecarboxymethyl)-L-phenylalanine-N- methylamide

[4-(N-Hydroxyamino)-2R-isobutyl-3S-hydroxysuccinyl]- 37 p<0.001 L-phenylalanine-N-methyiamide

[(N-formyl-N-hydroxy)-2R-isobutyl-3S-aminobutanoyl]-4- 50 p<0.005 amino-L-phenylalanine-N-methylamide

[4-(N-Hydroxyamino)-2R-isobutyl-3S-hydroxysuccinyl]- 26 p<0.05 L-phenylalanine

[4-(N-Hydroxyamino)-2R-isobutylsuccinyl]-L-alanine 48 p<0.005

[4-(N-Hydroxyamino)-2R-phenylmethylsuccinyl]-L- 53 p<0.05 alanine

Table 2

Compound % inhibition

[4-(N-Hydroxyamino)-2S-isobutyl-3S-methylsuccinyl]-4- 40% @ 100μM methoxy-L-phenylalanine-N-methylamide

[4-(N-Hydroxyamino)-2R-isobutylsuccinyi]-4-methoxy-L- phenylaianine-N-methylamide 35% @ 100μM

[4-(N-Hydroxyamino)-2R-isobutylsuccinyl]-L- 55% @ 100μM phenylalanine

[4-(N-Hydroxyamino)-2R-isobutylsuccinyl]-L- 50% @ 100μM phenylalanine-N-(3-pyridylmethyl)amide

[(N-formyl-N-hydroxy)-2R-isobutyl-3-aminopropanoyl]-4- 35% @ 100μM methoxy-L-phenylalanine-N-methylamide

[4-(N-Hydroxyamino)-2R-isobutylsuccinyl]-L- 35% @ 100μM phenylalanine-N-methylamide

[4-(N-Hydroxyamino)-2R-cyclopentylmethylsuccinyl]-L- 30% @ 100μM phenylalanine-N-methylamide

[4-(N-Hydroxyamino)-2S-isobutylsuccinyl]-L- 55% @ 100uM phenylalanine-N-methylamide

[4-(N-Hydroxyamino)-2R-isobutylsuccinyl]-4- 50% @ 100μM

(oxymethylenecarboxy)-L-phenylalanine-N- methylamide

[4-(N-Hydroxyamino)-2R-isobutylsuccinyl]-L-glutamic 40% @ 100μM acid-N-benzylamide

[4-(N-Hydroxyamino)-2R-isobutylsuccinyl]-4- 30% @ 100μM

(oxymethylenecarboxy-N-benzylamido)-L- phenylalanine-N-methylamide

[4-(N-Hydroxyamino)-2R-isobutylsuccinyl]-4- 35% @ 100μM

(oxymethylenecarboxyethyl)-L-phenylalanine-N- methylamide