NO154836B - ANALOGY PROCEDURE FOR PREPARING AN INHIBITOR FOR AN ANGIOTENSIN-CONVERSING ENZYM. - Google Patents

Description

The present invention relates to a preparation

of an angiotensin-converting enzyme inhibitor.

Angiotensin-converting enzyme (peptidyl dipeptidyl hy-

.drolase, hereinafter referred to as ACE) occupies a central role

in the physiology of hypertension. The enzyme can convert deca-

the peptide angiotensin I, which has the sequence

AspArgValTyrlleHisProPheHisLeu

to an octapeptide, angiotensin II, by removal of the KisLeu carboxy-terminal group. The symbols for the different chemical sub-groupings are explained in the table below:

Ala = L-alanine

Arg = L-arginine

Asp = L-aspartic acid

< Glu = pyro-L-glutamic acid

Gly = glycine

Hip = Hippuric acid (benzoyl-glycine)

His = L-histidine

Ile = L-isoleucine

Leu = L-leucine

Phe = L-phenylalanine

Pro = L-proline

A Pro = L-3i4-dehydroproiin

Ser = L-serine

Trp = L-tryptophan

Tyr = L-tyrosine

Val L-valine

ACE = Angiotensin converting enzyme

Hepes = N-2-hydroxyethylpiperazine-N'-2-ethane-

sulfonic acid

Angiotensin I is formed by the action of the enzyme

renin, an endopeptidase found in the kidney, other tissues and plasma, which acts on a serum α-2-globulin.

Blood pressure is affected by certain peptides such as

found in the blood. One of these, angiotensin II, is a powerful

pressor substance (blood pressure-increasing agent). Another, bradykinin, a nonapeptide with the sequence ArgProProGlyPheSerProPheArg is a powerful depressant (blood pressure lowering agent). In addition to a direct pressor effect, angiotensin II stimulates the release of aldosterone which tends to raise blood pressure by causing retention of extracellular salts and fluids. Angiotensin II is found in measurable amounts in the blood of normal people. However, it is found in elevated concentrations in the blood of patients with renal hypertension.

The level of ACE activity is usually in excess, in both normal and hypertensive people, relative to an amount necessary to maintain observed levels of angiotensin II. However, it has been found that significant blood pressure lowering is achieved in hypertensive patients by treatment with ACE inhibitors. [Gavras, I., et al., New Engl. J. Med. 291, 817 (1974)].

ACE is a peptidyl dipeptide hydrolase. It catalyzes the hydrolysis of the penultimate peptide bond at the C-terminal end in a variety of acylated tripeptides and larger polypeptides with an unblocked α-carboxyl group. The action of ACE results in hydrolytic cleavage of the penultimate peptide bond from the carboxyl terminal end and gives as reaction products a dipeptide and a residue.

The reactivity of the enzyme varies considerably depending on the substrate. At least one type of peptide bond, which has the nitrogen atom supplemented by proline, is not hydrogenated at all. The apparent Michaelis constant (Km) varies from substrate to substrate over several orders of magnitude. With regard to a general discussion of the kinetic parameters of enzyme-catalyzed reactions, reference should be made to Lehninger, A., Biochemistry, second edition, Worth Publishers, Inc.., New York, 1975, pages 189-195- Many peptides which are termed inhibitors for the enzymatic conversion of angiotensin I to angiotensin II are in fact substrates that have a lower Km value than angiotensin I. Such peptides are more correctly termed competitive substrates. Examples of competing substrates are bradykinin and the peptide BPP,b_a

(also designated SQ20475) from snake venom, whose sequence is

<GluLysTrpAlaPro.

Several synthetic peptide derivatives have been shown to be ACE inhibitors by Ondetti, et al. in US Patent 3,832,337-The role of ACE in the pathogenesis of hypertension has prompted a search for inhibitors of the enzyme that may act as antihypertensive drugs. See e.g. US Patents 3,891,616, 3-947-575, 4,052,511 and 4,053-651-A highly effective inhibitor, with high biological activity when administered orally, is D-3-mercapto-2-methylpropanoyl-L-

proline, designated SQ14225, described in US Patent No. 4,046,889,

and in scientific articles by Cushman, D.W. et al., Biochemisty 16, 5484 (1977), and by Ondetti, M. et al., Science 196, 441 (1977). The inhibitor SQ14225 has been reported to have a

eI t 5° a-lve.r, they r pkå on2s,e3 ntx ra1s0 j- o8nMe. n aIv .5-0 „i-nvheribdiiten or raspom porstkearl t taiv l Cfuor shmå angi,' 50% inhibition of the enzyme in a standard test system containing the substrate at a level substantially D above K m. It will be understood that I^Q values are directly comparable when all potential factors affecting the reaction are held constant. These factors include the source of enzyme, its purity, the substrate used and its concentration, and the composition of the sample buffer. All I^Q data reported herein were obtained with the same sample system and enzyme (human urine ACE) and with an approximate 1/2 K level of substrate and are therefore inherently uniform. Discrepancies with data obtained by other researchers can be observed. And such discrepancies do exist in the literature for unknown reasons. See e.g. The I^Q values for BPP^a reported by Cushman, D.W., et al., Experientia 29, 1032 (1973) and by Dorer, F.E., et al., Biochim.Biophys.Acta 429, 220 (1976).

The mode of action of SQ14225 has been based on a model of the active site of ACE developed by analogy with the better known related enzyme, carboxypeptidase A. The active site was postulated to have a cationic site for binding the carboxyl end group of the substrate and a pocket or gap that could bind the side chain in the C-terminal amino acid and provide a particularly strong bond for the heterocyclic ring in a terminal proline residue. A similar pocket for the penultimate amino acid residue was postulated, and the published data suggested a rather stringent steric condition, since the D form of the inhibitor was significantly more potent than its stereoisomer or 3-niethyl and unsubstituted analogues. The sulfhydryl group in the inhibitor, which is postulated to be bound at the active site near the catalytic center, was thought to play a central role in inactivating the enzyme by combining with the zinc moiety known to be essential for catalytic activity. Substituents on the sulfhydryl group, such as a methyl group,

and an S-acetyl derivative, significantly reduced the effectiveness of the inhibitor. See Cushman, D.W., et al., Biochemistry, supra.

In vitro studies of the mechanism by which SQ14225 and its analogues act to inhibit ACE

has been somewhat compromised by the instability of these molecules under ambient conditions. It is e.g. observed that a fresh aqueous solution with concentration, e.g. 1 mg per ml 3Q14225 at a pH value of approx. 8, becomes significantly less active when allowed to stand for as short as 30 minutes, and that the activity continues to decrease after the dissolution has been allowed to stand for a longer period of time. It is believed that this loss of activity is mainly the result of dimerization of SQ14225 occurring at the sulfhydryl end groups, whereby a disulfide is formed which is predominantly inactive as an inhibitor. Since the free sulfhydryl group is very reactive and can easily be oxidized to polar acidic groups such as sulfone and sulfoxide groups, it may also be that the observed in vitro loss of activity in aqueous solutions of SQ14225 upon standing is to some extent a consequence of one or more such oxidation reactions, with the formation of a sulfone or sulfoxide that does not act effectively as an inhibitor of ACE.

Such reports of SQli(225 clinical testing as

are available, some of which refer to the compound under the designation "Captopril", suggesting that the product is sufficiently stable in the normal gastrointestinal environments of most

patients to be an effective inhibitor of ACE when administered orally. However, it is not clear whether there may be a group of patients for whom SQ14225 is substantially ineffective. Due to the high reactivity of the free sulfhydryl group, SQ14225 could easily form mixed disulfides with serum, cellular proteins, peptides or other free sulfhydryl group-containing substances in the gastrointestinal tract,

in addition to the possibility of reactions with dimer formation or oxidative degradation. A mixed disulfide with protein can be antigenic and it is the case that allergic reactions have occasionally been observed clinically, see Gavras, et al.,

New England J. Med., 298, 991 (1978). Disulfides and oxidative degradation products of SQ14225, if formed, may

at best, are expected to be predominantly ineffective as inhibitors. It can therefore be postulated that the dose response to SQ14225 may vary with administration conditions and among individual patients. Moreover, at least in some patients, unwanted side effects may occur and maintaining an effective concentration of the inhibitor in the body may be difficult to regulate.

Thioester compounds are generally believed to be very reactive because the thioester bond is readily hydrolyzable into a sulfhydryl moiety and a carboxylic moiety. Thioesters are consequently often used as active ester intermediates for acylation under mild conditions. Such groups as e.g. acetylthio has been used as blocking groups in the above-mentioned patents. It is also postulated that thioester intermediates occur in the biosynthesis of cyclic peptides such as tyro-cidin or gramicidin S, cf. Lipmann, F. in Accounts Chem. Res. 6, 361 (1973).

Thioester compounds with potent ACE inhibitory activity and oral efficacy as anti-hypertensive agents are described in US Application Nos. 116,950, 116,951, and in applications from 064,897 to 064,903.

Compounds related to SQ14225 are described by Ondetti, et al., in US Patents 4,046,889, 4,052,511, 4,053,651, 5H3,715 and 5-154,840. Of interest, analogues of SQ14225 are described which have the five-membered hetero-

cyclic ring in proline replaced with a four- or six-

articulated ring. The inhibitory effects of such analogues in relation to SQ14225 have not been described. The use of D-proline instead of L-proline has been reported to drastically reduce the inhibitory potency of 3-mercaptopropanoyl amino acids (Cushman, D.W., et al., supra).

Use of L-3,4-dehydroproline instead of proline

have been studied in several systems. Substitution of L-3,4-

APro in the 7-position in bradykinin gives a bradykinin derivative

who have significantly reduced physiological activity, cf.

Fisher, G.H. et al., Arch. Biochem. Biophys. 189, 81 (1978).

On the other hand, substitution of L-3,4-APro at 3_, 5-,

The 8 or 9 position in the ACE inhibitor BPPQ promotes its inhibitory activity, cf. Fisher, G.H. et al., FEBS Letters 107, 273 (1979). In US application no. 116,951 it has been found that the compounds having APro, which are described in said application,

has high inhibitory efficiency and anti-hypertensive efficiency. At the moment, however, no logical justification can be given for the diversity in observed results after the introduction of APro instead of proline. There is also no clear picture of the effects of other proline derivatives or analogues substituted at different places on ACE inhibitors.

So far has the effect of the amino acid on the left

for the sulfur atom in the thioester compounds described in submitted applications, has not been determined. It is believed that this amino acid acts as an additional recognition site for the enzyme. If this is the case, it would be expected that a compound with an amino acid here would be a better inhibitor. It has been found that many amino acids, substituted amino acids or analogous compounds can be used for A in formula I below to provide effective anti-hypertensive agents and effective inhibitors of ACE. Reference should be made here to US applications 145,772. 145-773 and 146.107- It has been found that

the hydroxyprolines, proline, L- and D,D-3,4-dehydroproline, thiazolidine-4-carboxylic acid and L-5-oxoproline derivatives are all effective anti-hypertensive agents and have high inhibitory activity for ACE.

In US applications Nos. 064,897-064,903, 115-950, 116,951 and 121,188, it has been found that compounds of formula I were effective anti-hypertensive agents and have high inhibitory activity for ACE. At the time these applications were submitted, no salts of these compounds had been investigated and thus it was not known whether the salts would be effective. Salts of these compounds have now been prepared and determined to be effective anti-hypertensive agents and potent inhibitors of ACE.

According to the present invention, new inhibitors of angiotensin-converting enzyme are produced for the treatment of hypertension, and more the formula:

where R is hydrogen, lower alkanoyl, benzoyl, cycloalkanoyl, tert-butyloxycarbonyl, cyclopentylcarbonyl-L-lysyl, pyro-L-glutamyl; A is L-phenylalanyl, glycyl, L-alanyl, L-tryptophyll, L-isoleucyl, L-leucyl or L-valyl, the α-amino group therein being in amide mixture with R; R-1 is lower alkyl; B.^ is L-proline or L-3,4-dehydroproline, the imino group therein being in an imide mixture with the adjacent one and this method is characterized by (1) reacting with R„ to form where R, is acetyl and and R 2 has the meanings given above (2) removes R 2 from the product in step (1) to form wherein R 2 and R 2 have the meanings given above; (3) reacts the product of step (a) with R - A - OH to form

where R, A, R, and R2 have the meanings given above.

All of the above compounds of formula I are inhibitors of ACE and are useful as orally effective anti-hypertensive agents.

The invention will be better understood by reference to the following specific examples which describe the details of the synthesis and the efficiency of use of the above compounds. In these examples, thin layer chromatography (TLC) using silica gel plates was used. The numerical solvent systems used in the TLC methods are as follows:

(1) is methanol:chloroform, 1:1 (parts by volume).

(2) is benzene:water:acetic acid, 9:1:9 (parts by volume). (3) is acetic acid:water:n-butanol 26:24:150 (parts by volume). (4) is n-butanol:pyridine:acetic acid:water, 15:10:3:12 (parts by volume).

'5) is chloroform:methanol:ammonium hydroxide, 60:45:20 (v/v). The buffers for paper electrophoresis were: pH 1.9 - formic acid:acetic acid:water, 3 - 2: 25 (volume parts); pH 5.0 - diethylene glycol: acetic acid: pyridine: water, 100:6:8.5:885 (parts by volume).

■rt-buphyloxycarbonyl-, benzoyl-, acetyl-, " - 1-, propanoyl-,

the butanoyl-j phenylacetyl and phenylpropanoyl derivatives of the amino acids are commercially available.

Example_el_l

ACEzaktiyite^sbe^tame^se

For most of the experiments described, the enzyme was determined in 0.05 M Hepes buffer, pH 8.0 containing 0.1 M NaCl and 0.75 M Na0SO,.. The substrate used was benzoyl-glyHis--4 -4

Leu at a final concentration of 1 x 10 M, (Km « 2 x 10 M), together with approx. 130,000 cpm of [3H]benzoylGlyHisLeu (25 Ci/mmol). The enzyme was diluted in the above-mentioned buffer so that 40 µl of buffered enzyme could hydrolyze 13% substrate during a 15-minute incubation at 37°C. To initiate the sample determination, 40 µl of enzyme and 10 µl of water or inhibitor dissolved in water were pre-incubated for 5 minutes at 37°C. Substrate, 50 µl, was then added to initiate the reaction and the solution was incubated for 15 minutes at 37°C. To terminate the reaction, 1 ml of 0.1 M HCl was added, after which 1 ml of ethyl acetate was added. The mixture was stirred in a rotary mixer and briefly centrifuged to separate the phases.

An aliquot, 500 µl, of the ethyl acetate layer was transferred to a liquid scintillation sample bottle containing 10 ml of "Riafluor". To determine I^ values, enzyme activity in the presence of inhibitor at a number of different concentrations was compared with the activity in the absence of inhibitor. A graph of inhibitor concentration versus % inhibition gave the I^Q value.

Example_el_2

Svntese_ay_3^acetY^tiop^o^

3-acetylthio-2-D-methylpropanoic acid is dissolved in redistilled tetrahydrofuran (THF) and cooled to 0°C. A cooled solution of dicyclohexylcarbodiimide in THF is added, after which a cooled solution of L-proline-t-butyl ester is added. The reaction mixture is stirred at 0°C for 1 hour and then at 4°C overnight. The reaction mixture is then filtered, and the precipitate is washed with ethyl acetate. Solvents in the filtrates are removed under reduced pressure in a rotary

evaporates. The residue is dissolved in ethyl acetate which is then washed three times with cold 1 N citric acid, twice with saturated NaCl, twice with cold 1 N NaHCO^ and three times with saturated NaCl. The solution is dried over anhydrous MgSO^ and filtered. The solvent is removed under reduced pressure in a rotary evaporator at 30°C, giving a clear, colorless, oily product in about Qf% yield. The product will migrate as a single spot by thin layer chromatography in five solvent systems.

Example_el_3_

The product from example 2, 3-acetylthio-2-D-methyl-propanoyl-L-proline-t-butyl ester is mixed with 5.5 N methanolic ammonia at room temperature under nitrogen for 1 hour to remove the acetyl group. The solvent is then removed at 25°C in a rotary evaporator. After the product is taken up in 1 methanol and evaporated twice more in the rotary evaporator, the clear, oily residue is dissolved in ethyl ether, washed twice with 5% potassium bisulfate and once with saturated NaCl, dried over MgSO^ and filtered. The remaining solvent is then removed in vacuo to give a clear, oily product which migrates as a single spot by thin layer chromatography in three separate solvent systems. The t-butyl ester protecting group is removed by reaction with trifluoroacetic acid in anisole.

Eczema.£el_4

By using 3-acetylthio-2-D,L-methylpropane-

acid instead of 3-acetylthio-2-D-methylpropanoic acid in Example 2 and by essentially following the methods in Examples 2 and 3, 3-mercapto-2-D,L-methylpropanoyl-L-proline is obtained. By

removing the t-butyl ester protecting group with trifluoroacetic acid in anisole as a first step, the dicyclohexylamine salt can be formed to aid the resolution of isomers. The acetyl protecting group can be removed in a second step using methanolic ammonia, as described in Example 3.

Eczema gel__5

SY.ntese_ay_3_zmerka]3to-2^me^ L-3,4-dehydroproline (A3Pro), 1 mmol, is dissolved in DMF, and the solution is cooled to -15°C. The solution is neutralized by adding 1 equivalent of N-ethylmorpholine.

In a separate reaction container at -10°C, an equivalent of 3-acetylthio-2-methylpropanoic acid is mixed in an equal volume of DMF with 1.1 equivalents of 1,1'-carbonyldiimidazole, and the solution is stirred for 1 hour. The first solution containing A3Pro is mixed with the second containing 3-acetylthio-2-methylpropanoic acid while maintaining the temperature at -10°C. The combined solution is stirred for 1 hour at

-10°C. The solution is then allowed to warm slowly to room temperature. The solvent is removed in a rotary evaporator under reduced pressure at 40°C. Ethyl acetate (25 ml) is added and the solution is cooled to 0°C. 2 ml of 1 N citric acid is added, the two phases are mixed and then allowed to separate. The phases are separated with a separatory funnel, and the organic phase is washed twice more with 2 ml of 1 N citric acid, twice with saturated NaCl and finally dried over anhydrous MgSO 4 . MgSO^ is removed by filtration, and the solvent is removed with a rotary evaporator. The residue is dissolved in and recrystallized from a non-polar solvent such as benzene to form 3-acetylthio-2-D,L-methylpropanoyl-L-3,4-dehydro-proline. When the 2-D-methyl isomer is desired, the residue is dissolved in acetonitrile (about 3 mL) and the solution is heated to 40°C. An equivalent of dicyclohexylamine is added, and the solution is left at room temperature overnight. The crystals are collected by filtration and washed three times with acetonitrile. When further purification is required, the material can be recrystallized from isopropanol. The acetyl protecting group can be removed as in example 3-

Eczema Gel__6_

§^nS§S§_aY_^2l_0-(Na;acetyl-L-phenylal

A solution of 41.5 mg of Na-acetyl-L-phenylalanine

in 0.5 ml of redistilled dimethylformamide (DMF) was cooled in an ice-dry ice-acetone bath at -20°C. To this solution was added a cold solution of 35 mg of 1,1'-carbonyldiimidazole in 1.0 ml of DMF. The solution was stirred at -10°C for two hours and was then added to a cold solution of 48 mg of N-mercapto-2-D-methylpropanoyl-L-proline in 1 ml of DMF which was neutralized with N-ethylmorpholine . The reaction mixture was stirred at -10°C for a further 1 hour and then slowly warmed to room temperature. The solvent was removed under reduced pressure at 40°C, and ethyl acetate was added to the residue. The mixture was cooled in an ice bath and washed with 0.1 N HCl

and then three times with saturated NaCl solution. The organic solvent was removed with a rotary evaporator after drying over anhydrous MgSO 4 . The product was purified by "Sephadex LH-20<tm>" column chromatography using a 1.2 cm x 95 cm column and eluting with isopropanol. The top fractions were collected and the solvent removed under reduced pressure to give 35.5 mg of the desired product. This product was found to be homogeneous using TLC with solvent systems 1» 2, 3 and 5-

Example example is _ J-l4_

By using Not-acetylglycine, Na-acetylalanine, Na-acetyltryptophan, Na-acetyltyrosine, Na-acetylisoleucine, Na-acetylleucine, Na-acetylhistidine or Na-acetylvaline

instead of Na-acetylphenylalanine in example 6 and by essentially following the method in example 6, the following compounds are obtained:

Eczema<p>_<el_>15_

The L-3,4-dehydroproline derivative is obtained in the same way by using the product in Examples 2-5 instead of 3-mercapto-2-D-methylpropanoyl-L-proline in Examples 6-14 and by essentially following the method in example 6.

Example_el_l6

§^Qt§§§_§Y_Nci-I2li<N>a_ztert-butyloxy_k il2l m§ty_l]2ro£anoy.l]_-L-p.rolin

A solution of 133 mg of Na-tert-butyloxycarbonyl-L-phenylalanine (Na-Boc-L-Phe) in 0.5 ml of redistilled DMF was cooled in an ice-dry ice-acetone bath at -20°C. To this solution was added a cold solution of 87 mg of 1,1'-carbonyldiimidazole in 1.0 ml of DMF. The solution was stirred at -30°C for two hours and was then added to a cold solution of 119.5 mg of 3-mercapto-2-D-methylpropanoyl-L-proline in 1 ml of DMF

which was neutralized with N-ethylmorpholine. The reaction mixture was stirred at -10°C for a further 1 hour and then slowly warmed to room temperature. The solvent was removed

under reduced pressure at 40°C, and ethyl acetate was added to the residue. The mixture was cooled in an ice bath and washed with 0.1 N HCl and then three times with saturated NaCl solution. The solvent was removed with a rotary evaporator after drying over anhydrous MgSO. The product was purified by liquid chromatography on "Sephadex G-10 till" using a 1.2 cm x 95 cm column and eluted with THF:isopropanol, 3:7 (v/v). The top fractions were collected and the solvent removed under reduced pressure, yielding 65 mg of the desired product. This product was found to be homogeneous using paper electrophoresis at pH

5.0 and using TLC with solvent systems 1, 2 and 3.

Ex.ler_lY=24

By using Na-Boc-glycine, Na-Boc-alanine, Ncx-Boc-tryptophan, Na-Boc-tyrosine, Na-Boc-isoleucine, Na-Boc-leucine, Na-Boc-histidine or Na-Boc-valine instead of Na-Boc-Phe in Example 16 and by essentially following the methods

in Example 16, the following compounds are obtained::.

Eczema gel_25

The L-3,4-dehydroproline derivative is obtained in the same way

way by using the product in examples 3~5 instead of 3-mercapto-2-D-methylpropanoyl-L-proline in examples 16-24

and by essentially following the method in Example 16.

Example_el_26

Synthesis_of_Ng2Cy.klg2entylcarbonyl

A cold solution of 2.06 g of dicyclohexylcarbodiimide in 10 ml of dichloromethane was added to a solution of 1.4114 g of cyclopentanecarboxylic acid in 5 ml of dichloromethane at -5°C. This was followed by the addition of 4.28 g of L-phenylalanine benzoyl ester toluenesulfonate salt in 10 ml of DMF which was neutralized with 1.36 ml of N-ethylmorpholine. The reaction mixture was stirred at 0°C for 1 hour and then at room temperature for 3 hours. Dicyclohexylurea was removed by filtration and 50 ml of ethyl acetate was added to the filtrate. The organic phase was washed until neutral, dried over anhydrous MgSO 4 and filtered. The solvent was removed with a rotary evaporator. The residue was crystallized from isopropanol and hexane, yielding 2.35 g of white crystals with a melting point of 88-89°C. Elemental analysis of these crystals gave the following:

Calculated: C=75.19; H=7.17 and N=3.9855

Found : C=74.96; H=7.17 and N=4.09

The benzyl ester was removed by hydrogenolysis

with 2 g of 10% palladium-on-carbon in absolute alcohol. The catalyst was removed by filtration and the ethanol was removed with a rotary evaporator. The residue was crystallized from ether and hexane, and this gave 1.15 g of white crystals of the desired product with a melting point of 107-108°C. Elemental analysis of these crystals gave the following:

Calculated: C = 68.94 ; ."H=7.33; N=5.36

Found : C=68.90; H=7.32; N=5.34

The product was found to be homogeneous using paper electrophoresis at pH 1.9 and at pH 5.0

and using TLC with solvent systems 1, 2 and 3. The product prepared was shortened to Na-cpc-L-Phe.

Eczema_2_7_

§Y.I}£ §§§_§Y_N2ll2l iN°fl2y clovent y lcarbonyl;L-f eny_lalany_ltio); 2lD-metYl<p>.<ro>2<anoy>_<l>2z<L>;<p>_<rolin>

A solution of 52.5 mg of the compound from Example 26 in 0.5 ml redistilled DMF was cooled in an ice-dry ice-acetone bath at -20°C. To this solution was added a cold solution of 34 mg of 1,1'-carbonyldiimidazole

in 1.0 mL DMF. The solution was stirred at -10°C for two

hours and then mixed with a cold solution of 45.6 mg of 3-mercapto-2-D-methylpropanoyl-L-proline in 1 ml of DMF which was neutralized with N-ethylmorpholine. The reaction mixture was stirred at -10°C for a further 1 hour and then slowly warmed to room temperature. The solvent was removed under reduced pressure at 40°C and ethyl acetate was added to the residue. The mixture was cooled and washed with 0.1 N HCl and then three times with saturated NaCl solution. The solvent was removed with a rotary evaporator after drying

Example_el_25_

The L-3 y4-dehydroproline derivative is obtained in the same way

way by using the product in examples 3-5 instead of 3-mercapto-2-D-methylpropanoyl-L-proline in examples 16-24

and by essentially following the method in Example 16.

Example_el_26

Synthesis_of_Na-cyclop.entyl_lcarbo

A cold solution of 2.06 g of dicyclohexylcarbodiimide in 10 ml of dichloromethane was added to a solution of 1.4114 g of cyclopentanecarboxylic acid in 5 ml of dichloromethane at -5°C. This was followed by the addition of 4.28 g of L-phenylalanine benzoyl ester toluenesulfonate salt in 10 ml of DMF which was neutralized with 1.36 ml of N-ethylmorpholine. The reaction mixture was stirred at 0°C for 1 hour and then at room temperature for 3 hours. Dicyclohexylurea was removed by filtration and 50 ml of ethyl acetate was added to the filtrate. The organic phase was washed until neutral, dried over anhydrous MgSO 4 and filtered. The solvent was removed with a rotary evaporator. The residue was crystallized from isopropanol and hexane, yielding 2.35 g of white crystals with a melting point of 88-89°C. Elemental analysis of these crystals gave the following:

Calculated: C=75.19; H=7.17 and N=3.9855

Found : C=74.96; H=7.17; N=4.09

The benzyl ester was removed by hydrogenolysis

with 2 g of 10$ palladium-on-carbon in absolute alcohol. The catalyst was removed by filtration and the ethanol was removed with a rotary evaporator. The residue was crystallized from ether and hexane, and this gave 1.15 g of white crystals of the desired product with a melting point of 107-108°C. Elemental analysis of these crystals gave the following:

Calculated: C = 68.94 ; /H=7.33; N=5.36

Found : C=68.90; H=7.32; N=5.34

The product was found to be homogeneous using paper electrophoresis at pH 1.9 and at pH 5.0

and using TLC with solvent systems 1, 2 and 3. The product prepared was shortened to Na-cpc-L-Phe.

Eczema Gel_27_

§YQtese_av_Na-[^-(Na-cyclo2entylka ll5z<m>§t<y>lB^2E§D2Y<l>izL,ZBE2<l>iD

A solution of 52.5 mg of the compound from Example 26 in 0.5 ml redistilled DMF was cooled in an ice-dry ice-acetone bath at -20°C. To this solution was added a cold solution of 34 mg of 1,1'-carbonyldiimidazole in 1.0 ml of DMF. The solution was stirred at -10°C for two hours and then mixed with a cold solution of 45 }6 mg of 3-mercapto-2-D-methylpropanoyl-L-proline in 1 ml of DMF which was neutralized with N-ethylmorpholine. The reaction mixture was stirred at -10°C for a further 1 hour and then slowly warmed to room temperature. The solvent was removed under reduced pressure at 40°C and ethyl acetate was added to the residue. The mixture was cooled and washed with 0.1 N HCl and then three times with saturated NaCl solution. The solvent was removed with a rotary evaporator after drying

Eczema gel_25

The L-3,4-dehydroproline derivative is obtained in the same way by using the product in Examples 3~5 instead of 3-mercapto-2-D-methylpropanoyl-L-proline in Examples 16-24

and by essentially following the method in Example 16.

Example_el_26

Synthesis_of_Na-cyclo£entylcarbgylyL=phenylalanine

A cold solution of 2.06 g of dicyclohexylcarbodiimide in 10 ml of dichloromethane was added to a solution of 1.4114 g of cyclopentanecarboxylic acid in 5 ml of dichloromethane at -5°C. This was followed by the addition of 4.28 g of L-phenylalanine benzoyl ester toluenesulfonate salt in 10 ml of DMF which was neutralized with 1.36 ml of N-ethylmorpholine. reaction mix-

none was stirred at 0°C for 1 hour and then at room temperature for 3 hours. Dicyclohexylurea was removed by filtration

and 50 ml of ethyl acetate was added to the filtrate. The organic phase was washed until neutral, dried over anhydrous MgSO 4 and filtered. The solvent was removed with a rotary evaporator. The residue was crystallized from isopropanol and hexane, yielding 2.35 g of white crystals with a melting point of 88-89°C. Elemental analysis of these crystals gave the following:

Calculated: C=75.19; H=7.17 and N=3.9855

Found : C=74.96; H=7.17l N=4.09

The benzyl ester was removed by hydrogenolysis

with 2 g of 10% palladium-on-carbon in absolute alcohol. The catalyst was removed by filtration and the ethanol was removed

with a rotary evaporator. The residue was crystallized from ether and hexane, and this gave 1.15 g of white crystals of the desired product with a melting point of 107-108°C. Elemental analysis of these crystals gave the following:

Calculated: C = 68.94 ; .-H=7.33; N=5.36

Found : C=68.90; H=7.32; N=5.34

The product was found to be homogeneous using paper electrophoresis at pH 1.9 and at pH 5.0

and using TLC with solvent systems 1, 2 and 3- The product prepared was shortened to Na-cpc-L-Phe.

Example_el_27_

§Y2thesis_of_Na-[^-{Na;cyclop.entylkar iz!2zm§£Yl]2£2E§D2YllzLc]2£2iiQ

A solution of 52.5 mg of the compound from Example 26 in 0.5 ml redistilled DMF was cooled in an ice-dry ice-acetone bath at -20°C. To this solution was added a cold solution of 34 mg of 1,1'-carbonyldiimidazole in 1.0 ml of DMF. The solution was stirred at -10°C for two hours and then mixed with a cold solution of 45 >6 mg of 3-mercapto-2-D-methylpropanoyl-L-proline in 1 ml of DMF which was neutralized with N-ethylmorpholine. The reaction mixture was stirred at -10°C for a further 1 hour and then slowly warmed to room temperature. The solvent was removed under reduced pressure at 40°C and ethyl acetate was added to the residue. The mixture was cooled and washed with 0.1 N HCl and then three times with saturated NaCl solution. The solvent was removed with a rotary evaporator after drying

I) ksemp_el_25

The L-3,4-dehydroproline derivative is obtained in the same way

way by using the product in examples 3-5 instead of 3-mercapto-2-D-methylpropanoyl-L-proline in examples 16-24

and by essentially following the method in Example 16.

Example_el_26

Synthesis_of_Ng;cyclopentylcarbgnyl-

A cold solution of 2.06 g of dicyclohexylcarbodiimide in 10 ml of dichloromethane was added to a solution of 1.4114 g of cyclopentanecarboxylic acid in 5 ml of dichloromethane at -5°C. This was followed by the addition of 4.28 g of L-phenylalanine benzoyl ester toluenesulfonate salt in 10 ml of DMF which was neutralized with 1.36 ml of N-ethylmorpholine. The reaction mixture was stirred at 0°C for 1 hour and then at room temperature for 3 hours. Dicyclohexylurea was removed by filtration and 50 ml of ethyl acetate was added to the filtrate. The organic phase was washed until neutral, dried over anhydrous MgSO 4 and filtered. The solvent was removed with a rotary evaporator. The residue was crystallized from isopropanol and hexane, yielding 2.35 g of white crystals with a melting point of 88-89°C. Elemental analysis of these crystals gave the following:

Calculated: C=75.19; H=7.17 and N=3.9855

Found : C=74.96; H=7.17; N=4.09

The benzyl ester was removed by hydrogenolysis

with 2 g of 10% palladium-on-carbon in absolute alcohol. The catalyst was removed by filtration and the ethanol was removed with a rotary evaporator. The residue was crystallized from

ether and hexane, and this gave 1.15 g of white crystals of the desired product with a melting point of 107-108°C. Elemental analysis of these crystals gave the following: Calculated: 0 = 68.94 ; .'H=7.33» N=5.36

Found : C=68.90; H=7.32, N=5.34

The product was found to be homogeneous using paper electrophoresis at pH 1.9 and at pH 5.0

and using TLC with solvent systems 1, 2 and 3. The product prepared was abbreviated to Not-cpc-L-Phe.

<Eczema>2<el>_<27>_

§Y.2tese_ay_No<- [^ziNa^c y clovent ylkarbgnyl^L-f eny_lalany_ltio);

A solution of 52.5 mg of the compound from Example 26 in 0.5 ml redistilled DMF was cooled in an ice-dry ice-acetone bath at -20°C. To this solution was added a cold solution of 34 mg of 1,1'-carbonyldiimidazole

in 1.0 mL DMF. The solution was stirred at -10°C for two

hours and then mixed with a cold solution of 45.6 mg of 3-mercapto-2-D-methylpropanoyl-L-proline in 1 ml of DMF which was neutralized with N-ethylmorpholine. The reaction mixture was stirred at -10°C for a further 1 hour and then slowly warmed to room temperature. The solvent was removed under reduced pressure at 40°C and ethyl acetate was added to the residue. The mixture was cooled and washed with 0.1 N HCl and then three times with saturated NaCl solution. The solvent was removed with a rotary evaporator after drying

Example_el_25

The L-3,4-dehydroproline derivative is obtained in the same way

way by using the product in examples 3-5 instead of 3-mercapto-2-D-methylpropanoyl-L-proline in examples 16-24

and by essentially following the method in Example 16.

Example_el_26

Synthesis_of_Na-cyclggentylcarbgnYl enylalanine

A cold solution of 2.06 g of dicyclohexylcarbodiimide in 10 ml of dichloromethane was added to a solution of 1.4114 g of cyclopentanecarboxylic acid in 5 ml of dichloromethane at -5°C. This was followed by the addition of 4.28 g of L-phenylalanine benzoyl ester toluenesulfonate salt in 10 ml of DMF which was neutralized with 1.36 ml of N-ethylmorpholine. The reaction mixture was stirred at 0°C for 1 hour and then at room temperature for 3 hours. Dicyclohexylurea was removed by filtration and 50 ml of ethyl acetate was added to the filtrate. The organic phase was washed until neutral, dried over anhydrous MgSO 4 and filtered. The solvent was removed with a rotary evaporator. The residue was crystallized from isopropanol and hexane, yielding 2.35 g of white crystals with a melting point of 88-89°C. Elemental analysis of these crystals gave the following: Calculated: C=75.19, H=7.17; N=3.9855

Found : C=74.96; H=7.17" N=4.09

The benzyl ester was removed by hydrogenolysis

with 2 g of 10% palladium-on-carbon in absolute alcohol. The catalyst was removed by filtration and the ethanol was removed with a rotary evaporator. The residue was crystallized from ether and hexane, and this gave 1.15 g of white crystals of the desired product with a melting point of 107-108°C. Elemental analysis of these crystals gave the following:

Calculated: C = 68, 94, .'H=7.33, N=5.36

Found : C=68.90; H=7.32, N=5.34

The product was found to be homogeneous using paper electrophoresis at pH 1.9 and at pH 5.0

and using TLC with solvent systems 1, 2 and 3. The product prepared was shortened to Ncx-cpc-L-Phe.

Eczema_2£

§YDthesis_of_Na-[^-{Na-cyclop_entyl iz5zm§tYlE£22§D2yiIzLzE£2iiD

A solution of 52.5 mg of the compound from Example 26 in 0.5 ml redistilled DMF was cooled in an ice-dry ice-acetone bath at -20°C. To this solution was added a cold solution of 34 mg of 1,1'-carbonyldiimidazole in 1.0 ml of DMF. The solution was stirred at -10°C for two hours and then mixed with a cold solution of 45.6 mg of 3-mercapto-2-D-methylpropanoyl-L-proline in 1 ml of DMF which was neutralized with N-ethylmorpholine. The reaction mixture was stirred at -10°C for a further 1 hour and then slowly warmed to room temperature. The solvent was removed under reduced pressure at 40°C and ethyl acetate was added to the residue. The mixture was cooled and washed with 0.1 N HCl and then three times with saturated NaCl solution. The solvent was removed with a rotary evaporator after drying over anhydrous MgSO 4 . The product was purified by "Sephadex LH-20" column chromatography using a 1.2 cm x 95 cm column and eluted with isopropanol. The top fractions were collected and the solvent was removed under reduced pressure to give 60.5 mg of the desired product. This product was found to be homogeneous using TLC

in solvent systems 1, 2, 3 and 5-

Examples, 2$ =35

By using the benzoyl ester toluenesulfonate salts of glycine, L-alanine, L-tryptophan, L-tyrosine, L-isoleucine, L-leucine, L-histidine or L-valine instead of the L-Phe salt in Example 26 and by essentially following the methods of examples 26 and 27, the following compounds are obtained.

Eczema gel_3_6

The L-3,4-dehydroproline derivative is obtained in the same way

way by using the product in examples 3-5 instead of 3-mercapto-2-D-methylpropanoyl-L-proline in examples 27-35

and by essentially following the method in example 27-

Example_el_37

§YQt§§e_ay_Ng;;£3^L-f^nylal^

The product of Example 24 was treated to deprotect by stirring a mixture of 30 mg of the product, 50 µl of anisole and 200 µl of anhydrous trifluoroacetic acid (TFA) at room temperature for 1 hour. Anisole and TPA were removed under reduced pressure at 35°C and the residue was triturated with anhydrous ether. The white residue was purified by liquid chromatography on "Sephadex G-10<tm>" using a 1.2 cm x 95 cm column and eluted with 5% acetic acid. The top fractions were collected and freeze-dried, yielding 17.5 mg of the desired product. This product was found to be homogeneous using paper electrophoresis at pH 1.9 and 5.0

and by using TLC with solvent systems 4 and 5• Eksemgler__3_8__

By essentially following the procedure in Example 37, the protection is removed from the products in Examples 17-24 in the same way, and the following compounds are obtained!

Example_el_46

The L-3,4-dehydroproline derivative is obtained in the same way by removing the protection from the compound described in Example 25. This removal is carried out by substantially

follow the method in example 37-

Eczema gel_47

§Y£tese_ay_N-hydroxysuccinimide^ <silicic acid>

A cold solution of 11.4 g of dicyclohexylcarbodiimide in DMF was added dropwise to a mixture of 5.71 g of cyclopentanecarboxylic acid and 5.76 g of N-hydroxysuccinimide in DMF at 0°C. The reaction mixture was stirred at 0°C for 30 minutes and then at 4°C overnight. Crystalline dicyclohexylurea was removed by filtration and the precipitate was washed with ethyl acetate. Solvents from the combined filtrate were removed under reduced pressure and the residue was crystallized from benzene and hexane to give 5.77 g of white crystals with a melting point of 72.5~73<0>C. The infrared absorption spectrum in chloroform gave a typical spectrum for N-hydroxysuccinimide esters. Elemental analysis of the crystals gave the following results;

Calculated: C=56.86; H=6.20; N=6.63

Found : C=56.77; H=6.07; N=6.66

Example__48

§YDt§se_ay_N^-cv^logentyl^ L-lysine

A solution of 1.2316 g of Ne-tert-butyloxycarbonyl-L-lysine and 420 mg of NaHCO 3 in 10 ml of water and 5 ml of THF was cooled in an ice bath with stirring. To this solution was added a cold solution of 1.19 g of the product from Example 47 in 10 ml of THF. THF was removed with a rotary evaporator at 35°C after the reaction mixture was stirred overnight at room temperature. About 20 ml of water was added to the reaction mixture and the pH was adjusted to 9 with solid NaHCO 3 . The aqueous phase was extracted three times with ethyl acetate and the organic phase was discarded. The aqueous solution was cooled in an ice bath and then acidified to pH 2 with 1N HCl in the presence of ethyl acetate. The organic phase was washed twice with ice water and then twice with a solution of saturated NaCl. The organic solution was dried over anhydrous MgSO 4 and then filtered. The solvent was removed with a rotary evaporator and the residue was crystallized from ether and hexane to give 1.135 g of white crystals with a melting point of 104.5-105j5°C. Elemental analysis of the crystals gave the following;

Calculated: C=59.63; H=8.83; N=8.18

Found : C=59.74; H=8.85; N=8.24

The product was shown to be homogeneous using paper electrophoresis at pH 1.9 and 550 and using TLC with solvent systems 1, 2 and 3-Eksemgel_49

tlii§iDlN-hydroxy isuccininiide esters i

A solution of 1.027 g of the product from Example 48 and 346 mg of N-hydroxysuccinimide in 10 ml of CHCl was cooled to -5 oC. A cold solution of 680 mg of dicyclohexylcarbodiimide in 5 ml of CH 2 Cl 2 was added to this solution with stirring. The reaction mixture was stirred at 0°C for 30 minutes and then at 4°C overnight. Crystalline dicyclohexylurea was removed by filtration and washed with ethyl acetate. The combined filtrate was washed twice with 1 N NaHCO 3 , twice with water and finally with a solution of saturated NaCl. The organic phase was dried over anhydrous MgSO 4 , filtered and the solvent was removed on a rotary evaporator. The residue was crystallized from isopropanol, yielding 0.844 g of white crystals with a melting point of 140.5°C. The infrared absorption spectrum in chloroform gave a typical spectrum for N-hydroxysuccinimide esters. Elemental analysis of the crystals gave the following:

Calculated: C=57.39, H=7.57; N=9.56

Found : C=57.10; H=7.57, N=9.6l

Eczema gel_Yes?

§y^thesis_of_Na-cyclo£entylcarb^

A solution of 220 mg of the product from Example 49 in 2 ml of dioxane was added dropwise to a mixture of 99.1 mg of L-phenylalanine and 51 mg of NaHCO 3 in a mixture of 2 ml of water and 1 ml of DMF. The reaction mixture was stirred at room temperature overnight and dioxane was removed with a rotary evaporator at 35°C. Ethyl acetate (10 mL) was added to the reaction mixture, cooled, acidified to pH 2 with 0.1 N HCl, and the aqueous solution was discarded. The organic phase was washed with ice water, a solution of saturated NaCl and dried over anhydrous MgSO 4 ; and the solvent was removed with a rotary evaporator. The residue was crystallized from ether and petroleum ether (b.p. 30-60°C), giving 95 mg of a white solid with a melting point of 90-92°C. The product was shown to be homogeneous using paper electrophoresis at pH 1.9 and pH 5.0 and using TLC with solvent systems 1, 2, 3 and 4. Elemental analysis gave:

Calculated: C=63.78; H=8.03; N=8.58

Found : C=63.40; H=8.07; N=8.3^

Example_5I-§YDtese_of_Na-[ 2ziNg=cy_klop.enty.lka carbgnyl-L^lysyl^L-phenylalan^ EEolin.

A solution of 7355 mg of the product from Example 50 in 0.5 ml of redistilled DMF was cooled in an ice-dry ice-acetone bath at -20°C. To this solution was added a cold solution of 26 mg of 1,1'-carbonyldiimidazole in 1.0 ml of DMF. The solution was stirred at -10°C for two hours and then mixed with a cold solution of 36 mg of 3-mercapto-2-D-methylpropanoyl-L-proline in 1 ml of DMF which was neutralized with N-ethylmorpholine. The reaction mixture was stirred at

-10°C for a further 1 hour and then slowly warmed to room temperature. The solvent was removed under reduced pressure at 40°C and ethyl acetate was added to the residue. The mixture was cooled in an ice water bath and washed with 1 N citric acid and then three times with saturated NaCl solution. The solvent was removed with a rotary evaporator after drying over anhydrous MgSO|. The product was purified by "Sephadex LH-20" column chromatography using a 1.2 cm x 95 cm column and eluted with THF:isopropanol, 3:7 (v/v). The top fractions were collected and the solvent was removed under reduced pressure to give the desired product which can be abbreviated as Na-[3~(Ne-cpc-N-Boc-L-Lys-L-Phe-tio)-2- D-methylpropanoyl]-L-proline. Example_el_52

§YDt§§§_§Y_Nail3i(Ng-cyclogentyl_lcarbon The Ne-Boc group was removed from lysine by stirring

ing of a mixture of 30 mg of the product from Example 51 with 50 µl anisole and 200 µl anhydrous trifluoroacetic acid (TFA) at room temperature for 1 hour. Anisole and TFA were removed under reduced pressure at 35°C, and the residue was triturated with anhydrous ether. The residue was purified by liquid chromatography on "Sephadex G-10tm" using a 1.2 cm x 95 cm column and eluted with 5% acetic acid. The top fractions were collected and lyophilized to give the desired product which can be abbreviated as Na-[3-(Na-cpc-L-Lys-L-Phe-thio)-2-D-methyl-propanoyl]-L-proline.

<Examples>_5_3_i§<0>.

By using glycine, L-alanine, L-tryptophan, L-isoleucine, L-leucine or L-valine instead of L-phenylalanine in example 50 and by essentially following the procedures in examples 50, 51 and 52, the following is achieved connections :

Eczema gel_59

The L-3,4-dehydroproline derivative is obtained in the same way

way by using the product from examples 3-5 instead of 3-mercapto-2-D-methylpropanoyl-i-proline in examples 3-5 instead of 3-mercapto-2-D-methylpropanoyl-L-proline in examples 51 and 53-58 and by substantially following the procedures in Examples 51 and 52.

Example_el_60

By using pyro-L-glutamic acid instead of the cyclopentanecarboxylic acid in example 47 and essentially following the procedures in examples 47-49, Na-pyro-L-glutamyl-Ne-tert-butyloxycarbonyl-L-lysine-N-hydroxy is obtained -succinimide esters. By using this product instead of the Na-cpc-Ne-Boc-L-Lys-N-hydroxysuccinimide ester in example 50 and by substantially following the procedures in examples 50-59, the pyro-L-glutamyl derivatives are obtained.

Example_el_6l

Preparation_of_pyrg-L2glutamyl2L-phenylalanine benzyl ester.

A solution of 0.52 g of pyro-L-glutamic acid, 1.72 g of L-phenylalanine benzyl ester toluenesulfonic acid and 0.55 ml of N-ethylmorpholine in 5 ml of DMF and 20 ml of dichloromethane was cooled

in an ice bath with stirring. A solution of 0.826 g of dicyclohexylcarbodiimide in 2 ml of dichloromethane was added to the above reaction mixture. The reaction mixture was stirred in an ice-water bath for 1 hour and then at room temperature overnight. Dicyclohexylurea was removed by filtration, and the product was washed in ethyl acetate. The solvents in the combined filtrates were removed under reduced pressure with a rotary evaporator at 40°C. Ethyl acetate (25 ml) was added to the residue and the organic solution was washed until neutral. The organic phase was dried over anhydrous MgSO 4 , filtered and then the solvent was removed with a rotary evaporator. The material was crystallized from isopropanol and ether, yield 1.01 g of white needles, m.p. 103-104.5°C. The material was homogeneous on all TLC and electrophoresis systems. Elemental analysis gave:

Calculated: C=68.84; H=6.05; N=7.64

Found : 068.58; H=6.05; N=7.56

ExeniBel_ 62

_Preparation_of_2yro-L2glutamyl-L- .

The benzoyl ester protecting group on the compound of Example 6l (1.0 g) was removed by catalytic hydrogenolysis with 150 mg of 10% (by weight) Pd-on-carbon in 0.15 mL of glacial acetic acid and 15 mL of ethanol at 1.4 kg /cm 2 H2 at room temperature overnight. The catalyst was removed by filtration. Solvent was removed with a rotary evaporator. The material was crystallized from isopropanol and benzene, and this gave a total of 402 mg of white crystals, m.p. 147-149°C. The material was homogeneous by electrophoresis at pH 1.9 and pH 5.0 and by TLC in systems 1, 2 and 3 - Elemental analysis gave:

Calculated: C=60.86; H=5.84; N=10.14

Found: C=60.37, H=5.85, N=9.98

Example_62

Preparation_of_Ngrl^llEyro;L-glytamyl-L-phenylalanylthioJ-2ID-metYlp_rop_anoyl2-L2p_roline .

A solution of 87 mg of 1,1'-carbonyldiimidazole

in 1.0 ml DMF was added to a solution of 139 mg of the product of Example 62 in 0.5 ml DMF at -15°C. The reaction mixture was stirred at -10°C for 1 hour and then a mixture of 119.5 mg of 3-mercapto-2-D-methylpropanoyl-L-proline and 0.072 ml of N-ethylmorpholine in 1 ml of DMF was added. The reaction mixture was stirred at -10°C for an additional 1 hour and was then slowly warmed to room temperature. DMF was removed under reduced pressure with a rotary evaporator at 40°C and then 7 ml of ethyl acetate and 2 ml of 1 N citric acid were added. The organic phase was washed twice with 1 N citric acid and twice with saturated NaCl.

The organic phase was dried over anhydrous MgSO 4 and then filtered. The solvent was removed using a rotary evaporator. The residue was purified by "Sephadex G-25<tm>" (1.2 x 99 cm) column chromatography with n-butanol:acetic acid:H 2 O (4:1:5 by volume). The product was homogeneous by TLC (systems 1, 2 and 3) and by electrophoresis at pH 5a0.

Example_el_64_

By using the benzyl esters of Gly, Ala, Trp, Tyr, Ile, Leu, His or Val in the methods in example 6l, the corresponding pyro-L-glutamyl derivatives are obtained. Said benzyl esters are commercially available. Benzyl ester protecting groups are substantially removed as described in Example 62.

By essentially following the procedures

in Examples 2-5 and 61-64 a family of thiol ester compounds is obtained which has the general formula:

where

R is pyro-L-glutamyl;

A is L-phenylalanyl, glycyl, L-alanyl, L-tryptophyll, L-isoleucyl, L-leucyl or L-valyl, whose ct-amino group is in amide bond with R;

R 1 is hydrogen or methyl;

Rn is L-proline or L-3,4-dehydroproline, if

imino group is in mid-mixture with

Eczema<p>_<el_>65__

Say. r<n>tese<_>av_Na-l2liN<g>2benzoYl-L;tryp<_>tof<y>_ltio_};2-D-met<y_l>2ro-E§D2<Yl>lz<L-> gr<o>lin.

A solution of 62 mg of Na-benzoyl-L-tryptophan in 0.5 ml of redistilled dimethylformamide (DMP) was cooled in an ice-dry ice-acetone bath at -20°C. To this solution was added a cold solution of 35 mg of 1,1'-carbonyldiimidazole in 1.0 ml of DMF. The solution was stirred at

-10°C for two hours and then added to a cold solution of 48 mg of 3-mercapto-2-D-methylpropanoyl-L-proline in 1 ml of DMF

which was neutralized with N-ethylmorpholine. The reaction mixture was stirred at -10°C for a further 1 hour and then slowly warmed to room temperature. The solvent was removed under reduced pressure at 40°C and ethyl acetate was added to the residue. The mixture was cooled in an ice bath and washed with 0.1 N HCl and then three times with saturated NaCl solution. The solvent was removed with a rotary evaporator after drying over anhydrous MgSO 4 . The product was purified by liquid chromatography on "Sephadex LH-20<tm>" using a 1.2 cm x 95 cm column and eluted with isoproPanol. The top fractions were collected and the solvent removed under reduced pressure to give 60.5 mg of the desired product as a foamy material. This product was found to be homogeneous using TLC with solvent systems 1, 2, 3 and 5.

Examples_ §§- 11

By using Na-benzoyl-L-tyrosine, Na-benzoyl-L-histidine, Na-benzoyl-L-phenylalanine, Na-benzoyl-glycine, Na-benzoyl-L-alanine, Na-benzoyl-L-isoleucine, Na -benzoyl-L-leucine or Na-benzoyl-L-valine instead of Na-benzoyl-L-tryptophan in example 65 and by substantially following the procedures in example 65, the following compounds are obtained:

69 Na-[3~(Na-benzoyl-L-isoleucylthio)-2-D-methyl-propanoyl]-L-proline 70 Na-[3-(Na-benzoyl-L-leucylthio)-2-D-methyl- propanoyl]-L-proline 71 Na-[3-(Na-benzoyl-L-valylthio)-2-D-methyl-propanoyl]-L-proline

Example_el_72

The L-3,4-dehydroproline derivative is obtained in the same way by using the product in examples 3~5 instead of 3~ mercapto-2-D-methylpropanoyl-L-proline in examples 65-71 and v<p>d substantially to follow the methods in example 65.

An alternative method for the production of Na-[3-(Na-benzoyl-L-phenylalanylthio)-2-D-methylpropanoyl]-L-proline is described in the following two examples.

Example_73

Preparation_of_Na-benzoyl-L-phenylalanine-N-hy ester.

1.347 g of benzoylphenylalanine and 0.576 g of N-hydroxysuccinimide were mixed in a 1:1 (by volume) mixture of THF and DMF. The mixture was incubated at 4°C overnight in the presence of 1.133 g of dicyclohexylcarbodiimide.

The reaction mixture was filtered and the solvent was removed under reduced pressure at 30°C. A white residue remained and this was recrystallized from THF-iso-propyl alcohol to give 1.194 g (65.2%) of a white solid, m.p. 156-157°C. The infrared absorption spectrum in chloroform showed bands at 3440 cm indicating an NH group, at 1818 cm<-1>, 1790 cm<-1> and 1744 cm"<1>, which is characteristic of the N-carboxysuccinimide group and at 1669 cm <1>, which is characteristic of the N-benzoyl group Eczemagel_J3

§YDtese_ay_Na-X2ziNa-benzoyl-L3f eny lalanyl ioj^-D-methyl-BEoganoyl]-L-groline.

A reaction mixture containing 93.3 mg of 2-D-methyl-3-mercaptopropanoyl-L-proline, 62 mg of 1-hydroxybenzotriazole (HOBt), 165 mg of N-benzoyl-phenylalanyl-N-hydroxy-succinimide ester, prepared as described in example 73

was cooled in an ice bath at about 0°C, after which 0.0544 ml of N-ethylmorpholine was added. The reaction mixture was stirred

in an ice bath for three hours, incubated at 4°C overnight, and then at room temperature for 20 hours. The reaction was terminated by the addition of 25 µl of N,N-dimethyl-1,3-propanediamine and stirred for a further two hours. Ethyl acetate was added to the reaction mixture which was then washed by extraction with cold 0.1N HCl, followed by two washes with water and three washes with saturated NaCl. The mixture was then dried over anhydrous MgSO 4 and filtered. The solvent was removed under reduced pressure in a rotary evaporator to give a clear oily product.

The product was purified by chromatography on a column of "Sephadex LH-20<tm>", 1.2 cm x 96 cm. The column was eluted with THF and 2.5 mL fractions were collected. Fractions 30 and 31 were collected and, after solvent removal under high vacuum, gave 110 mg of product. Anhydrous ether was added and this gave a white gum-like material. The ether was decanted and the white gummy material was dissolved in THF and transferred to a sample bottle, dried in a stream of nitrogen, and then further dried over ^2^5 overnight. This gave 82 g of a foam-like product.

Fractions 29, 32 and 33 were chromatographed on

new under identical conditions. Fractions 33-36 from the second chromatography were collected, processed as described and yielded an additional 51 mg of product. Total yield was 71%.

Biological experiments

Example_el_75_

The inhibitory activity of different av

the above synthesized compounds in vitro were measured using the system described in Example 1. The enzyme preparation was purified from human urine as described by Ryan, J.W. et al., Tissue and Cell 10, 555 (1978). Table I shows the I^ value for various compounds. The I^ value is the concentration

of inhibitor required to give 50% inhibition of the enzyme under a standard test system containing substrate at a level substantially below K .

m

Eczema gel_76

Q£ai_§££§ktiYitet_for_different_connections

Rats (210-290 g body weight) were fasted overnight and then anesthetized with intraperitoneal pentobarital, 50-60 mg/kg. Tracheostomy was performed and the animals were mechanically ventilated. One cannula was inserted into a femoral vein for injection of angiotensin I, and another cannula was inserted into a common carotid artery for direct measurement of arterial blood pressure. Heparin, 1,000 units, was injected via the femoral vein to prevent coagulation. Blood pressure was measured using a pressure transducer connected to a polygraph. The rats were injected with 160-400 ng/kg angiotensin I in 20 µl of 0.9 g% NaCl; an amount of angiotensin I sufficient to raise mean arterial blood pressure by 27-50 mm Hg. After the response of an angiotensin I given above was established, the respective compound at 5.5-23 µmol/kg (drug dissolved in 0.15 ml H 2 O plus 10 µl 1 N NaHCO-j) was given via a gastric tube. At specific time intervals, the effects of 160-400 ng/kg angiotensin I

on the mean arterial blood pressure tested. The results are

shown in Table 2. The dose of the particular compound is shown in parentheses after the number of the compound in question. E.g. 6 (13.6) indicates that 13.6 µmol/kg of the compound from Example 6 was administered orally. The time, in minutes, after oral administration of the particular compound, when the effects of angiotensin I were tested, is indicated in parentheses after the "% of control" indication.

Exem2el_72

JD££aY§D^_eff §ktivity_f or_f or s kj^ ell ige_ connections.

Anesthetized rats were prepared as described in Example 1b. After the response of a given rat to angiotensin I was established, the respective compound in an amount of 2 µmol/kg in a volume of 15 µl of 0.01N sodium bicarbonate was injected via a femoral vein. At time intervals, the effects of angiotensin I, 400 ng/kg, on mean arterial blood pressure were tested. The results are shown in table 3 - The same set-up has been used in table 3 as in table 2.

<Eczema>P.<el>_j<8>

First illing_of_N-£ 3- (^ U^ 2Y. lzhz^^^ l^ U^ l^ i2lz?. Z^ Z^^.^. l~

a. First illing_of_N;^3.ll§22lL-feny E^22§22YiIltlE^2iin

A solution of 133 mg (0.5 mmol) of Boc-L-Phe-OH in 0.5 ml of redistilled dimethylformamide (DMF) was cooled

to -20°C in an ice-dry ice-acetone bath. A cooled solution of 87 mg (0.54 mmol) of 1,1'-carbonyldiimidazole in 1 mL of DMF was added and the resulting solution was stirred at -10°C for 2 hours. DMF, 1.0 mL, containing 119.5 mg (0.55 mmol) 2-D-methyl-3-mercaptopropanoyl-L-proline and 0.075 mL (0.55 mmol) N-ethylmorpholine, all at -10°C , was added, and the final solution was stirred at -10°C for 1 hour. The reaction mixture was slowly warmed to room temperature. Solvent was removed with a rotary evaporator. Ethyl acetate (10 mL) was added and the solution was cooled in an ice bath. The organic solution was washed twice with approx. 2 ml of 1 N citric acid and then washed twice with a saturated NaCl solution. The ethyl acetate phase was dried over anhydrous MgSO 4 and then filtered. The solvent in the filtrate was removed with a rotary evaporator. The residue was dissolved in a small volume of isopropanol/tetrahydrofuran (7^3 parts by volume) and applied to a column (1.2 x 99 cm) of "Sephadex LH-20" equilibrated with the same solvent. Each elution fraction from the column contained 2.55 ml. Fractions 31-36 were collected and solvent was removed with the rotary evaporator. The residue was redissolved in isopropanoyl/tetrahydrofuran solvent and applied to a column (1.2 x 98 cm) "Sephadex G-10" equilibrated and developed with the same solvent. Fractions, each of 2.55 ml, were collected. Fractions 19-23

were combined to give 165 mg of the desired product. Purification of side fractions from the "Sephadex LH-20" chromatography step yielded 40 mg of product. The product behaved as a pure substance by paper electrophoresis at pH 5.0 and by thin layer chromatography (silica gel plates) using three solvent systems. Amino acid analysis: Phe 1.00,

Pro 1.01.

b. Preparation_ay_N=[^ziHCl^^L^fenvlalanylthio)=2=D-m£tYilEI!°E§22Y.ll::3=!lE£2iiD

Anisole (0.2 mL) was added to 200 mg of N-[3~(Boc-L-phenylalanylthio)-2-D-methyl-propanoyl]-L-proline and the mixture was stirred at 0°C. Anhydrous trifluoroacetic acid (0.4 mL) was added and the solution was stirred at room temperature (~22°C)

for 45 minutes. Trifluoroacetic acid was removed with a rotary evaporator at 30°C and then 1 ml of ethyl acetate saturated with hydrogen chloride was added. Excess HCl and ethyl acetate were removed with the rotary evaporator and this gave an oily residue. Anhydrous ethyl ester was added and the mixture was left at 0°C

for 1 hour. A white solid was obtained by filtration and washed several times with anhydrous ether. The solid material was dried in a vacuum desiccator over NaOH and ^ 2^ 5' c. Preparation_of_N-£3iibenzoyl-L

groganoyl ]-L-p_roline

A solution of 93 mg (0.23 mmol) of N-[3-(HC1•H--L-phenylalanylthio)-2-D-methyl-propanoyl]-L-proline in 0.5 ml of dioxane (redistilled over Na ), was mixed with 0.029 mL (0.25 mmol) of benzoyl chloride in 0.099 mL (0.71 mmol) of N-ethylmorpholine. After 1 hour of reaction at room temperature, a further 0.029 ml of benzoyl chloride and 0.034 ml of N-ethylmorpholine were added. One hour after the second addition, glacial acetic acid (0.2 mL) was added. Solvent was removed with the rotary evaporator at 35°C. The crude product was purified by distribution chromatography [1.2 x 97 cm column of "Sephadex G-25" equilibrated with n-butanol/acetic acid/i 2 O (4:1:5)]. The sample was dissolved in a mixture of 0.3 ml lower phase and 0.3 ml upper phase. Elution was started with the upper phase. Fractions (each

of 2.75 ml) was collected. Fractions I6-18 were combined and solvent removed at 37°C with a rotary evaporator. The product was then applied to a column (1.2 x 98 cm) of silica gel equilibrated and developed with ethyl acetate. Fractions (5.15 mL each) were collected. Fractions 46-60 were combined to give 52.5 mg of product. The product behaved as a pure substance by thin layer chromatography using two solvent systems.

Example_el_79

The desired compound is prepared using the appropriate mercapto compound, whereby instead of L-proline in the method in example 78 above, L-3»4-dehydroproline is used.

The appropriate mercapto compounds can be prepared using the methods described in US Applications Nos. 64,897-64,903.

Example_el_80

By using the appropriate compound in place of benzoyl chloride in Examples 77-78 above and using well-known methods such as acetyl-, cyclopentanecarbonyl-, cyclopentanecarbonyl-L-lysyl- and pyro-L-glutamyl derivs.

Example_el_8l

The inhibitory action of various of the above synthesized compounds in vitro was measured in the test system described in Example 1. The enzyme preparation was purified from human urine as described by Ryan, J.W., et al., Tissue and Cell 10, 555 (1978). Table 4 below shows the I^Q value for "various compounds. The I^ value is the concentration of inhibitor required to produce 50% inhibition of the enzyme using a standard test system containing substrate at a level substantially below Km.

Salts_of_compounds_with_formula_I

Compounds of formula I form basic salts with various inorganic and organic bases. Such salts include ammonium salts, alkali metal salts, alkaline earth metal salts, salts with organic bases, salts with amino acids and the like. Examples of alkali metal salts are the sodium or potassium salts. Examples of alkaline earth metal salts are the calcium or magnesium salts. Examples of organic base salts are benzathine, N-methyl-D-glucamine or hydrabamine salts. Examples of amino acid salts are arginine and lysine. The sodium, potassium and lysine salts are preferred. Likewise

non-toxic, physiologically acceptable salts are preferred.

The salts are formed by reacting the free acid of the compounds of formula I with an equivalent of the suitable base which gives the desired cation in a solvent or medium in which the salt is insoluble, or in water and removing the water by freeze-drying.

The salts produced according to the invention inhibit the conversion of the decapeptide angiotensin I to angiotensin II and are therefore useful in reducing or alleviating angiotensin-related hypertension. The action of the enzyme renin on angiotensinogen, a pseudoglobulin in blood plasma, produces angiotensin I. Angiotensin I is converted by angiotensin-converting enzyme (ACE) to angiotensin II. The latter is an active pressor substance that has been implicated as the causative agent in various forms of hypertension in various mammalian species, e.g. rats and dogs. The connections in the present

invention intervenes in angiotensinogen

angiotensin I

the angiotensin II sequence by inhibiting angiotensin-

converting enzyme and reduce or eliminate the formation of

Eczema gel_79

Preparation_of_L-^A4-dehyde

The desired compound is produced using the appropriate mercapto compound whereby L-3,4-dehydroproline is used instead of L-proline in the method in example 78 above.

The appropriate mercapto compounds can be prepared using the methods described in US applications no.

64,897-64,903-

Example_el_80

By using the appropriate compound in place of benzoyl chloride in Examples 77-78 above and using well-known methods such as acetyl-, cyclopentanecarbonyl-, cyclopentanecarbonyl-L-lysyl- and pyro-L-glutamyl derivs.

Example_el_8l

The inhibitory action of various of the above synthesized compounds in vitro was measured in the test system described in Example 1. The enzyme preparation was purified from human urine as described by Ryan, J.W., et al., Tissue and Cell 10, 555 (1978). Table 4 below shows the I^Q value for different compounds. The I^^ value is the concentration of inhibitor required to produce 50% inhibition of the enzyme using a standard test system containing substrate at a level substantially 0 below the Km.

Compounds of formula I form basic salts with various inorganic and organic bases. Such salts include ammonium salts, alkali metal salts, alkaline earth metal salts, salts with organic bases, salts with amino acids and the like. Examples of alkali metal salts are the sodium or potassium salts. Examples of alkaline earth metal salts are the calcium or magnesium salts. Examples of organic base salts are benzathine, N-methyl-D-glucamine or hydrabamine salts. Examples of amino acid salts are arginine and lysine. The sodium, potassium and lysine salts are preferred. Likewise

non-toxic, physiologically acceptable salts are preferred.

The salts are formed by reacting the free acid of the compounds of formula I with an equivalent of the suitable base which gives the desired cation in a solvent or medium in which the salt is insoluble, or in water and removing the water by freeze-drying.

The salts produced according to the invention inhibit the conversion of the decapeptide angiotensin I to angiotensin II and are therefore useful in reducing or alleviating angiotensin-related hypertension. The action of the enzyme renin on angiotensinogen, a pseudoglobulin in blood plasma, produces angiotensin I. Angiotensin I is converted by angiotensin-converting enzyme (ACE) to angiotensin II. The latter is an active pressor substance that has been implicated as the causative agent in various forms of hypertension in various mammalian species, e.g. rats and dogs. The connections in the present

invention intervenes in angiotensinogen

angiotensin I

the angiotensin II sequence by inhibiting angiotensin-

converting enzyme and reduce or eliminate the formation of

Example_el_79

First llll^ S_ 3:Y._ hz^ i.^ Z§:t] ll^ 22£2li1^

The desired compound is prepared using the appropriate mercapto compound whereby L-3,4-dehydroproline is used instead of L-proline in the method in example 78 above.

The appropriate mercapto compounds can be prepared using the methods described in US applications no.

64,897-64,903-

Example_el_80

By using the appropriate compound instead of benzoyl chloride in Examples 77~78 above and using well-known methods such as acetyl-, cyclopentanecarbonyl-, cyclopentanecarbonyl-L-lysyl- and pyro-L-glutamyl derivatives.

Example_el_8l

The inhibitory action of various of the above synthesized compounds in vitro was measured in the test system described in Example 1. The enzyme preparation was purified from human urine as described by Ryan, J.W., et al., Tissue and Cell 10, 555 (1978). Table 4 below shows the I^Q value for different compounds. The I^Q value is the concentration of inhibitor required to produce 50% inhibition of the enzyme using a standard test system containing substrate at a level substantially below K

Compounds of formula I form basic salts with various inorganic and organic bases. Such salts include ammonium salts, alkali metal salts, alkaline earth metal salts, salts with organic bases, salts with amino acids and the like. Examples of alkali metal salts are the sodium or potassium salts. Examples of alkaline earth metal salts are the calcium or magnesium salts. Examples of organic base salts are benzathine, N-methyl-D-glucamine or hydrabamine salts. Examples of amino acid salts are arginine and lysine. The sodium, potassium and lysine salts are preferred. Likewise

non-toxic, physiologically acceptable salts are preferred.

The salts are formed by reacting the free acid of the compounds of formula I with an equivalent of the suitable base which gives the desired cation in a solvent or medium in which the salt is insoluble, or in water and removing the water by freeze-drying.

The salts produced according to the invention inhibit the conversion of the decapeptide angiotensin I to angiotensin II and are therefore useful in reducing or alleviating angiotensin-related hypertension. The action of the enzyme renin on angiotensinogen, a pseudoglobulin in blood plasma, produces angiotensin I. Angiotensin I is converted by angiotensin-converting enzyme (ACE) to angiotensin II. The latter is an active pressor substance that has been implicated as the causative agent in various forms of hypertension in various mammalian species, e.g. rats and dogs. The connections in the present

invention intervenes in angiotensinogen

angiotensin I

the angiotensin II sequence by inhibiting angiotensin-

converting enzyme and reduce or eliminate the formation of

Eczema gel_79

Mainly illing_of_L- 3_ A4-d^hv_drogroline

The desired compound is prepared using the appropriate mercapto compound whereby L-3,4-dehydroproline is used instead of L-proline in the method in example 78 above.

The appropriate mercapto compounds can be prepared using the methods described in US applications no.

64,897-64,903-

Example_el_80

By using the appropriate compound in place of benzoyl chloride in Examples 77-78 above and using well-known methods such as acetyl-, cyclopentanecarbonyl-, cyclopentanecarbonyl-L-lysyl- and pyro-L-glutamyl derivs.

Eczema gel_8l

The inhibitory action of various of the above synthesized compounds in vitro was measured in the test system described in Example 1. The enzyme preparation was purified from human urine as described by Ryan, J.W., et al., Tissue and Cell 10, 555 (1978). Table 4 below shows the I^Q value for different compounds. The 1,-Q value is the concentration of inhibitor required to produce 50% inhibition of the enzyme using a standard test system containing substrate at a level substantially below K]n

Compounds of formula I form basic salts with various inorganic and organic bases. Such salts include ammonium salts, alkali metal salts, alkaline earth metal salts, salts with organic bases, salts with amino acids and the like. Examples of alkali metal salts are the sodium or potassium salts. Examples of alkaline earth metal salts are the calcium or magnesium salts. Examples of organic base salts are benzathine, N-methyl-D-glucamine or hydrabamine salts. Examples of amino acid salts are arginine and lysine. The sodium, potassium and lysine salts are preferred. Likewise

non-toxic, physiologically acceptable salts are preferred.

The salts are formed by reacting the free acid of the compounds of formula I with an equivalent of the suitable base which gives the desired cation in a solvent or medium in which the salt is insoluble, or in water and removing the water by freeze-drying.

The salts produced according to the invention inhibit the conversion of the decapeptide angiotensin I to angiotensin II and are therefore useful in reducing or alleviating angiotensin-related hypertension. The action of the enzyme renin on angiotensinogen, a pseudoglobulin in blood plasma, produces angiotensin I. Angiotensin I is converted by angiotensin-converting enzyme (ACE) to angiotensin II. The latter is an active pressor substance that has been implicated as the causative agent in various forms of hypertension in various mammalian species, e.g. rats and dogs. The connections in the present

invention intervenes in angiotensinogen

angiotensin I

the angiotensin II sequence by inhibiting angiotensin-

converting enzyme and reduce or eliminate the formation of

Eczema gel_79

Preparation_of_L-^A4-dehyde

The desired compound is prepared using the appropriate mercapto compound whereby L-3,4-dehydroproline is used instead of L-proline in the method in example 78 above.

The appropriate mercapto compounds can be prepared using the methods described in US Applications Nos. 64,897-64,903.

Example_el_80

By using the appropriate compound in place of benzoyl chloride in Examples 77-78 above and using well-known methods such as acetyl-, cyclopentanecarbonyl-, cyclopentanecarbonyl-L-lysyl- and pyro-L-glutamyl derivs.

Example_el_8l

The inhibitory action of various of the above synthesized compounds in vitro was measured in the test system described in Example 1. The enzyme preparation was purified from human urine as described by Ryan, J.W., et al., Tissue and Cell 10, 555 (1978). Table 4 below shows the 1,-Q value for "various compounds. The 1,-Q value is the concentration of inhibitor required to produce 50% inhibition of the enzyme using a standard test system containing substrate at a level substantially below the Km .

Compounds of formula I form basic salts with various inorganic and organic bases. Such salts include ammonium salts, alkali metal salts, alkaline earth metal salts, salts with organic bases, salts with amino acids and the like. Examples of alkali metal salts are the sodium or potassium salts. Examples of alkaline earth metal salts are the calcium or magnesium salts. Examples of organic base salts are benzathine, N-methyl-D-glucamine or hydrabamine salts. Examples of amino acid salts are arginine and lysine. The sodium, potassium and lysine salts are preferred. Likewise

non-toxic, physiologically acceptable salts are preferred.

The salts are formed by reacting the free acid of the compounds of formula I with an equivalent of the suitable base which gives the desired cation in a solvent or medium in which the salt is insoluble, or in water and removing the water by freeze-drying.

The salts produced according to the invention inhibit the conversion of the decapeptide angiotensin I to angiotensin II and are therefore useful in reducing or alleviating angiotensin-related hypertension. The action of the enzyme renin on angiotensinogen, a pseudoglobulin in blood plasma, produces angiotensin I. Angiotensin I is converted by angiotensin-converting enzyme (ACE) to angiotensin II. The latter is an active pressor substance that has been implicated as the causative agent in various forms of hypertension in various mammalian species, e.g. rats and dogs. The connections in the present

invention intervenes in angiotensinogen

angiotensin I

the angiotensin II sequence by inhibiting angiotensin-

converting enzyme and reduce or eliminate the formation of

the pressor substance angiotensin II. By administration of a preparation containing one or a combination of salts of the compounds of formula I, angiotensin-dependent hypertension in the mammalian species suffering from such a condition will be attenuated. A single dose, or preferably two or four divided daily doses, provided on a basis of from approx. 0.1 to 100 mg per kilo per day, preferably from approx. 1 to 50 mg per kilo per day, is appropriate in reducing blood pressure as indicated in the animal model experiments described by S.L. Angel,

T.R. Schaeffer, M.H. Waugh and B. Rubin, Proe. Soc. Exp. Biol. With. 143 (1973). The substance is preferably administered

orally, but parenteral routes such as subcutaneous, intramuscular, intravenous or intraperitoneal can also be used.

The salts produced according to the present invention can be used to achieve a blood pressure reduction by incorporation into preparations such as tablets, capsules or elixirs for oral administration or in sterile solutions or suspensions for parenteral administration. About 10-500 mg of a salt or mixture of salts of the compounds of formula I is combined with a physiologically acceptable carrier, excipient, binder, preservative, stabilizer, flavoring agent, etc., in a unit dosage form according to accepted pharmaceutical practice. The amount of active substance in these preparations is such that a suitable dose in the specified range is achieved.

As an illustration of excipients which can be incorporated into tablets, capsules and the like, the following can be stated: a binder such as gum tragacanth, acacia, corn starch or gelatin; an excipient such as dicalcium phosphate; a disintegrant such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; a sweetener such as sucrose, lactose or saccharin; a flavoring such as peppermint, oil of wintergreen or cherry. When the dosage unit form is a capsule, it may contain, in addition to the above-mentioned materials, a liquid carrier such as fatty oil. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. Tablets can e.g. coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetener, methyl and propyl parabens as preservatives, a coloring agent and a flavoring agent such as cherry or orange flavor.

Sterile preparations for injection can be formulated

according to conventional pharmaceutical practice by dissolving or suspending the active substance in a carrier such as water for injection, a naturally occurring vegetable oil such as sesame oil, coconut oil, peanut oil, cottonseed oil, etc., or a synthetic fatty carrier such as ethyl oleate or the like. Buffers, preservatives and the like can be incorporated as required.

Physical data for prepared compounds

In the following, physical data and I^Q values are given for compounds prepared in the examples given above, and for compounds that are otherwise covered by formula I. The number preceding each named compound represents

the example that shows the preparation of the compound in question.

Solvent systems used for thin layer chromatography using silica gel plates were as follows: System 1 [R^(1)] = methanol:chloroform, 1:1 parts by volume.

System 2 [R^(D] = benzene:water:n.bitanol, 9:1:9 parts by volume.

System 3 [Rf(3)] = acetic acid:water:n-butanol, 26:24:150 parts by volume.

System 4 [R^(4)] = n-butanol:pyridine:acetic acid:water, 15:10:3:12 parts by volume.

System 5 [Rf(5)] = n-butanol:acetic acid:ethyl acetate:water, 1:1:1:1 parts by volume.

System 6 [R^(6)] = chloroform:methanol:acetic acid, 2:1:0.00 3 parts by volume.

System 7 [R^(7)] = n-butanol:acetic acid:water, 4:1:1 parts by volume.

System 8 [R^(8)] = ethyl acetate:acetic acid, 38:2 parts by volume.

Indicated values in NMR spectra were obtained at 60 MHz.

Ir_ value: concentration of 50% inhibitory ak-50 J

tivity against angiotensin-converting enzyme.

Analysis system: 1 ml incubation mixture consisting of

0.6 ml borate buffer, 0.2 ml 17.5 mM benzoyl-Gly-His-Leu borate buffer, compound borate buffer and enzyme solution, incubated at 37°C for 60 min.

Borate buffer: 0.1 M boric acid containing 0.8 M NaCl and 0.1 M Na 2 CO 3 containing 0.8 M NaCl were mixed and adjusted to pH 8.3.

ACE: Acetone powder of rabbit lung.

Measurement: UV absorption at 228 nm after extraction of benzoyl-Gly with ethyl acetate.

Claims (1)

Analogy method for making a angiotensin-converting enzyme inhibitor for treatment of hypertension, and with the formula: where R is hydrogen, lower alkanoyl, benzoyl, cycloalkanoyl, tert.butyloxycarbonyl, cyclopentylcarbonyl-L-lysyl, pyro-L- glutamyl; A is L-phenylalanyl, glycyl, L-alanyl, L-tryptophyll, L-isoleucyl, L-leucyl or L-valyl, the α-amino group therein is in an amide bond with R; R^ is lower alkyl; R^ is L-proline or L-3,4-dehydroproline, the imino group therein is in imide bond with the adjacent characterized by (1) reacting with R2 to form where R3 is acetyl and R1 and R2 have the meanings given above nings, (2) removes R 3 from the product in step (1) to form where and R 2 have the meanings given above; (3) reacts the product in step (2) with R - A - GH to form where R, A, R 1 and R 2 have the meanings given above.