NO850975L - RENIN INHIBITORS CONTAINING STATIN OR DERIVATIVES THEREOF - Google Patents

Abstract

Polypeptides of formula (I) and their salts are new. R-W-W'-NH-CHR2-CHR3-CH2-COR1 (I) W = Phe, His, Leu, Tyr, 1-naphthyl-Ala or 2-phenylmethyl 2-phenylmethylpropionylene; W' = Phe, His, Leu, Tyr or Nle; when W = Phe and W' = His, then the peptide link between W and W' may be replaced by -CH(1-4C)Alkyl-NH-; R = H, amino-protecting acyl of molecular wt. up to 500, or Pro or pGlu both amino protected; R2 = H, 1-6C alkyl, Ph, 4-7C cycloalkyl, Ph-(1-3C)alkyl or 5-10C cycloalkylalkyl; R3 = OH, NH2, NHR9, NHCOR9, OR9 or OCOR9; R9 = 1-4C alkyl; R1 = A-E-B; a gp. of formula (II); NH2; 1-4C alkoxy; 5-benzylipiperazin-1-yl; 1,2,3,4-tetrahydroquinolin-1-yl or -2-yl; 1,2,3,4-tetrahydro-3-aminocarbonyl isoquinolin-2-yl or the corresp. 3-methoxycarbonyl gp.; 1,2,3,4,5,6,7,8-decahydro-3-methoxycarbonyl isoquinolin-2-yl; 2-methoxycarbonylpyrrolidin -1-yl; 2-aminocarbonylpyrrolindin-1-yl; 4-phenylmethylpiperidin-1-yl; Pro-B; or Lys-B; -X-NH-CHZ-(CH2)n-Q-CHZ1-(CH2)m-Y (II) A = Lys or Pro or when R3 = NH2, Ile; E = Phe, Gly, Ala, Val, Leu, Ile, Lys, Orn, Arg, Asp opt. gamma-esterified or Glu opt. delta-esterified; B = OR4, NR4R5, glutamic acid (-OR4)2, glutamic acid (-OR4)(-NR4R5) or glutamic acid (-NR4R5)2; R4, R5 = H, 1-4C alkyl, Ph-(1-3C)alkyl or 5-10C cycloalkylalkyl; X = absent or Ala, Ile, Lys, Pro, Orn, Arg, N-(1-4C alkyl)- or N,N-(di-(1-4C) alkyl)-Lys or -Orn; Z, Z1 = H, 1-6C alkyl, 4-7C cycloalkyl, 5-10C cycloalkylalkyl or Ph-(1-3C)alkyl; n, m = 0-6; Q = CH2, CH=CH, O, NH, CH(OH) or CO; Y = Me, Ph, COOR6, CONR6R7, NH2, benzyloxycarbonylamino, CO-glutamic acid (-OR6)2, CO-glutamic acid (OR6)(NR6R7) or CO-glutamic acid (-NR6R7); R6, R7 = H, 1-4C alkyl, Ph-(1-3C)alkyl or 5-10C cycloalkylalkyl.

Description

The proteolytic enzyme renin, which has a molecular weight of

about. 40,000, produced in the kidneys and excreted in the blood. It is known to be active in vivo by cleaving the naturally occurring

upcoming plasma glycoprotein angiotensinogen, in the case of human angiotensinogen in the bond between leucine- (10.)

and the valine (11th) amino acid residue at the N-terminal end of the angiotensinogen:

The circulating N-terminal decapeptide (angiotensin I) which

formed by the above-mentioned splitting effect of renin, down-

is then broken down by the body into an octapeptide known as angiotensin II. Angiotensin II is known as a potent pressor-

substance, i.e. a substance capable of inducing a significant increase in blood pressure, and believed to act by causing constriction of the blood vessels and release of the sodium-retaining hormone aldosterone, from the adrenal cortex. The renin-angiotensinogenic system has therefore been seen as a contributing factor in certain forms of hypertension.

A way to ease the adverse effects of the impact

of the renin-angiotensinogen system, consists in the administration of a substance that can inhibit the angiotensinogen-splitting effect of renin. A number of such substances are known, including antirenin antibodies, pepstatin and naturally occurring phospholipids. European Patent Application No. 45,665 (published Feb. 2, 1982) discloses a series of renin-inhibiting polypeptide derivatives of the formula

where X can be hydrogen or an amino protecting group,

Y can be missing, B is a lipophilic amino acid residue, Z is an aromatic amino acid residue, W can be hydroxyl and A can i.a. be

wherein each of R 1 and R 2 is a lipophilic or aromatic side chain.

According to the definitions given in this published patent application, it is not included that A or Z can be statin or that B can be lycine.

European Patent Application No. 77,028 (published Apr. 20, 1983) discloses a series of renin-inhibiting polypeptide compounds having a non-terminal statin or statin derivative residue. These include compounds with a phenylalanine-histidine-statin sequence. However, the published patent application does not describe the placement of lycine immediately after the Phe-His-Sta sequence.

We have now discovered that certain new compounds are useful renin-inhibiting agents. This series of novel compounds consists of polypeptides and polypeptide derivatives of the formula

and the pharmaceutically acceptable salts thereof,

where

W is phenylalanine, histidine, leucine, tyrosine, 1-naphthyl-alanine or 2-phenylmethyl-propionylene;

W is phenylalanine, histidine, leucine, tyrosine or norleucine, where the peptide bond between W and W i is optionally replaced by -CH(alkyl with 1-4 carbon atoms)-NH- when W is phenylalanine and W 1 is histidine;

R is hydrogen, an amino-protecting acyl moiety with a molecular weight of less than 500, proline, amino-protected proline, pyroglutamic acid or amino-protected pyroglutamic acid;

R 2 is hydrogen, alkyl with 1-6 carbon atoms, phenyl, cycloalkyl with 4-7 carbon atoms, phenylalkyl with 7-9 carbon atoms or cycloalkyl (alkylene) with 5-10 carbon atoms;

R3 is hydroxyl, amino, -NHR<9>, -NHCOR<9>, -OR<9> or

9 9

-OCOR, where R is alkyl of 1-4 carbon atoms; and

R<1>'s

(a) -A-E-B,

where A is lysine or proline, or furthermore, only when R 3 is amino, isoleucine, E can be phenylalanine, glycine, alanine, valine, leucine, isoleucine, lysine, ornithine, arginine, asparagine-

acid, gamma-esterified aspartic acid, glutamic acid or

4 4 5 delta-esterified glutamic acid, B can be -OR , -NR R , 4 4 4 5

-glutamic acid(OR ) ~ , -glutamic acid(-OR )(NR R ) or

4 5 4 5

-glutamic acid (-NR R)2, and R and R can each be hydrogen, alkyl with 1-4 carbon atoms, phenylalkyl with 7-9 carbon atoms or cycloalkyl (alkyl) with 5-10 carbon atoms,

where X may be missing or be alanine, isoleucine, lysine, proline, ornithine, arginine, N-(alkyl of 1-4 carbon atoms)-lysine, N,N-di(alkyl of 1-4 carbon atoms)-lysine, N-( alkyl of 1-4 carbon atoms)-ornithine or N,N-di(alkyl of 1-4 carbon atoms)-ornithine, Z and Z in each are hydrogen, alkyl of 1-6 carbon atoms, cycloalkyl of 4-7 carbon atoms, cycloalkyl( alkylene) with 5-10 carbon atoms or phenylalkyl with 7-9 carbon atoms, n and m are both integers from 0 to 6, Q is

fi 6 7

Y is methyl, phenyl, -COOR , -CONR R , -NH~, (benzyloxy)-carbonylamino, -CO-glutamic acid(-OR 6)„, -CO-glutamic acid-

a cl ~i c "7 (OR )(-NR R ) or -CO-glutamic acid(-NR R )2, and R and R are each hydrogen, alkyl of 1-4 carbon atoms, phenylalkyl of 7-9 carbon atoms, or cycloalkyl( alkylene) with 5-10 carbon atoms,

(c) -NH 2 ,

(d) -Alkoxy having 1-4 carbon atoms,

(e) 4-benzylpiperazin-1-yl,

(f) 1,2,3,4-tetrahydroquinolin-1-yl,

(g) 1,2,3,4-tetrahydroisoquinolin-2-yl,

(h) 1,2,3,4-tetrahydro-3-aminocarbonylisoquinolin-2-yl, (i) 1,2,3,4-tetrahydro-3-methoxycarbonylisoquinolin-2-yl,

(j) 1,2,3,4,5,6,7,8-decahydro-3-methoxycarbonyl-isoquinolin-2-yl,

(k) 2-methoxycarbonyl-pyrrolidin-1-yl,

(1) 2-aminocarbonyl-pyrrolidin-1-yl,

(m) 4-phenylmethyl-piperidin-1-yl,

(n)-proline-B, where B is as above, or (o)-lysine-B, where B is as above.

Of particular interest are those compounds where R is an amino-protecting acyl moiety or amino-protected proline, W is phenylalanine, W i is histidine linked to W through a peptide-2 3

bond, R is isobutyl or cyclohexyl(methylene), R is hydroxy and R i is (a), (b) or (n), as defined above, A being lysine when R 1 is (a) and X being lysine or proline when R1 is (b) .

The compounds produced according to the invention show antihypertensive effects in in vivo tests in mammals, including humans. This is largely due to their ability to inhibit renin's cleavage of angiotensinogen. The mechanism of this renin-inhibitory effect of the compounds of formula I may, for example, depend on selective binding (compared to angiotensinogen) to renin. As a result of their low molecular weight, the compounds I have advantageous solubility conditions in aqueous media. This enables oral administration and synthesis at reasonable economic costs. The compounds produced according to the invention can also be used as diuretics.

"Pharmaceutically acceptable" salts here mean salts which are non-toxic in the given dosages. As the compounds of formula I can contain both basic and acidic groups, both acid addition and base addition salts are possible. Pharmaceutically acceptable acid addition salts include e.g. hydrochlorides, hydrobromides, hydroiodides, sulfates, bisulfates, phosphates, acid phosphates, acetates, lactates, maleates, mesylates, fumarates, citrates, acid citrates, tartrates, bitartrates, succinates, gluconates and saccharates. Pharmaceutically acceptable base addition salts include e.g. sodium, potassium, calcium and magnesium salts. Conventional methods for forming the acid addition and base addition salts can be used.

The group R at the N-terminal end of formula I, which is attached to the alpha-nitrogen of the residue W, is selected from hydrogen, amino-protecting acyl groups of molecular weight less than 500, proline, amino-protected proline, pyroglutamic acid and amino- protected pyroglutamic acid. The amino-protecting group of amino-protected proline or amino-protected pyroglutamic acid is also an amino-protecting acyl moiety with a molecular weight of less than 500. The term "amino-protecting acyl moiety" stands for acyl groups that can provide a significant in vivo inhibition of the reaction in the alpha nitrogen of W (or proline or pyroglutamic acid) after oral administration. R has a molecular weight of less than 500 to prevent unfavorable solubility conditions. Examples of suitable amino-protecting acyl moieties are well known, thus e.g. mention is made of t-butyloxycarbonyl, t-butylacetyl, benzyloxycarbonyl, t-butylureido, (tris-hydroxy)-(t-butylureido) and phenoxyacetyl groups. The group R is preferably of the type

where M is -O-, -CH2", -NH- or -SO2NH- and

R^ is alkyl with 1-6 carbon atoms, phenyl, phenylalkyl with 7-9 carbon atoms or cycloalkyl (alkylene) with 5-10 carbon atoms.

4 4 5

The expressions glutamic acid-(OR )(-NR R ) and -CO-glutamine-

6 6 7 acid(-OR )(NR R ) refers here to both C-terminal groups that are amidated in the delta carbon of glutamic acid and C-terminal groups that are esterified in the delta carbon of glutamic acid. As previously stated, a preferred group of compounds of formula I consists of those where R 3 is hydroxyl and R 2 is isobutyl or cyclohexyl (methylene), preferably the latter, so that

becomes -statin- or -cyclostatin-, R is an amino-protected acyl moiety of molecular weight less than 500 or amino-protected proline, W is phenylalanine, W1 is histidine linked to W through a peptide bond and R^ is -Lys-E-B, -Pro-B, or -(Light or

The placement of a lysine residue (instead of, for example, a leucine, isoleucine, valine or alanine residue) immediately after -phenylalanine-histidine-(statin or cyclostatin)- gives a marked and surprising increase in the duration of the renin-inhibitory effect in vivo . Another preferred group of compounds of formula I consists of compounds where R is an amino-protected acyl moiety with a molecular weight of less than 500 or amino-protected proline, W is phenylalanine, W 1 is histidine bound to W through a peptide bond, R 3 is hydroxyl, R 2 is isobutyl or cyclohexyl(methylene), preferably the latter, and R i is

Reduction of the peptide character of the molecular structure closest to the C-terminus appears to improve absorption into the bloodstream after oral administration.

It should be noted that when n and m are both zero, Z is

OH

isobutyl, Q is -C' H-, Z 2 is hydrogen and Y is carboxyl, becomes R<1>-X-statin.

Particularly valuable are the following compounds and their pharmaceutically acceptable salts: [N-(t-butyloxycarbonyl)-proline]-phenylalanine-histidine-cyclostatin-lysine-phenylalanine;

[N-(t-butyloxycarbonyl)-phenylalanine]-histidine-cyclostatin-lysine-phenylalanine;

[N-(t-butyloxycarbonyl)-phenylalanine]-histidine-statin-lysine-phenylalanine;

[N-(t-butyloxycarbonyl)-phenylalanine]-histidine-statin-alanine-statin-glutamic acid;

[N-(t-butyloxycarbonyl)-phenylalanine]-histidine-statin-isoleucine-[amino(n-butyric acid)];

[N-(t-butyloxycarbonyl)-phenylalanine]-histidine-cyclostatin-[amino(2-sec-butyl-ethylene)]-phenylalanine;

[N-(t-butyloxycarbonyl)phenylalanine]-histidine-statin-[amino(2-sec-butyl-ethylene)]-phenylalanine;

[N-(t-butyloxycarbonyl)-phenylalanine]-histidine-cyclostatin-1,2,3,4-tetrahydro-2-isoquinoline;

[N-(t-butyloxycarbonyl)-proline]-phenylalanine-histidine-cyclostatin-1,2,3,4-tetrahydro-2-isoquinoline;

[N-(t-butyloxycarbonyl)-phenylalanine]-histidine-cyclostatin-proline-[amino(n-pentylene)amine];

[N-(t-butyloxycarbonyl)-phenylalanine]-histidine-cyclostatin-proline-methyl ester;

[N-(t-butyloxycarbonyl)-phenylalanine]-histidine cyclostatin ethyl ester;

[N-(t-butyloxycarbonyl)-phenylalanine]-histidine-cyclostatin-proline-NH2;

[N(t-butyloxycarbonyl)-phenylalanine]-histidine-cyclostatin-Nt^;°^

[N(t-butyloxycarbonyl)-phenylalanine]-histidine-cyclostatin-lysine-statin.

The compounds of formula I can be prepared according to well-known methods. The basic part of the preferred synthesis consists in the acylation of the unprotected alpha-amino group of an amino acid residue with an amino acid having an activated (for the purpose of acylation) carboxyl function and an appropriate protecting group attached to its own alpha-nitrogen to form a peptide bond between the two amino acid residues, after which the protecting group is removed. This synthesis part, consisting of a coupling-deblocking, is carried out repeatedly to build up the polypeptide by starting from the C-terminus of the molecular structure and working its way to the N-terminus. Alpha-amino acids for the synthesis of compounds of formula I are commercially available (as free acids, salts, esters, etc.) both in alpha-amino-protected and alpha-amino-unprotected form. Statin is commercially available as N-(t-butyloxycarbonyl)-statin and can also be prepared (as free acid or ester) both in the gamma-amino-protected and gamma-amino-unprotected form according to methods described in the literature (see e.g. . US Patent 4,397,786 and Rich, D. H. et al., Jour, Org. Chem., 43, p. 3624 (1978). If desired, an appropriate N-unprotected amino acid analog (free acid, salt, ester, etc. .)

such as 4-aminobutyric acid, 4-amino-valeric acid or 4-amino-

4-sec-butyl-butyric acid as reactant in the first coupling step. Suitable derivatives of statin can be prepared by conventional synthetic methods. Thus, for example, the compound here designated aminostatin can be prepared in a form where both amino groups are protected, by reacting an amino-protected statin ester with a sulfonyl chloride so that a sulfonate ester is formed, which is reacted with sodium azide to an amino-protected 4- isobutyl-2-butenoic acid ester and an amino-protected 4-isobutyl-3-azidobutyric acid ester, whereupon the alkene compound is reacted with a primary amine and/or the azido compound is hydrogenated and the resulting intermediate is reduced with an alkali metal hydride in the presence of an aldehyde, under formation (in both cases) of a 4-isobutyl-3-sec-amino derivative of butyric acid, which is then hydrogenated and then reacted with acyl halide under basic conditions. N-protected compounds

which are referred to herein as N-protected cyclostatin compounds, can be prepared by hydrogenation of the corresponding N-protected-4-amino-4-phenylmethyl-3-hydroxybutyric acid compound, which itself can be prepared as described by Rich, D.H. et al., Jour. With. Chem., 23(1) pp. 27-33 (1980).

The reduced peptide1 bond (-C0- replaced by -CH-z-)

in new connection where R is

n is at least 1 and Q is -NH-, can be obtained by reducing an aldehyde with the formula

protective in the presence of an amino acid ester of formula

where R is different from hydrogen.

The action of the new compounds, as inhibitors of renin's angiotensinogen-splitting action, can be studied through (1) their ability to inhibit renin's angiotensinogen-splitting action in vitro and (2) their ability to antagonize exogenous renin-induced pressor- response in vivo.

The compounds produced according to the present invention can be given as antihypertensive agents, either orally or parenterally, for convenience reasons for the patient, preferably orally. These antihypertensive compounds are normally given in doses of 0.1-10 mg/kg body weight/day with certain variations depending on the patient's condition and which compound is given. It is started with a low daily dose which is increased if the attending physician deems it necessary. The compounds can, for both specified administration methods, be given in combination with pharmaceutically acceptable carriers, in a single or repeated treatment.

The new compounds can be given orally in many different dosage forms, i.e. they can be formulated with different pharmaceutically acceptable, inert carriers in the form of tablets, capsules, lozenges, drops, powders, sprays, aqueous suspensions, mixtures, syrups and the like. The carriers include solid diluents or fillers, sterile, aqueous media, various non-toxic, organic solvents, etc. The preparations may also have added sweeteners and/or flavoring agents of the commonly used type. The oral dosage forms generally contain the new compounds in concentrations of 0.5-90 percent by weight, in amounts sufficient to provide the desired unit dose.

For oral administration, tablets can be used containing different excipients, such as sodium citrate, calcium carbonate and calcium phosphate, together with different disintegrants, such as starch, preferably potato or tapioca starch, alginic acid and certain complex silicates, together with binding agents, such as polyvinylpyrrolidone, sucrose , gelatin and gum arabic. Smoothing agents such as magnesium stearate, sodium lauryl sulfate and talc are also often beneficial during tablet production. Solid compositions of a similar type can also be used as contents in soft and hard gelatin capsules. Preferred materials in this regard will also include lactose or milk sugar, as well as high molecular weight polyethylene glycols. In aqueous suspensions and/or mixtures for oral administration, the active substance can be combined with various sweeteners or flavoring agents, natural or artificial coloring agents and possibly with emulsifying and/or suspending agents, as well as with diluents such as water, ethanol, propylene glycol, glycerol and various combinations of these.

The following examples illustrate the invention.

Example 1

[ N-( t -butyloxycarbonyl)- Phe]- His- Sta- Lys- Phe

A. [N-alpha-(t-butyloxycarbonyl)-N-epsilon-benzyloxy-carbonyl-lysine]- phenylalanine benzyl ester

N-hydroxybenzotriazole (162 mg, 1.2 mmol), N-methyl-morpholine (101.2 mg, 1 mmol), L-phenylalanine benzyl ester p-toluenesulfonate (428 mg, 1 mmol), N-alpha-( t-butyloxycarbonyl)-N-epsilon-benzyloxycarbonyl-L-lysine (456 mg, 1.2 mmol) and 1-cyclohexyl-3-(2-morpholineethyl)-carbodiimide-metho-p-toluenesulfonate (635 mg, 80% pure , 1.2 mmol), was sequentially dissolved in methylene chloride (50 mL) at 0°C and the resulting solution stirred for 19 hours at 20°C. The reaction mixture was then washed successively with 75 mL of 5.5% aqueous HCl, 75 mL of saturated aqueous NaHCO 3 , and 75 mL of brine and dried over anhydrous MgSO 4 . After filtration and evaporation, 669 mg of crude product was obtained as a foam (H-NMR, CDCl 3 , 1.5 delta, 9H s [BOC]). The crude product was used without further purification in the next step.

B. (N-epsilon-benzyloxycarbonyl-lysine)-phenylalanine benzyl ester hydrochloride

[N-alpha-(t-butyloxycarbonyl)-N-epsilon-benzyloxy-carbonyl-lysine]-phenylalanine benzyl ester from Step A (650 mg,

1 mmol) was dissolved in 7 ml of 3.7N HCl/dioxane and set aside

for 1 hour at 20°C. The solution was then evaporated to dryness, yielding 583 mg of crude product as an oil which was used without purification in the next step (1H-NMR, CD₂OD, 5.2 delta, 2H s [benzyl CH₂₄).

C. [N-(t-Butyloxycarbonyl)-statin]-(N-epsilon-benzyl-oxycarbonyl- lysine)- phenylalanine- benzyl- ester

(N-epsilon-benzyloxycarbonyl-lysine)-phenylalanine benzyl ester hydrochloride from Step B (583 mg, 1 mmol), N-methyl-morpholine (101.2 mg, 1 mmol), N-(t-butyloxycarbonyl)-statin (330 mg, 1.2 mmol), N-hydroxybenzotriazole (162 mg, 1.2 mmol) and 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide-metho-p-toluenesulfonate (635 mg, 80% pure, 1.2 mmol), was successively dissolved in 50 ml of methylene chloride at 0°C and the resulting solution stirred for 19 hours at 20°C. The reaction mixture was worked up as in step A to 760 mg of crude product which was used without further purification in the next step. (1H-NMR, CDCl-3, 1.5 delta, 9H s [BOC]).

D. Statin-(N-epsilon-benzyloxycarbonyl-lysine)-phenylalanine benzyl ester hydrochloride

[N-(t-butyloxycarbonyl)-statin]-(N-epsilon-benzyloxy-carbonyl-lysine)-phenylalanine benzyl ester from Step C (760 mg,

1 mmol) was dissolved in 10 ml of 3.7N HCl/dioxane and set aside

for 1 hour at 20°C. The reaction mixture was worked up as in step B to 620 mg of crude product in the form of a foam (<1>H-NMR, CD3OD, 5.2 delta, 2H s [benzyl CH2]), which was used without purification in the next step.

E. [N-alpha-(t-butyloxycarbonyl)-N-im-(t-butyloxycarbonyl)-histidine]-statin-(N-epsilon-benzyloxycarbonyl-lysine)-phenylalanine benzyl ester

Statin-(N-epsilon-benzyloxycarbonyl-lysine)-phenylalanine-benzylester hydrochloride from Step D (600 mg, 0.857 mmol), N-methyl-morpholine (86.7 mg, 0.857 mmol), N-alpha-(t- butyloxycarbonyl)-N-im-(t-butyloxycarbonyl)-L-histidine (365 mg,

1.03 mmol), N-hydroxybenzotriazole (135 mg, 1.03 mmol) and 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide-metho-p-toluenesulfonate (545 mg, 1.03 mmol), were order dissolved in 50 ml of methylene chloride at 0°C and stirred for 19 hours at 20°C. The reaction mixture was worked up as in step A to 770 mg of crude product in the form of a foam (H-NMR, CDCl3, 1.5 delta, 2H s [BOC] and 1.6 delta, 9H s [BOC]), which was used in the next step without further purification.

F. Histidine-statin-(N-epsilon-benzyloxycarbonyl-lysine)-phenylalanine- benzyl ester- dihydrochloride

[N-alpha-(t-butyloxycarbonyl)-N-im-(t-butyloxycarbonyl)-histidine]-statin-(N-epsilon-benzyloxycarbonyl-lysine)-phenylalanine benzyl ester from Step E (770 mg, 0.85 mmol ) was dissolved in 10 ml of 3.7NHCl/dioxane and set aside for 1.5 hours at 20°C. The reaction mixture was worked up as in step B to 602 mg of crude product in the form of a foam (<1>H-NMR, CD3OD, 5.2 delta, 2H s [benzyl CH,,]), which was used in the next step without purification .

G. [N-(t-butyloxycarbonyl)-phenylalanine]-histidine-statin-( N- epsilon- benzyloxycarbonyl- lysine)- phenylalanine- benzyl ester

Histidine-statin-(N-epsilon-benzyloxycarbonyl-lysine)-phenylalanine-benzylester-dihydrochloride from Step F (602 mg, 0.689 mmol), N-methyl-morpholine (139 mg, 1.38 mmol), N-(t- butyloxycarbonyl)-L-phenylalanine (219 mg, 0.827 mmol), N-hydroxybenzotriazole (112 mg, 0.827 mmol) and 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide-metho-p-toluenesulfonate (438 mg, 0.827 mmol) ) were successively dissolved in 50 ml of methylene chloride at 0°C and the resulting solution stirred for 19 hours at 20°C. The reaction mixture was worked up as in step A to 555 mg of a foam which was purified by chromatography (silica gel, 95:5 CH2Cl2/MeOH) and gave 136 mg of purified product in the form of a foam (<1>H-NMR, CDCl3, 1 .5 delta, 9H s [BOC]).

H. Title connection

[N-(t-butyloxycarbonyl)-phenylalanine]-histidine-statin-(N-epsilon-benzyloxycarbonyl-lysine)-phenylalanine benzyl ester

(136 mg, 0.17 mmol) and 70 mg of 20%Pd(OH)2/C catalyst were added in the indicated order to 15 mL of methanol, whereupon the resulting mixture was hydrogenated for 4 h at 3.5 kg/cm ^ H2 and 20°C. The reaction mixture was then filtered through Super-Cel and evaporated to dryness to give 76 mg of a glassy product which was triturated with ether to give 63 mg of purified [N-(t-butyloxycarbonyl)-phenylalanine]-histidine-statin-lysine-phenylalanine as a powder ( iH-NMR, CD^OD,

1.5 delta, 9H s [BOC]).

Example . 2

[ N-( t- butyloxycarbonyl)- Phe]- His- Sta- Ile- Sta ( sodium salt)

A. [N-(t-Butyloxycarbonyl)-isoleucine]-statin ethyl ester

Statin ethyl ester hydrochloride (717 mg, 3 mmol), N-methyl-morpholine (304 mg, 3 mmol), N-(t-butyloxycarbonyl)-L-isoleucine 1/2 H2O (865 mg, 3.6 mmol) , N-hydroxybenzotriazole (486 mg, 3.6 mmol) and 1-cyclohexyl-3-(2-morpholinoethyl)-carbo-diimide-metho-p-toluenesulfonate (1.91 g, 80% pure, 3.6 mmol) was sequentially added to 100 ml of methylene chloride at 0°C and the resulting solution stirred for 19 hours at 20°C. The reaction mixture was then washed successively with 75 mL of 5.5% aqueous HCl, 75 mL of saturated aqueous NaHCO 3 , and 75 mL of brine and dried over anhydrous MgSO 4 . After filtration and evaporation, 1.39 g of crude product was obtained as a foam (H-NMR, CDCl 3 . 1.5 delta, 9H s [BOC]), which was used without further purification in the next step.

B. to G. [N-(t-butyloxycarbonyl)-phenylalanine]-histidine-statin- isoleucine-statin- ethyl ester

In a similar manner as described in Steps B to G of Example 1, [N-(t-butyloxycarbonyl)-isoleucine]-statin ethyl ester from Step A (1.37 g) was converted to the purified title product (121 mg, foam, <1>H-NMR, CDCl3, 1.5 delta, 9H s [BOC]). 95:5 CHCl3/MeOH was used as gradient in the chromatographic purification during step G.

H. Title connection

A solution of [N-(t-butyloxycarbonyl)-phenylalanine-histidine-statin-isoleucine-statin-ethyl ester from Step G (120 mg, 0.14 mmol) in 2 mL of dimethoxyethane was treated with 0.17 mL of 1N aqueous NaOH . After stirring for 2.5 hours at 20°C, a new 0.17 ml of 1N aqueous NaOH was added. After stirring for an additional 2 hours, the reaction mixture was treated in a rotary evaporator to remove dimethoxyethane, diluted with 5 mL of H 2 O and, after adjustment to pH 7.8, freeze-dried. The freeze-dried residue was slurried in ether. The supernatant was evaporated to dryness to give 88 mg of a foam which, after trituration with a small volume of ether, gave purified [N-(t-butyloxycarbonyl)-phenylalanine]-histidine-statin-isoleucine-statin-(sodium salt) as a yellowish powder (65 mg, H-NMR, CDC13,

1.5 delta, 9H s [BOC]).

Example 3

[N-(t-butyloxycarbonyl)-Phe]-His-Sta-Ile- [amino-(n-butyric acid)]

A. [N-(t-butyloxycarbonyl)-Isoleucine] - [ajnino (n-butyric acid) ] - benzyl ester

N-(t-butyloxycarbonyl)-L-isoleucine (2.4 g, 10 mmol), N-methyl-morpholine (1.0 g, 10 mmol), benzyl 4-aminobutyrate hydrochloride (1.93 g, 10 mmol), N-hydroxybenzotriazole (1.35 g, 10 mmol) and 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide-metho-p-toluenesulfonate (4.23 g, 80% pure, 10 mmol) were sequentially dissolved in 60 ml of methylene chloride at 0°C and the resulting solution stirred for 19 hours at 20°C. The reaction mixture was then evaporated to dryness and the residue dissolved in ethyl acetate. The solution was washed twice with 50 mL of 5% aqueous HCl, twice with 50 mL of 1N aqueous NaOH, once with H 2 O and

once with brine, dried over anhydrous MgSO 4 , and filtered and concentrated in a rotary evaporator to 2.87 g of crude product as a foam (<i>H-NMR, CDCl 3 , 1.5 delta, 9H s [BOC]), which was used in the next step without further purification.

B. Isoleucine-[amino(n-butyric acid)]- benzyl ester hydrochloride

[N-(t-Butyloxycarbonyl)-isoleucine]-[amino(n-butyric acid)]-benzyl ester from Step A (2.87 g, 7.1 mmol) was dissolved in 10 mL of 3.7N HCl/dioxane and the resulting solution set

aside for 1 hour at 20°C. The reaction solution was then evaporated to dryness, whereby after trituration, 2.4 g of crude product was obtained in the form of a white solid (H-NMR, CD3OD, 5.2 delta, 2H s [benzyl CH2]), which was used without purification in the next step.

C. [N-(t-butyloxycarbonyl)-statin]-isoleucine-[amino-(n-butyric acid)] benzyl ester

N-(t-butyloxycarbonylstatin (275.35 mg, 1 mmol), N-methylmorpholine (101 mg, 1.0 mmol), isoleucine-[amino(n-butyric acid)]benzyl ester hydrochloride from Step B ( 342 mg, 1 mmol), N-hydroxybenzotriazole (135 mg, 1.0 mmol) and dicyclohexylcarbodiimide (206 mg, 1.0 mmol) were sequentially added to 20 mL of methylene chloride at 0°C and the resulting solution stirred for 19 h at 20°C. The reaction solution was then filtered and evaporated to a residue which was slurried in ethyl acetate. The slurry was filtered and the filtrate evaporated to dryness. After chromatography of the resulting residue (silica gel, CHCl^/EtOAc gradient), 464 mg of purified product obtained (<1>H-NMR, CDCl3, 1.5 delta, 9H s [BOC]).

D. Statin-isoleucine-[amino(n-butyric acid) 3-benzyl ester hydrochloride

In a similar manner as described in step B, the protecting group was removed (deblocking) from [N-(t-butyloxycarbonyl)-statin]-isoleucine-[(amino(n-butyric acid)]-benzyl ester from step C (464 mg, 0, 82 mmol), whereby 416 mg of crude product in the form of a white solid was obtained. This was used without purification in the next step (iH-NMR, CD3OD,

5.2 delta, 2H s [benzyl CH2]).

E. [N-alpha-(t-butyloxycarbonyl)-N-im-(t-butyloxycarbonyl)-histidine]-statin-isoleucine-[amino(n-butyric acid)]-benzyl ester

In a similar manner as described in Step C, the statin isoleucine [amino(n-butyric acid)]benzyl ester hydrochloride from Step D (416 mg, 0.83 mmol) was coupled with N-alpha-(t-butyloxycarbonyl)-N -im-(t-butyloxycarbonyl)-L-histidine (295 mg,

0.82 mmol) to 391 mg of purified product (1 H-NMR, CDCl 3 , 1.5 delta, 9H s [BOC] and 1.6 delta, 9H s [BOC]).

F. Histidine-statin-isoleucine-[amino(n-butyric acid)]-benzyl ester dihydrochloride

In a similar manner as described in step B, [N-alpha-(t-butyloxycarbonyl)-N-im-(t-butyloxycarbonyl)-histidine]-statin-isoleucine-[amino(n-butyric acid)]benzyl ester from step E (391 mg, 0.49 mmol) deblocked, whereby 328 mg of crude product as a white solid (<1>H-NMR, CD3OD, 5.2 delta, 2H s [benzyl CH2]) was obtained. This was used without purification in the next step.

G. [N-(t-butyloxycarbonyl)-phenylalanine]-histidine-statin-isoleucine-[amino(n-butyric acid)] benzyl ester

Histidine-statin-isoleucine-[amino(n-butyric acid)]-benzyl ester dihydrochloride from Step F (328 mg, 0.487 mmol), N-methyl-morpholine (0.128 mL, 1.17 mmol), N-(t-butyloxycarbonyl )-L-phenylalanine (155 mg, 0.585 mmol), N-hydroxybenzotriazole (79 mg, 0.585 mmol) and dicyclohexylcarbodiimide (121 mg, 0.585 mmol) were sequentially dissolved in 20 mL of methylene chloride at 0°C and the resulting solution stirred in 19 hours at 20°C. The reaction mixture was then worked up and chromatographed as in step C, yielding 324 mg of purified product (1 H-NMR, CDCl 3 , 1.5 delta, 9H s [BOC]).

H. Title connection

A solution of [N-(t-butyloxycarbonyl)-phenylalanine]-histidine-statin-isoleucine-[amino(n-butyric acid)]-benzyl ester from Step G (324 mg, 0.38 mmol) in 5 mL of methanol was hydrogenated at room temperature below 3.5 kg/cm 2 H2 in 1 hour with 20% Pd(0H)2/C catalyst. The reaction mixture was then filtered to remove the catalyst and the filtrate evaporated to dryness. After trituration of the resulting residue with ether, 190 mg of pure [N-(t-butyloxycarbonyl)-phenylalanine]-histidine-statin-isoleucine[amino(n-butyric acid)] was obtained as a white powder (<1>H- NMR, CDCl 3 , 1.5 delta, 9H s [BOC]).

Example 4

[N-(t-butyloxycarbonyl)-Phe]-His-Sta-[amino(4-sec-butyl-butyric acid) 3

A. N-(t-butyloxycarbonyl)-isoleucinate

A solution of N-(t-butyloxycarbonyl)-L-isoleucine methyl ester (10.0 g, 40.7 mmol) in 100 mL of toluene was cooled to -75°C and then treated dropwise with 102 mL of 1M diisobutylaluminum hydride in toluene , so that the reaction temperature was kept lower than -65°C. After the addition was finished, the mixture was stirred for 10 minutes at -75°C and the reaction was slowly stopped by the addition of 10 ml of methanol, during which the temperature was kept lower than -65°C. The reaction solution was then poured into 200 ml of cold saturated aqueous Rochelle salt solution. The resulting mixture was layered with ether and stirred for 1 hour, after which the organic layer was separated, washed with brine, dried over anhydrous Na^O^, filtered, and evaporated to dryness to yield 7.0 g of crude product (^H-NMR, CDCl3 , 1.5 delta, 9H s [BOC]), which was used in the next step without purification.

B. Ethyl-L-(4-amino-4-sec.-butyl-butyrate)-hydrochloride

N-(t-Butyloxycarbonyl)-isoleucinate from Step A (1 g,

4.6 mmol) and carbethoxymethylenetriphenylphosphorane (1.93 g,

5.56 mmol), was dissolved in 50 ml of chloroform and the resulting solution set aside for 48 hours at 20°C. The reaction mixture was then evaporated to dryness and purified by chromatography (silica gel, CHCl 2 ) to give 1 g of purified ethyl L-[4-(t-butyloxycarbonylamino)-4-sec-butyl-delta-2-butyrate 3 -as a oil (1 H-NMR, CDCl 3 , 4.2 delta, 2H s [ethyl CH 2 3 ). Ethyl L-[4-(t-butyloxycarbonylamino)-4-sec-butyl-delta-2-butyrate] (300 mg, 1.05 mmol) was then hydrogenated for ca. 2 hours at 3.5 kg/cm^ H2 and 20°C in methanol containing a 20% Pd(OH)2/C catalyst. The reaction mixture was then filtered to remove the catalyst and the filtrate concentrated in a rotary evaporator to 300 mg of crude ethyl L-[4-(t-butyloxycarbonylamino)-4-sec-butyl-butyrate] in the form

of an oil (1 H-NMR, CDCl 3 , 4.2 delta, 2H s [ethyl CH 2 3 ). Ethyl L-[4-(t-butyloxycarbonylamino)-4-sec-butyl-butyrate]

(300 mg) was then dissolved in 4 ml of 3.7N HCl/dioxane and

the resulting solution stirred for 1 hour at 20°C. The reaction mixture was then evaporated to dryness

231 mg of crude product in the form of a foam, which was used without<0>purification in the next step (1H-NMR, CDCl 3 , 4.2 delta, 2H s [ethyl CH 2 ]).

C. [N-(t-butyloxycarbonyl)-statin]-[amino(4-sec-butyl-butyric acid)] ethyl ester

N-(t-butyloxycarbonyl)-statin (286 mg, 1.04 mmol), ethyl L-(4-amino-4-sec-butyl-butyrate) hydrochloride from Step B (232 mg, 1.04 mmol) , N-hydroxybenzotriazole (141 mg, 1.04 mmol), N-methyl-morpholine (105 mg, 1.04 mmol) and dicyclohexylcarbodiimide (215 mg, 1.04 mmol) were added successively to 15 mL of methylene chloride at 0 °C, after which the resulting solution was stirred for 19 h at 20 °C. The reaction mixture was then filtered and the filtrate evaporated to dryness. Chromatography of the residue (silica gel, CHCl 3 /EtOAc gradient) gave 400 mg of purified product (1 H-NMR, CDCl 3 , 1.5 delta, 9H s [BOC]).

D. to G. [N-(t-butyloxycarbonyl)-phenylalanine]-histidine-statin-[amino(4-sec-butyl-butyric acid)]-ethyl ester

In a similar manner as described under steps D to G of Example 3, [N-(t-butyloxycarbonyl)-statin]-[amino(4-sec-butyl-butyric acid)]-ethyl ester from step C (400 mg, 0, 94 mmol), converted to purified [N-(t-butyloxycarbonyl)-phenylalanine]-histidine-statin-[amino(4-sec-butyl-butyric acid)]ethyl ester (211 mg, (^H-NMR, CDCl 3 , 1, 5 delta, 9H s [BOC]).

H. Title connection

A solution of [N-(t-butyloxycarbonyl)-phenylalanine]-histidine-statin-[amino(4-sec-butyl-butyric acid)]ethyl ester from Step G (200 mg, 0.274 mmol) in 5 mL of dimethoxyethane was treated with 0.3 ml of 1N aqueous NaOH, and the resulting mixture stirred for 3 hours at 20°C. An additional 0.3 mL of 1N aqueous NaOH was then added and the reaction mixture stirred for 1 additional hour at 20°C. The reaction mixture was then concentrated in a rotary evaporator to remove dimethoxyethane, treated with ethyl acetate and then adjusted to pH 2 with 5% aqueous HCl. The organic layer was separated, washed with brine, filtered, dried over anhydrous MgSO 4 and concentrated to a white solid, which after trituration with ether gave 35 mg of purified [N-(t-butyloxycarbonyl)-phenylalanine]histidine-statin- [amino(4-sec-butyl-butyric acid)]

(^H-NMR, CDCl 3 , 1.5 delta, 9H s [BOC]).

Example 5

[ N-( t-butyloxycarbonyl)- Phe]- His- aminostatin- Ile- Phe- diacetate A. Ethyl 4(S)-(t-butyloxycarbonylamino)-3(S)-azido-6-methyl-heptanoate and ethyl -4(S)-(t-Butyloxycarbonylamino)-6-methyl-trans-2-heptenoate

A mixture of 3-epi-N-(t-butyloxycarbonyl)-statin (7.6 g, 25.05 mmol) and triethylamine (2.78 g, 27.55 mmol) in methylene chloride (200 mL) was added dropwise 3 .15 g (27.55 mmol) of methanesulfonyl chloride at 0°C. The resulting mixture was stirred for 1 h at 0°C, then allowed to warm to room temperature over 1 h and then washed with 1N aqueous HCl solution (2 x 200 mL) and saturated aqueous NaHCO 3 solution (1 x 200 ml). The organic layer was dried over MgSO 4 and evaporated to 9.75 g of a yellow oil. A solution of the oil in N,N-dimethylformamide (150 mL) was treated with sodium azide (2.45 g, 37.7 mmol) and the resulting mixture heated for 4 h at 60°C. The solvent was then removed under high vacuum and the residue diluted with 500 mL H 2 O and extracted with ether (2 x 150 mL). The combined ether extracts were washed with brine (1 x 100 mL), dried (MgSO 4 ) and evaporated to 6.6 g of a pale yellow oil. Separation of the oil by "flash" chromatography with 25-30% ether in hexane as eluent gave 334 mg (4% yield) ethyl-4(S)-(t-butyloxycarbonylamino)-3(S)-azido-6- methyl heptanoate as an oil tRf= 0.62 in ether-hexane, 1:1; 3 H-NMR (CDCl-j): delta 0.95 (d, J=6.6H), 1.30 (t, J=7.3H), 1.47 (s, 9H), 2.58 ( d, J=7f2H), 4.17 (q, J=7.2H), 4.53 (width, 1H);<13>C-NMR (CDCl3): delta 14.2; 22.2; 23.0; 24.9; 28.4;

37.2; 42.4; 51.6; 61.1; 62.8; 79.6; 155.8; 171.0; IR (CHC1-J: 3473, 2102 cm ]. The more polar chromatographic fraction,

4.9 g, gave a clear oil (Rf - 0.55 in ether-hexane, 1:1), which crystallized on standing. Trituration in ice-cold hexane gave 3.78 g (53% yield) ethyl-4(S)-(t-butyloxycarbonyl-

amino)-6-methyl-trans-2-heptenoate as a fluffy white solid [m.p. 56-58°C;<1>H-NMR (CDClg): delta 0.93 (d, J=6.6H), 1.30 (t, J=7.3H), 1.47 (s, 9H ), 4.18 (q, J=7.2H), 5.9(dd, J=15,1,1H), 6.83 (dd, J=15,5,1H); IR (KBr): 3350, 3307, 1720, 1701, 1684, 1659 cm"<1>].

B(1). Ethyl-3-benzylamino-4-(t-butyloxycarbonylamino)-3,4-(S,S)-6-methylheptanoate from Ethyl-4(S)-(t-butyloxycarbonyl-amino)- 6- methyl- trans- 2- heptenoate

A mixture of ethyl 4(S)-(t-butyloxycarbonylamino)-6-methyl-trans-2-heptenoate (3.52 g, 12.35 mmol), benzylamine (3.97 g, 37.0 mmol) and abs. EtOH (90 mL), was heated for 7 days at 58°C. The reaction mixture was then evaporated and separated by flash chromatography with 10-15% ethyl acetate in hexane as eluent. The less polar fractions yielded 963 mg

(20% yield) product in the form of an oil [Rf= 0.32 in 25%

EtOAc in hexane; 1 H-NMR (CDCl-j): delta 0.97 (d, J=5.6H),

1.25 (t, 3H, J=7), 1.45 (s, 9H), 2.43 (m, 2H), 3.32 d (m, 1H), 3.77 (s, 2H), 4.10 (q, 2HJ=7), 4.65 (d, 1H, J = 9), 7.22 (s, 5H), IR (CHC13): 3433, 1720 (shoulder), 1709 cm<-1 >; Mass spectrum

m/e 393 (M<+>+1), 319, 206 (base), 91]

B(2). Ethyl 3-benzylamino-4-(t-butyloxycarbonylamino)-3,4(S,S)-6-methylheptanoate from ethyl 4(S)-(t-butyloxycarbonylamino)-3(S)-azido-6 - methyl heptanoate

20 mg of 10% Pd/C catalyst was added to a solution of ethyl 4(S)-(t-butyloxycarbonylamino)-3(S)-azido-6-methyl-heptanoate (120 mg, 0.36 mmol) in 5 ml acetic acid:ethanol 1:1

and the resulting mixture hydrogenated for 3 hours at room temperature and one atmosphere of H 2 . After filtering off the catalyst, the reaction mixture was evaporated to an oil by means of reduced pressure and azeotropic distillation with ethanol. The oil (136 mg) was then dissolved in 3 mL of methanol and treated with 37 µL (38.2 mg, 0.36 mmol) of benzaldehyde at room temperature and then with 34 mg (54 mmol) of NaCNBH 3 at 0°C. The reaction mixture was stirred for \ hour at 0 C whereupon the

allowed to assume room temperature before dilution with H2O and saturated aqueous NaHCO3~ solution. An oily precipitate formed which was extracted with methylene chloride. The resulting organic layer was dried (MgSO 4 ) and evaporated to an oily

like residue. TLC of this showed only one epimer, corresponding to ethyl 3-benzylamino-4-(t-butyloxycarbonylamino)-3,4(S,S)-6-methylheptanoate. Purification of the oily residue by "flash" chromatography with 10-15% ethyl acetate:hexane, led to 68 mg of product which, according to NMR and TLC, was identical to the material prepared during step B(1).

C. 3-Benzyloxycarbonylamino-4-(t-butyloxycarbonylamino)-3,4(S,S)-6-methylheptanoic acid

60 mg of 10% Pd/C catalyst was added to a solution of ethyl 3-benzylamino-4-(t-butyloxycarbonylamino)-3,4(S,S)-6-methylheptanoate from step B(1) (530 mg , 1.35 mmol) in 12 ml of acetic acid:ethanol, 1:1, and the resulting mixture hydrogenated for 3 hours at room temperature under 3.5 kg/cm 2 H 2 . The catalyst was filtered off and the filtrate concentrated to an oil which was taken up in a mixture of 10 ml of dioxane and 10 ml of H 2 O. Solid NaHCO 3 was added to the reaction mixture (pH 7.8) followed by 289 µL (345 mg, 2.025 mmol) of benzyloxycarbonyl chloride (Chemalog). The resulting mixture was stirred for 1.5 hours at room temperature during which the pH was maintained at 8 with solid NaHCO 3 . The mixture was then stirred for a further 4 hours at

pH 12.5 by adding 4 ml of aqueous 1N NaOH solution and r^O-dioxane as needed to dissolve any precipitate. The mixture was then neutralized with 1N aqueous HCl at 0°C and the dioxane removed under reduced pressure. The residue was diluted with 50 mL H 2 O and extracted with ethyl acetate (2 x 50 mL). The combined ethyl acetate extracts were dried (MgSO₂) and evaporated to 588 mg of an oil which was purified by flash chromatography with 3% MeOH in CHCl₂ as eluent, resulting in 288 mg (41% yield) of product as an oil [<1>H-NMR (DMSO d<6>, 250 MHz): delta 0.81 (d, J=7.3H), 0.86

(d, J=7.3H), 1.38 (s, 9H), 2.2-2.4 (m, 2H), 5.02 (centroid of AB pattern, J=13, 2H), 6 .48 (d, J=9, 1H), 7.05 (d, J=9.1H), 7.30 (s, 9H);

Mass spectrum: m/e 186, 130, 91, 86 (base)]

D. Isoleucine phenylalanine benzyl ester hydrochloride

A solution of 14.05 g (58.5 mmol) N-(t-butyloxycarbonyl)-L-isoleucine hemihydrate (Chemalog) in 500 mL methylene chloride was dried (MgSO 4 >), filtered and treated with 6.43 mL (5, 92 g, 58.5 mmol) of N-methyl-morpholine.The resulting mixture was cooled to -16°C and then treated with 7.59 mL (7.99 g, 58.5 mmol) of isobutyl chloroformate at a rate ensuring that the exothermic reaction did not bring the temperature above -10° C. After 10 minutes of further stirring after the end of the addition, a solution of 24.8 g (58.0 mmol) of L-phenylalanine benzyl ester p-toluenesulfonate and 6.43 ml ( 5.92 g, 58.5 mmol) of N-methylmorpholine in 50 ml of methylene chloride added dropwise to the reaction mixture at a rate which ensured that the exothermic reaction did not exceed -10° C. After completion of the addition, the reaction mixture was allowed to reach room temperature within 1 hour, whereupon it was washed with 5% aqueous HCl solution (2 x 300 mL) and 10% aqueous H 2 CO 3 solution (2 x 300 mL).The resulting organic layer was dried (Mg SO4) and evaporated to 27.40 g of white solid. Trituration in hexane gave 24.97 g of purified [N-(t-butyloxycarbonyl)-L-isoleucine]-L-phenylalanine benzyl ester [42% yield; m.p. 131-132°C, Rf = 0.43 in ether: ethyl acetate, 1:1; <1>H-NMR (CDCl 3 ) delta: 0.78-0.9 (broad d, 6H), 1.43 (s , 9H), 3.08 (d,J=6), 3.87 (dd, J=5,9,1H), 4.75-5.1

(m, 1H), 5.06 (s, 2H), 6.3 (d, J=9.1H), 7.0-7.3 (m, 11H)].

A solution of 12.50 g (25.7 mmol) of [N-(t-butyloxycarbonyl)-L-isoleucine]-L-phenylalanine benzyl ester in 125 mL of 4N HCl in dioxane was stirred for 2 hours at room temperature under protection from moist atmosphere with a CaCl2~ tube. The solvent was then removed under reduced pressure and the residue triturated with ether, giving 11.16 g of product as a white solid [m.p. 178-179.5°C (decomp.); Rf= 0.73 in BuOH-H 2 O-AcOH, 4:1:1; (<1>H-NMR (CD3OD) delta: 0.9-1.1 (m, 6H), 3.1-3.0 (m, 2H), 3.73 (d, J=5.1H) , 5.08 (s, 1H), 7.18 (s, 5H), 7.24 (s, 5H)]

E. [ N-gamma-(t-butyloxycarbonyl)-benzyloxycarbonylamino-statin]- isoleucine- phenylalanine- benzyl ester

A solution of isoleucine-phenylalanine-benzyl ester hydrochloride (412 mg, 1.02 mmol) in 10 ml of methylene chloride was neutralized at 0°C with 142 µl of triethylamine (103 mg,

1.02 mmol). 3-Benzyloxycarbonylamino-4-(t-butyloxycarbonyl-amino)-3,4(S,S)-6-methylheptanoic acid (416 mg, 1.02 mmol) and N-hydroxybenzotriazole (205 mg, 1.52 mmol) were then added to the solution at 0°C. Finally, a solution of dicyclohexylcarbodiimide (210 mg, 1.02 mmol) in 5 ml of methylene chloride was added at 0°C. The reaction mixture was then stirred for 3 hours at 0°C and then for an additional 16 hours while warming to room temperature. The solvent was removed under reduced pressure and the residue stirred in 75 ml of ethyl acetate. The resulting mixture was filtered to remove some solid and the filtrate washed with 10% aqueous citric acid (1 x 75 mL) and saturated aqueous NaHCO 3 solution (1 x 75 mL), dried (MgSO 4 ) and evaporated to a solid ( 217 mg). The solid was combined with the filtered off solid from the aforementioned ethyl acetate mixture. The combined solid fractions (1.043 g), a mixture of product and dicyclohexylurea, were used in the next step without purification.

F. Benzyloxycarbonylaminostatin-isoleucine-phenylalanine-benzyl ester hydrochloride

The crude [N-gamma-(t-butyloxycarbonyl)-benzyloxycarbonyl-aminostatin]-isoleucine-phenylalanine benzyl ester from Step E (1.043 g) was stirred in 25 mL of 4N HCl in dioxane for 1 hour at room temperature under protection from a humid atmosphere with a CaC^ tube. Removal of the solvent under reduced pressure (by azeotropic distillation with methylene chloride and ether) and trituration in ether afforded 963 mg of solid, which consisted of a mixture of product and dicyclohexylurea. This crude solid was used in the next step without purification.

G. [N-alpha-(t-butyloxycarbonyl)-N-im-(t-butyloxycarbonyl)-histidine]-benzyloxycarbonylaminostatin isoleucine-phenylalanine benzyl ester

A suspension of the crude benzyloxycarbonylaminostatin isoleucine phenylalanine benzyl ester hydrochloride from step F

(963 mg) in 20 ml of methylene chloride was neutralized at 0°C with 177 µl of triethylamine (128 mg, 1.02 mmol). N-alpha-(t-butyloxycarbonyl)-N-im-(t-butyloxycarbonyl)-L-histidine (542 mg, 1.53 mmol) and N-hydroxybenzotriazole (206 mg, 1.53 mmol) were then added to the suspension at 0°C. Finally, a solution of dicyclohexylcarbodiimide (315 mg, 1.53 mmol) in 1 mL of methylene chloride was added at 0°C. The reaction mixture was then stirred for 3 hours at 0°C and then for an additional 16 hours at room temperature. About. half of the solvent was removed under reduced pressure and the residue stirred in 125 ml of ethyl acetate. The resulting mixture was filtered to remove some solids and the filtrate washed with aqueous 10% citric acid solution (2 x 100 mL) and aqueous saturated NaHCO 3 solution (1 x 100 mL), dried (MgSO 4 ) and evaporated to 920 mg solid. The solid was purified by flash chromatography using 0.5-0.75% MeOH in CHCl 2 as eluent, whereby 221 mg of purified product was obtained as a solid. The solid filtered off from the above ethyl acetate mixture contained a substantial amount of the product along with dicyclohexylurea. Most of the latter compound was separated from the product by filtration after trituration of the solid (with vigorous stirring) in 100 ml of methylene chloride. The filtrate was concentrated (1.1 g) and chromatographed as above, whereby a further 401 mg of product was obtained. The combined purified solids were triturated in ether to give 592 mg of a scaly white solid [58% yield; m.p. 229-231°C; Rf= 0.22 in 2.5% MeOH in CHCl 3 ;

<1>H-NMR (CDC13, 250 MHz): delta 0.6-0.85 (m, 12H), 1.42 (s, 9H),

1.58 (s, 9H) , 2.42 (centroid of AB pattern of ABX, 2H, J-=14,

Ari JAX"5'JBX=10)'2.94 (d'J=5, 2H)'3.13 (d'J=6, 2H)'3'55-3'7 (m, 1H), 4 .1-4.3 (m, 3H), 4.98 (q, J=7, 1H), 5.2-5.5 (m, 4H), 6.10 (d, J=7, 1H) , 6.37 (broad d, 1H), 6.80 (d, 1H, J=8), 7.0-7.4 (m, 17H), 7.5 (broad, 1H), 8.00 ( pp, 1H)].

H. Histidine benzyloxycarbonylaminostatin isoleucine phenylalanine benzyl ester hydrochloride

A solution of 204 mg of [N-alpha-(t-butyloxycarbonyl)-N-im-(t-butyloxycarbonyl)-histidine]-benzyloxycarbonylamino-statin-isoleucine-phenylalanine-benzyl ester in 7 ml of 4N HCl in dioxane was stirred for 5 hours at room temperature under protection from a humid atmosphere with CaCl2 tubes. The solvent was removed under reduced pressure (under azeotropic distillation with methylene chloride and ether), giving 153 mg of product as a white powder [68% yield;

m.p. 154-157°C; Rf= 0.55 in butyl alcohol:H2O:acetic acid (4:1:1); <1>H-NMR (CD30D): delta 1.05-1.1 (m, 12H), 2.3-2.5 (m, 2H), 3.1 (d,J=8.2H), 5.08 (s,4H), 7.18 (s, 5H), 7.30 (s, 10H), 7.53

(a, 1H) , 8.83 (s, 1H) ]

I. [N-(t-butyloxycarbonyl)-phenylalanine]-histidine-benzyl-oxycarbonylaminostatin-isoleucine-phenylalanine-benzyl ester

A solution of histidine-benzyloxycarbonylaminostatin-isoleucine-phenylalanine-benzyl ester hydrochloride (306 mg,

0.352 mmol) in 5 ml of dimethylformamide was neutralized at 0°C

with 123 µL of triethylamine (88 mg, 0.879 mmol). N-(t-butyloxycarbonyl)-L-phenylalanine (103 mg, 0.387 mmol) and N-hydroxybenzotriazole (79 mg, 0.580 mmol) were then added to the solution at 0°C. Finally, a solution of dicyclohexylcarbodiimide (980 mg, 0.387 mmol) in 1.5 mL of dimethylformamide was added at 0°C. The reaction mixture was then stirred for 3 hours at 0°C and then for an additional 16 hours at room temperature. The solvent was removed under high vacuum, the residue stirred in 50 mL of ethyl acetate and the resulting mixture filtered to remove some solids. The solvent was removed from the filtrate and the residue stirred in methylene chloride (50 mL) and the resulting mixture filtered to remove a new portion of solid. The filtrate after the latter filtration was concentrated, the residue dissolved in 50 ml of ethyl acetate and the resulting solution washed with 10% aqueous citric acid (2 x 40 ml) and saturated aqueous NaHCO 3 solution (2 x 40 ml).

The resulting organic layer was dried (MgSO 4 ) and concentrated to a solid (153 mg). This, along with the solid from the ethyl acetate and methylene chloride trituration, was combined and purified by flash chromatography, using 2-3% methanol in chloroform as eluent, yielding 261 mg of solid. Trituration of this in ether led to 218 mg of product in form

of a glassy solid [59% yield; m.p. 228-229°C;

Rf= 0.25 in 5% MeOH in chloroform; <1>H-NMR (CDCl2, 250 MHz);

delta 0.6-0.8 (m, 12H), 1.26 (s, 9H), 2.38 (broad d, 2H), 2.8- 3.0 (m, 2H), 3.15-3, 4 (m, 4H), 4.1-4.0 (m, 1H), 4.1-4.2

(m, 1H), 4.2-4.4 (m, 2H), 4.6 (broad d, 1H), 4.9-5.2 (m, 6H), 5.9- 6.1 (m, 1H), 6.78 (s, 1H), 7.2-7.4 (m, 22H), 7.49 (s, 1H)].

J. TitelverbindeIse

10% Pd/C catalyst (12 mg) was added to a solution of [N-(t-butyloxycarbonyl)-phenylalanine]-histidine-benzyloxycarbonylaminostatin-isoleucine-phenylalanine-benzyl ester (24 mg, 0.023 mmol) in 5 mL of acetic acid :ethanol (1:1) and the resulting mixture hydrogenated for 3 hours at room temperature and 2.8 kg/cm 2 H 2 . The reaction mixture was then filtered to remove the catalyst and the filtrate concentrated to a residue. Trituration of the residue in ether gave 17 mg of [N-(t-butyloxycarbonyl)-phenylalanine]-histidine-aminostatin-isoleucine-phenylalanine-diacetate as a white powder [m.p. 155-159°C; Rf= 0.58 in n-butyl alcohol:H2O:acetic acid (4:1:1);<1>H-NMR (CD3CO2D 250 MHz): delta 0.7-1.0 (m, 12H),

1.33 (s, 9H), 2.7-2.9 (m, 2H), 3.0-3.6 (m, 6H), 3.6-3.9

(m, 1H), 4.15-4.30 (m, 1H), 4.35-4.55 (m, 2H), 4.8-4.95 (m, 2H), 7.1-7 .4 (m, 10H), 7.38 (s, 1H), 8.78 (s, 1H)].

Example 6

[N-(t-butyloxycarbonyl)-Phe]-His-Sta-Ala-Sta-Glu dibenzyl ester

A. [N-(t-butyloxycarbonyl)-alanine]-statin glutamic acid dibenzyl ester

N-hydroxybenzotriazole (324 mg, 2.4 mmol), N-methyl-morpholine (202 mg, 2 mmol), L-glutamic acid dibenzyl ester p-toluenesulfonate (995 mg, 2 mmol), N-(t-butyloxycarbonyl) -statin (660 mg, 2.4 mmol) and 1-cyclohexyl-3-(2-morpholino-ethyl)-carbodiimide-metho-p-toluenesulfonate (1271 mg, 80% pure, 2.4 mmol) were sequentially dissolved into methylene chloride (100 mL) at 0°C and the resulting solution stirred for 19 hours at 20°C. The reaction mixture was then washed successively with 5.5% aqueous HCl, saturated aqueous NaHCO 3 solution and brine and then dried over anhydrous MgSO 4 . After filtration and evaporation, 1269 g of crude [N-(t-butyloxycarbonyl)-statin]-glutamic acid dibenzyl ester were obtained in the form of an oil (<1>H-NMR, CDC13, 5.2 delta, 2H s [benzyl CH2 ]). This oil (1269 g) was dissolved in 10 mL of 3.7N HCl/dioxane and the resulting solution set aside for 1 hour at room temperature. The solution was then evaporated under high vacuum to a residue which was triturated with ether and dried under nitrogen, giving 900 mg of statin glutamic acid dibenzyl ester hydrochloride as a foam (H-NMR, CDCl^, 5.2 delta, 2H s [benzyl CH,,]). N-hydroxybenzotriazole (186 mg, 1.38 mmol), N-methyl-morpholine (116 mg, 1.15 mmol), part of the above obtained statin glutamic acid dibenzyl ester hydrochloride (600 mg, 1.15 mmol) , N-(t-butyloxycarbonyl)-L-alanine (261 mg, 1.38 mmol) and 1-cyclohexyl-3-(2-morpholino-ethyl)-carbodiimide-metho-p-toluenesulfonate (732 mg, 80% pure , 1.38 mmol) was sequentially dissolved in methylene chloride (60 mL) at 0°C, whereupon the resulting solution was stirred for 19 hours at 20°C. The reaction mixture was then washed successively with 5.5% aqueous HCl, saturated aqueous NaHCO 3 solution and brine, and dried over anhydrous MgSO 4 . After filtration and evaporation, 743 mg of crude product was obtained as a foam (<1>H-NMR, CDCl 3 , 5.2 delta, 2H s [benzyl CH 2 ]).

B. to G. Title connection

In a similar manner as described in Steps B to G of Example 1, [N-(t-butyloxycarbonyl)-alanine]-statin-glutamic acid dibenzyl ester from Step A (743 mg) was converted to [N(t-butyloxycarbonyl)-phenylalanine ]-histidine-statin-alanine-statin-glutamic acid-dibenzyl ester (300 mg, foam,<1>H-NMR, CDC13, 5.2 delta 2H s [benzyl CH,,]). In each deblocking step, evaporation to dryness was followed by trituration with ether. After completion of step E, the crude foam product was purified by chromatography (silica gel 95:5 CHCl 3 /methanol) to give a purified foam. No chromatographic purification was performed at the end of step G.

Example 7

[ N-( t-butyloxycarbonyl)- Phe]- His- Sta- Ala- Sta- Glu A mixture of [N-(t-butyloxycarbonyl)-phenylalanine]-histidine-statin-alanine-statin-glutamic acid-dibenzyl ester (90 mg , 0.077 mmol), 20% Pd(OH) 2 /C catalyst (45 mg) and methanol (10 mL) were hydrogenated for 2 h at room temperature and 3.5 kg/cm 2 H 2 . The reaction mixture was then filtered to remove the catalyst, after which the filtrate was evaporated to a foam (72 mg). The foam was triturated with ether and dried to give a purified [N-(t-butyloxycarbonyl)-phenylalanine]-histidine-statin-alanine-statin-glutamic acid foam (51 mg, 72% yield,<1>H-NMR, CDC13, 1.5 delta, 9H s [BOC]).

Example 8

[N-(t-butyloxycarbonyl)-Phe]-His-sta-[amino(2-sec-butyl- ethylene)]- Phe- acetic acid salt

A. N-(t-butyloxycarbonyl)-L-isoleucine

A mixture of L-isoleucine methyl ester (24.3 g, 0.134 mmol) and triethylamine (13.5 g, 0.134 mmol) in methylene chloride (210 mL) was prepared and a solution of di-tert-butyl dicarbonate (Aldrich, 29.1 g, 0.134 mmol) in methylene chloride (25 mL) was added dropwise to the mixture at 0°C. After the addition was finished, the mixture was allowed to reach room temperature overnight, after which it was filtered. The filtrate was washed successively with H 2 O

(3 x 75 mL), aqueous 0.1N HCl solution, H 2 O (1 x 100 mL) and saturated aqueous NaHCO-j solution (1 x 75 mL), dried (MgSO 4 )

and evaporated to N-(t-butyloxycarbonyl)-L-isoleucine methyl ester as an oil [30.7 g; 93% yield; <1>H-NMR (CDCl 3 ): delta 0.92 (d, J=7.6H), 1.43 (s, 9H), 3.70 (s, 3H), 4.17 ( dd, J=5, 9.1H), 5.03 (d, J=9.1H)]. A solution of N-(t-butyloxycarbonyl)-L-isoleucine methyl ester oil (15.0 g, 61.1 mmol) in dry toluene (260 mL) was cooled to -78°C and a 1 M solution of diisobutylaluminum hydride was added dropwise. in hexane (153 ml), at a rate which ensured that the temperature of the exothermic reaction did not exceed -65°C. After the addition was complete, stirring was continued for 15 minutes at -78°C. The reaction was

then interrupted carefully with 15 ml of methanol (the temperature of the mixture was not allowed to exceed -65°C), followed by 200 ml of Rochelle salt solution. After warming to room temperature, the organic layer was separated and extracted with ether (3 x 200 mL), and more Rochelle salt solution was added as needed to dissolve the aluminum salts. The combined organic extracts were dried (Na 2 O 2 ) and evaporated to a crude oily product. The oil was stored at -78°C to prevent possible racemization, and then used without purification in the next step [132 g; 98% yield; Rf= 0.32 in 35% ether in hexane; <1>H-NMR (CDCl 3 ): delta 1.00 (d, J=7.6H), 1.46 (s, 9H), 9.65

(p, 1H)]

B. [Amino(2-sec-butyl-ethylene)]-phenylalanine benzyl ester dihydrochloride

A mixture of N-(t-butyloxycarbonyl)-L-isoleucinate (2.00 g, 9.29 mmol) and L-phenylalanine benzyl ester p-tosylate (Chemalog, 3.78 g, 8.84 mmol) in methanol (150 mL), was stirred for 35 min in the presence of molecular sieve (3 Å, Ventron) at room temperature. 0.73 g (11.6 mmol) of NaCNBH 3 was then added and the mixture stirred for an additional 1 hour at room temperature. The molecular sieve was then filtered off and the filtrate concentrated to an oily residue, which was diluted with saturated aqueous NaHCO 3 solution and extracted twice with ether. The combined ether extracts were dried (Na 2 SO 4 ) and concentrated to an oil which was purified by flash chromatography eluting with 12% ethyl acetate in hexane. 1.69 g of [(t-butyloxycarbonyl)amino(2-sec-butyl -ethylene)]-phenylalanine benzyl ester as an oil (40% yield; Rf= 0.28 in 25% ethyl acetate in hexane). The oil crystallized on standing [mp 53-55°C;<1>H-NMR (CDC13 ): delta 0.6-1.0 (m, 6H), 1.48

(s, 9H), 2.92 (d, J=7.2H), 5.07 (s, 2H), 7.17 (s, 5H), 7.25

(p, 5H)]. A solution of [(t-butyloxycarbonyl)amino(2-sec-butylethylene)]-phenylalanine benzyl ester (2.94 g, 6.47 mmol) in 1 4N HCl/dioxane (110 mL) was formed, and this was stirred in

2 hours at room temperature, protected from the atmosphere with a CaCl2 tube. The solvent was then removed under reduced pressure by azeotropic distillation with ether, after which the residue was triturated with ether to give a white solid [2.65 g; 95% unblocking yield; m.p. 183-184°C; Rf= 0.73 in BuOH:H2O:acetic acid (4:1:1); <1>H-NMR (CD30D): delta 1.00 (width, 6H), 5.13 (s, 2H), 7, 1-7.5 (m, 10H)]

C. [n-(t-butyloxycarbonyl)-statin]-[amino(2-sec-butyl-ethylene)]-( N- benzyloxycarbonyl- phenylalanine)- benzyl ester

A suspension of [amino(2-sec-butyl-ethylene)]-phenylalanine benzyl ester dihydrochloride (1.55 g, 3.63 mmol) in methylene chloride (40 mL) was cooled to 0°C and neutralized at this temp. -erature with triethylamine (0.808 g, 7.99 mmol). N-(t-butyloxycarbonyl)-statin (1.00 g, 3.63 mmol) and N-hydroxybenzotriazole (0.74 g, 5.45 mmol) were then added to the suspension at 0°C. Finally, dicyclohexylcarbodiimide (0.75 g, 3.63 mmol) in methylene chloride (2 mL) was added to the suspension at 0°C and the resulting mixture stirred for 3 hours at 0°C and then for 16 hours at room temperature. The resulting precipitate was then filtered off and the filtrate concentrated and stirred in 125 ml of ethyl acetate. After re-filtration of undissolved solid, the ethyl acetate layer was washed with saturated aqueous NaHCO 3 solution (135 mL), dried (Na 2 SO 4 ) and evaporated to a foam. The foam was purified by flash chromatography with 35% ethyl acetate in hexane as eluent to give a purified [N-(t-butyloxycarbonyl)-statin]-[amino(2-sec-butyl-ethylene)]-phenylalanine benzyl ester product as a foam [1.96 g; 88% yield; R^ = 0.58 in ethyl acetate:hexane, 1:1); 1 H-NMR (CDCl 3 ): delta 0.6-1.1

(m, 12H), 1.5 (s, 9H), 4.88 (d, J=9.1H), 5.06 (s, 2H), 7.15

(s, 5H), 7.25 (s, 5H)]. A solution of the purified [N-(t-butyloxycarbonyl)-statin]-[amino(2-sec-butyl-ethylene)]-phenylalanine benzyl ester foam (1.91 g, 3.12 mmol) in dioxane (16 ml), was acylated with benzyloxycarbonyl chloride (Chemalog, 67 µl, 0.80 g, 4.68 mmol) using saturated aqueous NaHCO 3 solution to maintain pH 8. After consumption of all the starting material (according to TLC), the dioxane removed under reduced pressure and the residue diluted with H 2 O and extracted with ethyl acetate. The ethyl acetate extract was dried (MgSO 4 ) and evaporated to a foam which was purified by flash chromatography to give a white foam [2.06 g; Rf= 0.7 in ethyl acetate:hexane, (1:1); 1H-NMR (CDCl 3 >: delta 0.6-1.0 (m, 12H), 1.48 (s, 9H), 7.0-7.4 (m, 15H)]

a

D. to G. [N-(t-butyloxycarbonyl)-phenylalanine]-histidine-statin-[amino(2-sec-butyl-ethylene)]-[N-benzyloxycarbonyl-phenylalanine]-benzyl ester

In a similar manner as described in steps F to I i

Example 5, the purified [N-(t-butyloxycarbonyl)-statin]-[amino(2-sec-butyl-ethylene)]-(N-benzyloxycarbonyl-phenylalanine)-benzyl ester from Step C (2.03 g, 2 .72 mmol), converted to purified [N-(t-butyloxycarbonyl)-phenylalanine]-histidine-statin-[amino(2-sec-butyl-ethylene)]-(N-benzyloxycarbonyl-phenylalanine)-benzyl ester [0.595 g; foam; R^- 0.36 in 5% methanol in chloroform; <1>H-NMR (CDCl 3 ): delta 0.6-1.0 (m, 12H), 1.38 (s, 9H), 6.75 ( s, 1H), 7.1-7.3 (m, 20H), 7.38 (s, 1H)].

H. Title connection

10% Pd/C catalyst (18 mg) was added to a solution of [N-(t-butyloxycarbonyl)-phenylalanine]-histidine-statin-[amino(2-sec-butyl-ethylene)]-(N-benzyloxycarbonyl- phenylalanine)-benzyl ester (150 mg, 0.150 mmol) in 4 ml ethanol:acetic acid (1:1), and the resulting mixture hydrogenated for 12 hours at room temperature and 2.8 kg/cm 2 H2 • The mixture was then filtered and the filtrate concentrated to a solid (128 mg) which was triturated with ether to give a purified [N-(t-butyloxycarbonyl)-phenylalanine]-histidine-statin-[amino(2-sec-butyl-ethylene)]-phenylalanine-acetic acid salt as of a solid product [109 mg; 84% yield; m.p. 117-126°C; R,-= 0.57 in BuOH-H-O-AcOH (4:1:1);<1>H-NMR (CD3OD, 250 MHz): delta 0.8-1.0 (m>12H), 1, 36 (s, 9H), I, 92 (s, 6H), 2.2-2.45 (m, 2H), 4.28 (dd,J=9,4,1H), 3.54 (broad t , 1H), 6.92 (s, 1H), 7.2-7.4 (m, 10H), 7.63 (s, 1H)]

Example 9

[ N-( t-butyloxycarbonyl)- Phe]- His- cyclostatin- Lys- Phe

A. N-(t-butyloxycarbonyl)-cyclostatin

Glacial acetic acid (6.5 mL) and 10% Rh/C catalyst (1.5 g) were added to a solution of N-(t-butyloxycarbonyl)-4(S)-amino-3(S)-hydroxy-5- phenylpentanoic acid (10.02 g, 9.70 mmol) in methanol (200 mL) and the resulting mixture hydrogenated for 8 h at room temperature under 3.2 kg/cm 2 H2 • The reaction mixture was then filtered and the filtrate concentrated to a white solid, which, after trituration in hexane, gave a purified product [8.46 g; 83% yield; m.p. 109-110°C;

Rf= 0.72 in CHCl3:MeOH:acetic acid, 18:2:1;<1>H-NMR (CDC13):

1.45 (s, 9H), 2.55 (d, J=6.2H)

B. Title connection

In a similar manner as described in Example 1, but with N-(t-butyloxycarbonyl)-cyclostatin instead of N-(t-butyloxycarbonyl)statin, the title compound was prepared (1H-NMR, CD30D, 1.50 delta, 9H s [ BOC]).

Example 10

[ N-( t-butyloxycarbonyl)- Pro]- Phe- His- cyclostatin- Lys- Phe In a similar manner as described under steps A to F in Example 1, but with N-(t-butyloxycarbonyl)-cyclostatin instead of N -(t-butyloxycarbonyl)statin was prepared histidine-cyclostatin-(N-epsilon-benzyloxycarbonyllysine)-phenylalanine benzyl ester dihydrochloride. In a similar manner as described under steps G and H of Example 1, but with [N-(t-butyloxycarbonyl)proline]-phenylalanine (which is commercially available) instead of N-(t-butyloxycarbonyl)-phenylalanine and using 95 :5 CHCl3:MeOH as eluent during the silica gel chromatography, the title compound was obtained as a purified solid product.

(<1>H-NMR, CD30D, 1.49 delta, 9H s [BOC]).

Example 11

[N-(t-butyloxycarbonyl)-Phe]-His-cyclostatin-[amino-(2-sec-butyl- ethylene)]- Phe- acetic acid salt

In a similar manner as described in Example 8, but with N-(t-butyloxycarbonyl)-cyclostatin instead of N-(t-butyloxycarbonyl)-statin, the title compound was prepared [m.p. 150-163°C (decomp.); Rf= 0.50 in butanol:acetic acid:water 4:1:1 (ninhydrin); <1>H-NMR (CD3OD). 0.6-2.4 (m, 23H), 1.37

(s, 9H), 7.1-7.4 (11H), 6.95 (s, 1H), 7.70 (s, 1H)].

Example 12

[ N-( t-butyloxycarbonyl)- Phe]- His- cyclostatin- Lys- Sta In a similar way as described in Example 1, but with statin benzyl ester hydrochloride instead of L-phenylalanine benzyl ester p-toluenesulfonate and N- (t-butyloxycarbonyl)-cyclostatin instead of N-(t-butyloxycarbonyl)statin, the title compound was prepared (1H-NMR, CD3OD, 1.5 delta,

9H s [BOC]).

Example 13

[N-(t-butyloxycarbonyl)-Phe]-His-cyclostatin-( 3- methoxycarbonyl- 1, 2, 3, 4- tetrahydro- 2- isoquinoline)

A. [N-(t-butyloxycarbonyl)-cyclostatin]-(3-methoxy-carbonyl-1,2,3,4-tetrahydro-2-isoquinoline)

In a similar way as described under step C i

Example 1, but with N-(t-butyloxycarbonyl)-cyclostatin

(190 mg, 0.60 mmol) in place of N-(t-butyloxycarbonyl)-statin and 3-methoxycarbonyl-1,2,3,4-tetrahydroisoquinoline hydrochloride (137 mg, 0.60 mmol) in place of ( N-epsilon-benzyloxycarbonyl-lysine)-phenylalanine-benzyl ester hydrochloride, [N-(t-butyloxycarbonyl)cyclostatin]-(3-methoxycarbonyl-1,2,3,4-tetrahydro-2-isoquinoline) was prepared as a foam which was used without purification in the next step (301 mg, <1>H-NMR, CDCl3, 1.5 delta, 9H s [BOC]).

Example 2 0

[N-(t-butyloxycarbonyl)-Phe]-His-cyclostatin-Pro-methyl ester

A. [N-(t-butyloxycarbonyl)-cyclostatin]-proline methyl ester

In a similar way as described under step C i

Example 1, but with N-(t-butyloxycarbonyl)-cyclostatin (3.15 g, 10 mmol) instead of N-(t-butyloxycarbonyl)-statin and proline methyl ester hydrochloride (1.65 g, 10 mmol) instead of (N-epsilon-benzyloxycarbonyl-lysine)-phenylalanine benzyl ester hydrochloride, [N-(t-butyloxycarbonyl)-cyclostatin]-proline methyl ester was prepared as a white solid (3.945 g).

B. Title connection

In a similar manner as described under steps D to H in Example 1, the compound [N(t-butyloxycarbonyl)-phenylalanine]-histidine-cyclostatin-proline-methyl ester was prepared.

Example 21

Inhibition of the angiotensinogen-splitting action of renin in vitro

Blood plasma from healthy laboratory personnel was pooled and stored deep-frozen until required. Before use, part of the plasma was thawed and centrifuged, and the supernatant liquid was mixed with protease inhibitors and buffered to pH 7.4. Renin inhibitors were added at various concentration levels to different aliquots of the centrifuged plasma and the resulting mixtures (310 µl) incubated for 3 hours at 37°C together with renin inhibitor-free control mixtures. After incubation, the reaction in the mixtures was stopped in ice water and examined for angiotensin I using angiotensin I antibodies. The formation of angiotensin I in the presence of a renin inhibitor was compared with what was formed without an inhibitor, and the percentage inhibition calculated. Using data obtained by incubating duplicate samples of different inhibitor concentrations, the required concentration in the incubation mixture to achieve 50% inhibition of renin's angiotensinogen-splitting action, i.e. the inhibitor's IC,-Q value, was calculated for different inhibitors.

Angiotensin I in mixtures where the incubation had been interrupted was determined by radioimmunoassay, using components from a renin radioimmunoassay kit from Becton Dickinson and Co. (Orangeburg, N.Y.). This radioimmunoassay was based on the technique developed by Haber et al., J. Clin. Endocrinol., 29^p. 1349-1355 (1969).

Using the stated procedure, the renin inhibition coefficients were determined for the compounds prepared in Examples 1-5 and Examples 7-20. In all cases the experimental ICC^ value was lower than 5 micromol/liter.

Example 22

Antagonism of exogenous renin-induced pressor response in vivo

Male Sprague-Dawley rats (230-300 g body weight) were anesthetized with sodium pentobarbital (65 mg/kg body weight, intraperitoneally), after which the femoral vein and carotid artery catheter was implanted in each animal. After the surgical intervention, the animals were placed in a recumbent position and the rectal temperature was continuously read. The mean arterial blood pressure (MAP) was recorded via the carotid artery catheter using a Statham P23 ID pressure transducer and a printer. After a stabilization period, control renin pressor response (dP) values of 20-30 mm Hg were achieved by administration of porcine renin (30-80 mU/kg body weight, intravenously). After MAP had returned to baseline, a renin inhibitor (10 mg/kg body weight, intravenous) was given. The animals were again stimulated with porcine renin (same dosage as for control response) 5, 15 and 30 minutes after administration of the renin inhibitor and the corresponding values for the renin pressor response (dP) measured. Percent antagonism is calculated as:

( control dP - experimental dP) x 100%,

control dP

where control dP and experimental dP are the pressor changes in MAP before and after administration of the renin inhibitor, respectively. At least three experimental animals were used in each test,

and the average results were calculated.

By using the described method, the percentage renin-induced pressor response antagonism can be determined for the compounds according to the invention.

Claims (23)

1. Procedure for preparing a compound of the formula and the pharmaceutically acceptable salts thereof, where W is phenylalanine, histidine, leucine, tyrosine, 1-naphthyl-alanine or 2-phenylmethyl-propionylene; W 1 is phenylalanine, histidine, leucine, tyrosine or norleucine, where the peptide bond between W and W i is optionally replaced by -CH(alkyl with 1-4 carbon atoms)-NH- when W is phenylalanine and W is histidine; R is hydrogen, an amino-protecting acyl moiety with a molecular weight of less than 500, proline, amino-protected proline, pyroglutamic acid or amino-protected pyroglutamic acid; R 2 is hydrogen, alkyl with 1-6 carbon atoms, phenyl, cycloalkyl with 4-7 carbon atoms, phenylalkyl with 7-9 carbon atoms or cycloalkyl (alkylene) with 5-10 carbon atoms; 3 9 9 9 R is hydroxyl, amino, -NHR , -NHCOR , -OR or 9 9 -OCOR, where R is alkyl of 1-4 carbon atoms; and R<1> is (a) -A-E-B, where A is lysine or proline, or furthermore, only when R 3 is amino, isoleucine, E can be phenylalanine, glycine, alanine, valine, leucine, isoleucine, lysine, ornithine, arginine, aspartic acid, gamma-esterified aspartic acid, glutamic acid or delta -4 4 5 esterified glutamic acid, B can be -OR , -NR R , -glutamine-4 4 4 5 acid(OR )~, -glutamic acid(-OR )(NR R ) or -glutamic acid-4 5 4 5 (NR R )2 , and R and R can each be hydrogen, alkyl with 1-4 carbon atoms, phenylalkyl with 7-9 carbon atoms or cycloalkyl (alkyl) with 5-10 carbon atoms, where X may be missing or be alanine, isoleucine, lysine, proline, ornithine, arginine, N-(alkyl with 1-4 carbon atoms)-lysine, N,N-di-(alkyl with 1-4 carbon atoms)-lysine, N- (alkyl of 1-4 carbon atoms) -ornithine or N,N-di(alkyl of 1-4 carbon atoms)-ornithine, Z and Z 1 are each hydrogen, alkyl of 1-6 carbon atoms, cycloalkyl of 4-7 carbon atoms, cycloalkyl( alkylene) with 5-10 carbon atoms or phenylalkyl with 7-9 carbon atoms, n and m are both integers from 0 to 6, Q is sr sr - i Y is methyl, phenyl, -COOR , -CONR R , -NH~ , (benzyloxy)-carbonylamino, -CO-glutamic acid(-0R 6 )~, -CO-glutamic acid(-OR 6 )-6 7 6 7 6 7 (NR R ) or -CO-glutamic acid(-NR R )2, and R and R are each hydrogen, alkyl of 1-4 carbon atoms, phenylalkyl of 7-9 carbon atoms or cycloalkyl(alkylene) of 5-10 carbon atoms, characterized by the following steps: (A) a compound of formula 12 3 1 or an acid addition salt thereof, where R , R , R and W are as indicated above and all possible reactive groups in 1 3 R and R are protected, acylated with a compound of formula R-W-OX, where -OX represents a carboxyl moiety that is activated for acylation purposes; (B) the optional protecting groups in R 1 and R 3 are removed; and (C) the resulting compound of formula I is optionally converted to a pharmaceutically acceptable salt. 2. Method according to claim 1, characterized in that R is an amino-protecting acyl part with a molecular weight of less than 500 or an amino-protected proline, W is phenylalanine, W 1 is histidine and W and W 1 are linked through a peptide bond. 3. Method according to claim 2, characterized 2 3 in that R is isobutyl and R is hydroxyl. 4. Process according to claim 2, characterized in that R 2 is cyclohexyl (methylene) and R <3> is hydroxyl. 5. Method according to claim 3 or 4, characterized in that R is -lysine-E-B. 6. Method according to claim 5, characterized in that E is phenylalanine and B is hydroxyl. 7. Method according to claim 6, characterized in that R is t-butyloxycarbonyl. 8. Method according to claim 6, characterized in that R is N-(t-butyloxycarbonyl)-proline. 9. Method according to claim 3 or 4, characterized in that R i is 10. Method according to claim 9, characterized in that X is not missing. 11. Method according to claim 10, characterized in that X is selected from alanine, proline, isoleucine and lysine. 12. Method according to claim 11, characterized in that Z is isobutyl, OH Q is -C' H- , Z 1 is hydrogen and n and m are both zero. 13. Method according to claim 12, characterized 6 6 7 in that Y is selected from -COOR , -CONR R , -CO-glutamic acid- f - f \ ft 7 (-OR )„, -CO-glutamic acid(-OR )(NR R ) and -CO-glutamic acid-(-nr6r') 2. 14. Method according to claim 11, characterized in that Z and 2? both are hydrogen, Q is -CH-, the sum of n and m is from 0 to 3 and Y is -COOR or -CONR R . 15. Method according to claims 12-14, characterized in that X is lysine. 16. Method according to claim 9, characterized in that X is missing. 17. Method according to claim 16, characterized in that Z is hydrogen, Q is -CH,,-, Y is -COOR , -CONR R , fi 6 6 7 -CO-glutamic acid(-OR ) -CO-glutamic acid(-OR )(NR R ) or -CO-glutamic acid(-NR R )2 , Z is alkyl with 1-6 carbon atoms and the sum of n and m is from 0 to 3. 18. Method according to claim 16, characterized in that Q is -NH-, m is zero, n is 0 or 1, Y is -COOR, 6 7 6 6 -CONR R , -CO-glutamic acid(-OR )9, -CO-glutamic acid(-OR )- 6 7 6 7 (-NR R ) or CO-glutamic acid (-NR R ) 2 and Z is alkyl with 1-6 carbon atoms. 19 Method according to claim 17 or 18, characterized by atYer -COOR or -CONR <6> R <7> . 20. Method according to claim 18, characterized in that Z <1> is benzyl. 21. Method according to claim 17 or 18, characterized by atZer sec-butyl. 22. Method according to claim 2, characterized in that R has the formula or where M is -0-, -CH.,-, -NH- g or -SO2 NH-, and R is alkyl with 1-6 carbon atoms, phenyl, phenylalkyl with 7-9 carbon atoms or cycloalkyl(alkyl) with 5-10 carbon atoms. 23. Method according to claim 22, characterized in that R is N-(t-butyloxycarbonyl)-proline.