US 4,609,643 AGrant
Renin Inhibitors, Treatments and Dipeptide Synthesis
Issue Date:1986-09-02
•7 Claims
Abstract
Preparation of renin inhibitors based on the structure of natural renin substrate at residues 6 to 13 from the amino terminal thereof, the inhibitors being polypeptide analogues having in particular an isosteric non-peptide link corresponding to the 10, 11 peptide link of the substrate, and preparation of dipeptide analogues.
Metadata
Assignee
- Aktiebolaget Hassle
Inventors
- Michael Szelke
- David M. Jones
- Allan Hallett
Application Information
Application Number:US 6706711
Filing Date:1984-11-13
Priority Date:1980-08-06
Art Unit:123
Classifications
IPC:
A61K 3764C07K 502C07K 702
Field of Search:
260424112.5 R177
Patent Drawings
This patent does not have any drawings.
Description
The invention relates to renin-inhibiting peptide analogues.
BACKGROUND
Renin is a natural enzyme, disorders in relation to which are implicated in many cases of hypertension. It is released into the blood from the kidney, and cleaves from a blood glycoprotein a decapeptide known as angiotensin-I. Circulating angiotensin-I is cleaved in lung, kidney and other tissues to an octapeptide, angiotensin-II, which raises blood pressure both directly by causing arteriolar constriction and indirectly by stimulating release of the sodium-retaining hormone aldosterone from the adrenal gland and thus causing a rise in extracellular fluid volume. The latter effect is caused by angiotensin-II itself or a heptapeptide cleavage product angiotensin-III.
Inhibitors of renin have therefore been sought, with two ends in view, first the provision of a diagnostic agent for identification of cases of hypertension due to renin excess, and secondly the provision of an agent for control of hypertension in such cases.
The present inventors' approach has been to consider the peptide sequence characterising the natural renin substrate at its binding site, and to seek peptide analogues sufficiently similar to bind to the enzyme, in competition with the natural substrate, but sufficiently dissimilar to it to be cleaved slowly or not at all. Such analogues will block the action of the enzyme and attack the hypertension at source.
Renin is specific to a particular bond in the substrate, the N-terminal sequence of which in the horse is for example: ##STR1## as found by L. T. Skeggs et al J. Exper. Med. 106 439 (1957). Human renin substrate has a different sequence recently discovered by D. A. Tewkesbury et al Biochem. Biophys. Res. Comm. 99 1311 (1981) ##STR2## the sequence to the left of the arrow A being as in formula (IA).
Cleavage at A gives angiotensin-I; subsequent cleavage at the Phe--His bond at B gives angiotensin--II; and cleavage subsequently again at the Asp--Arg bond at C gives angiotensin-III.
Peptides similar to certain partial sequences of the substrate have been shown to act as inhibitors of renin in vitro. An example is the tetrapeptide ester (the relation to the substrate residues being indicated by numbering): ##STR3## proposed by Kokubu, Nature, 217 456 (1968) but it is inactive in vivo, because of binding to plasma proteins and rapid attack by natural peptidases.
One of the present inventors undertook some years ago a development of Kokubu's work, seeking a renin inhibitor active in vivo, in which analogues of peptides similar to Kokubu's were made but having a methylene imino group --CH.sub.2 --NH-- in place of the peptide link --CO--NH-- between the leucine residues. One of these analogues was: ##STR4## which is the tetrapeptide (I) modified at the Leu--Leu link, leucine of course being ##STR5## This analogue (III) was the first effective in-vivo inhibitor of renin and was shown to have significant antihypertensive action in Goldblatt hypertensive rats (Parry, Russell and Szelke p. 541 in "Chemistry and Biology of Peptides" Ed. Meienhofer, Ann Arbor Science Publishers 1972). Little or no attention has however been paid to the work, which the authors themselves were unable to pursue, in spite of considerable activity in the general field of substrate-based inhibitors for renin, reviewed for example by Haber & Burton, Federation Proc. 38 No. 13 2768-2773 (1979).
THE INVENTION
The present invention is a development of the above work. Behind it is a concept of modifying peptide structures related to the peptide sequence at the site of action of renin on the natural substrate, by isosteric substitution at, at least, the site of cleavage. Optionally further there is isosteric substitution or other modification at other positions to increase stability or to modify the properties of the final peptide, for example its solubility under physiological conditions or its resistance to in vivo exopeptidase attack. Such modification may for example be by incorporation of residues other than those of the natural L-amino acids; by protection of the N-terminus with acetyl, pivaloyl, t-butyloxycarbonyl (Boc), benzoyl or other groups; or by conversion of the C-terminal carboxyl to another functional group, e.g. the corresponding alcohol, present as such or in ether or ester form.
General reference to amino acids and amino acyl residues and side chains in both the description and claims herein is to be taken as reference to such whether naturally occurring in proteins or not and to both D- and L-forms, and amino is to be taken as including imino.
The compounds of the present invention, showing desirable renin inhibitory action, are of the general formula: ##STR6## or the partial sequences:
where Pro, Phe and His may be in substituted form; e.g. Pro by OH preferably at the 4-position, Phe by OHFClBr or Me preferably at the 4-position, His by Me; or Phe is replaced by pGlu, Phe by Tyr or His by spinacin.
X=H; or an acyl or other N-protecting group e.g. acetyl, pivaloyl, t-butyloxycarbonyl (Boc), benzoyl or lower alkyl (primarily C.sub.1 -C.sub.5); or an L- or D-amino-acyl residue, which may itself be N-protected similarly and in particular may be Gly or D- or L-Pro, Val or Ile;
Y=D- or L-His or other D- or L-basic or aromatic amino-acyl residue, or is absent;
A= ##STR7## where the configuration at asymmetric centres * is either R or S, where in VIII the hydroxy group may be present as such or protected in ether --OR.sup.4 or ester ##STR8## form where R.sup.4 is as given under W below and where R.sup.1 and R.sup.2, the same or different=.sup.i Pro (isopropyl), .sup.i Bu (isobutyl), Bzl (benzyl) or other amino-acid side chain preferably lipophilic or aromatic
R.sup.3 =--H; lower alkyl (C.sub.1 -C.sub.5); or t-butyloxycarbonyl, benzyloxycarbonyl as such or ring substituted, --SO.sub.2 Ph, --SO.sub.2 --C.sub.6 H.sub.4 CH.sub.3 (p), formyl or other N-protecting group including lower acyl C.sub.1 -C.sub.5 generally;
B=D- or L-Val Leu or Ile or other D- or L-lipophilic amino-acyl residue;
Z=D- or L-Tyr, Phe, His or other L- or D-aromatic amino-acyl residue; and
W=(i)--OH as such or in protected ester form as --OR.sup.4 where R.sup.4 =lower alkyl primarily C.sub.1 -C.sub.5 and particularly .sup.t Bu, or cycloalkyl primarily C.sub.3 -C.sub.7, or Bzl, or other ester forming group; or (ii) --NH.sub.2 as such or in protected amide form as --NHR.sup.5 or --N(R.sup.5).sub.2 (where R.sup.5 =an N-protecting or other substituent group e.g. lower alkyl as for R.sup.4 and (R.sup.5).sub.2 =two such or e.g. cycloalkyl, primarily C.sub.3 -C.sub.7) or as --NH--(CH.sub.2).sub.n --Q or --NR.sup.5 --(CH.sub.2).sub.n --Q (where n=2 to 6 and Q=NH.sub.2 or ##STR9## and wherein any of the hydrogens attached to nitrogen may be substituted by R.sup.5 or (R.sup.5).sub.2); or (iii) an L- or D-serine or D- or L-lysine, arginine or other basic amino-acyl residue as such or in amide form substituted amide form or ester form e.g. containing a group or groups as given for R.sup.4 and R.sup.5 above as the case may be; or (iv) an amino alcohol residue derived therefrom as such or protected in ester or ether form e.g. containing a group as given for R.sup.4 above or
Z+W=an alcohol derived from L- or D-Tyr, Phe, His or other L- or D-aromatic amino-acyl residue as such or protected in ester or ether form as above;
such polypeptide being in the above form or modified by isosteric replacement of one or more remaining peptide bonds e.g. by reduced, --CH.sub.2 --NH--, keto, ##STR10## hydroxy, --CH(OH)--CH.sub.2 --, or hydrocarbon, --CH.sub.2 --CH.sub.2 -- isosteric links as set out above for A and further being in free form or in protected form at one or more remaining amino or amide (including peptide) nitrogen, carboxyl, hydroxy or other reactive groups, or in salt form at amino imidazole or carboxyl groups in particular as their physiologically acceptable acid addition salts at basic centres.
Reference to basic and aromatic amino acids above, and to amino acids with lipophilic side chains includes but is not restricted to the common amino acids of those classes, viz:
The above compounds may in particular be those related to the substrate sequence in the horse (B=Val at position 12) or those related to the substrate sequence in man (B=Ile at position 12). Particular groups of these compounds are set out in claims 2 and 3 respectively herein, as formulae VA and VB to which reference may be made but which are not repeated at this point.
The numbering of residues in formulae (V), (VA) and (VB) shows the correspondence with the renin substrates themselves, but without limitation of the generality of the formulae.
Where a peptide bond in addition to that corresponding to the Leu-Leu or Leu-Val bond in the natural renin substrate is isosterically substituted, the 7,8 and 8,9 positions i.e. the Pro--Phe and Phe--His bonds in formula V etc. are preferred, or possibly both of these positions, and it is further preferred that the substitution should be ##STR11## where R.sub.3 is as set out above. The alternative isosteric substitutions set out herein may however be used.
Protective or substituent groupings as mentioned above may be any of these known in the polypeptide art, amply disclosed in the literature and not requiring discussion at length here. Generally the selection of the groups is according to their function, some being primarily intended to protect against undesired reaction during synthetic procedures while the N- and C-terminal substituents are for example directed against the attack of exopeptidases on the final compounds or to increase their solubility and hence physiological acceptability. All these functions are generally within the term "protective group" or the like used herein, including the claims.
It is in particular possible for one or more remaining peptide bonds in the compounds of formula (V)etc, (VA) or (VB) to be N-substituted with protective groups.
The invention further lies (i) In a diagnostic test for high renin states, blood pressure falling most when renin is high, and as a surgical prognostic test for reno-vascular hypertension (renal artery stenosis), the administration of a polypeptide analogue as above followed by monitoring of blood pressure, and such polypeptide analogues when for such use, and (ii) In the long and short term treatment of heart failure and all forms of hypertension particularly those associated with high serum renin levels, the administration of a renin-inhibiting amount of a polypeptide analogue as above, and such polypeptide analogues when for such use.
The long and short term response of blood pressure to renin inhibitors is predictive of surgical outcome. In all cases single and repeated doses and any conventional form of pharmaceutical compositions may be used, for administration of intranasal or oral route, injection, or any other means as convenient. Amounts may for example be 0.001 to 10 mg/kg body weight daily more usually 0.01 to 1 mg, according to the potency of the analogue and the severity of the condition. Dosage unit compositions may contain such amounts or submultiples thereof to make up the daily dose. (Dosages herein and in the claims are related to the free base content where compounds are in salt form).
The invention still further extends to a product and method of making a hydroxy or keto isostere of a dipeptide wherein a derivative of a halohydrin preferably a bromohydrin or haloketone preferably a bromoketone ##STR12## wherein R.sup.6 is an amino acid side chain and the NH.sub.2 and OH groups are in protected form is subjected to an alkylation procedure to attach a group ##STR13## and gives the desired isostere as such or in protected form, R.sup.7 being the same or a different amino acid side chain.
In particular the alkylation procedure may be
(i) by reaction with an alkali metal carboxylic acid derivative preferably a lithium derivative ##STR14## where R.sup.7 is as above.
(ii) by reaction with an alkali metal malonic ester derivative preferably a sodium derivative ##STR15## where R.sup.8 is an esterifying group and a halide preferably an iodide
where R.sup.7 is as above to give intermediate ##STR16## in protected form which intermediate is then decarboxylated and if desired deprotected to give the desired isostere ##STR17## as such or in protected form.
The hydroxy isosteres so produced may further be oxidised to the corresponding keto isosteres, and such method and the isosteres produced fall within the invention.
In particular the methods may be applied to the production of a hydroxy dipeptide isostere of the formula ##STR18## or the correspondingf keto isosteres, where the significance of *, X.sup.1 and W.sup.1 is as above except that X.sup.1 and W.sup.1 do not represent amino-acyl.
The dipeptide isosteres given by all these methods may be incorporated in higher peptide analoges by the methods herein described or by the methods of peptide synthesis as generally known in the art, and the invention extends to the dipeptide whether as such or in the form of said higher analogues, in all cases as the compound itself or in protected form.
The dipeptide analogue syntheses are illustrated in detail herein, in the course of illustrating the preparation of the octapeptides and related compounds to which the invention chiefly relates.
Specific analogues within the invention, all as such or in protected form, are ##STR19## and the corresponding analogue (H-77) with R.sup.6 =hydrogen and D--His at position 6. A further analogue, with the same methylene-imino isosteric replacement of a Leu--Leu peptide bond, is: ##STR20## Further analogues within formula (VA) are given in the present disclosure in Examples VI to IX, XI and XII. Analogues within formula (VB) are given in Examples V and X.
SYNTHETIC METHODS
The inventors have developed synthetic methods for the isosteric replacement of the peptide bond --CO--NH-- with alternative groups, specifically --CH.sub.2 --NH-- (reduced), --CH.sub.2 CH.sub.2 -- (hydrocarbon), ##STR21## (keto) and --CH(OH)--CH.sub.2 -- (hydroxy) isosteres (see, e.g. Szelke, et al, pp. 57-70 in "Molecular Endocrinology" Vol. 1, Editors: MacIntyre and Szelke, Elsevier, Amsterdam 1977, and Hudson, Sharpe and Szelke, U.S. Pat. No. 4,198,398 "Enkephalin Analogues").
Reference may be made to these publications for general discussion of such isosteric replacement. A reaction sequence for the preparation in particular of the reduced isostere of leucyl leucine for incorporation in the analogues disclosed herein is however for example:
SCHEME 1
Synthesis of the protected reduced isostere of L--leucyl--L--leucine ##STR22## (1) Boc--Leucyl--leucine methyl ester
The dipeptide I' was prepared from Boc--leucine.H.sub.2 O (27.5 g, 0.11 mole) and leucine methyl ester.HCl (20 g, 0.11 mole) by mixed anhydride coupling using N-methyl morpholine and isobutylchloroformate. After a standard work-up procedure the dipeptide I' was obtained as white needles, 35.0 g (88%) from EtOAc/petrol bpt 40.degree.-60.degree., m.p. 132.degree.-133.degree..
(2) Preparation of compound II'
The dipeptide I' (7.2 g, 20 mmole) was dissolved in benzene (120 ml, Na-dried). A solution of sodium dihydro-bis(2-methoxyethoxy)aluminate (SDA, 70% in toluene, 41 ml) was added slowly with cooling. After addition, the solution was refluxed for 1/2 hr, cooled and poured into 0.5M ice-cold citric acid solution. At pH 2.5 the aqueous solution was extracted with ether (4.times.) and the combined extracts were discarded. The pH was adjusted to 9 with Na.sub.2 CO.sub.3 solution and the aqueous solution was saturated with sodium chloride. Extraction with ether (4.times.), followed by drying (Na.sub.2 SO.sub.4) of the combined organic phases, evaporation and crystallisation from petrol (40.degree.-60.degree.) at -20.degree. gave the reduced dipeptide II': 5.1 g (78%) as white needles.
Nmr (CDCl.sub.3) 9.05-9.15 (12H, d, 4.times.CH.sub.3) 8.75 (6H, m, 2.times.(CH.sub.3).sub.2 CH--CH.sub.2); 8.55 (9H, s, (CH.sub.3).sub.3 CO); 7.35 (5H, m, CH.sub.2 NH, CH.sub.2 OH); 6.05-6.85 (3H, m, 2.times..alpha.--CH and CH.sub.2 OH); 5.3 (1H, d, BocNH--).
(3) Protection of compound II' with benzenesulphonyl
The reduced compound II' (11.0 g, 34.7 mmole) in dioxan (100 ml) was added to a solution of KHCO.sub.3 (21 g., 6 equiv.) in H.sub.2 O (100 ml). This mixture was cooled in ice and benzene-sulphonyl chloride (9.0 ml, 2 equiv.) added in dioxan (25 ml) with vigorous stirring. Stirred at 22.degree. overnight. Poured into ether, washed with 2N NH.sub.4 OH (4.times.), H.sub.2 O (1.times.) 0.5M citric acid (2.times. to remove any unsulphonated material), H.sub.2 O (1.times.).
The protected compound III' was obtained as an oil. Nmr spectroscopy showed the presence of one benzenesulphonyl group. This material was used without further purification in the next stage:
(4) Oxidation of compound III'
The material from the preceding preparation was taken up in pyridine (50 ml), cooled in ice and KMnO.sub.4 (11.0 g 70 mmole) in H.sub.2 O (50 ml) and pyridine (100 ml) added. Stirred for 42 hrs at 20.degree.. The MnO.sub.2 precipitate was removed and the filtrate diluted with citric acid solution until acidic. Ether extraction at pH 5 removed product and starting material. The product IV' was obtained by (i) NaHCO.sub.3 extraction--to remove strongly acidic by-products (ii) extracted with 30% v/v 0.880 ammonia solution (6.times.). The ammonia washes contained essentially pure IV'. Starting material remaining in the ether was re-oxidised for 42 hrs and worked-up as above.
The total amount of IV' (isolated by acidifying the ammonia washes and extracting with CHCl.sub.3) obtained was 2.34 g (20% based on II').
The material was a colourless foam Rf 0.41 by TLC on silica in benzene-dioxan-acetic acid (95:25:4).
Nmr (CDCl.sub.3): 8.9-9.3 (12H, m, 4.times.CH.sub.3); 8.2-8.8 (15H, m, (CH.sub.3).sub.3 and 2.times.(CH.sub.3).sub.2 CH--CH.sub.2); 5.4-7.0 (4H, m, 2.times..alpha.--CH and --CH.sub.2 --N--); 2.0 and 2.4 (5H, m, C.sub.6 H.sub.5 SO.sub.2), 1.2 (1H, br.s, CO.sub.2 H).
Alternatively, the reduced Leu--Leu analogue IV may be synthesized by the following method:
SCHEME 2 ##STR23##
The following is a synthetic method for a reduced Leu--Val isostere by a preferred route.
SCHEME 3
Synthesis of the Protected Reduced Isostere of L--Leucyl--L--Valine ##STR24##
(1) Boc--L--Leucinal, 1
Boc--L--Leucine methyl ester (22.7 g, 90 mmoles) in dry toluene (250 ml) under N.sub.2 was cooled to -78.degree. and 25% di-isobutylaluminium hydride in toluene (130 ml, 225 mmoles) were added over 25 mins. keeping the temperature under -70.degree.. The mixture was stirred for 15 mins. at -78.degree. after completion of the addition, then MeOH (10 ml) was added cautiously. When effervescence ceased the mixture was poured into an ice-cold solution of Rochelle salt (100 ml of saturated solution+600 ml H.sub.2 O). This mixture was shaken until an extractable solution was obtained. The toluene was separated and the aqueous phase re-extracted with ether (2.times.300 ml). Toluene and ether extracts were combined and dried (Na.sub.2 SO.sub.4). The resulting oil was passed rapidly through a pad of silica gel in 15% EtOAc/petrol 40.degree.-60.degree.. The crude aldehyde was obtained as an oil, weight 18.68 g. Nmr showed aldehyde content to be 85%, therefore yield of aldehyde: 15.9 g (83%).
Nmr (CDCl.sub.3), .tau.: 0.45 (1H, s, CHO); 4.87 (H, br.d., BocNH): 5.83 (1H, br.m., NH--CHCHO); 8.43-8.93 (12H, m, (CH.sub.3).sub.3 C, (CH.sub.3).sub.2 CH.CH.sub.2); 9.0 and 9.1 (12H, 2.times.d, (CH.sub.3).sub.2 CH)
TLC: (solvent 30% EtOAc/petrol 60.degree.-80.degree.), Rf=0.43.
(2) Boc--L--Leucyl--L--valine benzyl ester reduced isostere, 2
L--Valine--OBzl (10 mmoles, from EtOAc/1N NaHCO.sub.3 partition of 3.8 g of p-toluene sulphonate salt) and Boc--L--Leucinal (2.54 g, 10 mmole aldehyde content) in dry tetrahydrofuran (20 ml) stood over 5 .ANG. molecular sieve (10 g) overnight. Sodium cyanoborohydride (630 mg, 10 mmoles) in MeOH (3 ml) was added with cooling, then left at room temperature for 30 mins. The mixture was diluted with methylene chloride (100 ml), filtered and evaporated to dryness. The residue was passed down a silica column in 20% EtOAc/petrol (60.degree.-80.degree.) to remove polar impurities. Isostere containing factions were combined. Crystallisation from petrol 60.degree.-80.degree. at -20.degree. gave large clusters of needles, 1.52 g (38%) m.p.
.tau.: 2.65 (5H, s, OCH.sub.2 C.sub.6 H.sub.5); 6.35 (1H, m, NHCHCO.sub.2 Bzl); 7.05 (1H, m, NH--CHCH.sub.2); 7.45 (2H, m, --CH.sub.2 NH--); 8.25-8.90 (13H, m, (CH.sub.3).sub.3 CO--.) (CH.sub.3).sub.2 CHCH.sub.2 and (CH.sub.3).sub.2 CH--); 9.05 and 9.15 (12H, 2.times.s, 2.times.(CH.sub.3).sub.2 CH).
TLC: (Solvent: 30% EtOAc/petrol 60.degree./80.degree.), Rf=0.39.
(3) N-(2S)-t-Butyloxycarbonylamino-4-methylpentyl, N-(3,4-dichlorobenzyloxycarbonyl)-L-valine, 4
Boc--L--Leucyl--L--valine, benzyl ester-reduced-isostere (1.5 g 3.68 mmoles) in dimethylformamide (60 ml) was hydrogenated at STP over 5% Pd/C (150 mg). After 31/2 hrs. the colloidal solution was flushed with nitrogen and 1M NaOH (3.8 ml, 1.05 equiv.) was added followed by 3,4-dichlorobenzyl pentachlorophenyl carbonate (1.92 g, 4.07 mmoles). The mixture was kept at 50.degree. in a stoppered flask for 24 hrs. and then evaporated to dryness. EtOAc was added and the Pd/C filtered off. The EtOAc solution was washed with 1M citric acid (2.times.), H.sub.2 O (1.times.), brine (1.times.), and dried (Na.sub.2 SO.sub.4).
The crude isostere 4 was chromatographed on silica-gel (Merck Keiselgel 60, 40-63 m) eluting with 2% MeOH/CHCl.sub.3 to give the title compound as a colourless oil.
Nmr(CDCl.sub.3), .tau.: 2.5-2.9 (3H, m, C.sub.6 Cl.sub.2 H.sub.3); 3.3-3.8 (2H, br, BocNH and CO.sub.2 H); 4.85 and 4.95 (2H, 2.times.s, OCH.sub.2 --C.sub.6 Cl.sub.2 H.sub.3): 5.5-6.3 (2H, m, NHCHCH.sub.2 and --NCHCO.sub.2 H); 6.5-7.2 (2H br, 2.times.d, CHCH.sub.2 N--); 8.2-8.9 (13H, m, (CH.sub.3).sub.3 CO, (CH.sub.3).sub.2 CHCH.sub.2 and (CH.sub.3).sub.2 CH--); 8.9-9.4 (12H, m, 2.times.(CH.sub.3).sub.2 CH)
TLC: (solvent 5% MeOH/CHCl.sub.3) Rf=0.32.
EXAMPLES
The following detailed Examples illustrate the invention.
The Examples are preceded by the preparation of Boc--Tyr[Bzl (2,6 Cl.sub.2)]--O--resin. (Reaction times marked * are convenient rather than necessary.)
Preparation of Resin
Boc--Tyr[Bzl(2,6 Cl.sub.2)]--OH (1.65 g, 3.75 mmol) was dissolved in ethanol (20 ml) and water (5 ml) added. The pH was brought to 7.0 with cesium bicarbonate solution and the solvent evaporated in vacuo. The residue was treated twice with toluene and evaporated to remove the last traces of water leaving a white powder which was dried for several hours over phosphorus pentoxide. The residue was dissolved in DMF (65 ml), chloromethylated resin (10 g, 7.5 mequiv.) added and the reaction stirred at 37.degree. for four days.
The resin was then filtered and washed thoroughly with DMF, DMF/water (9:1) and then DMF again. The resin was then resuspended in DMF (65 ml) and treated with acetic anhydride (2.36 ml, 25 mmol) and triethylamine (3.5 ml, 25 mmol) for 3 days.
The resin was filtered, washed thoroughly with DMF, DMF/water (9:1) and methanol and dried. The resin was then "defined" by shaking a suspension in dichloromethane and removing the particles slowest to float. The resin was then dried.
Yield 10.8 g.
Amino-acid analysis: (12N--HCl/propionic acid 1:1 130.degree., 2 hours) gave an incorporation of 0.11 mmol/g.
EXAMPLE I
H--His--Pro--Phe--His--Leu--reduced--Leu--Val--Tyr--OH (H-76)
Boc--Tyr[Bzl(2,6 Cl.sub.2)]--O--Resin (3 g, 0.6 mmol) was washed with reagents in the following sequence: CH.sub.2 Cl.sub.2 (3.times.) iPrOH (2.times.), CH.sub.2 Cl.sub.2 (3.times.), 40% TFA/CH.sub.2 Cl.sub.2 1 min then 20 min, CH.sub.2 Cl.sub.2 (3.times.), iprOH (2.times.), CH.sub.2 Cl.sub.2 (3.times.), 40% TFA/CH.sub.2 Cl.sub.2 1 min then 20 min, CH.sub.2 Cl.sub.2 (3.times.) iPrOH (2.times.) CH.sub.2 Cl.sub.2 (3.times.), 10% Et.sub.3 N/CH.sub.2 Cl.sub.2 (2.times.2 min), CH.sub.2 Cl.sub.2 (5.times.). Boc--Val--OH (0.65 g, 3 mmol) was then coupled using DCCI (0.68 g, 3.3 mmol) and HOBt (0.92 g, 6 mmol) in DMF/CH.sub.2 Cl.sub.2 (1:1) for 17 hours. The resin was then washed with DMF (3.times.), CH.sub.2 Cl.sub.2 (3.times.), iPrOH (2.times.), CH.sub.2 Cl.sub.2 (3.times.) 10% Et.sub.3 N/CH.sub.3 Cl.sub.2 (2 min), CH.sub.2 Cl.sub.2 (5.times.) then acetylated using acetylimidazole (0.66 g, 6 mmol) in DMF for 1 hour. The resin was then washed with DMF (3.times.) CH.sub.2 Cl.sub.2 (3.times.) iPrOH (2.times.) and finally CH.sub.2 Cl.sub.2 (3.times.).
This sequence of washes and reactions was repeated for the addition of each of the residues with the following modifications.
After deprotection of the Boc--Val--Tyr[Bzl(2,6 Cl.sub.2 ]--O--resin Boc--NH--CH(CH.sub.2 CHMe.sub.2)--CH.sub.2 --N(SO.sub.2 Ph)--CH(CH.sub.2 CHMe.sub.2)--CO.sub.2 H (0.42 g, 0.9 mmol) was coupled using DCCI (0.28 g, 1.35 mmol) and HOBt (0.275 g, 1.8 mmol) in DMF/CH.sub.2 Cl.sub.2 (1:1) for 18 hours, followed by acetylation using acetylimidazole (0.66 g, 6 mmol) in DMF for 1 hour.
After deprotection, Boc--His(Dnp)--OH (1.44 g, 3 mmol) was coupled using DCCI (0.68 g, 3.3 mmol) and HOBt (0.92 g, 6 mmol) for 2 hours, followed by acetylation for 1 hour.
Deprotection of the histidyl peptide was achieved using 50% TFA/CH.sub.2 Cl.sub.2 instead of the usual 40% TFA/CH.sub.2 Cl.sub.2. Boc--Phe--OH (0.796 g, 3 mmol) was coupled using DCCI (0.68 g, 3.3 mmol) and HOBt (0.92 g, 6 mmol) for 171/2 hours *, followed by acetylation for 1 hour.
Deprotection of the phenylalanyl peptide was achieved using the usual 40% TFA/CH.sub.2 Cl.sub.2. Boc--Pro--OH (0.646 g, 3 mmol) was coupled using DCCI (0.68 g, 3.3 mmol) and HOBt (0.92, 6 mmol) for 2 hours followed by acetylation for 1 hour.
After deprotection Boc--His(Dnp)--OH (1.44 g, 3 mmol) was coupled using DCCI (0.68 g, 3.3 mmol) and HOBt (0.92 g, 6 mmol) for 141/2 hours* followed by acetylation for 1 hour.
The resin was then washed with DMF (3.times.), CH.sub.2 Cl.sub.2 (3.times.) iPrOH (2.times.), CH.sub.2 Cl.sub.2 (3.times.) and finally MeOH (3.times.) and dried to give 3.5353 g of product.
1.2 g of this material was treated with HF at 0.degree. for 11/4 hours in the presence of anisol (1.5 ml) then dried overnight over potassium hydroxide. The resin was then washed with DMF/water (1:1), acetic acid and finally acetic acid/water (1:1) to remove the peptide. These washes were combined and evaporated in vacuo.
The residue was dissolved in DMF (15 ml) and water (6 ml) thioethanol (5 ml) added and the pH of the solution brought to 8.0 with sodium carbonate. The reaction was stirred overnight the solvent evaporated and the residue applied to a Sephadex G 25 column (72.times.2.5 cms) eluted with 50% acetic acid at 18 mls/hr collecting 6 ml fractions. Fractions 27-46 were combined and the solvent evaporated in vacuo and dried. Then 90% of the residue was taken (for the rest see Example III) and dissolved in anhydrous ammonia (100 ml) and small portions of sodium wire added until a permanent blue colour was achieved for 15 seconds. The ammonia was allowed to evaporate and the residue dried.
The residue was applied to a Sephadex SPC25 column (77.times.1.6 cms) eluted with 30% acetic acid at 40 mls/hr, with a sodium chloride gradient from 0.01M to 1M over two days collecting 6.6 ml fractions.
The product was contained in fractions 100-104. These were pooled, evaporated and the residue dissolved in glacial acetic acid and filtered to remove the sodium chloride. The solution was evaporated and desalted on a Sephadex G25 column (72.times.2.5 cms) eluted with 50% acetic acid at 18 mls/hr collecting 6 ml fractions. Fractions 32-6 were pooled, evaporated, transferred to a vial and lyophilised.
EXAMPLE II
H--Pro--Phe--His--Leu--reduced--Leu--Val--Tyr--OH (H-79)
Fractions 80-84 of the SPC 25 Sephadex column from the previous synthesis were combined, evaporated and the residue dissolved in glacial acetic acid and filtered to remove sodium chloride. The solution was evaporated and the product desalted on a Sephadex G25 column (72.times.2.5 cms) eluted with 50% acetic acid at 18 mls/hr collecting 6 ml fractions. Fractions 32-9 were pooled, evaporated, transferred to a vial and lyophilised.
The above example illustrates how Y in formulae (V), (VA) and (VB) may be absent.
EXAMPLE III
H--His--Pro--Phe--His--Leu--reduced (SO.sub.2 Ph)--Leu--Val--Tyr--OH (H-78)
In the synthesis of compound H76 10% of the residue from the Sephadex G25 column after the HP and thioethanol treatments of the resin was kept.
This material was applied to a Sephadex SPC25 column (77.times.1.6 cm) eluted with 30% acetic acid at 20 mls/hr with a sodium chloride gradient from 0.01M to 1M over 2 days collecting 6.6 ml fractions.
The product was contained in fractions 74-7. These were pooled, evaporated, dissolved in glacial acetic acid and filtered to remove sodium chloride. The solution was then evaporated and desalted on a Sephadex G25 column (72.times.2.5 cms) eluted with 50% acetic acid at 18 mls/hr collecting 6 ml fractions. Fractions 31-4 were pooled evaporated, the residue transferred to a vial and lyophilised.
EXAMPLE IV
H--DHis--Pro--Phe--His--Leu--reduced--Leu--Val--Tyr--OH, (H-77)
The Boc--Tyr[Bzl(2,6,Cl.sub.2)]--O--Resin (3 g, 0.6 mmol) was deprotected and Boc--Val--OH (0.65 g, 3.0 mmol) was coupled using DCCI (0.68 g, 3.3 mmol) and HOBt (0.92 g, 6 mmol) for 22 hours* then acetylated with acetylimidazole (0.66 g, 6 mmol) for 1 hour.
After deprotection, Boc--Leu--reduced (SO.sub.2 Ph)--Leu--OH, IV, (0.42 0.9 mmol) was coupled using DCCI (0.28 g, 1.35 mmol) and HOBt (0.275 g, 1.8 mmol) for 20 hours, then acetylated for 1 hour.
After deprotection Boc--His(Dnp)--OH (1.44 g, 3 mmol) was coupled using DCCI (0.68 g, 3.3 mmol) and HOBt (0.92 g, 6 mmol) for 22 hours*, then acetylated for 1 hour.
After deprotection, this time with 50% TFA/CH.sub.2 Cl.sub.2 Boc--Phe--OH (0.796 g, 3 mmol) was coupled with DCCI (0.68 g, 3.3 mmol) and HOBt (0.92 g, 6 mmol) for 19 hours* then acetylated for 1 hour.
After deprotection Boc--Pro--OH (0.646 g, 3 mmol) was coupled using DCCI (0.68 g, 3.3 mmol) and HOBt (0.92 g, 6 mmol) for 3 hours then acetylated for 1 hour.
The peptide was again deprotected and coupled with Boc--D--His(Boc)--OH (0.91 g, 2.56 mmol using DCCI (0.58 g, 2.82 mmol) and HOBt (0.78 g, 5.1 mmol) for 2 hours, then acetylated for 1 hour.
The resin was washed with DMF (3.times.) CH.sub.2 Cl.sub.2 (3.times.) iProH (2.times.) CH.sub.2 Cl.sub.2 (3.times.) and finally MeOH (3.times.) and dried to give 3.6563 g of product.
1.2 g of this material was treated with HF at 0.degree. for 11/4 hours in the presence of anisole (1.5 ml) then dried overnight over potassium hydroxide. The resin was then washed with DMF, DMF/H.sub.2 O (1:1), acetic acid and finally acetic acid/water (1:1) to remove the peptide. These washes were combined and evaporated in vacuo.
The residue was dissolved in DMF (15 ml) and water (6 ml), thioethanol (5 ml) added and the pH of the solution brought to 8.0 with sodium carbonate solution. The reaction was stirred overnight the solvent evaporated and the residue applied to a Sephadex G 25 column (72.times.2.5 cms) eluted with 50% acetic acid at 18 mls/hr collecting 6 ml fractions. Fractions 27-46 were combined and the solvent evaporated in vacuo and dried.
90% of the residue was dissolved in anhydrous ammonia (100 ml) and small portions of sodium wire added until a permanent blue colour was achieved for 15 seconds. The ammonia was allowed to evaporate and the residue dried.
The residue was applied to a Sephadex SPC 25 column (77.times.1.6 cms) eluted with 30% acetic acid at 20 mls/hr with a sodium chloride gradient 0.01M to 1M over 2 days collecting 6.6 ml fractions.
The product was contained in fractions 88-92. These were pooled, evaporated and the residue dissolved in glacial acetic acid and filtered to remove the sodium chloride. The solution was evaporated and desalted on a Sephadex G 25 column (72.times.2.5 cms) eluted with 50% acetic acid at 18 mls/hr collecting 6 ml fractions. Fractions 32-41 were pooled, evaporated, transferred to a vial and lyophilised.
EXAMPLE V
H--His--Pro--Phe--His--Leu--reduced--Val--Ile--His--OH (H 113)
The method is generally that of Example I above but illustrates formula (VB).
Preparation of Boc--His(DNP)--O--Resin (AH/30/83)
Boc--His(DNP)--OH. (4.74 g, 11.25 mmol) was dissolved in ethanol (60 ml) and a solution of cesium bicarbonate (2.18 g, 11.25 mmol) in water (15 ml) added. The solvent was evaporated in vacuo and the residue treated four times with toluene and evaporated to remove water before finally drying overnight over phosphorus pentoxide. The residue was dissolved in DMF (175 ml), chloromethylated resin (30 g, 22.5 m-equiv.) added and the reaction stirred at 37.degree. for five days.
The resin was filtered off and washed thoroughly with DMF, DMF/water (9:1) and then DMF again. It was resuspended in DMF (175 ml) and treated with acetic anhydride (7.08 ml, 75 mmol) and triethylamine (10.5 ml, 75 mmol) overnight.
The resin ester was filtered, washed thoroughly with DMF, DMF/water (9:1) and methanol and dried. It was then "de-fined" by shaking it in dichloromethane and removing the particles in the supernatant. Finally, the resin was dried. Yield: 30.45 g. A trial coupling with Boc--Ala--OH, followed by amino-acid analysis (after hydrolysis with 12N--HCl/propionic acid 1:1, 130.degree., 2 hrs) gave an incorporation of 0.22 mmol/.
Coupling to Resin Ester
Boc--His(DNP)--O--Resin (2.5 g 0.55 mmol) was deprotected with 50% TFA/CH.sub.2 Cl.sub.2 and Boc--Ile--OH (0.748 g 3 mmol) was coupled using DCCI (0.68 g, 3.3 mmol) and HOBt (0.92 g, 6.0 mmol) for 2 hours, then acetylated with acetyl imidazole (0.55 g, 5 mmol) overnight*.
After deprotection with 40% TFA/CH.sub.2 Cl.sub.2, Boc--Leu--reduced (3,4--Cl.sub.2 --Z)--Val--OH, 4 (0.343 g, 0.66 mmol) was coupled using DCCI (0.15 g, 0.73 mmol) and HOBt (0.202 g, 1.32 mmol) for 16 hours, then acetylated for 1 hour.
After deprotection, Boc--His(DNP)--OH (1.26 g, 3.0 mmol) was coupled using DCCI (0.68 g, 3.3 mmol) and HOBt (0.92 g, 6 mmol) for 2 hours, then acetylated for 1 hour.
After deprotection, again with 50% TFA/(CH.sub.2 Cl.sub.2, Boc--Phe--OH (0.796 g, 3 mmol) was coupled with DCCI (0.68 g, 3.3. mmol) and HOBt (0.92 g, 6 mmol) for 3 hours* then acetylated overnight*.
After deprotection, Boc--Pro--OH (0.646 g, 3 mmol) was coupled using DCCI (0.68 g, 3.3 mmol) and HOBt (0.92 g, 6 mmol) for 2 hours then acetylated for 1 hour.
The peptide was again deprotected and coupled with Boc--His(DNP)--OH (1.26 g, 3 mmol) using DCCI (0.68 g, 3.3 mmol) and HOBt (0.92 g, 6 mmol) for 2 hours, then acetylated overnight*.
The peptide resin ester was washed with DMF (3.times.), CH.sub.2 Cl.sub.2 (3.times.) iProH (2.times.), CH.sub.2 Cl.sub.2 (3.times.) and finally MeOH (3.times.) and dried. It was then treated with HF at 0.degree. for 11/4 hours in the presence of anisole (4 ml) and dried overnight over potassium hydroxide. The resin was washed with DMF, acetic acid and acetic acid/water (1:1) to remove the peptide. The washes were combined and evaporated in vacuo.
The residue was dissolved in DMF (60 ml) and water (24 ml), thioethanol (10 ml) was added and the pH of the solution brought to 8.0 with sodium carbonate solution. The reaction mixture was stirred overnight, the solvent evaporated and the residue applied to a Sephadex G25 column (77.times.2.5 cms). It was eluted with 50% acetic acid at 18 mls/hr collecting 6 ml fractions. Fractions 34-53 were combined and the solvent evaporated in vacuo and dried.
EXAMPLES VI-IX
These Examples illustrate formula (VA). The methods disclosed above are applied to condensing Boc--Phe--H or Boc--Leu--H with H--Phe--OBzl, reducing the imine link, deprotecting at the carboxyl terminus and protecting the nitrogen of the reduced peptide link to give: ##STR25## This Phe--reduced--Phe or Leu--reduced--Phe analogue is then used as follows:
VI Use of 12 (Phe--reduced--Phe) in an analogue otherwise as H-77 (see Example IV)
VII Use of 12a (Leu--reduced--Phe) in an analogue otherwise as H-77 (see Example IV)
VIII Use of 12 in an analogue as H-76 (Example I), viz: ##STR26## IX Use of 12a in an analogue as H-76 (Example I), viz: ##STR27##
EXAMPLE X
This Example illustrates formula (VB), the method of Example V being used but with the Tyr resin of Examples I to IV, to give: ##STR28##
EXAMPLE XI
Use of the methods disclosed herein to give: ##STR29## this being an analogue illustrating the non-criticality of the terminal portions of the chain allowing X and W in formulae (V), (VA) and (VB) to represent further residues. It is a further example of formula (VA).
EXAMPLE XII
Use of the methods disclosed herein to give: ##STR30## a compound of value in its relation to the Leu--Val structure at 11, 12 in human renin substrate.
The following Example illustrates the synthesis of hydroxy and keto isosteres.
EXAMPLE XIII
The reaction scheme below, also suitable for other hydroxy dipeptide analogues, was used to synthesise an N-terminal and hydroxy-group protected Leu--Leu hydroxy isostere 18.
SCHEME 4 ##STR31##
The resulting N-terminal phthaloyl protected, --OH protected hydroxy isostere of Leu--Leu can be coupled direct for example to valyl tyrosine, followed by removal of the phthaloyl group, coupling direct to a suitable tri or tetrapeptide, and deprotection at the --OH group by mild acid hydrolysis, to give for example analogues corresponding to H-76 (Example 1), H-79 (Example 2), H-77 (Example 4). Alternatively the phthaloyl group may be removed by treatment with hydrazine and a new protective group, e.g. benzyloxycarbonyl or t-butyloxycarbonyl attached prior to coupling. The methods used after the preparation of the protected hydroxy isostere are those of the peptide synthesis art, well known in themselves and exemplified in detail herein. The compounds specifically prepared are:
EXAMPLE XIV
The alternative and preferred reaction scheme below, also suitable for other dipeptide hydroxy isosteres was used to synthesise an N-terminal and hydroxy-group protected Leu-Val hydroxy isostere 23.
SCHEME 5 ##STR32##
In the above scheme the protected bromohydrin 19 is obtained in the same way as the corresponding intermediate 17 in Scheme 4, and is subjected to malonic ester synthesis and alkylation with isopropyl iodide to give the malonic ester derivative 20. Protection on the hydroxyl function is changed from Thp to Bzl to yield 21 and the latter is subjected to protonolysis and decarboxylation. In the resulting isostere acid 22 amino protection is changed from Pht to Boc yielding the protected isostere 23 which is suitable for incorporation into an octapeptide analogue by the usual methods of solid phase peptide synthesis to give for example the octapeptide analogue: ##STR33## This is a Leu--hydroxy--Val isostere.
EXAMPLE XV
Keto isosteres may be prepared for example by the method of published U.K. Specification No. 1 587 809 (U.S. Pat. No. 4,242,256) of R. Sharpe and one of the present inventors M. Szelke, to which reference may be made. Alternatively they may be prepared from hydroxy isosteres prepared as disclosed herein and in particular, in the present Example, from the final product 23 of scheme 5 as in the scheme below:
SCHEME 6 ##STR34##
In the above scheme the benzyl protection is selectively removed from the hydroxyl function of 23 and the free acid is is converted into its sodium salt 24. The latter is subjected to oxidation by pyridinium dichromate and acidification to give the partially protected keto isostere acid which is ready for incorporation into an octapeptide analogue by the usual methods of solid phase synthesis, for example the analogue: ##STR35## This is a Leu--keto--Val isostere.
Alternatively the keto isostere may be prepared directly by a modified version of scheme 5 wherein the bromohydrin 19 is replaced by the bromoketone 15 (scheme 4), which is: ##STR36## giving the compound ##STR37## which is successively treated with TsOH, toluene and (i) N.sub.2 H.sub.4, (ii) Boc.sub.2 O to give: ##STR38##
EXAMPLES XVI-XL
The tables at the end of the description herein show the structures and the activity, of further examples of compounds made by following the methods set out in detail for corresponding sequences in the preceding examples. Compounds H 157 and H 184 to H 188 in these examples of compounds represent partial sequences of formula V, as also shown in Example II. The activity of H 157 may be noted; H 184 to H 186 show lower but still potentially interesting activities.
The following examples are of the methods of diagnosis and treatment according to the invention.
EXAMPLE XLI
A person suffering from hypertension is given repeated doses of a nasal instillation preparation of 0.01 to 1 mg/kg body weight of the compound H 108 (Example 9), per dose, three times a day. Clinically significant maintained reduction of the hypertension is a positive indication of hypertension amenable to treatment by the method of the invention.
Comparable amounts of the compounds of other Examples such as Examples 1, 3, 4, 5, 8, 9 and 10 may be substituted and the method may be similarly applied to patients in heart failure, as referred to herein. In both the hypertension and the heart failure instances the plasma-renin may or may not be above normal.
EXAMPLE XLII
A person diagnosed as suffering from amenable hypertension by the method of Example 16 is treated by means of a continuing course of the compound H 108 (Example 9) or other compound as exemplified herein, in the amounts set out in Example 16.
ACTIVITY IN VITRO
Preliminary activity test results in the human renin-renin substrate reaction in vitro are given in the table below, with comparative figures for the tetrapeptide analogue (III). The test is based on the methods described by J. A. Millar et al. in Clinica Chimica Acta (1980) 101 5-15 and K. Poulsen and J. Jorgensen in J. Clin. Endocrinol. Metab. (1974) 39 816.
It is based on the measurement, by radioimmunoassay, of Angiotensin-I released from human renin substrate by human renin in human plasma. The inhibitor is dissolved in 0.01N HCl (10 .mu.l) and added to human plasma (75 .mu.l) containing EDTA, and angiotensin-I antibody (15 .mu.l) in 3M-Tris/HCl buffer (pH 6.9).
After incubation at 37.degree. C. for 0-120 mins., the enzymic reaction is quenched by the addition of ice-cold 0.25M Tris/HCl buffer (pH 7.4) containing 0.01% of bovine serum albumin. 125I-labelled angiotensin-I is added, followed by equilibration at 4.degree. C. for 48 hours. Free and bound ligand are separated by the addition of dextran-coated charcoal, and the amount of bound radio-ligand determined in a gamma counter.
The results for the renin inhibitory activities of the present compounds thus tested, expressed as the IC.sub.50 (the molar concentration required to cause 50% inhibition), are as follows:
These are most notable results, showing a potency, in the reduction of renin activity remaining in the plasma in the presence of the analogue, several orders of magnitude greater than the previously proposed tetrapeptide analogue.
The potency of the compound of Example XIV is most especially notable, and it is an example of a "hydroxy" isostere. Comparing its IC.sub.50 (0.0024 .mu.M) with that of the corresponding Leu--reduced--Val isostere of Example V (0.26 .mu.M) shows that high activity can be expected from this single change. Moreover the activity is highly specific, restricted to renin and not shown even in the closely related enzymes cathepsin D and renal acid protease.
ACTIVITY IN VIVO
The activity tests below are in animals but indicate corresponding activity in man.
In in vitro studies, in plasma from both normal and sodium-depleted dogs, the compound H-77 (Example IV) inhibited renin. In in vivo studies H-77 was infused into normal conscious sodium-depleted dogs at rates of 0.01, 0.1, 1 and 10 mg/kg/hr. A maximum fall in blood pressure, plasma renin (PR) angiotensin-I (A-I) and angiotensin-II (A-II) levels was obtained within 10 minutes at doses of 1 and 10 mg/kg/hr. When the infusion was stopped, blood pressure returned to baseline levels 30 minutes after the 1 mg/kg/hr. dose, but more slowly after the 10 mg/kg/hr. dose.
In the claims below it will be understood that compounds may be in the form shown or in protected or salt form at NH.sub.2, COOH, OH or other active groups and in particular as their physiologically acceptable acid addition salts at basic centres. Further as already noted herein general reference to amino acids and amino acyl residues and side chains is to be taken as reference to such whether naturally occurring in proteins or not and to both D- and L-forms, and amino is to be taken as including imino except where an aromatic acid, residue or side chain is specified.
Claims
We claim:
1. A polypeptide analogue of the formula: ##STR64## or the partial sequences: where: Pro, Phe and His are in substituted form; Pro by OH, Phe by OH, F, Cl, Br or Me, His by Me; or Phe is replaced by pGlu, Phe by Tyr or His by spinacin. X=H; or an N-protecting group; Y=D- or L-His or is absent; A= ##STR65## where the configuration at asymmetric centres * is either R or S, where in VIII the hydroxy group and protected in ether --OR.sup.4 or ester ##STR66## form where R.sup.4 is as given under W below and where R.sup.1 and R.sup.2, the same or different=.sup.i Pro (isopropyl), .sup.i Bu (isobutyl) or Bzl (benzyl); R.sup.3 =H or an N-protecting group; B=D- or L-Val Leu or Ile; Z=D- or L-Tyr, Phe, or His; and W=(i)--OH and in protected ester form as --OR.sup.4 where R.sup.4 =lower alkyl C.sub.1 -C.sub.5 ; or (ii)--NH.sub.2 and in protected amide form as --NHR.sup.5 or --N(R.sup.5).sub.2, wherein R.sup.5 =an N-protecting group; or (iii) an L- or D-serine or L- or D-lysine or arginine and in amide form, or (iv) an amino alcohol residue derived therefrom as such or protected in ester or ether form or Z+W=an alcohol derived from L- or D-Tyr, Phe or His residue and protected in ester or ether form; isosteric links as set out above for A being in free form or in protected form at one or more nitrogen, carboxyl, hydroxy or other reactive groups or in salt form at amino imidazole or carboxyl groups as their physiologically acceptable acid addition salts at basic centres.
2. A polypeptide analogue, according to claim 1, of the formula: ##STR67## where X, Y, Pro, Phe and His are as in claim 1 A is as in claim 1 except that R.sup.1 and R.sup.2, the same or different=.sup.i Bu (isobutyl) or Bzl (benzyl) R.sup.3 =--H; or --SO.sub.2 Ph, --SO.sub.2 C.sub.6 H.sub.4 CH.sub.3 (p), Boc or formyl Z=Tyr, Phe; W=is as in claim 1 Z+W is as in claim 1.
3. A polypeptide analogue, according to claim 1 of the formula: ##STR68## where X, Y, Pro, Phe and His are as in claim 1 A is as in claim 1 except that R.sup.1 =.sup.i Bu (isobutyl) or Bzl (benzyl) or other lipophilic or aromatic amino-acid side chain R.sup.2 =.sup.i Pr (isopropyl), and R.sup.3 =--H; or --SO.sub.2 Ph, --SO.sub.2 C.sub.6 H.sub.4 CH.sub.3 (p), Boc or formyl: Z is as in claim 1 W is as in claim 2 Z+W is as in claim 1.
4. A polypeptide according to claim 1 modified by isosteric replacement, as set out therein, at Pro-Phe or Phe-His links.
5. A polypeptide according to claim 1 wherein the isosteric replacement at 10,11 position is of the "reduced" kind.
6. A polypeptide according to claim 1 wherein the isosteric replacement at 10,11 position is of the "hydroxy" kind.
7. A composition for treating heart failure comprising an effective amount of a polypeptide of claim 1 in a pharmaceutically acceptable carrier.
Patent Citations (1)
| Patent | Date | Inventor | Cited By |
|---|---|---|---|
| US4424207 | 1984-01-01 | Szelke et al. |
Non-Patent Literature (3)
- Chemical Abstracts, 97, 624(1982), Abst. No. 39405p.
- Chemical Abstracts, 98, 726(1983), Abst. No. 54442n.
- Chemical Abstracts, 89, 122(1978), Abst. No. 100494v.