WO1993009134A1 - Phosphonamidate ester-containing pseudopeptides - Google Patents

Abstract

A 'capped' phosphonamide C1-C6 alkyl ester linkage unit for joining peptides is disclosed. The linkage unit is substantially isosteric and isocoulombic as compared to conventional peptide backbone structures. The 'capping' of the phosphonamide by the C1-C6 alkyl ester causes the linkage unit to be acid stable. When incorporated into a peptide, the phosphonamide C1-C6 alkyl ester linkage unit becomes resistant to cleavage by aspartic proteinase. Accordingly, the phosphonamide C1-C6 alkyl ester linkage unit enhances and/or facilitates the aspartic proteinase inhibition activity of peptides into which it is incorporated.

Description

Description

PHOSPHONAMIDATE ESTER-CONTAINING PSEUDOPEPTIDES Technical Field

The present invention relates to linkage units for joining peptide sequences and to the use of such linkage units for forming pseudopeptides, including pseudopeptides that inhibit aspartic proteinase enzymes. More

particularly, the invention relates to pseudopeptides that include a "capped" phosphonamidate ester linkage which is acid stable and which is substantially isosteric and isocoulombic with respect to conventional peptides and to the use of such "capped" phosphonamidate ester linkage units in place of amide bonds at the position in a peptide sequence that is cleavable by aspartic proteinase enzymes.

Background Art

Aspartic proteinase enzymes (EC 3.4.23] are a family of related enzymes that cleave (hydrolyze) protein and polypeptide chains. These enzymes have isoelectric points on the acid side of neutrality and molecule masses ranging from 35,000-45,000 Daltons (D) for fungal enzymes and about 35,500 D for pepsin.

Exemplary enzymes of this class include pepsin that is a mammalian gastric proteinase, cathepsin D that is the intracellular aspartic proteinase of the lysosomal system and whose level has been positively correlated with

recurring breast cancers [Tandon et al., N. Eng. J. Med., 322:297 (1990)] and with amyloid formation in Alzheimer's plagues [Cataldo et al., Brain Res., 513:181 (1991)], renin that regulates blood pressure by its cleavage of

angiotensinogen to form angiotensin I, and chymosin

(formerly called rennin) that cleaves milk proteins as a first step in cheese making. Penicillopepsin, a microbial enzyme from P. janthinellum is another member of this family, whereas nepenthesin, the digestive proteinase of the pitcher plant is exemplary of the plant aspartic proteinases.

Although the precise mechanism of action of this family of enzymes is not as well known as that of the serine proteinases, it is believed that two aspartic acid groups act with water in the active site to cause

hydrolysis of the peptide bond that is hydrolyzed. The hydrolyzed peptide bond is typically between hydrophobic residues. A covalent intermediate is not thought to be formed between this enzyme and its substrate as is the case with the serine proteinase family.

This family of enzymes forms an enzyme-substrate complex as is typical in enzyme-substrate reactions.

Binding is often found to be a two-step process even through no covalent bonds are formed.

Several peptide and peptidomimetic compounds have been reported in the literature that inhibit the action of aspartic proteinases. Exemplary discussions of such inhibitors are found in Rich, Proteinase Inhibitors,

Chapter 5, Volume 12, Barrett and Salveson eds., Dingle and Gordon general eds., Elsevier Science Publishers BV,

Amsterdam (1986), and Rich, Peptide Inhibitors.

Comprehensive Medicinal Chemistry, Chapter 8.2, Sammes, ed., Pergamon Press, Oxford, Volume 2 (1990).

Several inhibitors include polypeptides similar in sequence to a natural substrate of an enzyme that also include one or more D-amino acids in place of the naturally occurring L-amino acids. Another group of inhibitors contains the surrogate (3S,4R)-4-amino-3-hydroxy-6-methylheptanoic acid, designated AHMHA or statine (Sta), in place of the two residues between which the hydrolysis occurs, such as Leu and Ala. The statine-containing group of inhibitors were first found in the naturally occurring inhibitor known as pepstatin A that inhibits each of pepsin cathepsin D with a K_i value of about 10^-10-10^-11 M and renin with a K_i value of 10^-6 M.

Yet another group of inhibitors, reported in Luby et al., J. Med. Chem., 31:532 (1988), utilized an oligopeptide analog of angiotensinogen in which the cleavable Leu-Val dipeptide was replaced with an iso-propylsulfidoethanol derivative surrogate. Roberts et al., J. Med. Chem.,

33:2326 (1990) reported using 1,2,4-triazolo[4,3-a]pyrazine derivatives as surrogates to replace the amino-terminal three residues adjacent to the leucine of the cleaved angiotensinogen Leu-Val bond in renin inhibitory molecules.

In another recent paper, [Bartlett et al., J. Org.

Chem., 55:6268 (1990)] phosphonate-, phosphinate- and phosphinamide-containing pseudopeptide inhibitors of pepsin and penicillopepsin were reported. Those inhibitors were pseudopeptides that included a phosphorus-containing bond in place of the scissile amide bond that would normally be cleaved by those enzymes.

A phosphonate group has the linkage -P(O)(OH)O-, in which the shown valence of the phosphorus atom is bonded to a carbon and takes the place of an amide carbonyl group, and the free valence of the oxygen is bonded to a carbon atom, taking the place of the amido -NH- group. A

phosphinate group has the linkage -P(O) (OH)-, so that the phosphorus atom is bonded to two carbon atoms. A

phosphinamide has the linkage -P(O)(NH₂)- in which the -NH₂ group replaces the -OH of a phosphinate.

As can be seen from the above linkages, the

phosphonate- and phosphinate-containing compounds having pK_a values of about 1.5 and 3.0, respectively, would be

expected to bear an anionic charge at physiological pH values, e.g. pH 7.2-7.4. Bartlett et al., above, also disclosed preparation of compounds containing a phosphonate ester linkage [-P(O)(OCH₃)O-] as intermediates in the preparation of the phosphonate derivatives, but reported no inhibition data using those esters that were cleaved to form the assayed phosphonates.

Disclosure of Invention

The present invention contemplates a linkage unit for joining two peptide sequences, i.e. an amino terminal peptide sequence and a caiboxyl terminal peptide sequence. The linkage unit includes a "capped" surrogate amino acid residue. The "capped" surrogate amino acid residue has a backbone substantially isosteric with a peptide backbone. However, the backbone of the "capped" surrogate amino acid residue lacks a carbonyl group. Instead, "capped"

surrogate amino acid residue includes a -P(O)(OR)- group which substitutes for the lacking carbonyl group. "R" may be a C₁-C₆ alkyl group, but is preferably a methyl group. Furthermore, the "capped" surrogate amino acid residue lacks an ability to form a backbone peptide bond with the amino end of the carboxyl terminal peptide sequence.

Instead, the "capped surrogate amino acid residue forms a phosphonamide C₁-C₆ alkyl ester linkage with the amino end of the carboxyl terminal peptide sequence, i.e. -P(O)(OR)-N(H)-. The phosphonamide C₁-C₆ alkyl ester linkage is substantially acid stable.

The invention also contemplates an improved dipeptide or oligopeptide having a "capped" surrogate amino acid residue as described above and a second amino acid residue. The capped surrogate amino acid residue forming a

phosphonamide C₁-C₆ alkyl ester linkage with the amino end of the second amino acid residue, i.e. -P(O)(OR)-N(H)-.

Alternatively, the oligopeptide or polypeptide may include an amino terminal peptide sequence, a carboxyl terminal peptide sequence, and a "capped" surrogate amino acid residue as described above. The "capped surrogate amino acid residue joins the amino terminal peptide sequence and the carboxyl terminal peptide sequence. The amino terminal peptide sequence may be linked to the "capped" surrogate amino acid residue by means of a conventional peptide bond. However, the amino end of the carboxyl terminal peptide sequence is linked to the "capped" surrogate amino acid residue by means of the phosphonamide C₁-C₆ alkyl ester linkage described above.

The invention also contemplates a method for linking a "capped" surrogate amino acid residue with a second amino acid residue or to an amino end of a carboxyl terminal peptide sequence. The method includes a step for linking the amino end of the second amino acid residue (or the amino end of a carboxyl terminal peptide sequence) to the capped surrogate amino acid by means of a phosphonamide C₁-C₆ alkyl ester linkage. Alternatively, the method may also include a step for linking the "capped" surrogate amino acid residue or linkage unit to the carboxyl end of the amino terminal peptide sequence by means of a

conventional peptide bond.

The present invention also contemplates an inhibitor for an aspartic proteinase enzyme. The inhibitor is a pseudopeptide analog that includes a phosphonamidate ester linkage in place of the amide bond at the position in the pseudopeptide sequence that is cleaved by the enzyme.

Thus, an oligopeptide having a peptide bond at the position occupied by the phosphonamidate ester linkage of an

inhibitor of the invention is cleaved by an aspartic proteinase.

A pseudopeptide aspartic proteinase inhibitor has a length of 4 to about 15 amino acid residues, preferably 4 to about 10 residues, and contains a P₁ to P₁' bond that is constituted by a phosphonamidate C₁-C₆ alkyl ester in which the phosphorus atom is bonded to P₁ in place of the carbonyl carbon atom of a peptide bond. The C₁-C₆ alkyl group is preferably methyl (C₁). A particularly preferred pseudopeptide of the

invention can be represented by the formula

R¹-X₁-XaaΨ[P(O)(OC₁-C₆ alkyl)NH]X₂-Z wherein X₁ is an amino acid residue or oligopeptide containing a sequence of up to about ten amino acid

residues;

Xaa is a surrogate amino acid residue having an amino acid side chain;

X₂ is an amino acid residue or oligopeptide containing a sequence of up to about ten amino acid residues;

Ψ[P(O) (OC₁-C₆ alkyl)NH] represents the phosphonamide C₁-C₆ alkyl ester bond between X_aa and X₂. Note that the -P(O) (OC₁-C₆ alkyl)- portion of the bond belongs to X₁ and constitutes a substitution for a carbonyl group. The -NH- portion of the bond belongs to X₂ and constitutes the amino end of that amino acid residue or oligopeptide;

Z is selected from the group consisting of NH₂, NH-C₁-C₆ acyl, OH, O-C₁-C₆ alkyl and 2-amidoindanol; and

R¹ is selected from the group consisting of hydrogen, C₁-C₆ acyl, trifluoroacetyl and t-BOC.

The pseudopeptide has the length of 4 to about 15 amino acid residues.

In preferred practice the pseudopeptide has a length of 4 to about 10 amino acid residues. The C₁-C₆ alkyl group of the phosphonamide ester is preferably methyl. A

pseudopeptide preferably competitively inhibits the in vitro activity of an aspartyl proteinase with an inhibition constant of about 10^-6 to about 10^-11 M, and more preferably about 10^-8 to about 10^-10 M.

In preferred practice, Xaa has the side chain of a

Leu, Tyr or Phe residue, and the amino-terminal residue of X₂ is Tyr, Leu, Val, Met, Pro, Ala or Phe.

A pharmaceutical composition that contains an above pseudopeptide in an amount sufficient to inhibit an

aspartic proteinase dissolved or dispersed in a physiologically tolerable carrier or diluent is also contemplated.

Further contemplated is a method for inhibiting an aspartic proteinase. Here, an above composition is admixed in an aqueous medium with the enzyme and a substrate for the enzyme to form an inhibition mixture. The inhibition mixture so formed is maintained for a time sufficient for the aspartic proteinase activity of the enzyme to be inhibited.

Definitions

A compound of the invention is depicted using the "psi bracket" (Ψ [ ] ) nomenclature for oligopeptide analogs having backbone modification described in Spatola, Chemistry and Biochemistry of: Amino Acids, Peptides and Proteins,

Weinstein, ed., Volume 7, chapter 5, Marcel Dekker, Inc., New York, pages 267-357 (1983). In accordance with that nomenclature:

(a) hyphens between amino acid residues indicate the presence of a peptide bond joining the residues;

(b) absence of a hyphen, combined with the symbol ψ (psi) indicates the removal of the amide, peptide bond elements leaving the α-carbon and its side chain;

( c) the presence of brackets , [ ] , adj acent the ψ symbol and between residues, coupled with a structural group within the brackets indicates that the specified structural group within the brackets replaces the peptidyl amide bond;

(d) the word "surrogate" refers to an unnatural replacement for a naturally occurring entity, so that a psi-bracketed structural group is a surrogate for the peptidyl amide bond as is the residue containing the bond surrogate a surrogate for an amino acid residue;

(e) the term "pseudopeptide" refers to a peptide analog having a peptide backbone modification; (f) a "pseudodipeptide" is a modified dipeptide structural unit that contains a surrogate bond(s) or amino acid residue(s); and

(g) hyphens at pseudopeptide termini, not being part of the pseudopeptide backbone, merely refer to bonds; and

(h) substituents bonded to an atom within brackets are parenthesized.

A compound of this invention contains a pentavalent, tetrahedral phosphorus atom as part of a phosphonamidate C₁- C₆ ester surrogate for a peptidyl amide bond. That linkage is thus written Ψ[P(O) (OC₁-C₆ alkyl)NH] following the convention of parenthesizing carbonyj. oxygen atoms when depicted in single line notation.

The term "oligopeptide" is used in its usual sense to mean a peptide containing ten or fewer amino acid residues. Similarly, the term "oligopseudopeptide" refers to a pseudopeptide containing ten or fewer amino acid residues and surrogates therefor.

Another nomenclature system used herein is that of Schechter and Berger, Biochem. Biophys. Res. Commun.,

27:157 (1967) that was developed for describing peptide substrates for hydrolase enzymes. In accordance with that nomenclature system, a peptide substrate for a hydrolase enzyme is numbered in two directions from the point of hydrolytic cleavage. The amino acid residues of the

dipeptide portion that is cleaved are numbered P₁ and P₁ ' such that after cleavage, the P₁ residue becomes the

carboxy-terminal residue of one cleaved portion and the P₁' residue becomes the amino-terminal residue of the other portion. The remaining residues toward the carboxy-terminus of the P₁' -containing portion are numbered P₂', P₃' , P₄' ... and so on toward the carboxy-terminus. The

remaining residues of the portion containing the P₁ residue are numbered toward the amino-terminus of that portion as P₂, P₃, P₄ and so on. The dipeptide portion of a hydrolase substrate

oligopeptide that is cleaved is thus defined as P₁-P₁'. The Schechter and Berger nomenclature system is utilized whether or not the bond linking the P₁ and P₁' residues is a peptide bond or is capable of hydrolytic cleavage, and is therefore useful with pseudopeptides where P₁-P₁'

constitutes the before-defined pseudodipeptide.

The Schechter and Berger nomenclature system therefore not only identifies the bond normally cleaved, but also identifies the positions of residues on either side thereof regardless of whether the compound is an oligopeptide or a pseudopeptide. Following that nomenclature, residues P¹ and P₁' of a contemplated pseudopeptide described hereinafter are joined by the phosphonamide C₁-C₆ alkyl ester group.

Best Mode for Carrying Out the Invention

I. Background

The present invention relates to pseudopeptide

compounds that reversibly bind to and competitively inhibit the activity of an aspartic proteinase. A contemplated oligopeptide analog inhibits that enzymatic activity in the presence of a substrate for the enzyme and is therefore a competitive inhibitor.

A compound contemplated herein is referred to as a pseudopeptide because although most of the subunit amino acids are linked by peptidyl amide bonds, two such residues are linked by a phosphonamidate C₁-C₆ ester. Compounds containing peptide bonds and other bonds linking moieties having amino acid side chains are also sometimes referred to as peptidomimetic compounds.

A contemplated pseudopeptide reversibly binds to an aspartic proteinase to form an enzyme-inhibitor complex. Such binding and complex formation are familiar to those skilled in enzyme kinetics, and are to be distinguished from the interactions of materials that irreversibly bind to and react with an enzyme that are sometimes referred to as "suicide inhibitors". Thus, a contemplated

pseudopeptide binds to (or is bound by) an aspartic

proteinase, but does not chemically react with the enzyme.

Phosphonamidate-containing compounds have been used as transition state analog inhibitors for metallopeptidases such as carboxypeptidase A, thermolysin and angiotensin converting enzyme (ACE). See, for example Rich, Peptidase Inhibitors. Comprehensive Medicinal Chemistry, Chapter 8.2, Sammes, ed., Pergamon Press, Oxford, Volume 2 (1990). In addition, a number of phosphonamidate S- and O-esters have been investigated as irreversible phosphorylating agents of serine proteases. See, for example, Sampson et al.,

Biochem, 30:2255 (1991); Oleksyszyn et al., Biochem.

Biophys Res. Comm., 161:143 (1989); and Pratt, Science,

246:917 (1989); and Bartlett et al., Bioorg. Chem, 14:356 (1986).

Metallopeptidases and serine proteases act on their substrates in a different manner than do aspartic

proteinases. In addition, the non-esterified

phosphonamidates are unstable under the acid pH conditions at which aspartic proteinase enzymes act and are therefore poor candidates for use in inhibitors. The phosphonamidate esters contemplated, herein are stable at the acidic pH values at which aspartic proteinases act.

One strategy utilized in preparing aspartic proteinase inhibitors is to replace the scissile P₁-P₁' peptide bond with a non-hydrolyzable bond surrogate that is an isostere for the tetrahedral carbon atom of the transition state for amide bond hydrolysis. The previously noted phosphinic and phosphonic acid pseudopeptide derivatives described in Bartlett et al., J. Org. Chem., 55: 6268 (1990) and statine group of pepstatin A fulfill that function.

A phosphonamidate ester linkage of a compound

described herein also can be viewed as an isostere of an amide hydrolysis transition state. In addition, a

phosphonamidate ester linkage retains an important

recognition element, the nitrogen atom, of the scissile amide bond, as well as an extra hydrogen for hydrogen bonding, except where proline occupies position P₁'. A phosphonamidate ester is not susceptible to acid

hydrolysis. A phosphonamidate ester group contemplated herein is also electrically neutral (is free from ionic charge) at pH values encountered in living organisms and as such contributes to passage of an pseudopeptide through cell membranes.

II. The Pseudopeptides

A contemplated pseudopeptide aspartic proteinase inhibitor can have a length of 4 to about 15, and more preferably 4 to about 10, amino acid residues and has a P₁ to P₁' bond surrogate that is constituted by a

phosphonamidate C₁-C₆ alkyl ester in which the phosphorus atom is bonded to a carbon at P₁ in place of the carbonyl carbon atom of a peptide bond. The C₁-C₆ alkyl ester is preferably methyl (C₁).

Such an inhibitor typically inhibits the in vitro activity of an aspartic proteinase with an inhibition constant, K_i, of about 10^-6 to about 10^-11 M. The inhibition constant is readily ascertained in the presence of a usual, native substrate for the enzyme as discussed hereinafter.

The residue length of a contemplated inhibitor is determined as if the P₁-P₁' positions were linked by a peptide bond. The P₁ position surrogate residue is thus considered for this purpose to be an amino acid residue even though the carboxyl group normally present is replaced by a tetrahedral phosphorus-containing moiety.

A particularly preferred contemplated pseudopeptide has the formula

R¹-X₁-XaaΨ[P(O) (OC₁-C₆ alkyl)NH]X₂-Z wherein X₁ is an amino acid residue or oligopeptide containing a sequence of up to about ten amino acid

residues;

Xaa is a surrogate amino acid residue having an amino acid side chain;

X₂ is an amino acid residue or oligopeptide containing a sequence of up to about ten amino acid residues;

Z is selected from the group consisting of NH₂, NH-C₁-C₆ acyl, OH, O-C₁-C₆ alkyl and 2-amidoindanol; and

R¹ is selected from the group consisting of hydrogen, C₁-C₆ acyl, trifluoroacetyl and t-BOC.

A particularly preferred pseudopeptide has a length of 4 to about 15 amino acid residues, and competitively

inhibits the in vitro activity of the aspartyl proteinase with an inhibition constant of about 10^-8 to about 10^-10 M.

Exemplary C₁-C₆ acyl groups of R¹ and Z include formyl, acetyl, propionyl, butanoyl, iso-butanoyl, isovaleryl

(Iva), hexanoyl, cyclopentylcarbonyl and the like. A t-BOC group is a tertiary-butyloxy carbonyl group as is often used as a protecting group in solid phase peptide

syntheses, and is utilized herein bonded to an ammo-terminal amine or ∈-amino group of a lysine residue.

Exemplary C₁-C₆ alkyl groups of Z or the C₁-C₆ alkyl ester portion of the phosphonamide ester include methyl, ethyl, propyl, iso-propyl, butyl, iso-butyl, sec-butyl, tert-butyl, pentyl, 2-methylbutyl, cyclopentyl, hexyl,

cyclohexyl, and the like. Methyl is a preferred C₁-C₆ alkyl ester group.

The group Xaa is a surrogate amino acid residue having the side chain of an amino acid. Although Xaa can have any side chain of one of the twenty naturally occurring amino acids, Xaa preferably has the side chain of a Leu, Tyr or Phe amino acid. The Xaa group is in the P₁ position of a contemplated pseudopeptide.

Each of X₁ and X₂ can be an amino acid residue or an oligopeptide of up to about ten, and preferably up to about five amino acid residues. The sequences of X₁ and X₂ can contain any amino acid residues. Preferably, the amino-terminal residue of X₂ (the P₁' position residue) is

selected from the group consisting of Tyr, Leu, Val, Met, Pro, Ala and Phe.

The overall length of a pseαdopeptide, aside from an R¹ or Z group, is 4 to about 15 amino acid residues, and more preferably 4 to about 10 amino acid residues. Thus, although each of X₁ and X₂ can separately be up to about 10 amino acid residues in length, both cannot include 10 amino acid residues .

Nevertheless, X₁ can include 10 amino acid residues (occupying positions P₂-P₁₁ of the sequence) and X₂ can include four residues (occupying positions P₁'-P₄' of the sequence). X₂ can similarly include 10 residues with X₁ including 4 residues.

Thus, although any amino acid residue can be present in X₁ and X₂, reactive side chains such as the mercaptan of cysteine, carboxyl groups such as those of aspartic and glutamic acids, and the ∈-amino group of a lysine are typically absent from X₁ and X₂ sequences, and from Xaa group side chains. Those reactive side chains are also relatively hydrophilic. This absence of hydrophilic side chains is also a function of the active site of this family of enzymes exhibiting a preference for relatively

hydrophobic side chains, particularly for the P₁ and P₁' positions.

An amino acid residue of X₁ and X₂ or side chain of Xaa can be present in an oligopeptide analog sequence in either a D- or L-configuration, as is exemplified below wherein all residues are in the L-configuration unless preceded by a "D-". Not only are both D- and L-amino acid residues contemplated for use in an oligopeptide analog, but

modified and unusual amino acid residues, amines and

carboxylic acids are also contemplated, particularly at or adjacent the amino- and carboxy-termini of an pseudopeptide.

For example, a 2,2-diethylglycine or

2,2-dimethylglycine residue can be present at either or both termini, or a 2-aminoindanol can be amide-bonded to a carboxy-terminal residue to form a 2-amidoindanol group. Similarly, a C₁-C₆ acyl group as discussed previously such as 3-methylbutanoyl (isovaleryl; Iva) can be useful at the amino-terminus, whereas 3-methylbutylamine reacted at the carboxy-terminus to form a 3-methylbutylamide

(isoamylamide; Iaa) can also be useful as can an

aminovalaric acid forming a amylamide (Avl).

The above terminal substituent groups and modified amino acid residues serve at least two functions. First, their presence removes ionic charge from the pseudopeptide to help facilitate passage through membranes. Second, they help protect the pseudopeptide from degradation by other proteinase and peptidase enzymes such as trypsin and carboxypeptidase A that are present in vivo and can

otherwise cleave a pseudopeptide.

A contemplated pseudopeptide binds to an aspartic proteinase and inhibits the in vitro activity of the enzyme with an inhibitory constant, K_i, of about 10^-6 to about 10^-11, and preferably about 10^-8 to about 10^-10, in the presence of a usually assayed native substrate. The sequence of a contemplated pseudopeptide is that of an oligopeptide or oligopeptide analog that is reversibly bound by a given aspartic proteinase, except for the phosphonamidate ester linkage as is illustrated before.

The K_i value of a contemplated inhibitor can also be viewed relative to the dissociation constant of a usual, native substrate for the enzyme, as is angiotensinogen for renin. A contemplated inhibitor binds to its aspartic proteinase about 10⁵ to about 10⁶ times more tightly than does the usual, native substrate. Thus, where the

dissociation constant for the enzyme and its native substrate is about 10^-3, the dissociation constant for the same enzyme and a contemplated inhibitor is about 10^-8 to about 10^-9.

Although the mechanisms of action of the aspartic proteinase family of enzymes are substantially identical, the enzymes exhibit different binding and cleaving

properties with different substrates and inhibitors. Thus, different inhibitor sequences are utilized with different members of the aspartyl proteinase family. Each of those different sequences nevertheless contains a phosphonamidate C₁-C₆ ester link between the P₁ and P₁' residues of the inhibitor.

Table 1 below lists illustrative members of the aspartic proteinase enzyme family and exemplary inhibitors for each listed enzyme.

TABLE 1

Enzymes and Inhibitors

Cathepsin D

t-BOC-D-Phe-Pro-PheΨ[P(O)(OCH₃)NH]Phe-Val-D-Trp

(SEQ ID NO:1)

t-BOC-D-Phe-Pro-PheΨ[P(O) (OCH₃)NH]Phe-Avl

(SEQ ID NO:2)

Gly-Phe-Leu-Gly-Pheψ[P(O) (OC₂H₅)NH]Leu

(SEQ ID NO:3)

Gly-D-Phe-Leu-Gly-PheΨ[P(O) (OⁱC₃H₇)NH]Leu^*

(SEQ ID NO:4) Gly-Phe-D-Leu-Gly-PheΨ[P(O) (OC₆H₁₃)NH]Leu

(SEQ ID NO: 5)

Gly-Phe-Leu-Gly-D-PheΨ[P(O) (OⁱC₄H₉)NH]Leu^**

(SEQ ID NO: 6)

Renin

His-Pro-Phe-His-LeuΨ[P(O) (OCH₃)NH]Val-Ile-His

(SEQ ID NO: 7) t-BOC-His-Pro-Phe-His-LeuΨ[P(O) (OCH₃)NH]Val-Ile-His

(SEQ ID NO: 8)

Pro-His-Pro-Phe-His-Pheψ[P(O) (OCH₃)NH]Phe-Val-Tyr-Lys

(SEQ ID NO: 9)

Arg-Arg-Pro-Phe-His-LeuΨ[P(O) (OCH₃)NH]Val-Ile-His- Lys(t-BOC)-OCH₃

(SEQ ID NO: 10)

His-Pro-Phe-His-LeuΨ[P(O) (OCH₃)NH]Leu-Val-Tyr

(SEQ ID NO: 11)

His-Pro-Phe-His-PheΨ[ (O) (OCH₃)NH]Phe-Val-Tyr

(SEQ ID NO: 12)

Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His- Leuψ[P(O) (OCH₃)NH]Val-Ile-His

(SEQ ID NO: 13)

Chymosin

CH₃C(O)-Leu-Ser-PheΨ[P(O) (OCH₃)NH]Met-Ala-Ile-Pro- Pro-Lys-Lys

(SEQ ID NO: 14)

CH₃C(O)-Val-Val-LeuΨ[P(O) (OCH₃)NH]Ala-Leu

(SEQ ID NO: 15) Penicillopepsin

Iva-Val-Val-Leuψ[P(O) (OCH₃)NH]Phe-OCH₃

(SEQ ID NO: 16) Iva-Val-Val-Leuψ[P(O) (OCH₃)NH]Phe-Ala-Ala-OCH₃

(SEQ ID NO: 17)

Pepsin

Leu-Val-homoArg-Val-Pro-Leuψ[P(O)(OCH₃)NH]Val-Arg- homoArg-homoArg-Ser-Leu-Arg-Gln-Leu-Ile

(SEQ ID NO: 18) CH₃C(O)-Val-Val-Leuψ[P(O)(OCH₃)NH]Ala-Ala-Leu (SEQ ID NO:19)

Val-Val-LeuΨ[P(O) (OCH₃)NH]Ala-Ala-Leu

(SEQ ID NO: 20)

Iva-Val-LeuΨ[P(0) (OCH₃)NH]Ala-Ala-Iaa

(SEQ ID NO: 21)

Iva-Val-Val-LeuΨ[P(O) (OCH₃)NH]Ala-Ala-OEt

(SEQ ID NO: 22)

* ⁱC₃H₇ = iso-propyl

** ^lC₄H₉ = tertiary-butyl

III. Pseudopeptide Svntheses

Synthesis of a contemplated pseudopeptide is

relatively straightforward inasmuch as most of the

molecule's length is constituted by oligopeptides. One preferred method utilizes a first preprepared oligopeptide for the P₂-containing portion (X₁-Xaa) and a second

preprepared oligopeptide for the P₁'-containing portion (X₂). Those two portions are then joined to the phosphorus-containing segment to form the molecule.

An exemplary synthesis for a Cathepsin D

oligopseudopeptide inhibitor having the sequence

t-BOC-D-Phe-Pro-PheΨ[P(O)(OCH₃)NH]Phe-Val-D-Trp

(SEQ ID NO:1)

is provided below.

Scheme

As is shown in Scheme 1, above, benzyl aldehyde

(Compound 1) and dibenzylamine (Compound 2) are reacted with dimethyl phosphite (Compound 3) to form Compound 4. Catalytic reduction with hydrogen in the presence of palladium on charcoal (Pd/C) followed by reaction with trifluoroacetic anhydride provides Compound 5. Fields, J. Am. Chem. Soc., 74:1528 (1952).

Treatment of Compound 5 with phosphorus pentachloride (1.5 equivalents of PCl₅ in CHCl₃ at about 45°C) and then with prepared tripeptide Phe-Val-D-Trp (about 2 equivalents as the hydrochloride salt with about 3 equivalents of triethylamine) provides the pseudopeptide

PheΨ[P(O)(OCH₃)NH]Phe-Val-D-Trp, Compound 6. Reaction of Compound 6 with sodium borohydride (NaBK₄) followed by coupling of the previously prepared peptide t-BOC-D-Phe-Pro with dicyclohexylcarbodiimide (DCC) provides the desired pseudopeptide, Compound 7.

Where a phosphonamidate ester other than methyl is desired. Compound 5 of Scheme 1 can be reacted first with trimethylsilyl bromide (TMSBr in CH₂Cl₂ at about 40°C) and then oxalyl chloride (neat at about 45°C) to form Compound 8. Reaction of Compound 8 with a C₁-C₆ alkyl alcohol (about 2 equivalents of alcohol and about 3 equivalents of

triethylamine in CH₂Cl₂) provides the phosphonate diester Compound 9 of that C₁-C₆ alkyl alcohol. Compound 9 can then be used as was Compound 5 in the synthesis of a desired pseudopeptide. These reactions are illustrated in Scheme 2, below, wherein X-OH is the C₁-C₆ alkyl alcohol. Scheme 2

The above phosphorus-containing pseudopeptide

compounds are diastereomers due to the stereochemistry of the exemplary benzyl group shown as a wavy line (Xaa is the phenylalanine side chain) and the stereochemistry about the phosphorus atom. Where an optically pure pseudopeptide is desired, Compound 5 can be reacted with NaBH₄ to remove the trifluoroacetyl group and the resulting amine resolved using any appropriate resolving agent such as R-tartaric acid. Further reaction to form an pseudopeptide provides a set of enantiomers due to the chirality of the tetrahedral phosphorus of the phosphonamidate ester. Those enantiomers can be resolved as discussed below.

A completed pseudopeptide synthesized as described above can also be separated into diastereomers and then resolved. Here, the illustrated t-BOC group is removed, and a salt is formed with R-tartaric acid. The resulting diastereomers can be separated chromatographically and then resolved to enantiomeric purity. Resolution can also be achieved using a brucine salt of a carboxy-terminal

carboxylic acid.

The before-described preprepared oligopeptides can be prepared by any well known means. The solid phase techniques pioneered by Merrifield are preferred and are well known by those skilled in the art.

An R¹ group that is a C₁-C₆ acyl group can be added to the P₂-containing peptide portion (X₁-Xaa) while that peptide is on its synthesis resin. A trifluoroacetyl or t- BOC group is preferably added after cleavage of the peptide from its synthesis resin.

A Z group is preferably added to its P₁'-containing peptide portion (X₂) after the peptide is cleaved from the resin. Well known ester- and amide-forming reactions are used for addition of a Z group, and as such reactions are so well known, they will not be dealt, with herein.

IV. Pharmaceutical Compositions

A pharmaceutical composition is also contemplated that contains a before-described pseudopeptide of the invention as active agent dissolved or dispersed in a physiologically tolerable carrier or diluent. A pseudopeptide inhibitor is present in such a composition in an amount sufficient to inhibit the aspartic proteinase activity of a chosen aspartic proteinase (an effective inhibitory amount).

A pharmaceutical composition is prepared by any of the methods well known in the art of pharmacy all of which involve bringing into association the pseudopeptide active agent and the carrier therefor. For therapeutic use, a pseudopeptide utilized in the present invention can be administered in the form of conventional pharmaceutical compositions. Such compositions can be formulated so as to be suitable for oral or parenteral administration, or as suppositories. In these compositions, the agent is

typically dissolved or dispersed in a physiologically tolerable carrier or diluent.

A carrier or diluent is a material useful for

administering the active compound and must be

"pharmaceutically acceptable" in the sense of being

compatible with the other ingredients of the composition and not deleterious to the recipient thereof. Thus, as used herein, the phrases "physiologically tolerable" or "pharmaceutically acceptable" are used interchangeably and refer to molecular entities and compositions that do not produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a mammal. The physiologically tolerable carrier can take a wide variety of forms depending upon the preparation desired for administration and the intended route of administration.

As an example of a useful composit.ion, a compound of the invention (active agent) can be utilized, dissolved or dispersed in a liquid composition such as a sterile

suspension or solution, or as isotonic preparation

containing suitable preservatives. Particularly well-suited for the present purposes are injectable media constituted by aqueous injectable buffered or unbuffered isotonic and sterile saline or glucose solutions, as well as water alone, or an aqueous ethanol solution. Additional liquid forms in which these compounds can be incorporated for administration include flavored emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil, peanut oil, and the like, as well as elixirs and similar

pharmaceutical vehicles. Exemplary further liquid diluents can be found in Remmington's Pharmaceutical Sciences, Hack Publishing Co., Easton, PA (1980).

An active agent can also be administered in the form of liposomes. As is known in the art, liposomes are generally derived from phospholipids or other lipid

substances. Liposomes are formed by mono- or multilamellar hydrated liquid crystals that are dispersed in an aqueous medium. Any non-toxic, pharmaceutically acceptable and metabolizable lipid capable of forming liposomes can be used. The present compositions in liposome form can contain stabilizers, preservatives, excipients, and the like in addition to the agent. The preferred lipids are the phospholipids and the phosphatidyl cholines (lecithins), both natural and synthetic.

Methods of forming liposomes are known in the art. See, for example, Prescott, Ed., Methods in cell Biology, Vol. XIV, Academic press, New York, N.Y. (1976), p.33 et seq.

An active agent can also be used in compositions such as tablets or pills, preferably containing a unit dose of the compound. To this end, the agent (active ingredient) is mixed with conventional tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate, gums, or similar materials as non-toxic, physiologically tolerable carriers. The tablets or pills can be laminated or

otherwise compounded to provide unit dosage forms affording prolonged or delayed action.

It should be understood that in addition to the aforementioned carrier ingredients the pharmaceutical composition described herein can include, as appropriate, one or more additional carrier ingredients such as

diluents, buffers, flavoring agents, binders, surface active agents, thickeners, lubricants, preservatives

(including antioxidants) and the like, and substances included for the purpose of rendering the formulation isotonic with the blood of the intended recipient.

The tablets or pills can also be provided with an enteric layer in the form of an envelope that serves to resist disintegration in the stomach and permits the active ingredient to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, including polymeric acids or mixtures of such acids with such materials as shellac, shellac and cetyl alcohol, cellulose acetate phthalate, and the like. A particularly suitable enteric coating

comprises a styrene-maleic acid copolymer together with known materials that contribute to the enteric properties of the coating. Methods for producing enteric coated tablets are described in U.S. Patent 4,079,125 to Sipos, which is herein incorporated by reference.

The term "unit dose", as used herein, refers to physically discrete units suitable as unitary dosages for administration to warm blooded animals, each such unit containing a predetermined quantity of the agent calculated to produce the desired therapeutic effect in association with the pharmaceutically acceptable diluent. Examples of suitable unit dosage forms in accord with this invention are tablets, capsules, pills, powder packets, granules, wafers, cachets, teaspoonfuls, dropperfuls, ampules, vials, segregated multiples of any of the foregoing, and the like.

A pseudopeptide of the invention is present in such a pharmaceutical composition in an amount effective to achieve the desired inhibition. For example, where in vitro enzyme inhibition is the desired result, a compound of the invention can be utilized in an amount sufficient to provide a concentration of about 0.01 to about 2,000 nanomolar (nM) with enzyme concentration of about 1 nM to about 1 μM, and a substrate concentration of about 10 to about 2,000 micromolar (μM). The amounts of enzyme, substrate and inhibitor used are largely a function of convenience, with the substrate typically being in large excess over the enzyme (e.g. 100-10,00 fold excess). For in vivo use, an effective amount of a compound of the invention is about 0.1 to about 50 mg per kilogram of body weight or an amount sufficient to provide a concentration of about 0.01 to about 100 μg/mL to the bloodstream.

V. Methods

A method of inhibiting an aspartic proteinase is also contemplated. Here, a pharmaceutical composition as discussed before that contains an aspartic proteinase inhibiting amount of a before-discussed pseudopeptide is admixed in an aqueous medium with an aspartic proteinase in the presence of a substrate for the enzyme to form an inhibition mixture. The inhibition mixture is maintained for a time period sufficient for the inhibitor to inhibit the aspartic proteinase, typically five minutes to five hours.

When carried out in vitro, as where the binding properties or mechanism of action of the enzyme is studied, for example, the inhibition reaction is typically followed spectrophotometrically. The aqueous medium in such a case is typically a buffer solution. Exemplary in vitro

techniques are disclosed hereinafter.

When the inhibition reaction is carried out in vivo as where renin is the enzyme to be inhibited, the aqueous medium is constituted by a body fluid such as blood, lymph, stomach fluid or the like, and inhibition of the enzyme is assayed by a body function, as by blood pressure lowering for renin. An exemplary assay is discussed hereinafter.

VI. Assay Procedures

Assay procedures for aspartic proteinase enzymes are well known in the art. As a consequence, only a few such assays are described herein as exemplary.

A. In Vitro Pepsin Inhibition Assay

Porcine stomach mucosa pepsin (Sigma Chemical Co.) is chromatographically purified and is dissolved and diluted immediately prior to use in a 0.1 M sodium acetate buffer at pH 3.5. A useful substrate is the octapeptide analog:

Lys-Pro-Ala-Glu-Phe-p(NO₂)Phe-Arg-Leu (SEQ ID NO: 23) as discussed in Bartlett et al., J. Org. Chem., 55:6268

(1990). Initial rates of substrate hydrolysis are

determined by equilibrating substrate and pseudopeptide inhibitor solutions at 37°C in a cuvette for about three minutes, followed by initiation of the reaction by addition of 3.0 pmoles of pepsin per 1.00 mL final volume.

Substrate hydrolysis is measured by observing the decrease in absorbance at 310 nm. Initial rates are measured from 0.5 minutes until no more than 10 percent of the substrate is hydrolyzed. Inhibition constants are then determined by usual means.

B. In Vitro Renin Inhibition Assay

Purified human renin [Stein et al., J. Fed. Proc., Fed. Am. Soc. Exp. Biol., 44:1363 (1985)] is assayed using pure angiotensinogen [Dorer et al., Anal. Biochem., 87:11 (1978)] at pH 6.0 in maleate buffer. Inhibitors are dissolved in DMSO and diluted so that prior to addition of the assay system, the solutions contain 10 percent DMSO and 0.5 percent bovine serum albumin (BSA). The final

incubation mixture (100 μL) contains 0.135 M maleate buffer at pH 6.0, EDTA at 3 mM, phenylmethanesulfonyl fluoride at 1.4 mM, 0.21 μM angiotensinogen, 0.24 mGU [Bangham et al., Clin. Sci. Mole. Med., 48:1355 (1975)], BSA 0.44 percent and DMSO at 1 percent.

Several concentrations of inhibitor pseudopeptide that bracket the IC₅₀ value (the concentration that inhibits 50 percent activity) are preincubated with renin for about five minutes at 37°C. The substrate is then added and incubation is continued for about 10 minutes. The reaction is stopped by freezing the solution in a methanol/dry ice bath, and after thawing at 4°C, an aliquot is analyzed for angiotensin I by use of a commercial kit (NEN Research). The percent inhibition of the hydrolysis reaction is determined and an IC₅₀ value is calculated by regression analysis.

C. In Vivo Renin Inhibition Assay

The hypotensive activity of an inhibitor pseudopeptide in anesthetized, sodium depleted marmosets is used in this assay. The fall in mean arterial pressure (MAP) over a two-hour time period is used as the assayed value. An internal standard using a known hypotensive drug such as captopril (at 1.0 mg/kg, iv) or the compound designated CGP 385-60A [Buhlmayer et al., J. Med. Chem., 31:1839 (1988); DeGasparo et al., J. Clin. Pharmac., 27:587 (1989)] as used in this model can also be used.

For intravenous (iv) dosage, inhibitor is provided in a pharmaceutical composition at about 1-5 mg/kg. For peroral (po) administration, amounts on the order of 10-100 mg/kg are utilized.

The marmosets used for the study are depleted of sodium for two days by treatment with furosemide at 25 mg/kg/day po and a low sodium diet. A final dose of furosemide is administered one hour prior to anesthesia with Inactin at 120 mg/kg intraperitoneally (ip) followed by a 10 mg/kg/hour iv maintenance infusion.

Blood pressure is measured from a catheter in the carotid artery via a Gould Stratham P23 pressure transducer and Lectromed M19 chart recorder. The jugular vein is cannulated for inhibitor injection and anesthetic infusion. Each animal serves as it own blood pressure control.

Although the present invention has now been described in terms of certain preferred embodiments, and exemplified with respect thereto, one skilled in the art will readily appreciate that various modifications, changes, omissions and substitutions may be made without departing from the spirit thereof. SEQUENCE LISTING

(1 ) GENERAL INFORMATION:

(i) APPLICANT: Janda, Kim

Wirshing, Peter

(ii) TITLE OF INVENTION: ASPARTIC PROTEINASE INHIBITORS BASED ON PHOSPHOAMIDATE ESTER-CONTAINING PSEUDOPEPTIDES

(iii) NUMBER OF SEQUENCES: 23

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: The Scripps Research Institute, Office of

Patent Counsel

(B) STREET: 10666 North Torrey Pines Road, TPC 8

(C) CITY: La Jolla

(D) STATE: CA

(E) COUNTRY: USA

(F) ZIP: 92037

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1 .0, Version #1 .25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: PCT/US92/

(B) FILING DATE: 06-NOV-1 992

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 07/789,91 6

(B) FILING DATE: 06-NOV-1991

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Lewis, Donald G

(B) REGISTRATION NUMBER: SCR 1 223P

(C) REFERENCE/DOCKET NUMBER: 28,636

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 61 S-554-2937

(B) TELEFAX: 61 9-554-631 2

(2) INFORMATION FOR SEQ ID NO: 1 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6 amino acids (B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 1

(D) OTHER INFORMATION: /label = Xaa

/note = "Xaa is t-BOC-D-Phe"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 3

(D) OTHER INFORMATION: /label = Xaa

/note = "Xaa is a Phe whose carbonyl group is replaced by P(O)(OCH3) group."

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 6

(D) OTHER INFORMATION: /label = Xaa

/note = "Xaa is D-Trp"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1 :

Xaa Pro Xaa Phe Val Xaa

1 5

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 1

(D) OTHER INFORMATION: /label = Xaa

/note = "Xaa is t-BOC-D-Phe"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 3

(D) OTHER INFORMATION: /label = Xaa

/note= "Xaa is Phe whose carbonyl group is replaced by P(O)(OCH3)"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 4

(D) OTHER INFORMATION: /label = Xaa

/note = "Xaa is Phe amide bonded to aminovaleric acid"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Xaa Pro Xaa Xaa

1

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 5

(D) OTHER INFORMATION: /label = Xaa

/note = "Xaa is Phe whose carbonyl group is replaced by P(O)(OCH3)"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

Gly Phe Leu Gly Xaa Leu

1 5

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 2

(D) OTHER INFORMATION: /label = Xaa /note = "Xaa is D-Phe"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 5

(D) OTHER INFORMATION: /label = Xaa

/note = "Xaa is Phe whose carbonyl group is replaced by P(O)(O-isopropyl)"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

Gly Xaa Leu Gly Xaa Leu

1 5

(2) INFORMATION FOR SEQ ID NO:5:

(ϊ) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 3

(D) OTHER INFORMATION: /label = Xaa

/note = "Xaa is D-Leu"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 5

(D) OTHER INFORMATION: /label = Xaa

/note = "Xaa is Phe whose carbonyl group is replaced by P(OKO-hexyl)"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

Gly Phe Xaa Gly Xaa Leu

1 5

(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 5

(D) OTHER INFORMATION: /label = Xaa

/note = "Xaa is D-Phe whose carbonyl group is replaced by P(O)(O-tertiary-butyl)"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

Gly Phe Leu Gly Xaa Leu

1 5

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 8 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 5

(D) OTHER INFORMATION: /label = Xaa

/note = "Xaa is Leu whose carbonyl group is replaced by P(O)(OCH3)"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7 :

His Pro Phe His Xaa Val He His

1 5

(2) INFORMATION FOR SEQ ID NO:8 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 8 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 1

(D) OTHER INFORMATION: /label = Xaa

/note = "Xaa is t-BOC-His" (ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 5

(D) OTHER INFORMATION: /label = Xaa

/note = "Xaa is Leu whose carbonyl group is replaced by P(O)(OCH3)"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

Xaa Pro Phe His Xaa Val He His

1 5

(2) INFORMATION FOR SEQ ID NO:9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 6

(D) OTHER INFORMATION: /label = Xaa

/note = "Xaa is Phe whose carbonyl group is replaced by P(O)(OCH3)"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:

Pro His Pro Phe His Xaa Phe Val Tyr Lys

1 5 10

(2) INFORMATION FOR SEQ ID NO:10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 6

(D) OTHER INFORMATION: /label = Xaa

/note = "Xaa is Leu whose carbonyl group is replaced by P(O)(OCH3)" (ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 1 0

(D) OTHER INFORMATION: /label = Xaa

/note = "Xaa is epsilon-t-BOC-Lys methyl ester"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1 0:

Arg Arg Pro Phe His Xaa Val lie His Xaa

1 5 1 0

(2) INFORMATION FOR SEQ ID NO: 1 1 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 8 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 5

(D) OTHER INFORMATION: /label = Xaa

/note = "Xaa is Leu whose carbonyl group is replaced by P(O)(OCH3)"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1 1

His Pro Phe His Xaa Leu Val Tyr

1 5

(2) INFORMATION FOR SEQ ID NO: 1 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 8 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 5

(D) OTHER INFORMATION: /label = Xaa

/note = "Xaa is Phe whose carbonyl group is replaced by P(O)(OCH3)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:

His Pro Phe His Xaa Phe Val Tyr

1 5

(2) INFORMATION FOR SEQ ID NO: 13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 13 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 10

(D) OTHER INFORMATION: /label = Xaa

/note = "Xaa is Leu whose carbonyl group is replaced by P(O)(OCH3)"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:

Asp Arg Val Tyr He His Pro Phe His Xaa Val lie His 1 5 10

(2) INFORMATION FOR SEQ ID NO:14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 1

(D) OTHER INFORMATION: /label = Xaa

/note = "Xaa is acetyl-Leu"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 3

(D) OTHER INFORMATION: /label = Xaa

/note= "Xaa is Phe whose carbonyl group is replaced by P(O)(OCH3)" (xi) SEQUENCE DESCRIPTION:. SEQ ID NO: 1 4:

Xaa Ser Xaa Met Ala lie Pro Pro Lys Lys

1 5 1 0

(2) INFORMATION FOR SEQ ID NO: 1 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 1

(D) OTHER INFORMATION: /label = Xaa

/note = "Xaa is acetyl-Val"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 3

(D) OTHER INFORMATION: /label = Xaa

/note = "Xaa is Leu whose carbonyl group is replaced by P(O)(OCH3)"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1 5 :

Xaa Val Xaa Ala Leu

1 5

(2) INFORMATION FOR SEQ ID NO: 1 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 1

(D) OTHER INFORMATION: /label = Xaa

/note = "Xaa is 3-methylbutanoyl-Val"

(ix) FEATURE:

(A) NAME/KEY: Modified-site (B) LOCATION: 3

(D) OTHER INFORMATION: /label = Xaa

/note = "Xaa is Leu whose carbonyl group is replaced by P(O)(OCH3)"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 4

(D) OTHER INFORMATION: /label = Xaa

/note = "Xaa is Phe methyl ester"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:

Xaa Val Xaa Xaa

1

(2) INFORMATION FOR SEQ ID NO:17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 1

(D) OTHER INFORMATION: /label = Xaa

/note = "Xaa is 3-methylbutanoyI-VaI"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 3

(D) OTHER INFORMATION: /label = Xaa

/note= "Xaa is Leu whose carbonyl group is replaced by P(O)(OCH3)"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 6

(D) OTHER INFORMATION: /label = Xaa

/note= "Xaa is Ala methyl ester"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:

Xaa Val Xaa Phe Ala Xaa

1 5 (2) INFORMATION FOR SEQ ID NO: 1 8 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1 6 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 3

(D) OTHER INFORMATION: /label = Xaa

/note = "Xaa is homo-Arg"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 6

(D) OTHER INFORMATION: /label = Xaa

/note = "Xaa is Leu whose carbonyl group is replaced by P(O)(OCH3)"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 9

(D) OTHER INFORMATION: /label = Xaa

/note = "Xaa is homo-Arg"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 1 0

(D) OTHER INFORMATION: /label = Xaa

/note = "Xaa is homo-Arg"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1 8 :

Leu Val Xaa Val Pro Xaa Val Arg Xaa Xaa Ser Leu Arg Gin Leu He

1 5 1 0 1 5

(2) INFORMATION FOR SEQ ID NO: 1 9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 1

(D) OTHER INFORMATION: /label = Xaa

/note= "Xaa is acetyl-Va!"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 3

(D) OTHER INFORMATION: /label = Xaa

/note = "Xaa is Leu whose carbonyl group is replaced by P(O)(OCH35"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:

Xaa Val Xaa Ala Ala Leu

1 5

(2) INFORMATION FOR SEQ ID NO:20:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 3

(D) OTHER INFORMATION: /label = Xaa

/note = "Xaa is Leu whose carbonyl group is replaced by P(O)(OCH3)"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:

Val Val Xaa Ala Ala Leu

1 5

(2) INFORMATION FOR SEQ ID NO:21 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 1

(D) OTHER INFORMATION: /label = Xaa

/note = "Xaa is 3-methylbutanoyl-Val"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 2

(D) OTHER INFORMATION: /label = Xaa

/note = "Xaa is Leu whose carbonyl group is replaced by P(O)(OCH3)"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 4

(D) OTHER INFORMATION: /label = Xaa

/note = "Xaa is Ala isoamylamide"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21

Xaa Xaa Ala Xaa

1

(2) INFORMATION FOR SEQ ID NO:22:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 1

(D) OTHER INFORMATION: /label = Xaa

/note = "Xaa is 3-methylbutanoyl-Val"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 3

(D) OTHER INFORMATION: /label = Xaa

/note = "Xaa is Leu whose carbonyl group is replaced by P(O)(OCH3)"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 5

(D) OTHER INFORMATION: /label = Xaa /note = "Xaa is Ala ethyl ester"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:

Xaa Val Xaa Ala Xaa

1 5

(2) INFORMATION FOR SEQ ID NO:23:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 8 amine acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 6

(D) OTHER INFORMATION: /label = Xaa /note= "Xaa is p-nitro-Phe"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:

Lys Pro Ala Glu Phe Xaa Arg Leu

1 5

Claims

Claims

1. A linkage unit for joining two peptide sequences, i.e. an amino terminal peptide sequence and a carboxyl terminal peptide sequence, the linkage unit comprising:

a capped surrogate amino acid residue, said capped surrogate amino acid residue having a backbone

substantially isosteric with a peptide backbone, the backbone of said capped surrogate amino acid residue lacking a carbonyl group and having, instead, a -P(O)(OR)- group substituting for the lacking carbonyl group, where R is a C₁-C₆ alkyl group,

said capped surrogate amino acid residue lacking an ability to form a backbone peptide bond with the amino end of said carboxyl terminal peptide sequence and forming, instead, a phosphonamide C₁-C₆ alkyl ester linkage with the amino end of said carboxyl terminal peptide sequence, i.e. -P(O)(OR)-N(H)-, the phosphonamide C₁-C₆ alkyl ester linkage being substantially acid stable.

2. A linkage unit as described in claim 1 wherein the improvement further comprises:

R is a methyl group.

3. An improved linkage unit for joining two peptide sequences, i.e. an amino terminal peptide sequence and a carboxyl terminal peptide sequence, the linkage unit including a surrogate amino acid residue having a backbone substantially isosteric with a peptide backbone, the backbone of said surrogate amino acid residue lacking a carbonyl group and having, instead, a -P(O)(OH)- group substituting for the lacking carbonyl group, said surrogate amino acid residue lacking an ability to form a backbone peptide bond with the amino end of said carboxyl terminal peptide sequence and forming, instead, a phosphonamide linkage with the amino end of said carboxyl terminal peptide sequence, i.e. -P(O)(OH)-N(H)-, the phosphonamide linkage being acid labile, wherein the improvement

comprises:

said surrogate amino acid residue being capped, i.e. the -P(O) (OH)- group being substituted by a -P(O) (OR)- group,

wherein R is a C₁-C₆ alkyl group, and

the phosphonamide linkage between said surrogate amino acid residue and the amino end of said carboxyl terminal peptide sequence being substituted by a phosphonamide C₁-C₆ alkyl ester linkage, the phosphonamide C₁-C₆ alkyl ester linkage being substantially acid stable.

4. An improved linkage unit as described in claim 3 wherein the improvement further comprises:

R is a methyl group.

5. An improved polypeptide having an amino terminal peptide sequence, a carboxyl terminal peptide sequence, and a surrogate amino acid residue for joining said amino terminal peptide and said carboxyl terminal peptide, said surrogate amino acid residue having a backbone

substantially isosteric with a peptide backbone, the backbone of said surrogate amino acid residue lacking a carbonyl group and having, instead, a -P(O) (OH)- group substituting for the lacking carbonyl group, said surrogate amino acid resiude lacking an ability to form a backbone peptide bond with the amino end of said carboxyl terminal peptide sequence and forming, instead, a phosphonamide linkage with the amino end of said carboxyl terminal peptide sequence, i.e. -P(O) (OH)-N(H)-, the phosphonamide linkage being acid labile, wherein the improvement

comprises:

said surrogate amino acid residue being capped, i.e. the -P(O) (OH)- group being substituted by a -P(O) (OR) - group, where R is a C₁-C₆ alkyl group, and the phosphonamide linkage between said surrogate amino acid residue and the amino end of said carboxyl terminal peptide sequence, i.e. -P(O)(OR)-N(H)-, being substituted by a phosphonamide C₁-C₆ alkyl ester linkage, i.e.

-P(O)(OR)-N(H)-, the phosphonamide C₁-C₆ alkyl ester linkage being substantially acid stable.

6. An improved polypeptide as described in claim 5 wherein the improvement further comprises:

R is a methyl group.

7. A peptide comprising:

a capped surrogate amino acid residue and

a second amino acid residue,

said capped surrogate amino acid residue having a backbone substantially isosteric with a peptide backbone, the backbone of said capped surrogate amino acid residue lacking a carbonyl group and having, instead, a -P(O)(OR)- group substituting for the lacking carbonyl group, where R is a C₁-C₆ alkyl group,

said capped surrogate amino acid residue lacking an ability to form a backbone peptide bond with the amino end of said second amino acid residue and forming, instead, a phosphonamide C₁-C₆, alkyl ester linkage with the amino end of said second amino acid residue, i.e. -P(O)(OR)-N(H)-, the phosphonamide C₁-C₆ alkyl ester linkage being

substantially acid stable.

8. A method for linking a capped surrogate amino acid residue with a second amino acid residue, the method comprising the following step:

Step A: linking the amino end of the second amino acid residue to the cappped surrogate amino acid by means of a phosphonamide C₁-C₆ alkyl ester linkage.

9. A method for linking an amino terminal peptide sequence and a carboxyl terminal peptide sequence by means of a linkage unit, the method comprising the following steps:

Step A: linking the carboxyl end of the amino terminal peptide sequence to the linkage unit by means of a peptide bond; and

Step B: linking the amino end of the carboxyl terminal peptide sequence to the linkage unit by means of a

phosphonamide C₁-C₆ alkyl ester linkage.

10. An aspartic proteinase inhibitor pseudopeptide having a length of 4 to about 15 amino acid residues and containing a P₁ to P₁' bond constituted by a phosphonamidate C₁-C₆ alkyl ester in which the phosphorus atom is bonded to P, in place of the carbonyl carbon atom of a peptide bond.

11. The inhibitor of claim 10 wherein said C₁-C₆ alkyl ester is a methyl ester.

12. The inhibitor of claim 10 wherein said inhibitor has a length of 4 to about 10 amino acid residues.

13. A pseudopeptide aspartic proteinase inhibitor of the formula

R¹-X₁-XaaΨ[P(O)(OC₁-C₆ alkyl)NH]X₂-Z

wherein X₁ is an amino acid residue or oligopeptide containing a sequence of up to about ten amino acid

residues;

Xaa is a surrogate amino acid residue having an amino acid side chain;

X₂ is an amino acid residue or oligopeptide containing a sequence of up to about ten amino acid residues;

Z is selected from the group consisting of NH₂, NH-C₁-C₆ acyl, OH, O-C₁-C₆ alkyl and 2-amidoindanol; and

R¹ is selected from the group consisting of hydrogen, C₁-C₆ acyl, trifluoroacetyl and t-BOC; said oligopeptide analog having the length of 4 to about 15 amino acid residues.

14. The pseudopeptide of claim 13 wherein Xaa is a

surrogate amino acid having an amino acid side chain selected from the group consisting of Leu, Tyr and Phe, and the amino-terminal residue of X₂ is selected from the group consisting of Tyr, Leu, Val, Met, Pro, Ala and Phe.

15. The pseudopeptide of claim 14 wherein said C₁-C₆ alkyl is methyl.

16. An oligopseudopeptide aspartic proteinase inhibitor of the formula

R¹-X₁-XaaΨ[P(O)(OC₁-C₆ alkyl)NH]X₂-Z wherein X₁ is an amino acid residue or oligopeptide containing a sequence of up to about ten amino acid

residues;

Xaa is an amino acid surrogate having a side chain selected from the group consisting of Leu, Try, Val and Phe;

X₂ is an amino acid residue or oligopeptide containing a sequence of up to about ten amino acid residues whose amino-terminal residue is selected from the group

consisting of Tyr, Leu, Val, Met, Pro, Ala and Phe;

Z is selected from the group consisting of NH₂, NH-C₁-C₆ acyl, OH, O-C₁-C₆ alkyl and 2-amidoindanol; and

R¹ is selected from the group consisting of hydrogen, C₁-C₆ acyl, trifluoroacetyl and t-BOC

said oligopeptide analog having the length of 4 to about 10 amino acid residues.

17. The oligopseudopeptide of claim 16 wherein said

aspartyl proteinase is cathepsin D and said peptide has the sequence

t-BOC-D-Phe-Pro-PheΨ[P(O)(OCH₃)NH]Phe-Val-D-Trp

(SEQ ID NO: 1).

18. The oligopseudopeptide of claim 16 wherein said aspartyl proteinase is cathepsin D and peptide has the sequence

t-BOC-D-Phe-Pro-Pheψ[P(O)(OCH₃)NH]Phe-Avl

(SEQ ID NO: 2).

19. The oligopseudopeptide of claim 16 wherein said

aspartyl proteinase is renin and said peptide has the sequence

His-Pro-Phe-His-LeuΨ[P (O) (OCH₃) NH]Val-Ile-His

(SEQ ID NO: 7) .

20. The oligopseudopeptide of claim 16 wherein said

aspartyl proteinase is renin and said peptide has the sequence

t-BOC-His-Pro-Phe-His-LeuΨ[P (O) (0CH₃) NH]Val-Ile-His

(SEQ ID NO : 8 ) .

21. The oligopseudopeptide of claim 16 wherein said

aspartyl proteinase is renin and said peptide has the sequence

Pro-His-Pro-Phe-His-PheΨ[P(O)(OCH₃)NH]Phe-Val-Tyr-Lys

(SEQ ID NO: 9).

22. The oligopseudopeptide of claim 16 wherein said

aspartyl proteinase is renin and said peptide has the sequence

Arg-Arg-Pro-Phe-His-LeuΨ[P(O)(OCH₃)NH]Val-Ile-His- Lys(t-BOC)-OCH₃

(SEQ ID NO: 10).

23. The oligopseudopeptide of claim 16 wherein said aspartyl proteinase is renin and said peptide has the sequence

His-Pro-Phe-His-LeuΨ-[P(O)(OCH₃)NH]Leu-Val-Tyr

(SEQ ID NO: 11).

24. The oligopseudopeptide of claim 16 wherein said aspartyl proteinase is renin and said peptide has the sequence

His-Pro-Phe-His-PheΨ[P(O)(OCH₃)NH₂]Phe-Val-Tyr

(SEQ ID NO: 12).

25. The oligopseudopeptide of claim 16 wherein said aspartyl proteinase is chymosin and said peptide has the sequence

CH₃C(O)-Leu-Ser-PheΨ[P(O)(OCH₃)NH]Met-Ala-Ile-Pro- Pro-Lys-Lys

(SEQ ID NO: 14).

26. The oligopseudopeptide of claim 16 wherein said aspartyl proteinase is chymosin and said peptide has the sequence

CH₃C(O)-Val-Val-LeuΨ[P(O)(OCH₃)NH]Ala-Leu

(SEQ ID NO: 15).

27. The oligopseudopeptide of claim 16 wherein said

aspartyl proteinase is penicillopepsin and said peptide has the sequence

Iva-Val-Val-LeuΨ[P(O)(OCH₃)NH]Phe-OCH₃

(SEQ ID NO:16).

28. The oligopseudopeptide of claim 16 wherein said

aspartyl proteinase is penicillopepsm and said peptide has the sequence

Iva-Val-Val-LeuΨ[P(O)(OCH₃)NH]Phe-Ala-Ala-OCH₃

(SEQ ID NO: 17).

29. The oligopseudopeptide of claim 16 wherein said

aspartyl proteinase is pepsin and said peptide has the sequence

Val-Val-Leuψ[P(O)(OCH₃)NH]Ala-Ala

(SEQ ID NO: 20).

30. The oligopseudopeptide of claim 16 wherein said

aspartyl proteinase is pepsin and said peptide has the sequence

Iva-Val-LeuΨ[P(O)(OCH₃)NH]Ala-Ala

(SEQ ID NO:21).

31. A pharmaceutical composition comprising an aspartic proteinase inhibitor pseudopeptide present in an effective inhibitory amount dissolved or dispersed in a

physiologically tolerable diluent, said pseudopeptide inhibitor having a length of 4 to about 15 amino acid residues and containing a P₁ to P₁' bond that is constituted by a phosphonamidate C₁-C₆ alkyl ester in which the

phosphorus atom is bonded to P₁ in place of the carbonyl carbon atom of a peptide bond.

32. The pharmaceutical composition of claim 31 wherein said phosphonamide C₁-C₆ alkyl ester is a methyl ester.

33. A method of inhibiting the activity of an aspartic proteinase that comprises admixing in an agueous medium an aspartic proteinase, a substrate for that enzyme and a pharmaceutical composition containing an effective

inhibitory amount of a pseudopeptide inhibitor for that aspartic proteinase to form an inhibitory mixture, said pseudopeptide inhibitor having a length of 4 to about 15 amino acid residues and containing a P₁ to P₁' bond that is constituted by a phosphonamidate C₁-C₆ alkyl ester in which the phosphorus atom is bonded to P₁ in place of the carbonyl carbon atom of a peptide bond; and maintaining said

inhibitory mixture for a time period sufficient for said pseudopeptide inhibitor to inhibit the activity of said aspartic proteinase.

34. The method of claim 33 wherein said phosphonamide C₁-C₆ alkyl ester is a methyl ester.