IE43869B1 - Peptide enzyme inhibitors - Google Patents

Description

This invention relates to the nonapeptide H-PyrogluTrp-Pro-Arg-Pro-Gln-Ile-Pro-Pro-oH.

The action of the enzyme renin on renin substrate, a pseudoglobulin in blood plasma, produces a polypeptide angiotensin I, also known as hypertensin X. 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 individuals 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. 0. S. Patent Specification No. 3,832,337 discloses various peptides and acylated peptides which inhibit 1-' enzymatic conversion of angiotensin I into angiotensin II, among such peptides being H-Pyroglu-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro -oH.

Studies on structure-activity relationships concerning peptides as disclosed in U.S. Patent Specification No.3,832,337 indicate that a free terminal carboxyl group is needed to obtain · potent inhibitors in vitro and in vivo. For example, see the paper by Cushman et al., Inhibition of AngiotensinConverting Enzyme by Analogs of Peptides from Bothrops jararaca Venom, Experienta 29, 1032 (1973), Birkhauser Verlag, Basel, Switzerland. However, it has now been found that not only is the above nonapeptide an inhibitor of angiotensin I induced hypertension, but esters and amides of such nonapeptide are useful for such purpose as well. This is -243869 surprising inasmuch as such esters and amides show very low or no detectable enzyme inhibition activity in vitro. Generally, in vitro activity of peptides in this area usually 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 H-Pyroglu-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.

The present invention also provides a process for preparing a peptide having the formula H-Pyroglu-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 which comprises coupling or reacting suitable amino acids and peptides or reactive derivatives thereof to form the peptide.

H-Pyroglu-Trp-Pro-Arg-Pro-Gln-Xle-Pro-Pro-oH and before, during or after the formation of this peptide, providing an alkyl ester group or an amide group to the e-terminal Pro.

Unless otherwise indicated in the following specification and claims, all amino acids are of the L-con£iguration.

In describing the peptides of the present invention, the following abbreviations will be used throughout the specification and claims: - 3 43869 Arg - Arginine Gin - Glutamine lie - Isoleucine Pro - Proline Pyroglu-Pyroglutamic acid Trp - Tryptophane The following peptides of the present invention have been found to be effective in inhibiting angiotensin I induced hypertension: □ Ia - The esters having the formula H-Pyroglu-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro-OR, wherein R is alkyl II - The amide having the formula H-Pyroglu-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro-NHj The alkyl group of the above esters is a straight or branched chain hydrocarbon fragment containing 1 to 10 carbon atoms, and preferably 1 to 6 carbon atoms, such as methyl, ethyl, n-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 invention are preferably prepared by reaction of a di-, tri-, tetra-, penta-, hexa-, heptaor octa-peptide including a Pro- terminal group protected by an alkyl ester with one or more peptides, as is necessary, employing conventional peptide preparatory technigues, 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 ) (IIDH-Pyroglu-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro - ©H - 4 4386Q The amide of the invention is preferably prepared by reaction of a di-, tri-, tetra-, penta-, hexa-, heptaor oota-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 will 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 procedures as outlined in U.S. Patent Specification No. 3,832,337 and thereafter 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 capable of inhibiting the hypertensive effect of angiotensin 1 when administered to mammals such as rats, mice or dogs, in a dosage level of from about 0.5 to about 10 mg/kg. For the latter purpose they may be administered parenterally by incorporating the appropriate dosage with a physiologically acceptable carrier. The invention thus extends to a pharmaceutical composition comprising a peptide of the invention and a physiologically acceptable carrier. -5The following examples illustrate the present invention without, however, limiting the same thereto.

All temperatures are expressed in degrees Centigrade. All amounts and proportions are by weight unless or otherwise indicated.

Example 1 Benzyloxycarbonyl-isoleucyl-prolyl -proline Prolyl-proline hydrobromide (4.24 g) is dissolved in a mixture of dimethylformamide (40 ml) and triethylamine (2.8 ml). Benzyloxycarbonyl-isoleucine p-nitrophenyl ester (9.27 g) and 1-hydroxybenzotriazole (3 g) are addtd and cne mixture is stored at room temperature for 16 hours. Dimethylaminopropylamine (1 ml) is added and after 2 hours the solvent is removed in vacuo. The residue is dissolved in ethyl acetate (200 ml) and washed sequentially with 0.1 H hydrochloric acid, and water. The organic phase is dried over magnesium sulfate and concentrated to dryness in 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 Benzyloxycarbonyl-isoleucyl-prolyl-proline methyl ester.

The tripeptide of Example 1 (4.6 g) is dissolved in methanol and treated with an ethereal solution of diazomethane until a persistent yellow color is obtained. After 0.5 hour a few drops of acetic acid are added to discharge - 6 438G9 . the yellow color; the solvent is ramovct. in vacuo and replace:) with ethyl acetate. This solution is washed sequentially with 0.1 N IiCl, water, saturated sodium bicarbonate and water.

The organic phase is dried over magnesium sulfate and concentrated to dryness to yield the title compound.

Example 3 Benzyloxycarbonyl-glutaminyl-isoleucyl-prolyl-prollne methyl ester 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). Benzyloxycarbonyl-glutamine p-nitrophenyl ester (1.6 g) and 1-hydroxybenzotriazole (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 ensuing solution is washed neutral. The organic phase is dried over magnesium sulfate and the solvent is removed in vacuo to yield the title compound. 3 8 60 Example 4 Benzyloxycarbonyl-w -nitroarginyl-prolyl-glutaminyl-isoleucylprolyl-proline methyl ester The tetrapeptide of Example 3 (14 g) is dissolved in absolute ethar.cl (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). Benzyloxycarbonyl nitroarginyl-proline 2,4,5--trichlorophenyl ester (15.7 g) and 1-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 in vacuo. The residue is dissolved in ethyl acetate and the solution is washed neutral. The ethyl acetate is concentrated in vacuo to ca 70 ml and the solution poured into 1 liter of vigorously stirred diethyl ether. The precipitate is filtered and dried to give the title compound.

Benzyloxycarbonyl-tryptophyl-prolyl-arginyl-prolyl-glutaminylisoleucyl-prolyl-prollhe methyl ester 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 g) is added and the mixture is· stirred under a positive hydrogen pressure until the ultraviolet absorption of the nitroguanidine chromophore can no longer - 8 £2869 be detected. The catalyst is filtered off and the filtrate is concentrated to dryness in 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 pyrocrlutantyl-tryptophyl-prolyl-arginyl-prolyl-clutaminyl-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 in vacuo. The residue is dissolved in dimethylformamide (40 ml) and l-hydroxybenzotriazole (1.2 g), triethylamine (1.2 ml) and pyroglutamic acid 2,4,5-trichlorophenyl ester are added in rapid succession. The reaction is allowed to proceed until the ninhydrin test is negative. The triethylamine hydrochloride precipitate is 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 materia.1 is purified by 4S869 chromatography on Sephadex (Trade Mark) G-25 in 0.01 M ammonium bicarbonate.

Amino Acid Analysis - Molar Ratios: Found (Calculated): NH^ 0.92 (1.0),- Arg 0.86 (1.0); Pro 4.0 (4.0); Trp 0.78 (l.o); Gin 2.12 (2.0); lie 0.96 (1.0) Thin layer chromatography - Silica gel (Butyl alcohol 30, Pyridine 20, Acetic Acid 6, Water 24) Rf = 0.53.

Example 7 Benzyloxycarbonyl-isoleucyl-prolyl-proline-n-butyl-ester A solution of benzyloxycarbonyl-isoleucyl-prolylproline (4.5 g) in chloroform (30 ml) is added slowly to a solution of l-n-butyl-3-p-tolyltriazene (1.9 g) in chloroform (15 ml). When the reaction is complete, the chloroform solution is washed neutral and the organic phase is dried over magnesium sulfate and concentrated to dryness in vacuo to give the title compound.

Example 8 Benzyloxycarbonyl-glutaminyl-lsoleucyl-prolyl-proline n-butyl ester This compound is prepared by the procedure of Example 3, starting with the tripeptide in Example 7.

Example 9 Benzyloxycarbonyl-tii-nitroarglnyl-prolyl-glutamlnylprolyl-proline butyl ester This compound is prepared by the procedure of Example 4, starting with the tetrapeptide obtained in Example 8. -1043860 Example 10 Benzyloxycarbonyl-tryptophyl-prolyl-arginyl-prolyl-glutamlnylisoleucyl-prolyl-proline n-butyl ester.

This compound is prepared by the procedure of Example 5, starting with the hexapeptide of Example 9.

Example 11 pyroglutaroyl-tryptophyl-prolyl-arginyl-prolyl-glutaminyl isoleucyl-prolyl-proline, n-butyl ester This compound is prepared by the procedure of Example 6, starting with the ootapeptide of Example 10.

Example 12 Benzyloxycarbonyl-isoleucyl-prolyl-proline amide Benzyloxycarbonyl-isoleuoyl-prolyl-proline (4.5 g) is dissolved in a mixture of tetrahydrofuran (30 ml) and triethylamine (1.4 ml). The solution 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 (10 ml) is added. After four hours stirring at room temperature, the reaction is concentrated in vacuo, diluted with ethyl acetate and washed sequentially with saturated sodium bicarbonate, water, 0.1 N hydrochloric acid and water. The organic phase is dried over magnesium sulfate and concentrated to dryness in vacuo to give the title compound.

Example 13 Benzyloxycarbonyl-glutamlnyl-isoleucyl-prolyl-proline amide This compound is prepared by the procedure of Example 3, starting with the tripeptide of Example 12. - 11 43869 Example 14 Bcnzyloxycarbonyl-az-nitroarcrinyl-prolyl-glutaminyl-isoleucylprolyl-proline amide This cowpoi-'.d is prepared by the procedure of Example 4, starting with the tetrapeptide of Example 13.

Example 15 Βοηζν1οχγο3Γΐ)οην1-ίΓνρίορ1ιν1-ρΓθ1ν1-Β^ίην1-ρΓο1τ/1-ίϊ1ιιί&Γηίην1isoleucyl-prolyl-proline amide This compound is prepared by the procedure of Example 5, starting with the hexapeptide of Example 14.

Example 16 Pyroglutamyl-tryptophyl-prolyl-arginyl-prolyl-glutaminyl·isoleucyl-prolyl-prolinamide This compound is prepared by the procedure of Example 6, starting with the octapeptide of Example 15.

Example 17 Benzyloxycarbonyl-tryptophyl-prolyl-arginyl-prolylglutaminyl-isoleucyl-prolyl-proline, tert-butyl ester Benzyloxycarbonyl nitroarginyl-proline-glutaminyl-isoleucylproline-proline t-butyl ester (9.42 g,»v 10 mmoles) is dissolved in 135 ml absolute ethanol and 20 ml Ef HCl and 1.9 g 10% palladium on carbon with stirring under positive hydrogen pressure for 22 hours. It is filtered through hyflo and taken to dryness in vacuo. The crude residue is dissolved in 50 ml of dimethylformamide and 1.4 ml triethylamine followed immediately by (12 mmoles) 7.4 g of benzyloxycarbonyl-tryptophyl-prolyl-2,4,5-trichlorophenyl ester. Over a 30 hour period an additional 1.5 mmoles of banzyloxycarbonyltryptophyl-prolyl-2,4,5-trichlorophenyl ester and 1.5 mmoles of triethylamine are added. After 50 hours the triethylamine· HCl is filtered and the filtrate 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 Pyrpglutamyl-tryptophyl-prolyl-arqinyl-prolyl-qlutaminyl isoleucyl-prolyl-proline, t-butyl ester g (ό 8.5 mmoles) 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 stirred under positive hydrogen pressure for eight hours. It is filtered through hyflo and the filtrate concentrated to dryness in vacuo to yield 9.2 g of crude residue.

The crude residue is dissolved in 40 ml of dimethylformamide and 1.14 g (8.5 mmoles) of 1-hydroxy benzotriazole and (8.5 mmoles) 1.2 ml of triethylamine followed immediately by 3.14 g pyroglutamic acid 2,4,5-trichlorophenyl ester (20% excess). An 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 through paper#42 and washed with ethyl aoetate to yield 9.0 g of the title compound. 3 8 θ 9 Amino Acid Analysis - Molar 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); lie O.S (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 NH4KCO3, Example 19 pyroglutamyl-tryptophyl-prolyl-arginyl-prolyl-glutaminylisoleucyl-prolyl-proline, methyl ester Pyroglutamyl-tryptophyl-prolyl-arginyl-prolyl-glutaminyl-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 effecteci on a Sephadex G-25 column eluting with 0.01 M (NH^lHCOg, followed by a DEAE Sephadex :θ A-25 column eluting with 0.005 M NH4HCO3 to yield 1.1 g of the title compound.

Example 20 Pyroglutamyl-tryptophyl-prolyl-arginyl-prolyl-glutaminylisoleucyl-prolyl-prolinamide Pyroglutamyl-tryptophyl-prolyl-arginyl-prolylglutaminyl-isoleucyl-prolyl-proline (1 g) is dissolved in 6 ml of dimethyl sulfoxide with gentle warming, The solution is cooled to room temperature and 0.14 ml of triethylamine and 0.16 ml of isobutyl chloroformate are added. The 33 69 reaction mixture ia 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 which is filtered and washed with ethyl acetate to yield 1.375 g of solids.

This material is purified on a 225 ml column 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: Pound (Calculated): NH3 1.71 (2.0); Arg 0.88 (1.0); Pro 4.13 (4.0); Trp 0.80 (1.0); Gin 2.12 (2.0)» lie 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 values (concentration of peptide expressed in micrograms/ml producing a 50% inhibition of angiotensin-converting enzyme), varying concentrations of 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 potassium phosphate buffer, pH 7.5, 30 mM NaCl, and 0.3 mM angiotensin I. Enzymatic reactions are initiated by addition of enzyme and incubation is carried out at 37®. The concentration of the peptide of Example 19 of the present invention which inhibits conversion of 50% of angiotensin 1 into angiotensin IX is found to be 7 pg/ml as opposed to an Ι5θ (pg/ml) of 0.9 for the parent peptide H-Pyroglu-Trp-Pro-Arg-ProGln-Ile-Pro-Pro-CH.This in vitro test clearly demonstrates that the Example 19 peptide of the invention is substantially £3869 . 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 H-Pryoglu Pyr-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro-oH are administered intravenously in two atropinized anesthetized male rats infused with pentolinium followed by an injection of 0.10 ug/kg of angiotensin I. On the baoi.. of the degree oi inhibition of angiotensin I induced pressor response, it is found that the in vivo activity of the Example lg peptide of the invention is roughly equivalent to that of the parent control peptide.

Claims (9)

1. CLAIMS :1. A peptide having the formula I H-Pyroglu-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro-Y wherein Y is -NHj or -OR where R is an alkyl group containing 1 to 10 carbon atoms.

2. 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 peptide as defined in claim 2 wherein R is methyl.

4. A peptide as defined in claim 2 wherein R is t-butyl.

5. A peptide as defined in claim 1 wherein Y is -NH 2 .

6. A process for preparing a peptide having the formula H-Pyroglu-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro-Y wherein Y is -nh 2 or -OR where R is an alkyl group containing 1 to 10 carbon atoms which comprises coupling or reacting suitable amino acids and peptides or their reactive derivatives thereof to form the peptide H-Pyroglu-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro and before, during or after the formation of this peptide, providing an alkyl ester group or an amide group on the cterminal Pro. 17 43869

7. A peptide according to Claim 1, when prepared by a process according to Claim 6.

8. A peptide according to Claim 1, as named in any of the Examples.

9. A pharmaceutical composition comprising a peptide according to any one of Claims 1 to 5, 7 and 8 and a physiologically acceptable carrier.