NO169172B - ANALOGY PROCEDURE FOR THE PREPARATION OF THERAPEUTICALLY ACTIVE SUBSTITUTED DIPEPTIDES - Google Patents

Description

Enkephalin is a natural opiate receptor agonist and is thought to be a mixture of two pentapeptides: H-Tyr-Gly-Gly-Phe-Met-OH (methionine-enkephalin), and H-Tyr-Gly-Gly-Phe-Leu-OH (leucine-enkephalin). Hereafter, the name enkephalin is used generically to include all such compounds.

It has been reported by Beluzzi et al., Nature, 260, 625 (1967) that when enkephalin is injected into the cerebral ventricle of rats, a profound analgesic effect is obtained. It is also known in the art that enkephalin is affected by a group of enzymes known generically as enkephalinases, which are also naturally occurring, and which are thereby inactivated.

European Patent Application 79105015.6, Publication No. 12401, describes certain dipeptide derivatives which are described to exhibit antihypertensive activity.

European Patent Application 81110337.3, Publication No. 54862, discloses certain dipeptide compounds that exhibit enkephalinase inhibitory activity.

The present invention relates to an analogue method for the production of therapeutically active compounds of the formula

and pharmaceutically acceptable salts thereof, wherein

R-1 is phenyl-lower alkyl or diphenyl-lower alkyl;

1*2 is hydroxy, -OCF^OCO-lower alkyl, the group

wherein R 7 and R 8 are lower alkyl, or R 2 is -O-lower alkyl-O-phenyl; 0-CH 2 -CHOH-CH 2 -OH, phenyl-lower alkoxy or amino-phenyl alkoxy;

R 1 is benzyl or phenylbenzyl;

R 4 is hydrogen, lower alkyl or hydroxy;

R 1 is hydrogen;

Rg is hydroxy, phenyl-lower alkoxy, -OCf^OGO-lower alkyl, the group

lower alkoxy or phenoxy-lower alkoxy;

R 7 and R 8 are lower alkyl;

p is 1

In formula I, the asterisks indicate the carbon atoms which may be asymmetric (chiral) centers. The invention encompasses the preparation of all isomers at these centers, both in pure form and in mixture.

A group of compounds of formula I of interest are those in which R 4 does not denote hydrogen, i.e. lower alkyl or hydroxy, preferably methyl. Preferred meanings for the other groups represented in formula I when R4 has such a meaning are as follows: Rx is benzyl optionally para-substituted with phenyl or 2-phenylethyl, and preferably benzyl or p-phenylbenzyl, R2 and R6 are the same or different and is hydroxy, methoxy, ethoxy, benzyloxy, 2-phenylethoxy, 1-glyceryl, and is preferably selected from hydroxy, 2-phenoxyethoxy, 1-glyceryl,

R 3 is benzyl or p-phenyl-benzyl, and

R 5 is hydrogen.

Another group of compounds of interest are those in which one of R2 and Rg is selected from 2-phenoxyethoxy, 1-glyceryl, or

and the other is selected from the preceding groups or is hydroxy, methoxy, ethoxy or benzyloxy: and when R 2 and R 6 have such meanings they are the preferred meanings for R 3 and R 5 as indicated in the preceding paragraph, and preferred meanings for R 4 are hydrogen and methyl .

p is 1.

As used herein unless otherwise indicated, the terms alkyl and alkoxy denote such groups having straight or branched carbon chains of from 1 to 6 carbon atoms.

Certain of the compounds of structural formula I form salts with pharmaceutically acceptable acids. Hydrochloric acid, sulfuric acid, acetic acid, maleic acid, fumaric acid etc. can be used.

Compounds of structural formula I in which R 2 and/or

R6 is hydroxy forming salts with pharmaceutically acceptable bases. Sodium, potassium and calcium hydroxide as well as sodium and potassium carbonate are examples of suitable bases for this purpose. In addition, consideration is given to salts formed with pharmaceutically acceptable amines such as e.g. ammonia, N-methylglucamine, benzylamine and morpholine.

The compounds of structural formula I can be prepared using reactions and reactants well known in the polypeptide art.

The analogue method according to the present invention is characterized by using a suitable method chosen from the following methods in which p, R1r R2, R3, R4, R5 and R6 are as defined above, including suitable protection of any reactive group;

(a) reduction of a compound of formula XX at the C=N double bond

(b) linking an amino acid of formula VIII to an amino acid of formula V

followed by removal of any protecting group if necessary, to form the desired compound of formula I, and, if desired, converting it to a salt thereof.

The following general procedure can be used to prepare the compounds of formula I from readily available or easily prepared starting materials:

Procedure

In the preceding reaction sequence, the amino function in compound VI is protected with an amino-protecting group that is usually used in the field (G) such as benzyloxycarbonyl, t-butyloxycarbonyl, etc. Compound VI is condensed with an aminoester derivative V in which Rg is protected if necessary or desired, and for example such a protected Rg group can be benzyloxy, t-butyloxy, lower alkoxy etc. Condensing agents such as dicyclohexylcarbodiimide or diphenylphosphoryl azide can be used. Activating agents such as 1-hydroxybenzotriazole can also be used in the reaction.

The resulting dipeptide IV is deprotected at the amine end by treatment with acids or by hydrogenation using e.g. hydrogen and a metal catalyst. The resulting product (III) is then condensed (according to method (b) as defined above) with a keto acid or keto ester (II) in a suitable solvent such as water or acetonitrile at a substantially neutral pH in the presence of a reducing agent such such as sodium cyanoborohydride or equivalent reducing agent. Alternatively, the Schiff base resulting from the initial condensation of II and III can be reduced (according to method (a) as defined above), e.g. by catalytic reduction to form I using hydrogen at a pressure of 1-4 atmospheres. The catalytic reduction can be carried out using Raney nickel catalyst or 10% palladium on carbon or the like. The compounds of formula I having a carboxyl end group can be prepared from a corresponding ester by hydrolysis or hydrogenolysis.

In the preceding reaction series, the substituents R 1 , B- 2' R 3 r R 4 1 R 5 and R 8 and p are as previously defined, G is a suitable protecting group, e.g. benzyloxycarbonyl or t-butyloxycarbonyl.

In the preceding reaction sequences in which water is formed from the reactants (e.g. the condensation of compounds II and III in method a), the reaction can be carried out by azotropic distillation of the water formed with suitable high-boiling solvents such as toluene or xylene. Alternatively, the reactions can be carried out in the presence of dehydrating agents such as molecular sieves or the like.

Various intermediates for use in the described methods and procedures can be obtained by directly following the procedures or analogous procedures described in European patent application 5486 2.

Furthermore, a number of the intermediate products for the preparation of the compound according to the invention are commercially available, or they can be easily prepared according to known methods. Intermediate products for the preparation of a significant number of the compounds according to the invention are described, or the preparation thereof is covered in publications and treatises relating to peptide chemistry, such as J.H. Jones, in "Comprehensive Organic Chemistry", Vol.2, D. Barton and W.D. Ollis, Publishers, Pergamon Press, 1979 pages 819-823 and references 2, and 29-31.

The following examples illustrate the preparation of the compounds according to the invention.

Example 1

N- In- [L-1-carboxy-2-(4-phenyl)-phenylethyl]-L-phenylalanyl]-Q-alanine benzyl ester

A. 4- Phenylphenylpyruvic acid

40 g of potassium t-butoxide was added to 150 ml of diethyl oxalate in small portions with stirring. After the initial exothermic reaction had subsided, the reaction mixture was heated on a steam bath under nitrogen to dissolve the solids

materials. After cooling to room temperature, 79 g of 4-biphenylacetic acid methyl ester was added in one portion. The mixture was stirred at 60-70°C for two hours while low boiling material was removed under vacuum. After cooling to room temperature, the viscous residue was stirred with 200 ml of ether and 350 ml of water while cooling. The ether layer was separated and extracted once with 100 ml of water. The aqueous layers were combined, extracted once with ether, acidified with concentrated HCl (cooling) and extracted with 2 x 300 mL ether. Some solid material did not dissolve in the ether layer and was filtered. The ether layer was then evaporated to dryness and the semi-solid residue was combined with the preceding solid materials. A mix of 160

ml of concentrated HCl and 350 ml of acetic acid were added to the solid/semi-solid residue mixture, and the resulting mixture was heated under reflux for 2.5 hours. After cooling to 50°C, a solid material precipitated which was filtered and washed with 150 ml of water. The wet solid was stirred with 150 mL of acetonitrile for 5 minutes, then filtered and dried under high vacuum at room temperature for 3 hours. 42.2 g of the title compound with melting point 215-218°C were obtained.

B. N-[N- pST-tert-butyloxycarbonyl]-L-phenylalanyl]-$-alanine benzyl ester

To a stirred mixture of 0.0 23 mol each of di-t-butyldicarbonate, L-phenylalanine, 8-alanine benzyl ester-p-toluene-sulfonate, hydroxybenzotriazole, N-dimethylaminopropyl-N<1->ethyl-carbodiimide hydrochloride in 75 ml of dry N,N-dimethylformamide in an ice bath was added 5 ml of N-ethylmorpholine, and the mixture was stirred at room temperature for 3 hours and then poured into 600 ml of ice water and extracted with 3 x 150 ml of ether. The ether layers were combined and extracted 1 time with 150 ml of 0.3N .HCl, then 2 times with 300 ml of water and were then dried over Na 2 SO 4 , filtered and evaporated to dryness at 28°C in vacuo. 9.0 g of a gummy solid residue was obtained.

C. N-(L-phenylalanyl)-g-alanine benzyl ester hydrochloride

4.0 g of the material from Example 1B in 25 ml of ethyl acetate at 0°C was stirred with gaseous HCl for 10 minutes. The mixture was stirred at 0°C for 1.5 hours and then at 10°C for 30 minutes. A stream of N 2 was passed through the solvent to drive off the excess HC 1 . The solution was poured into 200 mL of ether with vigorous stirring, and the precipitated solid was filtered and then dried at room temperature under high vacuum for 2 hours to give 3.35 g of product.

D. N-[N-[1-1-carboxy-2-(4-phenyl)-phenylethyl]-L-phenylalanyl]-B-alanine benzyl ester

A mixture of 9.10 g of N-(L-phenylalanyl)-8-alanine benzyl ester hydrochloride and 8.40 g of the compound from Example 1A in 500 ml of a mixture of tetrahydrofuran-ether (9:1) was treated with triethylamine to pH 6 ,6 and was stirred at room temperature for 1.5 hours. A solution of 2.0 g of sodium cyanoborohydride in a mixture of 100 ml of tetrahydrofurane ethanol was added dropwise over 2 hours with stirring. Stirring was continued at room temperature overnight. The reaction mixture was concentrated to 75 mL at 40°C in vacuo. The residue was stirred with 400 ml each of 0.5N HCl and ether for 1 hour. The ether layer was dried over sodium sulfate, filtered and evaporated to dryness in vacuo. A yellow viscous acid was obtained which was dissolved in 25 ml of ethanol. Solid material formed on standing overnight in a refrigerator and was filtered off, washed with cold ethanol (4.2 g) and chromatographed over a 300 g column of silica gel using a solvent mixture consisting of CHC13:CH3OH:CH3CO2H (200:10:2) . The eluate was divided into fractions which were evaporated to dryness in vacuo. In this way, 600 mg of a solid product consisting of the title compound with melting point 19 2-194°C was obtained.

Example 2

N-[N-[L-1-carboxy-2-(4-phenyl)-phenylethyl]-L-phenylalanyl]-3-alanine

A suspension of 590 mg of the product from Example 1 in 100 ml of ethanol was shaken overnight with 200 mg of 10% Pd/C in a Parr apparatus. The resulting mixture was diluted with 40 mL of ethanol and 10 mL of water and then heated briefly on a steam bath until all of the white precipitate had dissolved. After cooling, the catalyst was filtered off. The filtrate was evaporated at 50°C under vacuum to 5 ml. The solid was filtered, washed with ethanol and dried at room temperature under high vacuum for 6 hours to give 155 mg of the title compound, mp 226-228°C.

[ct]D26 = 16.8 (C = 0.5, DMF)

Analysis, calculated: C 70.40, H 6.13, N 6.08 Found: C 70.54, H 6.26, N 5.97

Example 3

N-[N-[L-[1-[pivaloyloxymethoxy]-carbonyl]-2-phenylethyl)]-L-phenylalanyl]-g-alanine, pvaloyloxymethyl ester

A. N-(tert-butyloxycarbonyl)-g-alanine, pivaloyloxymethyl ester

32.4 ml of triethylamine was added to 40 g of N-tert-butyloxycarbonyl-g-alanine in N,N<1->dimethylformamide at room temperature under a nitrogen atmosphere in a 1 liter round bottom flask. The solution was stirred for 15 minutes and 36.6 g of chloromethyl pivalate [m. Rasmussen & N.J. Leonard, J. Amer. Chem. Soc, 39, 5439

(1967)] was added dropwise at 0°C. The solution was stirred at room temperature overnight. The mixture was then diluted with ethyl acetate, filtered, washed with water and then brine and evaporated to give 70 g of crude material which was chromatographed (silica gel) and eluted with 15% ethyl acetate-hexane to give 62.5 g of product.

B. g-alanine, pivaloyloxymethyl ester, trifluoroacetate

100 ml of trifluoroacetic acid was added to a solution of 62 g of the product from Example 3A in 180 ml of methylene chloride at 0°C. The mixture was stirred at room temperature for 2 hours and the solvent was removed in vacuo to give 100 g of product as a pale yellow oil.

C. N-[1-1-(phenylmethoxy)-carbonyl-2-phenylethyl)]-L-phenylalanine

19 0.4 g (0.652 mol) of L-phenylalanine benzyl ester hydrochloride was suspended in 960 ml of absolute methanol, 6.7 l of dry (3A sieve) tetrahydrofuran was added and the slurry was stirred while approx. 50 ml of triethylamine was added to bring

pH to 6.5-7.0. pH was checked with EM Reagents ColorpHast indicator strips, range pH 5-10, moistened with water before use. To the neutral slurry was added 200 g (0.98 mol) of sodium phenylpyruvate hydrate (Sigma), followed by 240 g of crushed 3A molecular sieves. (The sieves can be crushed in a mortar and do not need to be finely pulverized. If these are too fine they are difficult to remove by filtration.) The slurry was stirred at ambient temperature while a solution of 61.6 g (0.98 mol) sodium cyanoborohydride in 40 ml of methanol plus 300 ml of dry tetrahydrofuran were added dropwise over 5 hours. The reaction mixture

was stirred at ambient temperature for 48 to 72 hours while the disappearance of benzyl ester was monitored by thin layer chromatography. The reaction mixture was filtered to remove sieves, the sieves were washed thoroughly with hot methanol as some product had precipitated on them.

The filtrate was concentrated on a rotary evaporator at 50°C to a syrup. This syrup was dissolved in 2.4 1 ether in a 12 l round bottom flask and was stirred in an ice bath while 2.4 1 2.5% aqueous HCl was added. The large volume of HCN that was evolved was passed through a sodium hydroxide trap. The mixture was stirred for 2.5 to 3 hours while a white solid gradually formed at the interface and evolution of HCN stopped. The 2-phase mixture was filtered (most of the aqueous phase can be removed in a separator before filtration as the product remains at the interface) and the solid material was washed thoroughly with ether and dried in vacuo below 50°C. Yield 80-90 g with melting point 175-180°C.

This material is 90-95% pure L,L isomer as determined by thin layer chromatography. The impure product was redissolved in approx. 10 L of boiling absolute methanol, some fine white inorganic insoluble material was filtered and the filtrate was concentrated to 5 L at the boiling point after which flocculating white crystals appeared. The product was allowed to cool slowly to room temperature and then to 0°C for 2-3 hours. The solid material was collected and dried in vacuo at 50°C. Yield 57-60 g, m.p. 185-186°C, [α]D26 5.9 to 6.3° <*> (DMSO, C=1). The product comprised more than 98% of the L,L isomer as determined by thin layer chromatography and high pressure liquid chromatography. <*> (result of several attempts). D. N-(L-1-benzyloxycarbonyl-2-phenylethyl)-L-phenylalanine,

pivaloyloxymethyl ester

A solution of 4.03 g (10 mmol) of the product from Example 3C and 1.5 ml (11 mmol) of triethylamine in 20 ml of DMF was treated at ambient temperature with 1.6 ml (11 mmol) of chloromethylpivalate, was stirred at 50 -60°C for 24 hours and the resulting slurry was poured into water and extracted with 3 x 100 ml of ether. Some insoluble material was filtered off and the ether phase was washed with water, dried and concentrated to an oil. Yield 4.8 g. NMR was consistent with the structure of the title compound.

E. N-(L-carboxy-2-phenylethyl)-L-phenylalanine, pivaloyloxy methyl ester

The crude diester from Example 3D (4.8 g) was hydrogenated in a Parr apparatus at 4.2 kg/cm 2 in 50 mL methanol plus 5 mL H 2 O in the presence of 0.4 g 10% Pd/C for 3 h . The resulting reaction mixture was filtered and concentrated to a moist solid which was recrystallized from methanol/H 2 O. White fluffy needles were filtered off and dried in vacuo. Yield 3.0 g with melting point 122-124°C. TLC showed mainly one spot, Rf 0.2, in CHCl3/MeOH/HOAc 100:1:0.0.5.

F. N-(L-1-pivaloyloxymethylcarbonyl-2-phenylethyl)-L-phenylalanine-g-alanine, pivaloyloxymethyl ester

A mixture of 1.0 g (2.3 mmol) of the product from Example 3E and 0.89 g (2.3 mmol) of the product from Example 3B in 25 mL DMF was treated with 1.01 mL (8 mmol) N -ethylmorpholine, followed by 352 mg (2.3 mmol) of 1-hydroxybenzotriazole hydrate and 439 mg (2.3 mmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and was stirred at ambient temperature overnight. Thin-layer chromatography showed that the starting material was still present. The reaction mixture was heated to 40-50°C for 6 hours and allowed to stand at room temperature overnight. The mixture was poured into water and extracted with several portions of ether, the ether phase was then washed thoroughly with water, dried and concentrated to an oil (1.2 g). The oil was chromatographed on silica gel 60-G from Merck of thin layer chromatography quality, yielding 0.9 g of oil showing a single spot, Rf 0.4 in EtOAc/Hexane 1:2 (same system as used for column chromatography). Analysis: Calculated for C33<H>44<N>2°9: C 64.69, H 7.24, N 4.57.

Found: C 64.47, H 7.20, N 4.29.

[a] D26 -21.7° (DMF, C = 1.0) .

Example 4

N-[N-[L-[1-[(2,2-dimethyl-1-oxopropoxy)-methoxy]-carbonyl]-2-(4-phenyl)-phenylethyl]-L-phenylalanylj-g-alanine A. N-Q},L-1-carboxy-2-(4-phenyl)-phenylethyl]-L-phenylalanine,

benzyl ester

A stirred suspension of 24 g of 4-phenylphenylpyruvic acid and 23.2 g of L-phenylalanine benzyl ester hydrochloride in 1 1 THF/ethanol (9:1) was brought to pH 6.6 by gradual addition of triethylamine. During this process all solid material was dissolved. After stirring the resulting solution for 2 hours at room temperature, a solution of 3.5 g of sodium cyanoborohydride in the same solvent was added dropwise with stirring. The reaction mixture was stirred at room temperature overnight.

The reaction mixture was then concentrated to 200 ml under reduced pressure. The residue was poured into 600 ml of 0.3N HCl while cooling and stirring. A gummy solid separated. The aqueous material was decanted off and the remaining solid material was stirred with 120 ml of ethanol.

The resulting solid was filtered and the wet solid was stirred with 100 ml of fresh ethanol. After standing overnight, the solid material was filtered and dried to form 22.3 g of solid product.

B. N- [p,L- [1- [(2, 2-dimethyl-1-oxopropoxy)-methoxy]-carbonyl] -

2-(4-phenyl)-phenylethyl]-L-phenylalanine, benzyl ester

3.05 ml of triethylamine was added to a solution of 9.6 g of N-[p,L-1-carboxy-2-(4-phenyl)-phenylethyl]-L-phenylalanine benzyl ester in 30 ml of dimethylformamide. The mixture was stirred at room temperature for 20 minutes and 3.15 ml of chloromethylpivalate was added. The resulting mixture was heated in a bath to 45-55°C

for 4 hours with stirring, and was then stirred at room temperature overnight. The resulting mixture was diluted with 300 mL of water and extracted with 3,150 mL portions of ether. The combined ether layers were extracted twice with 100 mL portions of water and the ether solution was dried over anhydrous Na 2 SO 4 . Filtration and evaporation in vacuo gave 7.5 g of a syrupy product.

This material was chromatographed on 135 g of silica gel eluting with a mixture of ethyl acetate-cyclohexane (85:15). Fractions containing the desired diastereomeric mixture of products were identified by thin layer chromatography, combined and evaporated to dryness in vacuo to give 5, 9 g of product as a syrup.

C. N- [D,L- [1 - |]( 2, 2-dimethyl-1 -oxopropoxy) -methoxy] -carbonyl] -

2-(4-phenyl)-phenylethyl]-L-phenylalanine

A solution of 5.9 g of the above product in 175 ml of ethanol was hydrogenated at 1.05-2.1 kg/cm over 750 mg of 10% Pd/C for 2 hours. The reaction mixture was diluted with an additional 250 ml of ethanol and was heated to 45°C to dissolve the precipitated product. The catalyst was filtered from the hot solution and the filtrate was evaporated to give a total of 4.4 g of product.

D. N-[n-[l-jj-[(2,2-dimethyl-1-oxopropoxy)-methoxy^-carbonyl]-2-(4-phenyl)-phenylethyl]-L-phenylalanyl]-3-alanine , benzyl ester

1.2 ml of N-ethylmorpholine was added to a stirred mixture of 3.25 g of the product obtained above, 1.7 g of N-(N,N-dimethylaminopropyl)-N<1->ethylcarbodiimide hydrochloride, 1.3 g of hydroxybenzotriazole and 3.0 g of 8-alanine benzyl ester p-toluenesulfonate in 25 ml of dimethylformamide. The mixture was stirred at room temperature for 3 hours, diluted with 200 ml of ice water and extracted into 2 125 ml portions of ether. The combined extracts were washed with 250 ml water and dried over anhydrous MgSO 4 . Filtration and evaporation gave 5.25 g of residue. Thin layer chromatograph in (silica gel, CHCl₂/EtOAc - 10:1) showed two major products, R₂ = 0.36 and R₂ = 0.32 (partially overlapping). This material was chromatographed on 350 g silica gel (thin layer chromatography quality), and was eluted with CHCl-j-EtOAc (100:5). Fractions containing the pure individual components were . identified by thin-layer chromatography, were combined and evaporated. In this way, 650 mg of the faster moving component (L,L-diastereoisomer) was obtained together with 590 mg of the L,D-diastereoisomer.

The final product was obtained by hydrogenating a solution of 650 mg of the L,L-diastereoisomer in 50 ml of ethanol over 50 mg of 10% Pd/C at 1.05-3.15 kg/cm<2> for 3 hours. The catalyst was filtered off and the filtrate was evaporated to dryness in vacuo at 30°C. The residue was chromatographed on 50 g thin layer chromatography quality silica gel and was first eluted with 300 ml CHCl3/EtOAc (10:1) and then with CHCl3/CH3OH/AcOH

(600:10:2). Fractions containing pure product were identified by thin layer chromatography (silica gel) CHCl3/CH3OH/AcOH (600:10:2), Rf = 0.31. These fractions were combined, evaporated and the residue was dried at room temperature under high vacuum overnight. The residue was recrystallized from ether and the product was dried at 45°C for 3.5 hours in high vacuum. 250 mg of solid material was obtained, m.p. 101-103°C, [a]D26 -22° (c = 0.5,

DMF).

Example 5

N-[N-[l-[1-(2,2-dimethyl-1-oxopropoxy)-methoxy]-carbonyl]-2-phenylethyl]-L-phenylalanyl]-g-alanine

A mixture of 0.9 g (2.1 mmol) N-(L-1-carboxy-2-phenylethyl)-L-phenylalanine, pivaloyloxymethyl ester (Example 3E) and equimolar amounts of 73 mg of g-alanine benzyl ester-p-toluene- sulfonate, 321 mg of 1-hydroxybenzotriazole hydrate and 401 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide HCl were dissolved in 20 ml of DMF containing 0.8 ml (6.3 mmol) of N-ethylmorpholine and the mixture was stirred at ambient temperature over the night.

The yellow solution was poured into 150 ml of water and extracted with 3 x 100 ml of ether. The ether phase was washed with water, dried and concentrated to 1.1 g of an oil which showed one major spot with minor contamination by thin layer chromatography.

The total product was chromatographed on 150 g of silica gel and was eluted with ethyl acetate/hexane (30:70). The fractions were combined and concentrated to an oil [α]D26 - 20.0° (DMF, c = 1).

1.5 g of the above described product was hydrogenated at 2.8 kg/cm<2> in 100 ml of absolute EtOH over 0.2 g of 10% Pd/C for 3 hours.

The catalyst was filtered and the filtrate was concentrated to an oil which was crystallized by drying under high vacuum overnight. On standing in hexane, fine colorless crystals were obtained which, on filtration and drying, gave 1.2 g of product m.p. 93-95°C, [α] D26 - 27.3° (DMF, c = 1).

By following the procedures described in the preceding description and examples, but using suitable intermediates, each of the previously specified compounds 1-63 can be prepared.

In the following table, the abbreviations CgH^CH2 and C^H^ indicate the benzyl groups and CgHj. indicates the phenyl group. By following the methods described above, the following compounds were prepared. Except where specifically stated, all compounds L have absolute stereochemistry at the chiral centers, and Rj- is equal to hydrogen.

Example 6

A. N-(L-1-(phenylmethoxycarbonyl)-2-phenylethyl)-L-phenylalanine,

cyanomethyl ester

A solution of 4.03 g (10 mmol) of the product from Example 3C and 2.1 ml (15 mmol) of triethylamine in 150 ml of acetone was treated dropwise at room temperature with 0.95 ml (15 mmol) of chloroacetonitrile. The resulting solution was heated to reflux overnight. It was then concentrated to dryness and partitioned between water and ether. The ether phase was washed with several portions of water, dried and concentrated to an oil. 4.5 g of the material was thus obtained, and was used directly in the next step.

B. N-(L-1-(phenylmethoxycarbonyl)-2-phenylethyl)-L-phenylalanine, 2,2-dimethyl-1,3-dioxolan-4-yl-methyl ester 4.4 g (10 mmol) of the product from previous step and 25 mL (20 mmol) of 2,2-dimethyl-1,3-dioxolan-4-yl-methanol (Aldrich Chemical Co.) were combined, then 1.4 mL (10 mmol) of triethylamine and 20 mg of N, N-dimethyl 1-4-aminopyridine was added. The resulting mixture was stirred under a nitrogen atmosphere at 55-60°C for 16 hours. The reaction mixture was then poured into water and extracted with several portions of ether. The combined ether extracts were washed with several portions of water, dried and concentrated to give 5.7 g of an oil. This product was chromatographed on 400 g of Merck silica gel 60G, and was eluted with ethyl acetate-hexane mixtures. Fractions shown to be pure by thin layer chromatography were combined and evaporated to dryness to give 4.2 g of oil,

La] <26> -6.2° (c 1.3, DMF) .

C. N-(L-1-(2,2-dimethyl-1,3-dioxolan-4-yl-methoxycarbonyl)-2-phenylethyl)-L-phenylalanine

The product from the previous step was dissolved in 50 ml of tetrahydrofuran and was hydrogenated in a Parr apparatus over 0.4 g of 10% palladium on carbon at room temperature and 4.2 kg/cm<2>

for 4 hours. The catalysts were filtered and the filtrate was evaporated in vacuo to give 3 g of a waxy residue which was crystallized from ethyl acetate to give 2.6 g of crystals of melting point 140-2°C.

D. N-(N-(L-(1-(2,2-dimethyl-1,3-dioxolan-4-yl)-methoxy)-carbonyl)-2-phenylethyl)-L-phenylalanyl)-g-alanine , benzyl ester

A mixture of 2.5 g (5.8 mmol) of the product from the previous step, 2.2 g (6.4 mmol) of g-alanine benzyl ester p-toluene sulfonate, 887 mg (5.8 mmol) of 1-hydroxybenzotriazole hydrate , 1.1 g (5.8 mmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 23 ml (18 mmol) of N-ethylmorpholine in 20 ml of dimethylformamide were stirred at room temperature overnight.

The resulting yellow solution was poured into water and extracted with several portions of ether. The combined ether extracts were washed repeatedly with water, dried over anhydrous potassium carbonate and concentrated to 3.3 g of an oil. This material was chromatographed on 300 g of Merck silica gel 60, and eluted with hexane-ethyl acetate (2:1). Fractions which by thin layer chromatography proved to be homogeneous (silica gel ^254' ethynyl acetate-nexane (1:1)) were combined and the solvent was evaporated to give 2.7 g of an oily product which was used directly in subsequent steps.

E. N-(N-(L-(1-(2,2-dimethyl-1,3-dioxolan-4-yl)-methoxy)-carbonyl)- 2- phenylethyl)- L- phenylalanyl)- g- alanine

The product from the previous step (2.2 g) was dissolved in

50 ml of absolute ethanol and hydrogenated in a Parr apparatus using 0.2 g of 10% palladium/carbon at 3.5 kg/cm 2 for 4 hours at room temperature. The catalyst was filtered and the filtrate was concentrated to an oil (1.9 g) which solidified. Recrystallization from ether at -80°C gave 1.8 g of solid material, m.p. 80-2°C, [α] 26 = -22.7° (c 1, DMF). Thin layer chromatograph i showed a single spot, R^= 0.35 (chloroform-methanol-acetic acid 500:25:2.5).

F. N-(N-(L-(1-(2,2-dimethyl-1,3-dioxolan-4-yl)-methoxy)-carbonyl)-2-phenylethyl)-L-phenylalanyl)-g-alanine , maleate

(1:1)

5.0 g of the product from the previous step was dissolved

in 200 ml of ether and was treated with a solution of 1.16 g of maleic acid in 200 ml of ether. Crystals were slowly formed on standing overnight at 5-10°C in a refrigerator. These were filtered

and dried to form 4.7 g of salt, m.p. 127-9°C, [α] ^ = -16.3° (c 1, DMF). Analysis calculated for c3 •] H38N2°i 1 : c 60.58, H 6.23, N 4.55. Found: C 60.33, H 6.31, N 4.48.

The compounds of structural formula I inhibit the activity of the enzymes designated as enkephalinases. The compounds are particularly useful for inhibiting enkephalinase A, which is derived from both rat and human striata. In in vitro tests, it was found that selected compounds of structural formula I in a concentration range from 10 -9 to 10 -6 M inhibited the activity of the above-mentioned enzyme by 50% or more.

The following test procedure was used to determine the enkephalinase A inhibition of the compounds of structural formula I.

Enkephalin (ENK) degrading activity was divided into

the subsequent three fractions according to the method of Gorenstein and Snyder, (Life Sci., 25, 2065 (1979): Enk'ase A

3 4 12

(Gly -Phe ), Aminopeptidase, (AP) (Tyr -Gly ), and Enk'ase B (Gly<2->Gly<3>)).

Enzyme activity was separated by taking brain tissue (minus cerebellum) from Sprague-Dawley rats and homogenizing it in 30 volumes of 50 mM Tris buffer, pH 7.4, using a Brinkmann Polytron. The resulting homogenate was centrifuged at 50,000 x g for 15 minutes. The pellet consisting of membrane-bound enzyme material was washed by resuspending it in tris and recentrifuging it 4 times.

After washing, solubilization of the membrane pellet was achieved by incubating it for 45 minutes at 37°C in the presence of 15 volumes (based on the original brain weight) of 50 mM tris-1% triton X-100 buffer, pH 7.4 . After centrifugation at 100,000 x g for 60 minutes to remove unsolubilized material, the triton-soluble supernatant was applied to a 1.5 x 30 cm DEAE Sephacel column pre-equilibrated with 50 mM tris-0.1% triton , pH 7.4. The material was eluted from the column using a 1 liter linear NaCl gradient from 0.0 to 0.4 M. The eluent was collected in 7 ml<1>s fractions where each was determined for enkephalin degrading activity. Under these conditions, Enk'ase A activity was found to be eluted between 120 and 200 ml, followed by AP activity (260 to 400 ml) and finally Enk'ase B activity between 420 and 450 ml.

Enkephalin-degrading activity was monitored using a radiometric assay. The substrate is 3 H-Met 5-ENK (50.1 Ci/mmol, New England Nuclear), diluted in 0.05 M Tris buffer, pH 7.4, so that the final reaction mixture concentration is 40 nM. The total reaction mixture volume including enzyme and substrate is 250 µl. The incubation was carried out for 90 minutes at 37°C. To stop the reaction, the tubes were transferred to a boiling water bath for 15 minutes.

The sample products were separated from each other using thin layer chromatography. A 4 µl aliquot of the reaction mixture was placed on a Baker-flex Silica Gel 1B plate (20 x 20 cm) along with unlabeled standards (Met <5->ENK, tyrosine, tyrosylglycine, tyrosylglycylglycine) and the components were co-chromatographed in a isopropanol:ethyl acetate:5% acetic acid solvent system (2:2:1) which was able to dissolve Met -ENK from its degradation products. The total test time was approx. 17 hours. The TLC vessels were flushed with nitrogen before the start of the experiment. After the experiment, the markers were clarified with ninhydrin spray. These spots, along with remaining plate regions, were cut from the plate and the radioactivity corresponding to each was monitored using liquid scintillation counting. IC^Q values were determined using the linear regression technique.

Using this procedure, the following nano-molar (nM) concentrations of the specified compounds were found to inhibit the action of enkephalinase A by 50% (IC^q).

The following test procedure was used to determine the properties of the indicated compounds in potentiating the analgesic action of (DAla 2 -Met 5 )-enkephalin amide (DAEAM). Background to the application of this procedure is provided in Chipkin, R.E., Iorio, L.C., Barnett, A., Berger, J., and Billard, W., Regulatory Peptides: From Molecular Biology to Function, edited by E. Costa and M. Trabucchi, Raven Press, New York, 1982, pp. 235-242.

Male CF1 mice (19-23 g) from Charles River Breeding Labs, Massachusetts, were used (N=10/dose or dose combination). Tail flick testing was performed similar to that described by Dewey and Harris, Methods in Narcotic Research, published by S. Ehrenpreis and A. Neidle, pages 101-109, Marcel Dekker, Inc., New York 1975 using a noxious irradiation heat stimulus.

After determining control latency times (typically 2-3 seconds), the mice were first injected (subcutaneously or orally) with either vehicle or drug, and after a suitable interval, injected intracerebroventricularly (icv) with either vehicle (10 µl saline) or DAEAM according to Haley and McCormick, Br., J. Pharmacol., 12, 12 (1957). Tail-flick latencies were re-determined 30 minutes later, having previously been determined to be the time of peak analgesic effect for DAEAM, where a cut-off time of 10 seconds was used.

Using this procedure, the following EDj-q values (the dose at which half of the test animals exhibited analgesic activity) were obtained for the selected compounds.

It should be noted that compounds A, B, C, F and H are ester derivatives, compound H is an ester derivative of compound G and compound L is an amide derivative.

Such derivatization has been found to contribute to

oral activity of the parent compound which exhibits poor absorp-

tion from the gastrointestinal tract. These derivatives which show no activity in vitro at 10 3 nM (see Table A) are bioactivated in vivo to deliver the parent compound (in vivo enkephalinase A inhibitors) to a site of action within the central nervous system (see Table B).

Claims (4)

1.. Analogy method for the preparation of therapeutically active compounds of the formula and pharmaceutically acceptable salts thereof, wherein is phenyl-lower alkyl or diphenyl-lower alkyl; R2 is hydroxy, -OCH2OCO-lower alkyl, the group wherein R7 and R8 are lower alkyl, or R2 is -O-lower alkyl-O-phenyl; 0-CH 2 -CHOH-CH 2 -OH, phenyl-lower alkoxy or amino-phenyl alkoxy; R 1 is benzyl or phenylbenzyl; R 4 is hydrogen, lower alkyl or hydroxy; R 1 is hydrogen; R 1 is hydroxy, phenyl-lower alkoxy, -OCH 2 OGO-lower alkyl, the group lower alkoxy or phenoxy-lower alkoxy; R 7 and R 8 are lower alkyl; p is 1; characterized in that a suitable method selected from the following methods is used in which p, R^, R2, R3' R4' R5 °9 R6 is as defined above, including suitable protection of any reactive group; (a) reduction of a compound of formula XX by The C=N double bond (b) linking an amino acid of formula VIII to a amino acid of formula V followed by removal of any protecting group if necessary, to form the desired compound of formula I, and, if desired, converting it to a salt thereof. 2. Process according to claim 1 for the production of N-[N-[L-[l-t(2,2-dimethyl-1,3-dioxolan-4-yl)-methoxy]-carbonyl]-2-phenylethyl]-L- phenylalanyl]-g<->alanine, characterized in that corresponding starting materials are used. 3. Process according to claim 1 for the preparation of N-[N-[L-Cl-[(2,2-dimethyl-1-oxopropoxy)-methoxy]-carbonyl]- 2-phenylethyl]-L-phenylalanyl]-g-alanine, characterized in that corresponding starting materials are used. 4. Process according to claim 1 for the production of N-[(L)-1-carboxy-2-phenylethyl]-L-phenylalanyl-L-phenylalanine, characterized in that corresponding starting materials are used.