CA1065858A - Peptide enzyme inhibitors - Google Patents

Abstract

Abstract Esters and an amide of the nonapeptide Pyr-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro having the formula Pyr-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro-Y

wherein Y is -NH2 or -OR where R is an alkyl group con-taining 1 to 10 carbon atoms have been found to inhibit the hypertensive effect of angiotensin I.

Description

ii8 This invention relates to novel esters and amides of the nonapeptide Pyr-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro which have been found to inhibit the hypertensive effect of angiotensin I.
The action of the enzyme renin on renin substrate, a pseudoglobulin in blood plasma, produces a polypeptide angiotensin I, also known as hypertensin I. The latter is converted by an enzyme to angiotensin II, also known as hypertensin II or angiotonin. Angiotensin II is an active pressor substance which is present in the plasma of indivi~
duals with hypertension in quantities sufficient to maintain elevated blood pressure. Inhibition of the enzyme responsible for the conversion of angiotensin I to angiotensin II serves to remove a cause of essential hypertension.
U. S. Patent No. 3,832,337 to Ondetti et al. dis-closes various peptides and acylated peptides which inhibit enzymatic conversion of angiotensin I into angiotensin II, among such peptides being Pyr-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro.

Studies on structure-activity relationships concerning pep-tides as disclosed in U. ~. Patent. No. 3,~32,337 indicate that a ~ree terminal carboxyl group is needed to obtain potent inhibitors ln vitro and ln vivo. For example, see the paper by Cushman et al., "Inhibition of Angiotensin-Converting Enzyme by Analogs o~ Peptides from Bothrops jararaca Venom", Experienta 29, 1032 (1973), Birkhauser Verlag, Basel, Switzerland. However, it has now been ~ound that not only is the above nonapeptide an inhibitor of angio-tensin I induced hypertension, but esters and amides of such nonapeptide are useful for such purpose as well. This is ~6~3S~3 surprising inasmuch as such esters and amides show ver~ low or no detectable enzyme inhibition activity in vitro.
Generally, ln vitro activity of peptides in this area usu-ally corresponds to in vivo activity thereof. Accordingly, .
it is indeed unexpected that such esters and amides are potent inhibitors of angiotensin I induced hypertension.
The present invention provides a peptide having the formula Pyr-Trp-Pxo-Arg-Pro-Gln-Ile-Pro-Pro-~

wherein Y is -NH2 or -OR where R is an alkyl group contain-ing 1 to 10 carbon atoms.
qlhe present invention also provides a process for preparing a peptide having the formula Pyr-l'rp-Pro-Arg-Pro-Gln-Ile-Pro-Pro-Y

wherein Y is -NH2 or -OR where R is an alkyl group contain-ing 1 to 10 carbon atoms which comprises coupling or reacting suitable amino acids and peptides or their reactive deri~a-~ives in a per se conventional procedure adopted for peptide

2.0 s~nthesis to form the peptide Pyr-'rrp-Pro-Arg-Pro-(31n-~le-Pro-Pro and before, during or after the formation of this peptide, adding an alkyl ester group or an amide group to the terminal Pro by conventional procedures.
Unless otherwise indicated in the following speci~i-cation and claims, all amino acids are of the L-configuration.
In describing the peptides of the present invention, the following abbreviations will be used throughout the specification and claims:

~L~6~ 8 Arg - Arginine Gln - Glutamine Ile - Isoleucine Pro - Proline Pyr - Pyroglutamic acid Trp - Tryptophane The following peptides of the present invention have been found to be effective in inhibiting angiotensin I in~

duced hypertension:

Ia - The esters having the formula Pyr-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro-OR, ~

wherein R is alkyl :

II - The amide having the formula Pyr-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro-NEI2 The alkyl group o the above esters may comprise a straight or branched chain hydrocarbon fragment containing 1 to 10 carbon atoms, and preferably 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, n-butyl, s-butyl, -t-butyl, pentyl, hexyl, heptyl, octyl, nonyl and decyl, as well as the various isomers of the latter six groups. The methyl and t-butyl esters are particularly preferred.

The esters of the lnvention are pre~erably prepared by reaction o~ a di-, tri-, tetra-, penta-, hexa-, hepta-or octa-peptide including a Pro- terminal group protected by an alkyl ester with one or more peptides, as is necessary, ~
employing conventional peptid0 preparatory techniques, to :
form the nonapeptide alkyl ester (Ia) of the invention. In this manner, the ester of the invention may be formed directly without having to first form the parent peptide ~:

(III) Pyr-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro . .
", . ' . ' ~0~58S8 KX37 The amide of the invention is preferably prepared by reaction of a di-, tri-, tetra-, penta-, hexa-, hepta-or octa-peptide including a Pro-terminal group protected by an amide group with one or more peptides, as is necessary, employing conventional peptide preparatory techniques, to form the nonapeptide amide (II) of the invention. In this manner, the amide of the invention may be formed directly - without having to first form the parent peptide (III).
However, it wlll be appreciated that the esters as well as the amide of the invention may be formed by first forming the parent peptide III in accordance with the pro-cedures as outlined in U. S. Patent No. 3,832,337 and there-after adding the alkyl ester or amide group to the terminal Pro employing conventional procedures.
The compounds of the present invention inhibit the conversion of angiotensin I into angiOtensin II in vivo and thereby antagonize the hypertensive effect of angiotensin I.
The present compounds are capablé of inhibiting the hyper-tensive effect of angiotensin I when administered to mammals such as rats, mice or dogs, in a dosa~e level of from about 0.5 to about 10 mg/ky. For the latter purpose they may be administered parenterally by incorporating the appropriate dosage with a physiologically acceptable carrier.

~ KX37 ~5~
he following examples illustrate the present invention without, however, limiting the same thereto.
All temperatures are expressed in degrees Centigrade.

Example 1 Benzyloxycarbonyl-isoleucyl-proline-proline Prolyl-proline hydrobromide (4.24 g) is dissolved in a mixture of dimethylformamide (40 ml) and triethylamine (2O8 ml). Benzyloxycarbonyl-isoleucine p-nitrophenyl ester (9.27 g) and l-hydroxybenzotriazole (3 g) are added and the mixture is stored at room temperature for 16 hours. Dimethyl-aminopropylamine (1 ml) is added and after 2 hours the solvent is removed ln vacuo. The residue is dissolved in ethyl acetate (200 ml) and washed se~uentially with 0.1 N hydrochloric ' acid, and water. The organic phase is dried over magnesium sulfate and concentrated to dryness ln vacuo to yield 13.8 g solid. This material is applied to a silica gel column ~-~
(300 g) in ethyl acetate and eluted with the same solvent.
The fractions containing the desired above-titled tripeptide are pooled and concentrated to dryness.

Example 2 Methyl benzyloxYcarbonyl-isoleucyl-proly-l-prolinate The tripeptide acid oE Example 1 (~.6 g) is dissolved in methanol and treated with an ethereal solution of diazo-methane until a persistent yellow color is obtained. After 0,5 hour a few drops of acetic acid are added to discharge . ,, , , ~

6~
the yellow color; the solvent is removed in vacuo and replaced with ethyl acetate. This solution is washed sequen-tially with 0.1 N ilCl, water, sa-turated sodium bicarbonate and water.
The organic phase is dried over magnesium sulfate and concentrated to dryness to yield the title compound.

xample 3 ~thyl benzyloxycarbonyl-c3lutaminyl-isoleucyl-prolyl-prolinate The tripeptide of Example 2 (1.4 g) is dissolved in 95 ethanol (30 ml) and N hydrochloric acid (3 ml.) Palladium on charcoal (0.3 g) is added and the suspension is stirred under a positive hydrogen pressure until no more carbon dioxide is detected in the outcoming gases. The catalyst is removed by filtration and the filtrate is concentrated to dryness in vacuo. The residue is dissolved in a mixture of .. ...
dimethylformamide (6 ml) and triethylamine (0.5 ml). Benzyloxy-carbonyl-glutamine p-nitrophenyl ester (1.6 g) and l-hydroxy-benzotriazole (0.5 g) are added and the reaction is allowed to proceed at room temperature until the ninhydrin test is negative. Dimethylaminopropylamine (0.5 ml) is added and the reaction is allowed to proceed for another hour. Ethyl acetate (200 ml) is added and -the enSuinCJ solu-tion is washed neutral. The organic phase is dried over magnesium sulfate and the solvent :is removed in vacuo to yield thc title co~pound.

31L(~6~
Example 4 Methyl benzyloxycarbonyl-~-nitroarqinyl-prolyl-qlutaminyl-isoleucyl-prolyl-prolinate The tetrapeptide of Example 3 (14 y) is dissolved in absolute ethanol (325 ml) and N hydrochloric acid (23 ml).
Palladium on charcoal (3.0 g) is added and the mixture~is stirred under positive hydrogen pressure until no more carbon dioxide is liberated. The catalyst is filtered off and the filtrate is concentrated to dryness. The residue is dissolved in dimethylformamide (43 ml) and triethylamine (3.2 ml).
senzyloxycarbonyl nitroarginyl-proline 2,4,5-trichlorophenyl ester (15.7 g) and l-hydroxybenzotriazole (3.1 g) are added immediately and the reaction is allowed to proceed until the ninhydrin test is negative. Dimethylamino propylamine (5.4 ml) is added and after two hours the solvent is removed ln vacuo.
The residue is dissolved in ethyl acetate and the solution is washed neutra].. The e-thyl acetate is concentrated ln vacuo to ca 70 ml and the solution poured into 1 liter of vigorously stirred ether. The precipitate is filtered and dried to give the title compound.

Example 5 ~ethyl benzyloxycarbonyl-tryptophyl-proLyl-arqinyl-prolyl-ql~
The hexapeptide of Example 4 (1.8 g) is dissolved in absolute ethanol (27 ml) and N hydrochloric acid (4 ml).
Palladium on charcoal (0.38 y) is added and the mixture is stirred under a positive hydrogen pressure until the ultraviolet absorption of the nitroguanidine chromophore can no longer ~65~5~ KX37 be detected. The catalyst is filtered off and the filtrate is concen-trated to dryness ln vacuo. The residue is dissolved in dimethylformamide (10 ml) and triethylamine (0.28 ml).
Benzyloxycarbonyl-tryptophyl-proline 2,4,5-trichlorophenyl ester (1.5 g) and l-hydroxybenzotriazole (0.3 g) are added immediately and the reaction is allowed to proceed until the ninhydrin test is negative. The triethylamine hydrochloride precipitate is filtered off and the precipitate is poured into 250 ml of vigorously stirred ethyl acetate. The precipitate is filtered and dried to give the title compound.

Example 6 Pyroqlutamyl-tryptophyl-prolyl-arqinyl-prolyl-qluta-minyl-isoleucyl~prolyl-proline, methyl ester The octapeptide of Example 5 (10 g) is dissolved in absolute ethanol (150 ml) and N hydrochloric acid (9 ml).
Palladium on charcoal (2 g) is added and the suspension is stirred under positive hydrogen pressure until no more carbon dioxide is evolved. The catalyst is removed by filtration and the filtrate is concentrated to dryness ln vacuo. The residue is dissolved in dimethylformamide (~0 ml) and l-hydroxybenzotriazole (1.2 y), triethylamine (1.2 ml) and pyro~lutamic acid 2,~,5-tri-chlorophenyl ester are added in rapid succession. The reaction is allowed to proceed until the ninhydrin test is negative. The triethylamine hydrochloride precipitate i~s removed by filtration and the filtrate is poured into 1.1 liters of vigorously stirred ethyl acetate. The precipitate is filtered and dried to give the title compound. This material is purified by :. :
~;
. .

1~65858 KX37 chromatography on Sephadex G-25 in 0.01 M ammonium bicar-bonate.
Amino Acid Analysis - Molar Ratios:
Found (Calculated): NH3 0.92 (1.0); Arg 0.86 (1.0);
Pro 4.0 (4.0); Trp 0.78 (1.0); ; ~ -Gln 2.12 (2.0); Ile 0.96 (1.0) Thin layer chromatography - Silica gel (Butyl alcohol 30, Pyridine 20, Acetic Acid 6, Water 24) Rf = 0.53.
~ xample 7 n-Butyl benzyloxycarbonyl-isoleucyl-proline-prolinate A solution of benzyloxycarbonyl-isoleucyl-prolyl- ;
proline (4.5 g) in chloroform (30 ml) is added slowly to a solution of l-n-butyl-3-p-tolyltriazene (1.9 g) in chloro-form ~15 ml). When the reaction is complete, the chloroorm solution is washed neutral and the organic phasa is dried over magnesium sulfate and concentrated to dryness in vacuo to give the title compound.

Example 8 n-Butyl-benzyloxycarbonyl-glutaminyl-isoleucyl-prolyl-prolinate This compound is prepared by the procedure o~ Exam-ple 3, starting with the tripeptide in Example 7.

, Example 9 n-Butyl benzyloxycarbonyl-~-nitroarginyl-prolyl-glutaminyl-isoleucyl-prolyl-prolinate This compound is prepared by the procedure of Exam , ple 4, starting with the tetrapeptide obtained in Bxample 8.
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de Mark I . , - ~ ' ~0658S8 KX37 ~xample 10 n-Butyl benzyloxycarbonyl-tryptophyl-prolyl-arginy;l-prol~l-.
glutaminyl-isoleucyl-prolyl-prolinate This compound is prepared by the procedure o Exam- ' ple 5, starting with the hexapeptide of Example 9.

Example ll Pyroglutamyl-tryptophyl-prolyl-arginyl-prolyl-glutaminyl isoleucyl-prolyl-proline, n-butyl ester This compound is prepared by the procedure of Exam-ple 6, starting with the octapeptide of Example lO.

' Example 12 Benzyloxycarbonyl-isoleucyl-prolyl-proline amide Benzyloxycarbonyl-isoleucyl-prolyl-proline (4.5 g) is dissolved in a mixture o tekrahydrofuran (30 ml) and triethylamine (l.4 ml). The so1ution is chilled in a -5 cooling bath and isobutyl chloroformate (1.6 ml) is added.
The solution is allowed to warm to room temperature (10 minutes) and concentrated aqueous ammonia (lO ml) is added.
After Eour hours stirring at room temporature, the reaction is concentrated ln vacuo, diluted with ethyl acetate and wa~hed 9equentially with ~aturated sodium bicarbonate, water, O.l N hydrochloric acid and water. The organic phase is dried over magnesium sulate and concentrated to dryness in vacuo to give the title compound.

Example 13 Benzyloxycarbonyl-glutaminyl-isoleucyl-prolyl-proline amide '' This compound is prepared by the procedure of Exam~
30 ple 3, starting with the tripeptide of Example 12. , ~ 10--1~X37 5~
Example 14 Benzyloxycarbonyl-~nitroarqinyl-prolyl-qlutaminyl-isoleucyl-prol~l-proline amide This compound is prepared by the procedure of Example 4, startiny with the tetrapeptide of Example 13.

Example 15 BenzYloxYcarbonyl-tryptoPhyl-Prolyl-arqinyl-prolyl-qlutamin '~
isoleucyl-pro~y~-proline amide This compound is prepared by the procedure of Example 5, starting with the hexapeptide of Example 14.

Example 16 Pyroqlutamyl-tryptophYl-prolYl-arqinYl-prolYl-qlutaminyl-isoleucyl-prolyl-prolinamide This compound is prepared by the procedure of Example 6, starting with the octapeptide o:E Example lS.

Example 17 .

BenzyloxycarbonYl-tryptophyl-prolyl--arqinyl-prolyl-qlutaminyl-isoleucyl-prolyl~proline, ter-t-butyl es-ter t-Butyl benzylo~ycarbonyl nitroarginyl-proline-glutaminyl-isoleucyl-proline-prolinate (9.42 g,~ 10 mmoles) is dissolved in 135 ml absolute ethanol and 20 ml N HCl and 1.9 g l~/o palladium on carbon with stirring under positive hydrogen pressure for 22 hours. It is filtered through hyflo and taken to dryness ln vacuo. The crude residue is dissolved in 50 ml of dimethylformamide and 1.4 ml triethylamine followed : :

: . . . : :
. .
- .... .

~ KX37 ~651~5~

immediately by (12 mmoles) 7.4 g of benzyloxycarbonyl-tryp-tophyl-prolyl-2,4,5-trichlorophenyl ester. Over a 30 hour period an additional 1.5 mmoles of benzyloxycarbonyl-tryptophyl-prolyl-2,4,5-trichlorophenyl ester and 1.5 mmoles of triethylamine are added. After 50 hours the triethyl-amine-HCl is filtered and the ~iltrate added to 1.2 1 of vigorously swirling ethyl acetate. The precipitate is filtered and washed with ethyl acetate to give the title compound. Yield ~ 10.64 g.

Example 18 Pyroqlutamyl=trypto~hy---prolyl-arqinyl-prolyl-qlutamin isoleucyl-prolyl-proline, t-butyl ester 10 g (~ 8.5 mrnoles) of the peptide of Example 17 is dissolved in 150 ml of absolute ethanol and 8.5 ml N HCl and 2 g of 10~/~ palladium on carbon and s-tirred under positive hydrogen pressure for eight hours. It is filtered through hyflo and the filtrate concentrated to dryness ln vacuo to :
yield 9.2 g of crude residue.
The crude residue is dissolved in 40 ml of dimethyl-formamide and 1.14 g (8.5 mmoles) of l-hydroxy benzotriazole .
and (8.5 mmoles) 1.2 ml of triethylamine followed immediately by 3.14 g pyroglutamic acid 2,4,5-trichlorophenyl ester (2~/q excess~. ~n additional 0.6 ml triethylamine is added~
The reaction is allowed to run overnight. The triethylamine HCl .
.is filtered and the filtrate poured into 1.1 1 of vigorously swirling ethyl acetate. The recovered precipitate is filtered throuyh paper~ 42 and washed with ethyl acetate to yield 9.0 g of the title compound.

1~65858 ,, Amino Acid Analysis - ~olar Ratios;
Found (Calculated: NH3 0.85 (1.0); Trp 0.66 (1.0);
Arg 1.0 (1.0); Glu 2.03 (2.0);
Pro 4.0 (4.0); Ile 0.9 (1.0) Thin layer chromatography - Silica gel (Butyl alcohol 30, pyridine 20, acetic acid 6, water 24) Rf = 0.58.
Purification is achieved on a DEAE Sephadex A-25 column by elution with 0.005 M NH4HCO3.

Example 19 Pyroglutamyl-tryptophyl-prolyl-arginyl-prolyl-glutaminyl-isoleucyl-prolyl-proline, methvl ester Pyroglutamyl-tryptophyl-prolyl-arginyl-prolyl-gluta-minyl-isoleucyl-prolyl-proline (1.6 g) is dissolved in 80 ml of methanol. To this, excess ethereal diazomethane is added and the solution is kept for seven hours at room temperature.
Acetic acid is added to destroy the excess diazomethane and the resulting mixture is concentrated to dryness in vacuo.
Purification is effected on a ~ephadex G-25 column eluting with 0.01 M (NH4)HCO3, followed by a DEAE*Sephadex A-25 column eluting with 0.005 M NH~HCO3 to yield 1.1 g of the title compound.

Example 20 Pyroglutamyl-tryptophyl-prolyl-arginyl-prolyl-glutaminyl-isoleucyl-prolyl-prolinamide Pyroglutamyl-tryptophyl-prolyl-arginyl-prolyl-glutaminyl-isoleucyl-prolyl-proline (1 g) is dissolved in 6 ml of dimethyl sulfoxide with gentle warming. The solu-tion is cooled to room temperature and 0~14 ml of triethyl-amine and 0.16 ml of isobutyl chloroformate are added. The *Trade Mark `- xx37 reaction mixtuxe is stirred for 15 minutes. One ml of concentrated ammonium hydroxide is added and the reaction proceeds for four hours prior to slow addition to 200 ml of vigorously swirling ethyl acetate. A precipitate is formed wh.ch is ~iltered and washed with ethyl acetate to yield 1.375 g of solids.
This material is purified on a 225 ml coluDu~ of DEAE Sephadex A-25 by elution with 0.005 M NH4HCO3 to yield 924 mg of the title compound~
Amino Acid Analysis - Molar Ratios:
Found (Calculated): NH3 1.71 (2.0); Arg 0.88 (1.0);
Pro 4.13 (4.0); Trp 0.80 (1.0);
Gln 2.12 (2.0); Ile 0.93 (1.0) Thin layer chromatography - Silica gel (Butyl alcohol 30, pyridine 20, acetic acid 6, water 20) Rf = 0.53.
.'.""' ' .
Example 21 For determination of I50 values (concentration of peptide expressed in micrograms/ml producing a 50% inhibition of angiotensin-converting enzyme), varying concentrations of 2~ the peptide of Example 19 are added to 13 x 100 mm assay tubes containing a final volume of 0.25 ml containing 100 mM potas- ~ ;
sium phosphate buffer, pH 7.5, 30 mM NaCl, and 0.3 mM angio- ;~
tensin I. En~ymatic reactions are init~ated by addition of enzyme and incubation is carried out at 37. The concentra-tion of the peptide of Example 19 of the present invention which inhibits conversion of 50% of angiotensin I into angiotensin II is found to be 7 ~g/ml as opposed to an I50 (~g/ml) of 0.9 for the parent peptide Pyr-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro. This ln vitro test clearly demonstrates that the Example 19 peptide of the invention is substantially B~
r --*Trade Mark ~L~6585~?, less potent than the parent control peptide in vitro in inhibiting conversion of angiotensin I into angiotensin II.

Example 22 The peptide of Example 19 and the parent control peptide Pyr-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro are administered intravenously in two atropinized anesthetized male rats infused with pentolinium followed by an injection of 0.10 ~g/kg of angiotensin~I. On the basis of the degree of inhibition of angiotensin I induced pressor response, it is found that the in vivo activity of the Example lg pep-tide of the invention is roughly equivalent to that of the parent control peptide.

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Claims (10)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. Process for preparing a peptide having the formula Pyr-Trp-Pro-Axg-Pro-Gln-Ile-Pro-Pro-y wherein Y is -NH2 or -OR where R is an alkyl group containing 1 to 10 carbon atoms which comprises condensing an octapeptide having the formula Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro with pyroglutamic acid to form the peptide Pyr-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro and before, during or after the formation of this peptide, adding an alkyl ester group or an amide group to the terminal Pro by conventional procedures.

2. A process for preparing a peptide as defined in claim 1 wherein Y is -OR and R is an alkyl group containing 1 to 6 carbon atoms.

3. A process for preparing a peptide as defined in claim 2 wherein R is methyl.

4. A process for preparing a peptide as defined in claim 2 wherein R is t-butyl.

5. A process for preparing a peptide as defined in claim 1 wherein Y is -NH2.

6. A peptide having the formula I Pyr-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro-Y
wherein Y is -NH2 or -OR where R is an alkyl group containing 1 to 10 carbon atoms, whenever prepared according to the process of claim 1.

7. The peptide as defined in claim 6 wherein Y is -OR and R is an alkyl group containing 1 to 6 carbon atoms, whenever prepared according to the process of claim 2.

8. The peptide as defined in claim 7 wherein R is methyl, whenever prepared according to the process of claim 3.

9. The peptide as defined in claim 7 wherein R is t-butyl, whenever prepared according to the process of claim 4.

10. The peptide as defined in claim 6 wherein Y is -NH2, whenever prepared according to the process of claim 5.