The Synthesis of Cyclopropane Amino Acids and Peptides

The present application is a Continuation In Part Application of pending Application Serial No. 523,808 filed August 16, 1983. The United States Government has a non-exclusive license right in the present invention for government purposes pursuant to N.I.H. Grant No. DA 02938 awarded by the Department of Health and Human Services.

Technical Field

The field of the invention is a new class of amino acid derivatives which in large measure have never been described in the literature and which were first synthesized into a peptide which exhibited unexpected stability against hydrolytic cleavage and degradation by organic acids and enzymes. A specific field of use for the new product is in a beverage composition which includes a novel peptide sweetener which is stable to acid hydrolysis by such organic acids as citric and fumaric acids and enzyme degradation and cleavage to give prolonged sweetness to the product. Food compositions are also described which employ the new peptides as ingredients.

Background Art

The only process described in the literature and related to the novel process disclosed herein is the addition of compound (1) wherein R =R ₂ =hydrogen to

compound ( 2) to form a cyclopropylanine acid ( 4) to wit :

wherein the R , R ² , R ³ and R ⁴ are as defined below, as reported by Bregovec and T. Jakovcic, Monats fur

Chemie, 1972 103, 288. The commonly known addition of CH ₂ N2 to unsaturated azlactones (M. Bernabe, et al, Ann de Quimica 1972, 68, 501, 1055; Eur. J. Med. Chem. 1979, 14, 33 (1979); and Synthesis Co m. 1977, 191; J. Hetrocyclic Chem. 1983, 20, 607; Pages, R.A. Burger, A; J. Medicinal Chemistry 1966, 9, 766; and 10, 435, (1967); Awad, W. I. et al Tetrahedron, 1964, 20, 891) is similar but not the same process and product of the present invention. The present synthesis requires the addition of a substituted diazomethane specifically to a dehydroalanine derivative which must be synthesized for this purpose to form a 5-substituted pyrazoline. Contrary to this the initial product formed in the process described in the prior art literature is a 4- substituted pyrazoline which is then converted into the cyclopropyl amino acid.

Other reported synthesis of cyclopropyl amino acid analogs of the present invention are reported by V. Schollkopf, R. Harms and D. Hoppe, Liebigs Ann. Chem. 611, (1973); also Martinez-Garcia, F.H. Cano and S.

Garcia-Blanco, Acta Crystallographia, 31 (1980), Sect. A. Suppl. S103, also A. Ichihara, K. Shiraishi and S. Sakamura, Tetrahedron Letters 269, (1977) for synthesis of coronamic acid.

As to prior synthesis attempts at peptides

containing cyclopropyl amino acids see F.H.C. Stewart, Austrian Jour, of Chemistry, _34_ pp 2431 (1981).

As to foreign patents Spanish Patent No. 448,771 issued July 16, 1977 discloses a cyclopropyl phenylalanine and cyclopropyl tyrosine.

A cyclopropylphenylalanine has been described in the literature by King et al "Synthesis of Racemic (E) and (Z)-l-Amino-2-phenylcyclopropanecarboxylic Acid: (E) and (Z) Cyclopropylphenylalanine" Jour, of Organic Chem. 1982, 47, 3270-3273. In addition United States Patent 3,313,842 to Kaiser et al issued in 1967 discloses phenylcyclopropanecarboxylic acids and esters as hypotensive agents. U.S. Patent 3,050,559 to Burger discloses cyclopropyl amines. Also U.S. Patent 4,298,760 describes an improved process for preparation of 1-aminocyclopropane-l-carboxylic acid for use as a plant growth regulator. Kimura et al in Biochemical and Biophysical Research Communications, Vol. 115, p. 112-115 No. 1 (1983) describes the synthesis of cyclopropyl phenylalanine and its use in preparation of a stabilized peptide.

As can be observed from the above prior art background several of the cyclopropyl amino acids are known materials and a peptide which employs one of these known amino acid analogues is reported in the prior art.

Disclosure of the Invention

The process aspects of the present invention describe a method which generates chiral cycloalkyl amino acids directly. This means that if chirality is

present in a dehydroalanine derivative optically active cycloalkyl amino acids can be prepared directly without the necessity of resolution. This is not possible in other reported prior art processes.

In the product aspects of the invention new stereo specific cycloalkyl amino acids are disclosed.

Another process aspect of the invention comprises a process for synthesizing an acid or enzyme stable peptide containing at least one stereo specific cycloalkyl amino acid residue. These totally unique peptides for the most part retain their initial biological activity but because of steric blocking or hinderance effected by the inclusion of the new claimed amino acid analogs as a substitute for one or more normal amino acid residues in the peptide chain the peptide becomes cleavage resistant.

As a result of their altered amino acid structure the peptide products of the invention do not degrade upon contact with hydrolytic enzymes or organic acids. Their enhanced stability is manifest by their new resistance to cleavage of the peptide linkages and methyl ester bonds. This stability has pharmacological advantages.

There are at least three product aspects to the invention. The first is the new and unique cycloalkyl substituted amino acids described. These variant amino acids when substituted for normal amino acids in a peptide chain act to stabilize the peptide against cleavage by enzymes and hydrolysis by acids. The peptides so formed are themselves unique products having a distinctly different molecular structure and

an added property of long term stability. A third product aspect of the invention resides in the preparation of a food product, for example, a beverage with long term sweetness when its peptide sweetener is replaced by the cycloalkyl amino acid modified peptide sweetener of the present invention.

Specific examples of each of these process and products aspects of the present invention will be set forth hereinbelow. It is of course clear that because of the magnitude of peptides known for a multitude of purposes in many end use areas of food chemistry, pharmacology, herbicide and pesticide end use applications, etc. the concrete embodiments to support all of the possible broad implications of the present invention are difficult to express in a single document. However, the basic concept and application of the same to a new food composition has been set forth herein to exemplify the broad notion of the invention.

It is therefore an object of the present invention to provide new stereo specific cyclopropyl amino acids. It is a further object to provide new peptides containing at least one stereo specific cyclopropyl amino acid residue. It is a further object to provide an end product containing the peptide as an ingredient. These and other objects, aspects and advantages of this invention will become apparent from a consideration of the accompanying specification and claims:

Brief Description of the Drawings

No drawing is submitted in this case since the

OMPI

I/. ^~ W >

subject matter does not warrant such disclosure.

Best Mode for Carrying Out the Invention

The process embodiment of the present invention is carried out by allowing a diazo compound (1), in the presence or absence of a catalyst or light, to react with a dehydroalanine derivative (2). The initial reaction product may be a pyrazoline derivative (3) which is pyrolyzed, photolyzed or treated with a catalyst to give the cyclopropyl amino acid derivative (4) which is one of the product embodiments. The reaction is as shown in equations A and B, below:

_R 1_ (Equation A); (1) (2 ) (3 )

(3) (4)

The product (4), a cyclopropyl amino acid derivative, may consist of a mixture of stereoisomers which are separable by physical means into the E- and Z diastereomers. Each of these diastereomers consists of a pair (2R, 2S) of enantiomers which can be separated by standard resolution methods. Separation into E- and Z- diastereomers and separation of the enantiomers may occur either before or after deblocking the product (4).

The diazo compound (1), R ¹ and R ² can be hydrogen, an alkyl (aliphatic) group, an aromatic group (aryl such as phenyl, indolyl, imidazolyl or the like), an

alkyl group substituted by a halogen, oxygen, nitrogen, or sulfur, an alkyl group substituted by an aromatic group, or an aromatic group substituted by a halogen, oxygen, nitrogen, sulfur, aromatic or aliphatic group.

•3 In dehydroalanine compound (2), R can be any alkyl or aromatic group or alkoxy or aryloxy group. R ⁴ can be any alkyl or aryl group. Preferred lower alkyl groups are methyl, ethyl, propyl , isopropyl and butyl.

The solvent used in the reaction (A) can be any aprotic solvent, such as CHCI3, CH ₂ Cl ₂ , tetrahydrofuran, dioxane, diethyl ether, etc. or protic solvent such as methanol or ethanol.

The reaction temperature (first step) is 0-30°C and that of the second step may be 0°-150°C. A solvent such as benzene or. toluene or the like may be used in the second step.

For purposes of obtaining the free cyclopropyl amino acid (AA), compound (4) may be C-terminal deblocked, depending on the nature of R-. , by standard procedures such as saponification or hydrogenolysis giving the acid (5) as shown in equation C, below:

R ¹ NHCOR ³ R ¹ NHCOR ³ X - AA (Equation C) ;

R ² C0 ₂ R ⁴ R ² C0 ₂ H

(4) (5) and N-terminal deblocking of (5) by the use of anhydrous acid, dry HCl or CF3CO2H, by hydrogenolysis

OMPI °

or by hydrolysis, depending on the nature of R , can be accomplished by standard procedures as shown in equation O _f below:

(Equation D)

(5) (AA)

Deblocking of the amino group (N-terminal deblocking) as shown in equation E, below, may precede deblocking of the carboxyl group (C-terminal deblocking) (equation C) resulting in a cyclopropyl amino acid.

A - PREFERRED EMBODIMENT - METHOD OF PREPARING CYCLOPROPYL AMINO ACID

One facet of this process invention thus discloses a process for synthesizing a cyclopropyl amino acid selected from the group consisting of (2S)-E-, (2R)-E-, (2S)-Z-, (2R)-Z-, (2S)-, (2R)-, (2RS)-E-, and (2RS)-Z- isomers wherein the cyclopropyl amino acid is selected from the group consisting of cyclopropyl amino acids, analogs, derivatives, and cogeners thereof comprising the following steps:

(a) reacting a diazo compound having the formula i n ~ ^X R' ^& CN ₂ wherein R ^x is selected from the group consisting of hydrogen, an alkyl group, an aromatic group, an alkyl group substituted by a halogen, oxygen, nitrogen, or sulfur, an alkyl group substituted by an aromatic group, and an aromatic group substituted by a halogen, oxygen, nitrogen, sulfur aromatic or aliphatic group, n wherein R ^ώ is selected from the group consisting of hydrogen, an alkyl group, an aromatic group, an

alkyl group substituted by a halogen, oxygen, nitrogen, or sulfur, an alkyl group substituted by an aromatic group and an aromatic group substituted by a halogen, oxygen, nitrogen, sulfur aromatic or aliphatic group, with a dehydroalanine derivative having the formula (3)

NHCOR ³

CH = C

C0 ₂

(3)

wherein R° is selected from the group consisting of an alkyl group, an aromatic group, an alkoxy group, and an aryloxy group and wherein R ⁴ is selected from the group consisting of an alkyl group and an aryl group to produce an initial reaction product;

(b) decomposing the initial reaction product (3) to produce a cyclopropyl amino acid derivative having the formula

wherein the cyclopropyl amino acid derivative is a mixture of stereoisomers; (c) separating the mixture of stereoisomers by physical means into E- and Z-diastereomers wherein the E- and Z-diastereomers comprise a pair of enantiomers;

(d) separating the pair of enantiomers by standard resolution means to produce a stereo specific cyclopropyl amino acid derivative;

(e) deblocking the initial reaction product to

produce a stereo specific cyclopropyl amino acid having the formula

If R or R contain acidic groups selected from the group consisting of carboxyl, mercapto, and phenolic hydroxyl, R 1 and R2 are blocked by standard means to protect such groups during the process. Synthesis of the following cyclopropyl amino acids requires such blocking; aspartic acid, tyrosine, 3-4- dihydroxyphenylalanine (DOPA) , 5-hydroxytryptophan, cysteine, and homocysteine. If R ³ or R of the dehydroalanine derivative is optically active, an optically active stereo specific cyclopropyl amino acid can be produced without step (d) of the process. The diazo compound is reacted with the dehydroalanine derivative in the presence or absence of a catalyst and in the presence or absence of light. Pyrolysis, photolysis, or catalytic decomposition can be used to decompose the initial reaction product. A solvent selected from the group consisting of an aprotic solvent and a protic solvent can be used in the process. The step producing the initial reaction product is carried out at a temperature range of 0°C through 30°C, and the step producing the cyclopropyl amino acid derivative is carried out at a temperature range of 0°C through 150°C. When pyrolysis is used to decompose the initial reaction product, a solvent selected from the group consisting of benzene, toluene, and a similar solvent can be used in the step producing the cyclopropyl amino acid derivative. If the stereo specific cyclopropyl amino acid derivative is C- terrainal deblocked to produce a stereo specific cyclopropyl acid having the formula

R ¹ NHCOR ³

(5) saponification or hydrogenolysis can be used to C- terminal deblock the stereo specific cyclopropyl acid derivative. After C-terminal deblocking, the stereo specific cyclopropyl acid is N-terminal deblocked to produce the stereo specific cyclopropyl amino acid. Anhydrous acid, the stereo specific cyclopropyl acid, dry hydrogen chloride, trifluoroacetic acid, hydrogenolysis, the stereo specific cyclopropyl acid, or hydrolysis can be used to N-terminal deblock the stereo specific cyclopropyl acid. N-terminal deblocking can precede carboxyl terminal deblocking to produce the stereo specific amino acid. Steps (c) and (d) of the process described above can be carried out before or after deblocking.

PEPTIDE SYNTHESIS USING NEW AMINO ACIDS

For purposes of preparing peptides, compound (5), R ³ = OCH ₂ Ph or OC(CH ₃ )3, is prepared. Standard coupling methods, mixed anhydride, carbodiimide, etc., ar used to couple (5) with C-terminal blocked amino acids or peptides. N-terminal deblocking of (4) to give (6) is accomplished using anhydrous acids, dry HCl or CF3C0 ₂ H, or hydrogenolysis depending on the natures of R and R as shown in equation E, below:

⁽ Equation E ⁾

(4) (6)

Compound (6) is useful in coupling with N-blocked carboxyl-activated amino acids or peptides to form desired peptides.

r

PREFERRED EMOBODIMENTS - METHODS OF PEPTIDE SYNTHESIS

This invention discloses a process for synthesizing peptides having at least two amino acid residues selected from the group consisting of D- or L- isomers of amino acid residues wherein amino acid residues are selected from the group consisting of amino acid residues, analogs, derivative, and congeners thereof and wherein at least one amino acid residue is a stereo specific cyclopropyl amino acid residue selected from the group consisting of cyclopropyl amino acid residues, analogs, derivatives, and congeners thereof comprising the following steps:

(a) synthesizing the cyclopropyl amino acid derivative utilizing steps (a) and (b) of the process described above for synthesizing stereo specific cyclopropyl amino acid;

(b) separating the stereo specific cyclopropyl amino acid utilizing steps (c) and (d) of the process described above for synthesizing stereo specific cyclopropyl amino acids;

(c) deblocking the stereo specific cyclopropyl amino acid derivative by standard means to produce an N-terrainal blocked stereo specific cyclopropyl amino acid; (d) coupling the N-terminal blocked stereospecific cyclopropyl amino acid with a C- terminal blocked amino acid or peptide; and (e) repeating the above steps as necessary to produce a desired peptide. Step (c), the deblocking, can be carried out before step (b), the separation step.

An alternate process for synthesizing peptides utilizes stereo specific cyclopropyl acids generated as

described above comprises the following steps:

(a) N-terminal blocking the stereo specific cyclopropyl amino acid by standard means to produce the N-terminal blocked stereo specific cyclopropyl amino acid;

(b) coupling the N-terminal blocked stereo specific cyclopropyl amino acid with a C-terminal blocked amino acid or peptide; and

(c) repeating the above steps as necessary to produce a desired peptide having at least two and not more than twenty amino acid residues selected from the group consisting of D- or L-isomers of amino acid residues wherein amino acid residues are selected from the group consisting of amino acid residues, analogs, derivatives, and congeners thereof and wherein at least one amino acid residue is a stereo specific cyclopropyl amino acid residue selected from the group consisting of cyclopropyl amino acid residues, analogs, derivatives, and congeners thereof.

A second alternate process for synthesizing peptides having at least two amino acid residues selected from the group consisting of D- or L-isomers of amino acid residues wherein amino acid residues are selected from the group consisting of amino acid residues, analogs, derivatives, and congeners thereof and wherein at least one amino acid residue is a stereo specific cyclopropyl amino acid residue selected from the group consisting of cyclopropyl amino acid residues, analogs, derivatives, and congeners thereof comprises the following steps:

(a) synthesizing a cyclopropyl amino acid utilizing steps (a), (b), and (e) of the process described above for synthesizing stereo specific cyclopropyl amino acid;

(b) N-terminal blocking the cyclopropyl amino acid by standard means to produce a N-terminal blocked cyclopropyl amino acid;

(c) separating the stereo specific cyclopropyl amino acid utilizing steps (c) and (d) of the process described above for synthesizing stereo specific cyclopropyl amino acids;

(d) coupling the N-terminal blocked stereospecific cyclopropyl amino acid with a C- terminal blocked amino acid or peptide; and

(e) repeating the above steps as necessary to produce a desired peptide.

Step (c) of this second alternate process for synthesizing peptides can be carried out before step (b) of same.

A third alternate process for synthesizing peptides having at least two amino acid residues selected from the group consisting of D- or L-isomers of amino acid residues wherein amino acid residues are selected from the group consisting of amino acid residues, analogs, derivatives, and congeners thereof and wherein at least one amino acid residue is a stereo specific cyclopropyl amino acid residue selected from the group consisting of cyclopropyl amino acid residues, analogs, derivatives, and congeners thereof comprises the following steps:

(a) synthesizing the cyclopropyl amino acid derivative utilizing steps (a) and (b) of the process described above for synthesizing stereo specific cyclopropyl amino acids;

(b) separating the stereo specific cyclopropyl amino acid utilizing steps (c) and (d) of the process described above for synthesizing stereo specific cyclopropyl amino acids;

UiY-r-.

(c) N-terminal deblocking the stereo specific cyclopropyl amino acid derivative by standard means to produce a C-terminal blocked amino acid;

(d) coupling the C-terminal blocked stereo specific cyclopropyl amino acid with a N-terminal blocked amino acid or peptide; and

(e) repeating the above steps as necessary to produce a desired peptide.

Step (c) of this third alternate process for synthesizing peptides can be carried out before step (b) of same.

A fourth alternate process for synthesizing peptides utilizing stereo specific cyclopropyl amino acids generated as described above and comprising the following steps:

(a) C-terminal blocking the stereo specific cyclopropyl amino acid by standard means to produce the C-terminal blocked stereo specific cyclopropyl amino acid; (b) coupling the C-terminal blocked stereo specific cyclopropyl amino acid with a N-terminal blocked amino acid or peptide; and (c) repeating the above steps as necessary to produce a desired peptide having at least two and not more than twenty amino acid residues selected from the group consisting of D- or L-isomers of amino acid residues wherein amino acid residues are selected from the group consisting of amino acid residues, analogs, derivatives, and congeners thereof and wherein at least one amino acid residue is a stereo specific cyclopropyl amino acid residue selected from the group consisting of cyclopropyl amino acid residues, analogs, derivatives, and congeners thereof.

' A

- w7?

A fifth alternate process for synthesizing a peptide having at least two and not more than twenty amino acid residues selected from the group consisting of D- or L-isoraers of amino acid residues wherein amino acid residues are selected from the group consisting of amino acid residues, analogs, derivatives, and congeners thereof and wherein at least one amino acid residue is a stereo specific cyclopropyl amino acid residue selected from the group consisting of cyclopropyl amino acid residues, analogs, derivatives, and congeners thereof comprises the following steps:

(a) synthesizing a cyclopropyl amino acid utilizing steps (a), (b), and (e) of the process described above for synthesizing stereo specific cyclopropyl amino acids;

(b) C-terminal blocking the cyclopropyl amino acid by standard means to produce a C-terminal blocked cyclopropyl amino acid;

(c) separating the C-terminal blocked stereo specific cyclopropyl amino acid utilizing steps

(c) and (d) of the process described above for synthesizing stereo specific cyclopropyl amino acids;

(d) coupling the C-terminal blocked stereo specific cyclopropyl amino acid with a N-terminal blocked amino acid or peptide; and

(e) repeating the above steps as necessary to produce a desired peptide.

Step (c) of this fifth alternate process for synthesizing peptides can be carried out before step (b) of same.

A sixth alternate process for synthesizing peptides utilizing stereo specific amino acids generated as described above, wherein R or R of the

s

dehydroalanine derivative is optically active and step (d) of such process described above for synthesizing stereo specific cyclopropyl amino acids is not required, comprises the following steps: (a) N-terminal blocking the stereo specific cyclopropyl amino acid by standard means to produce the N-terminal blocked stereo specific cyclopropyl amino acid;

(b) coupling the N-terminal blocked stereo specific cyclopropyl amino acid with a C-terminal blocked amino acid or peptide; and

(c) repeating the above steps as necessary to produce a desired peptide having at least two and not more than twenty amino acid residues selected from the group consisting of D- or L-isomers of amino acid residues wherein amino acid residues are 'selected from the group consisting of amino acid residues, analogs, derivatives, and congeners thereof and wherein at least one amino acid residue is a stereo specific cyclopropyl amino acid residue selected from the group consisting of cyclopropyl amino acid residues, analogs, derivatives, and congeners thereof. A seventh alternate process for synthesizing peptides utilizes stereo specific amino acids generated as described above, wherein R or R of the dehydroalanine derivative is optically active and step (d) of such process described above for synthesizing stereo specific cyclopropyl amino acids is not required, comprises the following steps:

(a) C-terminal blocking the stereo specific cyclopropyl amino acid by standard means to produce the C-terminal blocked stereo specific cyclopropyl amino acid; (b) coupling the C-terminal blocked stereo

OMPI . IPO ~ *

specific cyclopropyl amino acid with a N-terminal blocked amino acid or peptide; and (c) repeating the above steps as necessary to produce a desired peptide having at least two and not more than twenty amino acid residues selected from the group consisting of D- or L-isomers of amino acid residues wherein amino acid residues are selected from the group consisting of amino acid residues, analogs, derivatives, and congeners thereof and wherein at least one amino acid residue is a stereo specific cyclopropyl amino acid residue selected from the group consisting of cyclopropyl amino acid residues, analogs, derivatives, and congeners thereof.

PREFERRED EMBODIMENTS - CYCLOPROPYL AMINO ACIDS

This invention- discloses novel stereo specific cyclopropyl amino acids selected from the group consisting of (2S)-E-, (2R)-E-, (2S)-Z-, (2R)-Z-, (2S)-, (2R)-, (2RS)-E-, and (2RS)-Z-isomers wherein the cyclopropyl amino acids are selected from the group consisting of cyclopropyl amino acids, analogs, derivatives, and congeners thereof having the formula

wherein R is selected from the group consisting of hydrogen, a carboxylic group or lower alkyl ester thereof an alkyl group, an aromatic group, an alkyl group substituted by a halogen, oxygen, nitrogen, or sulfur, an alkyl group substituted by an aromatic group, and an aromatic group substituted by a halogen, oxygen, nitrogen, sulfur aromatic or aliphatic group," wherein R is selected from the group consisting of

hydrogen, a carboxylic group or lower alkyl eter thereof an alkyl groups, an aromatic group, an alkyl group substituted by a halogen, oxygen, nitrogen, or sulfur, an alkyl group substituted by an aromatic group and an aromatic group substituted by a halogen, oxygen, nitrogen, sulfur aromatic or aliphatic group, wherein R ^x and R are not both hydrogen, wherein R and R- are not CgHg and H, respectively, or H and CgHg, respectively, wherein R 1 and R2 are not 4-H0C _g H ₄ and H, respectively, or H and 4-H0CgH ₄ , respectively, and wherein R 1 and R2 are not 4(5)-imidazolyl and H, respectively, or H and 4(5)-imidazolyl, respectively.

Cyclopropyl amino acids (AA) which can be made according to this invention are shown in Table I, below.

Table I CYCLOPROPYL AMINO ACIDS

CH ₃ CH ₃ *Valine (CH ₃ ) ₂ CH H *Leucine H (CH ₃ ) ₂ CH *Leucine

C0 ₂ H H *Aspartic acid H C0 ₂ H *Aspartic acid

CH ₂ C0 ₂ H H *Glutamic acid

H CH ₂ C0 ₂ H *Glutamic acid

CH ₂ CO H *pyroGlutamic acid

CHnSCH H *Methionine

H CH SCH^ *Methionine

NH ₂ (CH ₂ ) ₃ H *Lysine

H NH ₂ (CH ₂ ) ₃ *Lysine

^NH -sk *Arginine

H *Arginine NH ₂ (CH ₂ ) ₂ H *Ornithine

H NH ₂ (CH ₂ ) ₂ *Ornithine

^C 6 ^H 5 H Phenylalanine

H H Alanine

H ^C 6 ^H 5 Phenylalanine

4-H0C ₆ H ₄ H Tyros ine

H 4-HOC ₆ H ₄ Tyros ine

3,4(H0) ₂ C _e i ^H 3 H *3,4 Dihydroxyphenylalanine

H 3,4(HO) ₂ C ₆ H ₃ *3,4 Dihydroxyphenylalanine

3-indolyl H *Tryptophan

H 3-indolyl *Tryptophan

5-hydroxy- ^■ 3- indoyl H *Hydroxytryptophan

H 5-hydroxy-3-indoyl *Hydroxytryptophan

HO H *Serine

H HO *Serine

HS H ^♦ Cyst ine

H HS *Cysteine

HSCH ₂ H *Homocysteine

H HSCH ₂ *Homocysteine

HOCH ₂ H *Homoserine

H HOCH ₂ *Homoserine

4(5)-imidazo lyl H *Histidine

H 4(5)- ^■ imidazolyl *Histidine

(CH ₂ ) _n H *Proline wherein n = 1, 3, or 4

CHo) H *Proline wherein n = 2

HO (CHCH ₂ ) H *Hydroxyproline

Except for valine, pyroGlutamic acid, proline, and proline analogs, the above cyclopropyl amino acids are shown as E- and Z-diastereoraers. It is understood that all of the above listed cyclopropyl amino acids have

(2R)- and (2S)-enantiomers.

Alternative General Structures of Other Cycloalkyl Amino Acid

From known starting materials cycloalkyl amino acid derivatives of the following general structure can also be prepared in the manner of the present invention: These compounds are generally represented by formula:

Wherein R, and R2 are as above defined as n represents an integer from 1 to 4. More specifically the compounds wherein R-^ is H phenyl or COOH and R2 is H or wherein R- _j _ is hydrogen and R2 is phenyl H, or COOH are preferred species of compounds with the general structure as especially stable amino acids for peptide synthesis.

For purposes of peptide synthesis the above compound AA' would be treated in an analogous fashion to compound AA above and its successor derivatives as noted in instructions for peptide synthesis.

PREFERRED EMBODIMENTS - POLYPEPTIDES

Peptides having at least two amino acid residues up to about 20 such residues with at least one of these being a cyclopropyl derivative are produced by the practice of one of the several methods of this invention as noted hereinabove. The amino acid residues which make up the peptide chain can be either D or L-isomers. The amino acid residue can be selected

from the group consisting of (2S)-E, (2R)-E, (2S)-Z, (2R)-Z, (2S)- and (2R)-isomers of the stated amino acids in the peptide chain.

Selected examples of peptides capable of synthesis by means of the present invention and their specific utility is noted in the following Table II. The several stabilized peptides have a diverse field of use ranging from the food additive dipeptide sweeteners of Group I to the complex bradykin or shock inhibitor peptides of Group VII or antihypertensives of Group

V. The quantity of each peptide composition employed will vary with the specific intended use and mode of administration. In the case of the dipeptides VAsp-Phe OCH ₃ or Asp-V Phe OCH ₃ employed as a beverage sweetner a relatively large quantity will be required. These orally ingested peptide sweetners have from 100 to 200 times the sweetening power of sucrose when substituted for the same. On the other hand relatively small amounts (50-100 mulligrams/kilo of body weight) of stabilized peptides of Group V to VIII principally employed in intravascular pharmaceutical administration are normally administered to achieve a therapeutic result.

In the following Table a brief compilation of selected new stabilized peptides ranging from R -R ² ( a simple short chain sweetener bipeptide) to R ^A -R ⁹ (a more complex central central nervous system regulatory hormone) is described. This list is only intended to illustrate the types of variety of polypeptides now capable of synthesis using the new and unique concepts described in the several specific preparations illustrated in the following Examples of a preferred mode of practice of the invention.

Table II

Group Peptide Chain Biological Function

I R ^x -R ²

VAsp-Phe OCH ₃ Food and Sweetener

Asp- VPhe OCH ₃ Beverage Sweetener

VAsp- VPhe OCH 3 Food & Beverage Sweetener

Asp- VAla OC 3H 7 Food & Beverage Sweetener

Asp-cbPhe-OCH ₃ Food and Beverage Sweetener cbAsp-cbPhe-OCH ₃ Food and Beverage Sweetener Asp-cpPhe-OCH ₃ Food and Beverage Sweetener cpAsp-cpPhe-OCH3 Food and Beverage Sweetener Asp-cbAla OCH ₃ Food and Beverage Sweetener

I R ¹ -R ² -R ³ VMet-Leu-Phe Bacteriostatic Agent Met- VLeu-Phe Bacteriostatic Agent pGln-His- VPro-NH _ Als therapeutic pGln- VHis-Pro-NH ₂ Als therapeutic VpGln-His-Pro-NH ₂ Als therapeuti c Met-Leu-Phe Bacteriostatic Agent

R ¹ -R ² -R ³ -R ⁴ -R ⁵ Analgetics CNS Regulators Tyr-Gly-Gly-cbPhe-Leu Analgetics CNS Regula tors Tyr-Gly-Gly-cpPhe-Leu Analgetics CNS Regulators Tyr-Gly-Gly- VPhe-Leu Analgetics CNS Regulators Tyr-Gly-Gly- VPhe-Met Analgetics CNS Regulators Tyr-D-Ala-Gly- VPhe-Met Analgetics CNS Regulators Tyr-D-Ala-Gly- VPhe-Leu Analgetics CNS Regulators VTyr-Gly-Gly-Phe-Leu Analgetics CNS Regulators VTyr-D-Ala-Gly-Phe-Leu Analgetics CNS Regulators VTyr-D-Ala-Gly-Phe-Met Analgetics CNS Regulators

VTyr-Gly-Gly- VPhe-Leu Analgetics CNS Regulators

VTyr-Gly-Gly- VPhe-Met Analgetics CNS Regulators

Tyr-Gly-Gly-Phe-VLeu Analgetics CNS Regulators

Tyr-D-Ala-Gly-Phe- VLeu Analgetics CNS Regulators

VTyr-D-ala-Gly-Phe- VLeu Analgetics CNS Regulators

Tyr-Gly-Gly-Phe- VMet Analgetics CNS Regulators

Tyr-D-Ala-Gly-Phe- VMet Analgetics CNS Regulators

VTyr-D-Ala-Gly-Phe- VMet Analgetics CNS Regulators

VTyr-Gly-Gly-Phe- VMet Analgetics CNS Regulators

VTyr-Gly-Gly-Phe- VLeu Analgetics CNS Regulators

R ¹ -R ² -R ³ -R ⁴ -R ⁵ -R ⁶

Ile-His-Pro-VPhe-His-Leu Blood Pressure Regulator

Ile-His-Pro-Phe-VHis-Leu Blood Pressure Regulator

R ¹ -R ² -R ³ -R ⁴ -R ⁵ -R ⁶ -R ⁷

Pro-Phe-His- VLeu-Leu-Val-Tyr Renin Inhibitors Pro-Phe-His-Leu- VLeu-Val-Tyr Anti-hypertensives Pro- VPhe-His-Leu- VLeu-Val-Tyr Pro- VPhe-His- VLeu-Leu-Val-Tyr

R^-R^R^R^R ^S -R^R^R ⁸ Asp-Arg-Val-Tyr-Ile-His-Pro-VPhe Antihypertensive Asp-Arg-Val-Tyr-Ile-VHis-Pro-Phe Antihypertensive Asp-Arg-Val-VTyr-Ile-His-Pro-Phe Antihypertensive Asp-Arg-Wal-Tyr-Ile-His-Pro-Phe Antihypertensive Asp-VArg-Val-Tyr-Ile-His-Pro-Phe Antihypertensive VAsp-Arg-Val-Tyr-Ile-His-Pro-Phe Antihypertensive Asp-Arg-Val-VTyr-Ile-His-Pro-VPhe Antihypertensive Asp-Arg-Val-VTyr-Ile-VHis-Pro-Phe Antihypertensive Asp-Arg-Val-Tyr-Ile-VHis-Pro-VPhe Antihypertensive Sar-Arg-Val-VTyr-Ile-His-Pro-Phe Antihypertensive Sar-Arg-Val-Tyr-Ile-VHis-Pro-Phe Antihypertensive Sar-Arg-Val-Tyr-Ile-His-Pro-VPhe Antihypertensive

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Sar-Arg-Val-Tyr-Ile-VHis-Pro-VPhe Antihypertensive

Sar-Arg-Val-VTyr-Ile-VHis-Pro-Phe Antihypertensive

Sar-Arg-Val-VTyr-Ile-VHis-Pro-VPhe Antihypertensive

Sar-Arg-Val-VTyr-Ile-His-Pro-VPhe Antihypertensive

VII R ¹ -R ² -R ³ -R ⁴ -R ⁵ -R ⁶ -R ⁷ -R ⁸ -R ⁹

Arg-Pro-Pro-Gly-Phe-Ser-Pro-VPhe-Arg Bradykin Inhibitors

Shock Treatment Arg-Pro-Pro-Gly-VPhe-Ser-Pro-Phe-Arg Bradykin Inhibitors

Shock Treatment Arg-Pro-Pro-Gly-VJ=he-Ser-Pro-VPhe-Arg Bradykin Inhibitors

Shock Treatment

In the above Table II the following list of abbreviations were employed :

V = cyclopropyl cb = cyclobutyl cp = cyclopentyl ch = cyclohexyl

Phe ^* OCH ₃ = phenylalanine methyl ester

Arg = arginine

Ala = alanine

Asp = aspartic acid p Glu = pyroglutamic acid

Gly = Glycine

His = Histidine

He = isolencine

Leu = leucine

Met = methionine

Phe = phenylalanine

Pro = proline

Phe = Phenylalanine

Pro = proline

Pro NH ₂ = proline amide

Sar = sarcosme

Ser = serine

Tyr = tyrosine

Val = valine

The following several specific Examples of preparations of the cyclopropyl (V) amino acids as well as peptides.-where at one or more points in the chain they have replaced normal amino acids will serve to illustrate the invention claimed. Finally, the example of at least one improved food composition and one pharmaceutical composition will serve to illustrate the end use of the new modified amino acids and peptide compositions sought to be patented.

EXAMPLE I Boc-SerOBzl(N0 ₂ ) (1)

Boc-Ser ^* 0H ().097 mol) was dissolved in ethyl acetate (140 ml) and p-nitrobenzyl bromide (21 g, 0.097 mol) followed by triethylamine (9.7 g, 0.097 mol) were added to the mixture, which was refluxed for 18 hr. After cooling, water (200 ml) was added to the reaction mixture, the layers were separated and the aqueous layer was extracted with AcOEt. The extracts were washed with 5% NaHC0 ₃ soln. (100 ml), sat. NaCl soln, dried over Na ₂ S0 and evaporated in vacuo. The resulting pale yellow oil was dissolved in Et ₂ 0 (100 ml) and kept in refrigerator for 4 hr. The colorless crystals were filtered by suction to give Boc- Ser ^* OBzl(N0 ₂ ) (1) (18.0 g, 54.5%) as prisms; from the filtrate, a 2nd crop weighing 3.5 g was obtained (total yield 65%); mp 92-93°C; IR: (KBr) 3320-3420 (NH,0H),

1745 (C=0), 1660 (C=0). NMR (CDCl ₃ )δ: 8.2 and 7.2 (AB d, J = 12 Hz, 4H, ArH). 5.5 (br, d, 1H, NH), 5.25 (s.

-

2H, OCH ₂ ) 4.25-4.5 ( m, 1H , CH ) , 3.90 ( d , 2H , CH ₂ 0H ) , 2.60 (br , 1H , OH) , 1.38 ( s , 9H , Boc ) .

EXAMPLE II

Boc-Dehydroalanine-p-nitrobenzyl ester ( 2 )

a ) Using-EDC

To a suspension of Boc-Ser ^* OBzl(N0 ₂ ) (1) (6.0 g, 0.0176 mol) and CuCl (1.8 g, 0.018 mol) in CHCI3 (180 ml) was added l-ethyl-3(3-dimethylamino-propyl)- carbodiimide hydrochloride (EDC, 4.14 g, 0.0216 mol) at room temperature. The mixture was stirred overnight at room temperature during which time a brown oil separated. Water (150 ml) was added and the CHCI3 layer was separated and washed with water, dried over Na ₂ S0 ₄ , and evaporated in vacuo. The resulting solid was recrystallized from ethyl acetate-n-hexane to give colorless prisms 4.5 g (79.3%) of Boc-dehydroalanine-p- nitrobenzyl ester (2), mp 94-95°C; IR; (KBr): 3420 (NH), 1710 (C=0), 1632 (C=0), 1600 (C=C). NMR (CDCl ₃ )δ: 8.19 and 7.5 (d, J = 12 Hz, 4H, ArH), 6.93 (br, s, 1H, NH), 6.20 (s, 1H, H-C=C) , 5.75 (s, 1H, H- C=C), 5.30 (s, 2H, OCH ₂ ).

Anal. Calcd. for ₁₅ H _ιa N ₂ 0 ₆ C: 55.0: H, 5.63: N: 8.69. Found: C: 55.85; H: 5.67; N: 8.65.

b) Using DCC

To a suspension of Boc-Ser*0Bzl(N0 ₂ ) (1) (6.0 g, 0.0176 mol) and CuCl (1.8 g, 0.018 mol) in CHCI3 (180 mol) was added dicyclohexylcarbodii ide (DCC: 4.9 g, 0.0216 mol) with ice cooling and the mixture was stirred for 3 days at room temperature. Water (200 ml) was added to the reaction mixture and additional

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stirring was continued for 3 min when the CHCI3 layer was separated and the aqueous layer was extracted with CHCI3. The combined CHCI ₃ layer was washed with water, dried over Na ₂ S0 and evaporated in vacuo. The resulting residue was chroraatographed by elution with benzene (silica gel 60-200 mesh, 40 g. Baker Analyzed) to give 4.1 g, (72.3%) of Boc-dehydroalanine-p- nitrobenzyl ester (2), mp 94-96°C.

EXAMPLE III p-Nitrobenzyl-3-t-butoxycarbonylaminopyrazoline-3- carboxylate (3)

To a solution of Boc-dehydroalanine-p-nitrobenzyl ester (2) (700 mg, 2.2 mmol) in CH ₂ Cl ₂ (10 ml) was added dropwise ethereal CH ₂ N ₂ solution (prepared from Diazald, 4.12 g, 19 mmol) in Et ₂ 0 (40 ml) over a period of 40 min with ice cooling. After stirring for 1 hr at 0 5°C, excess of CH ₂ was decomposed by addition of CaCl ₂ at room temperature and the mixture was filtered. The filtrate was evaporated in vacuo to give a white solid and the solid was triturated with n- hexane and then filtered by suction to give 3 (710 mg: 88.5%), mp 79-80°C. Recrystallizatiion from AcOEt-n-hexane gave pure p-Nitrobenzyl-3-t- butoxycarbonylaminopyrazoline-3-carboxylate (3) as colorless prisms, mp 84°C (dec); IR: (KBr) 3270 (NH), 1700-1740 (C=0), 1600 (N=N). NMR(CDCl ₃ )δ: 8.2 and 7.45 (dd, 4H, ArH H), 6.38 (s, 1H, NH), 5.29 (s, 2H, OCH ₂ ), 4.4-52 (m, 2H, NCH ₂ ) , 1.95-2.25 (m, 2H, CH ₂ ) , 1.40 (s, 9H, Boc).

Anal. Calcd. for C _lg H ₂₀ N ₄ O ₆ : C: 52.74; H: 5.53; N: 15.38. Found: C: 52.62; H: 5.59; N: 15.34.

EXAMPLE IV

Boc-Cyclopropylalanine p-nitrobenzyl Ester (4)

A mixture of the pyrazoline, p-nitrobenzyl-3-t- butoxycarbonylamino-pyrazoline-3-carboxylate (3) (550 mg), and benzene (10 ml) was refluxed for 1.5 hr (bath temp., ca 90°C) and the benzene was removed in vacuo to give a solid, which was recystallized from AcOEt-n- hexane to afford 508 mg of Boc-cyclopropylalanine p- nitrobenzyl ester (4) (100%); mp 117-118°C; IR (KBr) 3350 (NH), 1730 (C=0) , 1680 (C=0) . NMR (CDCI3) δ: 8.2 and 7.48 (AB d, 4H, ArH), 5.2 (s, 2H, 0CH ₂ ), 5.15 (br, s, 1H, NH), 1.00-1.75 (m, 4H, CH ₂ x2) , 1.44 (s, 9H, Boc) .

Anal. Calcd. for C ₁₆ H ₂₀ N ₂ O ₆ : C: 57.14; H: 5.99; N: 8.33. Found: C: 57.05; H: 5.99; N: 8.31.

EXAMPLE V

Boc-Cyclopropyl Alanine (5)

To a solution of Boc-cyclopropylalanine p- nitrobenzyl Ester (4) (450 mg, 1.34 mmol) in MeOH (20 ml) was added IN NaOH (2.6 ml, 2.6 mmol) at room temperature. After the mixture was stirred for 3 hr, water (10 ml) was added and the MeOH was removed in vacuo. The aqueous residue was washed with Et ₂ 0 to remove p-nitrobenzyl alcohol and the aqueous layer was separated, cooled in an ice bath and 10% citric acid solution was added to pH 3. The mixture was saturated with NaCl and extracted with AcOEt and the extracts were washed with sat. NaCl solution, dried over Na ₂ S0 and evaporated in vacuo to give a white solid which was recrystallized from AcOEt-n-hexane to afford 260 rag of Boc-cyclopropyl Alanine (5) (96.3%) as colorless

needles, rap 176-177°C (dec); IR (KBr): 3230(NH), 1630-1680 (C=0). NMR (CDCI3+DMSO) : 9.45 (br, s, 1H, COOH), 5.88 (br, s, 1H, NH) 1.3-1.7 (m, 2H, CH ₂ ), 1.0- 1.2 (m, 2H, CH ₂ ) , 1.50 (s, 9H, Boc) .

Anal. Calcd. for C ₉ H ₁₅ 0 ₄ C: 53.72; H: 7.51; N: 6.96. Found: C: 53.59; H: 7.58; N: 6.88.

EXAMPLE VI Diazoisobutane (6)

To a solution of isobutylurea (2.0 g, 0.017 mol) in HOAc/H ₂ 0 (6:1) (6 ml) was added dropwise 4.8 M NaN0 ₂ solution (6 ml) with ice cooling over 1 hr. After stirring was continued for an additional 1 hr, water (20 ml) was added to the reaction mixture and the yellow crystals were extracted into CHCI3. The extract was washed with water and evaporated at 25°C to dryness giving a yellow solid. The resulting crude nitroso compound was dissolved in Et ₂ 0 (20 ml) and the solution was added dropwise to the mixture of 40% KOH solution (5.4 ml) and Et ₂ 0 (20 ml) at -15°C to -20°C over a 1 hr period. The reaction mixture was stirred for 1 hr at the same temperature and the Et ₂ 0 layer containing diazoisobutane (6) was separated and used in the next reaction immediately.

EXAMPLE VII p-Nitrobenzyl 3-t-Butoxycarbonylamino-5- ispropylpyrazoline-3-carboxylate (7)

The ethereal diazoisobutane (6) was gradually added to a solution of Boc-dehydroalanine-p-nitrobenzyl ester (2) (967 mg, 3 mmol) in CH ₂ C1 ₂ (15 ml) at -10°C to -15°C with stirring. After stirring for 1 hr at the

same temperature, the solvent was evaporated in vacuo and the resulting residue was triturated with hexane and filtered by suction to give 1.2 g of p-nitrobenzyl p-nitrobenzyl 3-t-butoxycarbonylamino-5- isopropylpyrazoline-3-carboxylate (7) (98.4%) mp 78- 79°C (dec). Recrystallization from AcOEt-n hexane gave colorless prisms having mp 87-89°C (dec); IR (KBr) 3390(NH), 1745(C=0), 1690(C=0), 1605 (N=N) .NMR (CDC1 ₃ ) δ: 8.2 and 7.5 (d, d, 4H, ArH), 6.2 (br s, IH, NH); 5.30 (s, 2H, OCH ₂ ), 4.8-5.2 (m, IH, CH-N=N) , 1.5- 2.3 (m, 3H, (CH ₃ ) ₂ CH and CH ₂ ), 1.35 (s, 9H, Boc), 1.00- 1.30 (m, 3H, CH ₃ ), 0.8-1.10 (ra, 3H, CH3).

Anal. Calcd for C ₁₉ H ₂₆ N ₄ 0 ₆ : C: 56.15; H: 6.45 N: 13.79. Found: C: 55.93; H: 6.53; N: 13.75.

EXAMPLE VIII

Boc-Cyclopropyl Leucine p-Nitrobenzyl Ester (8)

The pyrazoline, p-nitrobenzyl 3-t- butoxycarbonylamino-5-isopropylpyrazoline-3-carboxylate (7) (1.1 g, 2.7 mmol), was dissolved in benzene (20 ml), the solution was refluxed for 2 hr and evaporated in vacuo to give a white solid which was recrystallized form AcOEt-n-hexane giving co ^' lorless prisms of Boc- cyclopropyl leucine p-nitrobenzyl ester (8), 950 mg (95%), mp 139-143°C, IR (KBr) 3360(NH), 1725(C=0), 1680 (C=0). NMR (CDCl ₃ δ: 8.20 and 7.52 (d,d, 4H, ArH), 5.25 (s, 2H, 0CH ₂ )/ 5.22 (br, s, 1H,NH), 1.2-1.8 (m, 4H, CH ₂ / CHx2), 1.4 (s, 9H, Boc), 0.8-1.15 (m, 6H, (CH ₃ ) ₂ CH).

Anal. Calcd. for C ₁₉ H ₂₆ N ₂ 0 ₆ : C: 60.30; H: 6.93; N: 7.40. Found: C: 59.73; H, 7.05; N, 8.27.

Λ

EXAMPLE IX

Boc-Cyclopropyl-Leucine (9)

To a suspension of Box-cyclopropyl leucine p- nitrobenzyl ester (8) (300 mg, 7.9 mmol) in MeOH (20 ml) was added 2N NaOH soln. (7 ml) under ice cooling and the mixture was stirred for 3 hr at room temperature, the starting material gradually dissolved and the mixture turned yellow. Water (10 ml) was added and the MeOH was evaporated in vacuo. The aqueous residue was washed with AcOEt, cooled in an ice bath and acidified by the addition of 10% citric acid to pH 3. The resulting white precipitate was extracted with AcOEt (3x20 ml) and the extract was washed sat. NaCl soln, dried over Na ₂ S0 ₄ , and evaporated in vacuo. The resulting of Boc-cyclopropyl-leucine (9) was recrystallized from AcOEt-n-hexane to give 140 mg, (72.5%) as colorless prisms; mp 196-7°C (dec); IR: (KBr) 3230(NH), 1690(C=0), 1645(C=0). NMR (CDCI3+DMSO- d ₆ ) : 5.78 (s, IH, NH), 1.2-1.8 (m, 4H, CH ₂ , CHx2), 1.4 (s, 9H, Boc), 0.9-1.1 (m, 6H, (CH ₃ ) ₂ CH).

Anal. Calcd for C ₁₂ H ₁ N0 ₄ : C: 59.24; H: 8.70; N: 5.76. Found: C: 59.28; H: 8.74; N: 5.72.

EXAMPLE X

Benzaldehyde p-Toluenesulfonyl Hydrazone (10)

A mixture of benzaldehyde (5.25 g, 0.05 mol), p- toluenesulfonyl hydrazide (9.3 g, 0.05 mol) an dAcOH (20 ml) was stirred for 15 min at 70°C, and allowed to stand overnight at room temperature. Et ₂ 0 (20 ml) was added to the mixture, the precipitated solid was tritrated with ether and the crystals were filtered by suction and washed with Et2θ to give Benzaldehyde p-

Toluenesulfonyl Hydrazone (10), 9.7g (70.7%), mp 126- 128°C (dec).

EXAMPLE XI

Boc E-Cyclopropyl Phenylalanine p~Nitrobenzyl Ester (11)

Sodium (138 mg, 6 mmol) was dissolved in ethylene glycol (10 ml) and the tosylhydrazone, benzaldehyde p- toluenesulfonyl hydrazone (10), 923 mg, 3 mmol) was added to the solution. When dissolution was completed, hexane (20 ml) was added and the mixture was refluxed for 20 min (bath temp. 85-90°C) with vigorous stirring. Then the mixture was cooled in an ice bath and the resulting red colored product was extracted with cold n-hexane (20 ml x 3). The combined extracts were washed with IN NaOH soln. (20 ml), sat. NaCl soln. (20 ml) and then dried over Na ₂ S0 ₄ . After filtering, the pink filtrate was added to a mixture of Boc-dehydroalanine-p-nitrobenzyl ester (2) (322 mg, 1 mmol) in CH ₂ Cl ₂ (10 ml) over a period of 15 min at 0°C. The mixture was stirred overnight at room temperature and the red color disappeared. The solvent was evaporated in vacuo and the residue was triturated with n-hexane-ether. The resulting solid was filtered by suction to give Boc E-cyclopropyl Phenylalanine p- nitrobenzyl ester (11) (380 mg, 92.2%); mp 115-6°C; IR: (KBr) 3390(NH), 1715(C=0). NMR (CDCl ₃ )δ: 8.1 and 7.1 (dd, 4H, ArH), 7.1-7.50 (m, 5H, ArH), 5.45 (br. s, IH, NH), 4.9 (s, 2H, CH ₂ 0) , 2.8-3.1 (m, IH, CH) , 2.0-2.4, 1.2 1.8 (m, 2H, CH ₂ ), 1.45 (s, 9H, Boc).

EXAMPLE XII Boc E-Cyclopropyl Phenylalanine (12)

A mixture of Boc E-cyclopropyl phenylalanine p-

nitrobenzyl Ester (11) (200 mg, 0.485 mmol), MeOH (10 ml) and 2N NaOH soln. (3 ml) was stirred overnight at room temperature. Water (10 ml) was added and the MeOH was evaporated in vacuo. The residue was washed with AcOEt and the aqueous layer was cooled in an ice bath and acidified with 10% citric acid to pH 3. The mixture was saturated with NaCl and extracted with AcOEt. The extract was washed with sat. NaCl soln, dried over Na ₂ S0 ₄ and evaporated in vacuo. The resulting solid was recrystallized from AcOEt-n-hexane to give Boc E-cyclopropyl phenylalanine (12) (90 mg, 67.2%) as colorless prisms, mp 158-160°C (dec); NMR (CDC1 ₃ ) δ: 7.2-7.4 (m, 5H, ArH), 2.7-2.9 (m, IH, CH), 2.0-2.3 and 1.5-1.7 (m, 2H, CH ₂ ), 1.5 (s, 9H, BOC). NMR identical with that of earlier sample.

EXAMPLE XIII p-Nitrobenzyl 3-t-Butoxycarbonylamino-5—(N-tosylindol- 3-yl)-pyrazoline-3-carboxylate (13)

A solution of N-tosylindol-3-yl diazomethane (3 mml) in CH ₂ Cl ₂ (20 ml) is added to a solution of Boc- dehydroalanine-p-nitrobenzyl ester (2) in 20 ml CH C1 ₂ at -15°C. After stirring 1 hr at -15°C and 4 hr at 25°C, the solution is evaporated to dryness and the residue is triturated with hexane. The solid product, p-nitrobenzyl 3-t-butoxycarbonylamino-5-(N-tosylindol- 3-yl)-pyrazoline-3-carboxylate (13), is recrystallized from ethyl acetate-hexane to constant melting point. NMR (DCDI3) δ: 4.8-5.4 (m, IH, CH-N=N).

EXAMPLE XIV Boc Cyclopropyl Tryptophan p-Nitrobenzyl Ester (14)

The pyrazoline, p-nitrobenzyl 3-t-

"gJR£

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butoxycarbonylamino-5-(N-tosylindol-3-yl)-pyrazolline- 3-carboxylate (13), (1 mmole) is suspended in toluene (50 ml) and the mixture is refluxed until its NMR spectrum shows the presence of cyclopropane protons (δ3.0-3.5 and 0.8-1.2 ppm) and the loss of the pyrazoline peak at δ 5.0. (2 hr). The solution is evaporated and the residue of Boc cyclopropyl tryptophan p-nitrobenzyl ester (14) is crystallized from ethylacetate-hexane. NMR (DCDI3) δ: 2.7-3.0 (m, IH, CH), 2.0-2.3 and 1.5-1.7 (m, 2H, CH ₂ ).

EXAMPLE XV p-Nitrobenzyl 3-t-Butoxycarbonyl-5-(3-chloropropyl)- pyrazoline-3-carboxylate (15)

4-Chlorobutyraldehyde tosylhydrazone (3 mmole) is added to a solution of sodium (6 mmole) in ethylene glycol (15 ml). Hexane (25 ml) is added and the mixture is stirred 30 min at 90°C. After cooling, the diazo-compound is extracted into cold hexane (3 x 20) ml). The combined extracts are washed with IN NaOH (20 ml) and saturated NaCl solution (25 ml) and dried over anhyd. Na ₂ S0 ₄ . After filtration, the filtrate is added to a mixture of Boc-dehydroalanine-p-nitroenzyl ester (2) (1 mmole) in CH ₂ C1 ₂ (10 ml) at 0°C over a 15-minute period. After stirring at 25°C for 16 hr, the solution is evaporated and the residue is triturated with Et ₂ o- hexane. The solid pyrazoline, p-nitrobenzyl 3-t- butoxycarbonyl-5-(3-chloropropyl)-pyrazoline-3- carboxylate (15), is crystallized from ethyl acetate- hexane. NMR (CDCI3) 4.5-5.4 (m, IH, CH-N=N) .

EXAMPLE XVI

Boc 3-(3-Chloropropyl) Cyclopropyl Alanine p- Nitrobenzyl Ester (16)

The pyrazoline, p-nitrobenzyl 3-t-butoxycarbonyl- 5-(3-chloropropyl)-pyrazoline-3-carboxylate (15), (1 mmole) is suspended in toluene (50 ml) and the mixture is refluxed until its NMR spectrum shows the presence of cyclopropane protons ( δ3.0-3.5 and 0.8-1.2 pmm) and the loss of the pyrazoline peak at δ5.0 (2 hr) . The solution is evaporated and the residue of Boc 3-(3- Chloropropyl) cyclopropyl alanine p-nitrobenzyl ester (16) is crystallized from ethyl acetate-hexane. NMR (CDC1 ₃ ) δ:2.7-3.0 (m, IH, CH) , 2.0-2.3 and 1.5-1.7 (m,

EXAMPLE XVII

Boc Cyclopropyl Lysine p-Nitrobenzyl Ester (17)

The cyclopropyl alanine derivative Boc 3-(3- Chloropropyl) cyclopropyl alanine p-nitrobenzyl Ester (16) is treated with a 1M solution of ammonia in isopropanol at 50°C in a sealed pressure bottle for 48 hrs. Evaporation of the solution gives a solid residue which is dissolved in warm ethyl acetate (5 ml) and the solution is washed with saturated NaCl solution (3x25 ml) and dried over anhyd. Na S0 ₄ . Evaporation of the solution gives Boc cyclopropyl lysine p-nitrobenzyl ester (17) which recrystallized from ethyl acetate- hexane. NMR (CDCI3) : 1.2-1.8 (m, 5, CH ₂ CH ₂ CH), 0.8- 1.1 (m, 2H, CH ₂ ), 2.5 (m, 2H, CH ₂ NH ₂ ) .

Example XVIII p-Nitrobenzyl 3-t-Butoxycarbonylamino-5-(4-[2,4- dinitrophenoxyl]-phenyl)-pyrazoline-3-carboxylate (18) .

A solution of 4-(2,4-dinitrophenoxy)- phenyldiazomethane in CH ₂ Cl2 (20 ml) is added to a solution of Boc-dehydroalanine-p-nitrobenzyl ester (2) in 20 ml CH ₂ C1 ₂ at -15°C. After stirring 1 hr at -15°C and 4 hr at 25°, the solution is evaporated to dryness and the residue is triturated with hexane. The solid product, p-nitrobenzyl 3-t-butoxycarbonylamino-5-(4-

[2,4-dinitro-phenoxyl]-phenyl)-pyrazoline-3-carboxylate (18), is recrystallized from ethyl acetate-hexane to constant melting point. NMR (CDCI3) : 4.8-5.4 (m, IH, CH-N=N) .

Example XIX

Boc-0-2,4-dinitrophenyl Cyclopropyl Tyrosine p- Nitrobenzyl Ester (19).

The pyrazoline, p-nitrobenzyl 3-t- butoxycarbonylamino-5-(4-[2,4-dinitrophenoxyl]-phenyl)- pyrazoline-3-carboxylate (18), is suspended in toluene (50 ml) and the mixture is refluxed until its NMR spectrum shows the presence of cyclopropane protons (63.0-3.5 and 0.8-1.2 ppm) and the loss of the pyrazoline peat at 65.0 (2 hr) . The solution is evaporated and the residue of Boc-0-2,4-dinitrophenyl cyclopropyl tyrosine p-nitrobenzyl ester (19) is crystallized from ethylacetate-hexane. NMR (CDCL3 6: 2.7-3.0 (m, IH, CH), 2.0-2.3 and 1.5-1.7 (m, 2H, CH ₂ ).

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Ξxample XX Boc Cyclopropyl Tyrosine p-Nitrobenzyl Ester (20).

A solution of Boc 0—2,4-dinitrophenyl cyclopropyl tyrosine p-nitrobenzyl ester (19) (1 mmole) in DMF (2 ml) is treated with 2-raercaptoethanol (22 mmole). After 1 hr. at 25°C the DMF was evaporated and the residue of Boc cyclopropyl tyrosine p-nitrobenzyl ester (20) is crystallized from ethyl acetate-hexane. NMR (CDC1 ₃ ) 6 : 2.7-2.9 (m, IH, CH) , 2.0-2.3, 1.5-1.7 (m, 2H, CH ₂ ).

Example XXI

A solution of isobutyl chloroforraate (546 mg, 4 mmol) in chloroform (10 ml) was added dropwise to a solution of Boc-Ser ^* OBzll(N0 ₂ ) (1) (1.24 g, 4 mmol) and N-methylraorpholine (404 mg, 4 mmol) in chloroform (30 ml) at 0.5°C. After stirring for 20 min, a solution of Leu*OMe*HCl (1.45 g, 8 mmol) and N-methylraorpholine (0.81 g, 8 mmol) in chloroform (20 ml) was added at 10°C. After stirring for 2 hr at 0.5°C and then overnight at room temperature, the reaction mixture was washed with H ₂ 0, 5% citric acid and 5% aHCθ3 successively, and dried over Na ₂ S0 ₄ .~ The solvent was evaporated in vacuo and the residue was recrystallized from ethylacetate-hexane to give Z-(2RS)- V Phe-

Leu'OMe, '21) (1.13 g, 64.4%) as colorless needles, mp 94-97°C. R _f (l) = 0.85, R _f (III) = 0.12.

Anal. Calcd. for C ₂₅ H3 _{0 2} Og: C, 68.47; H, 6.90; N, 6.39. Found: C, 68.50; H, 6.99; N, 6.38.

Separation of Z-(2RS)- V ⁵ Phe-Leu'OMe, (21) into Diastereomers by HPLC.

Z-(2RS)- V ^s Phe-Leu ^* OMe, (21) was separated into its diastereoisomers using HPLX (C^g Lichrosorb, 20 cm x 0.46 cm, CH ₃ CN-H ₂ 0 (55:45), 2 ml/min) . The (2S) V^Phe isomer showed t _R =6.2 min and the (2R)V^Phe isomer showed t _R =8.1 min. This compound has utility in synthesis as an intermediate to form an enkephalin peptide which inhibits muscle contraction.

Example XXII

(2S)-V ^e Phe-Leu OMe TFA (22)

A solution of (Z-(2S)-V ^E Phe-Leu OMe (1.31 g, 3 mmol) and thioanisole (2 ml) in trifluoroacetic acid (TFA) (20 ml) was stirred at 0° for 3 hr, and then room temperature overnight. The TFA was removed under reduced pressure, and the residue "was triturated with ether (30 ml) and the precipitated crystals were collected by suction and washed with ether to give (2S)-V ^E Phe-Leu OMe TFA (22) (1.14 g, 91.2%), mp 251-2° (dec); [c-] ^D -89.6 ^ϋ (C=0.5 H ₂ 0). NMR (CF ₃ C0 ₂ H- CDC1 ₃ (1:) δ : 0.67 (6H, br s (CH3)), 0.78-1.40 (3H, m, CH ₂ CH), 2.28 (2H, d, J = 10 Hz, ), 3.50 (IH, t, J = Hz, _H -Δ), 3.85 (3H, s, CH3O), 4.20-4.50 (lH; m, CHC0 ₂ Me), 5.66-5.88 (IH, m, NH) , 7.53 (5H, s, ArH), 7.70-8.20 (2H, br, NH3). R _f (IV) = 0.74.

Anal. Calce. for C ₁₉ H ₂ gF3 ₂ θ5: C, 54.54; H, 6.02; N, 6.70. Found: C, 54.61; H, 6.05; N, 6.66.

This compound has utility as an intermediate in synthesis of an enkephalin peptide which inhibits

^•

muscle contraction.

Example XXIII Z-(2S)-V ^E Phe-Leu OMe (23).

A solution of Z(2S)V ^E Phe (1.24 g, 4 mmol) and Leu OMe HCl (1.09 g, 6 mmol) in the THF (50 ml) was chilled to 0°C and triethylamine (0.61 g, 6 mmol), HOBt (0.54 g, 4 mmol) and DCC (0.83 g, 4 mmol) and DCC (0.83 g, 4 mmol) were added successively at 0° with stirring. After stirring for 4 hr at 0°C and room temperature overnight, the precipitated crystals were filtered and the filtrate was evaporated in vacuo. The residue was extracted with ethyl acetate and the extract was washed with 5% citric acid, 5% NaHCθ3 and water successively, and then dried over Na ₂ S0 . The solvent was removed under reduced pressure and the resulting solid was recrystallized from ethyl acetate- hexane to give Z-(2S)-V ^E Phe-Leu OMe (23) (1.48 g, 84.5%) as a colorless needles, mp 114-115°C; [α] ² - 132.1° (c=1.0, MeOH). NMR (CDCI3) δ : 0.78 6H, d, J « 6 Hz, (CH ₃ ) ₂ CH), 1.10-1.70 (4H, m, CH-CH ₂ and H), 2.18 (IH, d of d, J = 9 Hz, and 6 Hz, VH), 2.80 (IH, t, J = 9 Hz, Ph ^H Δ) , 3.56 (3H, s, CH3O), 4.17-4.45 (IH, m, CHC0 ₂ Me), 5.25 (2H, s, PhCH ₂ 0) , 5.56-5.80 (IH, br, NH) , 6.60-6.95 (IH, br, NH), 7.33 (5H, br s, Ph V), 7.47 (5H, s, PhCH ₂ ). R _f (l) = 0.85, R _f (IV) = 0.12.

Anal. Calcd. for C ₂₅ H3 ₀ N ₂ O ₅ : C, 68.471 H, 6.90; N, 6.39. Found: C, 68.53; H, 6.93; N, 6.35.

This compound has utility as an intermediate in synthesis of an enkephalin peptide which inhibits muscle contraction.

Example XXIV

Z-(2S)-V ⁱⁱ Phe-Leu OMe (24)

Following the same procedure as above for Z-(2S)-V ^E Phe-Leu OMe (23), Z-(2R)-V ^E Phe (1.24 g, 4 mmol) and Leu OMe HCl (1.09 g, 6 mmol) was treated with Et ₃ N (0.61 g, 6 mmol), HOBt (0.54 g, 4 mmol) and DCC (0.83 g, 4 mmol) in THF (50 ml) to give Z-(2S)-V ^E Phe-Leu OME (24) 1.41 g, 80.5%) as prisms (ethyl acetate-hexane); [α] ^ 87.6° (C=1.0, MeOH); NMR (CDC1 ₃ ): 0.57 (3H, d, J = 6 Hz, CH3), 0.68 (3H, d, J = 6 Hz, CH ₃ ), 0.70-1.45 (4H, m, CH ₂ CH, _H_), 2.25 (IH, d of d, J = 9 Hz and 6 Hz, E ) , 2.77 (IH, t, J - 9 Hz 3,65 (3H, s, CH3O), 4.15-4.50 (IH, m, CHC0 ₂ Me), 5.27 (2H, s, PhCH ₂ 0), 5.73 (IH, s, NH), 6.83-7.15 (IH, br, NH), 7.32 (5H, br s Ph-CH-) , 7.55 (5H, s, PhCH,). P _f (l) = 0.85, R _f (III) - 0.12.

Anal. Calcd. for C ₂₅ H _{30 2} O ₅ : C, 68.47; H, 6.90; N, 6.39. Found: C, 68.30; H, 6.96; N, 6.32.

Example XXV (2R)-V ^E Phe-Leu OMe TFA (25).

Following a procedure similar to that above for (25)-V ^E Phe-Leu OMe TFA (22), Z(2R)V ^E Phe (1.31 g, 4 mmol) was treated with thioanisole (2 ml) and TFA (20 ml) to give (2R)-^Phe-Leu OMe TFA (25) (1.09 g, 87%), mp 256-257°C (dec); C ] ²² 24,2°, NMR (CDC1 ₃ -CF ₃ C0 ₂ H

(1:1)) 6 : 0.83 (6H, d, J = 4 Hz, CH3), 1.05-1.53 (3H, m, CH ₂ CH), 2.03-2.45 (2H, n, 3.53 (IH, t, J = 10 Hz, 3.75 (3H, s, CH3O), 4.23-4.52 (IH, m, CHC0 ₂ Me), 5.80 (IH, d, J = 8 Hz, NH), 7.52 (5H, s, ArH), 7.80- 8.20 (2H, br NH). R _f (lV) = 0.77, R _f (V) ^~~ 0.80.

Anal. Calcd. for C ₁₉ H ₂₅ F _{3 2} 0 ₅ : C, 54.54, H, 6.02; N, 6.70. Found: C, 54.56; H, 6.06; N, 6.66.

This compound has utility as an intermediate in synthesis of an enkephalin peptide which inhibits muscle contraction.

Example XXVI Z-D-Ala-Gly-OMe (26).

A solution of Z-D-Ala (2.23 g, 10 mmol) and Gly OMe HCl (1.26 g, 10 mmol) in THF (50 ml) was chilled to 0°C and triethylamine (1.01 g, 10 mmol), HOBt (1.35 g, 10 mmol) and DCC (2.06 f, 10 mmol) was added successively at O±C with stirring. After stirring for 4 hrs at 0°C, the reaction mixture was stirred at room temperature overnight and the precipitted crystals were filtered and the filtrate was evaporated in vacuo. The residue was extracted with ethyl acetate and the extract was washed with 5% citric acid, 5% NaHCθ and water successively, and dried over anhyd. Na ₂ S0 ₄ . The solvent was removed in vacuo and the resulting solid was recrystallized from ethyl acetate-hexane to give Z-D-Ala-Gly-OMe (16) (2.48 g,

22 84.4%) as colorless needles, mp 96-97°C; [«]" 23.3° (c

= 1.0, MeOH): NMR (CDC1 ₃ ) δ : 1.39 (3H, d, J = 8 Hz,

CH3), 3.76 (3H, s CH3O), 4.03 (2H, d, J = 6 Hz, CH ₂ NH) , 4.15-4.50 (IH, m, -CH-NH), 5.15 (2H, s, CH ₂ 0), 5.58

(IH, d, J = 7 Hz, NH), 6.70-6.95 (IH, br, NH), 7.42

(5H, s, Ar-H). R _f (l) = 0.62, R _f (Vl) = 0.38.

Anal. Calcd. for C ₁ H ₁₈ N ₂ 0 ₅ : C, 57.14; H, 6.16; N, 9.52. Found: C, 57.19; H, 6.210; N, 9.50.

This compound has utility as an intermediate in

synthesis of an analgetic peptide which inhibits muscle contraction.

Example XXVII Z-Try-D-Ala- _G ly-OMe (27).

A suspension of Z-D-Ala-Gly-OMe (26) (5.88 g, 0.02 mmol) and 10% Pd-C (0.4 g) in methanol (300 ml) was stirred under a hydrogen atmosphere at room temperature for 1.5 hr. The catalyst was fintered and the filtrate was evaporated in vacuo. The residue and Z-Tyr (6.30 g, 0.02 mol) was dissolved in dry THF (300 ml), cooled to 0°C and HOBt (2.70 g, 0.02 mol) and DCC (4.12 g. 0.02 mol) were added successively at 0°C. The solution was stirred for 3 hr at 0°C and overnight at room temperature. The precipitated crystals were filtered. The filtrate was evaporated under reduced pressure, the residual crystals were dissolved in EtOAc and the solution was washed with 5% NaHO^, 0.2 N Hcl and water, and dried over anhyd. Na ₂ S0 ₄ . The solvent wamoved in vacuo and the resulting solid was purified by silica gel column chromatography using CHCI3-CH3OH

(98:2) as eluant to give Z-Tyr-D-Ala-Gly-OMe (17) (8.62 g, 94.3%), mp 154-155°C (AcOEt-hexane) ; [ α] ²² 33.4° (c = 1.0, MeOH); NMR (CDCl ₃ -DMSO-d ₆ (4:1) δ: 1.25 (3H, d, J = 7 Hz, CH3), 2.82-3.03 (2H, m, CH ₂ ,), 3.70 (3H, s, CH3O), 3.80-3.95 (2H, m, CH ₂ PhOH), 4.15-4.60 (2H, m, 2 CH), 5.05 (2H, s, CH ₂ 0), 6.70-7.25 (4H, m, ArH), 6.70- 6.90 (IH, br, NH), 7.36 (5H, s, ArH), 7.70-8.10 (2H, br, NH). R _f (D = 0.48; R _f (VI) = 0.08.

Anal. Calcd for G23H ₂₇ N ₃ 0 ₇ : C, 60.39; H, 5.95; N, 9.18. Found: C, 60.23; H, 6.00; N, 9.08.

This compound has utility as an intermediate in

synthesis of an analgetic peptide which inhibits muscle contraction.

Example XXVIII Z-Tyr-D-Ala-Gly.OH (28) .

To a solution of Z-Tyr-D-Ala-Gly-OMe (27) (4.57 g, 0.01 mol) in MeOH (10 ml) was added 1 N NaOH (20 ml, 0.02 mol) at 0° was stirring. The suspension was stirred for 2 hr at 0°C, diluted with water (80 ml), and neutralized with 1 N HCl (20 ml). The precipitated crystals were collected by suction, washed with water and dried under reduced pressure to give Z-Tyr-D-Ala- Gly.OH (18) (3.59 g, 81.0%), mp 102-104°C (AcOEt) (lit. ⁺ mp 1241C); [c-] ²² , 18.0° (C = 1.0, DMF, (lit. ^1" [α] ^E _Q 16.7° (c = 0.54, DMF)); NMR (DMSO- D ₆ ) 6: 1.16 (3H, d, J = 7 Hz, CH3), 2.60-2.90 (2H, m, CH2-), 3.80 (2H, d, J = 6 Hz, CH ₂ PHOH), 4.10-4.45 (2H, m, 2CH), 5.02 (2H, s, CH ₂ 0), 6.63-7.20 (4H, m, HOPh-) , 7.40 (5H, s, ArH), 8.10-8.30 (2H, br, 2 NH), 9.10-9.40 (IH, br, OH). R _f (IV) = 0.20.

Anal. Calcd. for C ₂₂ H _{25 3} 0 ₇ : C, 59.59; H, 5.68; N, 9.48. Found: C, 58.32; H, 5.79; N, 9.39. ^* S m Shinagawa, M. Fujini, H. Ishii and K. Kawai, Chem. Pharm. B ull., 29, 3630 (1981).

This compound has utility as an intermediate in synthesis of an analgetic peptide which inhibits muscle contraction.

Ξxample XXIX Z-Tyr-D-Ala-Gly(2S)-V ^E Phe-Leu OMe (29).

To a solution of Z-Tyr-D-Ala-Gly OH (887 mg, 2 mmol) and (2S)-V ^E Phe-Leu TFA (836 mg, 2 mmol) in THF (80 ml) was added N-methylmorpholine (202 mg, 2 mmol) HOBt (270 rag, 2 mmol) and EDC (l-ethyl-3(3- dimethylaminopropyl)-carbodimide HCl) (384 mg, 2 mmol) successively at 0°C. After stirring for 3 hr at 0°C and overnight at room temperature, the solvent was removed under reduced pressure, and the residue was extracted three with 100 ml of AcOEt. The combined AcOEt extracts were washed with 5% NaHCθ , 0.2 N HCl, water, and dried over anhyd. Na ₂ S0 ₄ . The solvent was removed under reduced pressure, and the solid was recrystallized from ethylacetate-hexane to give 1.212 g (83%) of Z-Tyr-D-Ala-Gly(2S)- V ^E Phe-Leu OMe (29) as a colorless powder, mp 162-163°C; [ ] _D -56.8° (c = 0.5, DMF). NMR (DMSO-d ₆ ) wδ: 0.78 (6H, t, J = 5 Hz, CH ₃ x2), 1.00-1.67 (4H, m, CH ₂ CH and cyclopropyl-H), 1.18 (3H, d, J = 7 Hz, CH ₃ ), 1.90-2.15 (IH, m, cyclopropyl-H),

2.46-3.00 (3H, m, CH ₂ and cyclopropyl-H), 3.48 (3H, s, CH ₃ 0), 3.70-3.80 (2H, br, CH ₂ ) 3.95-4.43 (3H, m, 3 CH), 5.01 (2H, s, CH ₂ ), 6.67-7.18 (4H, m, HOPh-) , 7.32 (5H, s, ArH), 7.40 (5H, s, Ar-H), 7.30-7.60 (IH, m, NH), 7.72 (IH, d, J = 8 Hz, NH), 8.10-8.40 (2H, m, 2 NH), 8.68 (IH, s, NH), 9.28 (IH, s, OH). R _f (Il) = 0.79; R _f (IV) = 0.63.

Anal. Calcd, for C ₃₉ H ₄₇ N ₅ 0 ₉ : C, 64.18; H, 6.49; N, 9.60. Found: C, 64.04; H, 6.54; N, 9.56.

This compound has utility as an intermediate in synthesis of an analgetic peptide which inhibits muscle contraction.

V- !Λ".'•'

Example XXX

Z-Tyr-D-Ala-Gly-(2R)-V ^E Phe-Leu OMe (30).

Following a procedure similar to that described for Z-Tyr-D-Ala-Gly (2S)- ^Phe-Leu OMe (29), Z-Tyr-D-Ala-Gly OH (887 mg,

2 mmol); (R)- V ^E Phe-Leu OMe TFA (836 mg, 2 mmol), N-methylmorpholine (202 mg, 2 mmol), HOBt (270 mg, 2 mmol), EDC (384 mg, 2 mmol), and THF (80 ml) gave 1.287 g (88.2%) of Z-Tyr-D-Ala-Gly-(2R)-V ^E Phe-Leu OMe (30) as a colorless powder, mp 160-170°C (AcEOt-hexane) . NMR (DMSO- d ₆ ) δ: 0.53 (3H, d, J = 5 Hz, CH ₃ ), 0.73 (3H, d, J = 5 Hz, CH ₃ ), 1.20 (3H, d, J = 6 Hz, CH3), 1.05-1.45 (4H, ra, CH ₂ CH and VH) , 1.87-2.13 (IH, m, VH) , 2.43-2.60 (IH, m, VH), 2.60-2.90 (3H, m, CH3), 3H, s, CH3O), 3.70-3.85 (2H, br, CH ₂ ), 3.90-4.45 (3H, m, CHx3), 5.03 (2H, s, CH ₂ ), 6.66-7.23 (4H, m, HOPh)7.31 (5H, s, ArH), 7.40 (5H, s, ArH), 7.40-7.85 (2H, m, NHx2) , 8.15-8.40 (2H, br, NHx2), 8.70 (IH, s, NH), 9.25 (IH, s, OH). R _f (Il) = 0.81, R _f (IV) 0.63.

Anal. Calcd, for C ₃₉ H ₄₇ N ₅ 0 ₉ : C, 64.18; H, 6.49; N, 9.60. Foyund: C, 63.99; H, 6.51; N, 9.56.

This compound has utility as an intermediate in synthesis of an analgetic peptide which inhibits muscle contraction.

Example XXXI Z-Tyr-D-Ala-Gly-(25) V ^E Phe-Leu OH (31).

Z-Tyr-D-Ala-Gly-(2S) V ^E Phe-Leu OMe (29) (365 mg, 0.5 mmol) was dissolved in methanol (1 ml), the

solution was cooled in an ice-water bath and IN NaOH (1 ml, 1 mmol) at 0°C was added. After stirring for 2 hr at 0°C, the solution was neutralized with IN HCl and diluted with water. The precipitated solid was collected by suction and dried under reduced pressure. The solid was purified by silica gel column chroma¬ tography using chlorofoirm-methanol (19:1) and chloroform-methanol-aceit acid (95:5:1) as eluants. After starting material was removed using CHC^/MeOH (19:1), Z-Tyr-D-Ala-Gly-(2S) V ^E Phe-Leu OH (31) 230 mg

(64%), mp 153-155°C (AcOEt). Intermediate to Example XXXIV. R _f (lV) = 0.48; Rf(V) = 0.88; [α] ²² -98.2° (co.5, DMF), was eluded with CHCl ₃ :MeOH:HOAc (95:5:1)

Anal. Calcd. for C ₃₈ H 5 ₅ 0 ₉ .H ₂ 0: C, 62.20; H, 6.46; N, 9.54. Found: C, 62.39; H, 6.50, N, 9.30.

Example XXXII Z-Tyr-D-Ala-Gly-(2R) V ^E Phe-Leu OH (32).

According to a procedure similar to that described above, Z-Tyr-D-Ala-Gly-(2R) V ^E Phe-Leu OMe (30) (365 mg, 0.5 mmol) gave 270 mg (75%) of

Z-Tyr-D-Ala-Gly-(2R) ^Phe-Leu OH (32), mp 204-205°C. (AcOEt); Rf(lV)=0.40; Rf(V)=0.80; (α) ²² -41.2° (c.0.5, DMF) .

Anal. Calcd. for C ₃₈ H ₄₅ N ₅ H ₂ 0: C, 62.20; H, 6.46 / N. 9.54. Found: C, 62.28; H, 6.46; N, 9.38.

Example XXXIII Tyr-D-Ala-Gly-(2S) ^Phe-Leu (33).

A solution of Z-Tyr-D-Ala-Gly-(2S) ^Phe-Leu OH (31) (143 rag.

- * ∑A£ _{/ m} CMP1 _. _ ^7v. ^ϊr' ° - ^ £i ΛTϊC ' ^'

0.2 mmol) and thioanisole (0.3 ml) in trifluoroacetic acid (TFA) (3 ml) was stirred 1 hr at 0°C, and 4 hr at room temperature. The solvent was removed under reduced pressure and the residue was triturated with ether to give a TFA salt. This salt was dissolved in 5% AcOH and passed through a column containing a large excess of Amberlite CG400 (acetate form), followed by a column of BioGel P-2 (1.9x8.9 cm, 200 400 mesh) (4ml/20 rain) . The fractions containing Tyr-D-Ala-Gly-(2S) ^Phe-Leu (33) were pooled and lyophilized to give 70 mg (60.1%) of (S)-10, mp 197 ^β C (dec); ret. time = 4.4 min (HPLC, C^g-Lichrosorb (20 cm x 0.46 cm), CH3CN: H ₂ 0:TFA (30:70:1), 1 ml/min; R _f (IV)=0.05; R _f (VI)0.38 [o] ² ^ -7.12° (c o.25 AcOH). Amino acid ratios in acid hydrolyzate: Tyr .94; Ala

0.92; Gly 1.0; Leu 1.01; C(2S)V ^E Phe was not detected].

Anal. Calcd. for C ₆ H _{39 5} O ₇ .0.8H O C, 60.45; H, 6.86; N, 11.74. Found: C, 60.62; H, 6.89; N, 11.42.

This compound has utility as a final peptide in synthesis of an analgetic peptide which inhibits muscle contraction.

Example XXXIV Tyr-D-Ala-Gly-(2R) ^Phe-Leu (34).

Following a procedure similar to that described above, Z-Tyr-D-Ala-Gly-(2R) V ^E Phe-Leu OH (32) (0.43 mg, 0.2 mmol) gave 82 mg (70.4%) of (2R)-10, mp 185°C (dec); ret. time 8.1 min [HPLC, C^g-Lichrosorb (20 cm x 0.46 cm), CH ₃ CN:H ₂ 0:TFA (30:70:1), 1 ml/min]; Rf(IV)=0.03; Rf(V)=0.35; Cα] ²² 89.6 (c 0.25, AcOH). Amino acid ratios in acid hydrolazate: Tyr 0.91, Ala 0.98, Gly 1.0, Leu 0.95. [(2SR)V ^E Phe was not detected].

OMPI K. WIPO ^~ .-

Anal. Calcd. for C3 ₀ H3 ₉ N ₅ O ₇ .CH3CO ₂ H: 1.5 H 0; C, 57.47; H, 6.93; N, 10.47. Found: C, 57.56; H, 6.67; N, 10.08.

This peptide is an active analgetic agent also.

These new peptides are extremely refractory towards hydrolysis. The cyclopropane ring of the variant amino acid sterically hinders reactions at the adjacent carbonyl and amino functions and the β-groups C0 ₂ H and phenyl will retard the enzyme splitting or cleavage of the peptide linkages.

Short chain dipeptides where both amino acids have been cyclopropylized will be particularly stable to acid hydrolysis at the raethylester group and the peptide linkage both. Unless the peptides themselves are toxic to humans no added toxicity due to the modified unnatural amino acid components should be exhibited by the sweetener.

The following example illustrates a specific end product embodiment within the scope of this invention and is not to be construed as limiting said scope. Where the invention has been described herein with regard to a certain specific embodiment, it is not so limited. It is to be understood that variations and modifications thereof may be made by those skilled in the art without departing from the scope of the invention.

It must be remembered that the sweetening properties of the aspartame type of peptide is dependent upon the stereochemistry of the individual amino acid components of the peptide. Each of the

amino acids can exist in either dextro (D) or leavo (L) rotatory form. The L form of the compounds possess the sweetness characteristic as best can be determined at this point. The D form of isomers are usually bitter or tastless. Combinations of the D and L forms of isomers are sometimes sweet also but usually not as intense in their sweetness as the L-isomers.

The new dipeptide cyclopropyl derivatives of the following example are water soluble substances which can be used in a number of applications where a non toxic non sugar type sweetener is desired. They are 100-200 times as sweet as sucrose and exhibit long term retention of that sweetening capacity. Moreover, the new dipeptides do not exhibit the unpleasant after- taste characteristic of saccharine and cyclamates.

These dipeptide compositions are particularly valuable as sweetening agents for carbonated and non- carbonated beverages, chewing gum edible materials such as fruit juices, vegetables, fruit, dairy products such as egg product, milk drinks, ice cream, syrups, cake mixes, instant beverage mixes, soda pop formulae, iceing and dessert toppings, sherbert, meat products, wines and salad mixes, and dressings.

Example XXXV N-CBZ-g-Benzyl-L-aspartyl-V -phenylalanine methyl ester (27)

A total of 1.57 g (4.1 mmoles) of N-CBZ-aspartic acid-β-benzyl ester dissolved in 50 ml of dry THF was cooled to -20°C in a Dry Ice-carbon tetrachloride bath and 0.58 ml (5.3 mmoles) of N-methylmorpholine and 0.68 ml (5.3 mmoles) of isobutyl chloroformate were added.

After 20 minutes a solution of 1.0 g (4.4 mmoles) of 25

V

in 20 nil of dioxane-water (7:3) containing 0.61 ml (4.4 mmoles) of triethylamine was added. The mixture was allowed to stir at room temperature overnight, the THF was removed in vacuo, 50 ml of ether and 10 ml of H ₂ 0 were added and the solution was extracted. The aqueous portion was extracted with additional ether (2 x 25 ml), the ether layers combined, washed with 5% citric acid (2 x 25 ml), 5% NaHC0 ₃ (2 x 25 ml), saturated NaCl (1 x 2 ml), and dried over anhydrous Na ₂ S0 ₄ . The solution was filtered, concentrated to ca 25 ml and hexanes added until cloudy. Crystallization at room temperature gave 1.73 g of 27 as a white crystalline solid. The filtrate was stored at 5 ^β C to affored an additional 0.22 g of _27_ for a total yield of 84%, m.p. 99-104°; R _f (IV) = 0.80; IR (KBr) 3310 (amide NH), 1750- 1650 cm ^-1 (CBZ C=0, ester C=0, amide C=0) ; NMR (CD1 ₃ ) 6 7.5-7.3 (m, 15H, ArH), 6.5 (br, s, IH, NH_) , 5.9-5.7 (m, IH, NH) , 5.2-5.0 (dd, 4H, PhCH^OCO-) , 4.5 (, IH, ce-jl) , 3.7 (2s, 3H, diastereomeric COOCH_, ₃ ), 3.1- 2.9 (, IH, ), 2.8-2.6 (, 2H, COO-CH^-) , 2.3-2.1 and 1.9-1.8 (m, 2H, ).

Anal. Calcd. for C ₃₀ H ₃₀ O _{7 2} : C, 67.92; H, 5.66; H, 5.28. Found: C, 67.98, H, 5.73; N, 5.30.

L-Aspartyl-V -phenylalanine methyl ester (29).

A total of 1.0 g (1.9 mmoles) of 27 was dissolved in 50 ml of absolute methanol, 70 mg of 10% Pd/C was added, and hydrogen bubbled through the solution for 5 hours. The solution was filtered through celite, and the filtrate concentrated in vacuo. Partition chromatography of this material on Sephadex G-10 using n-BuOH/HOAc/H ₂ 0 4:1:5 upper phase as eluant afforded one pure diastereomer of 29 (now termed 29A) in

fractions 41-90, the other pure diastereomer in fractions 112-150 (now termed 29B) , and a mixture of the two diastereomers in fractions 91-111. Fractions 41-90 were combined, concentrated in vacuo, 40 ml of 5% acetic acid was added, and the solution was lyophilized to give 150 mg of 29A. Fractions 91-111 were treated similarly to give 147 mg of 29A and 29B. Fractions 112-150 were treated similarly to give 210 mg of 29B, for a total yield of 88%, m.p. _29B_ 126-180°1T; [ α] ²⁵ 29A=35° (c.0.7, in lN_Hcl), [α] ² p 29B = 1.3° (c 1.1, in 1N_HC1); R _f =29A (V) = 0.36, R _f 29B (V) = 0.30; NMR (CD ₃ 0D) 29A and 29B 67.5-7.4 (br s, 5H, ArH), 5.3 (29B) and 5.1 (29A) (amide NH_) , 4.6-4.2 (m, IH, ce-H), 3.8 (2s, 3H, COOCH3), 3.2-3.0 (ra, IH, AL-OH), 2.6-2.4 (ra, 2H, OOC-CH2-), 2.3-1.8 (m, 2H, cp-CH ₂ )

This, peptide is a potent long term sweetener for carbonated beverages when used in a dose of 373 " milligrams per 12 ounce can of carbonated orange soda, or cola beverage.

Anal. Calcd. for C ₁₅ H ₁₈ 0 _{5 2} .1 H ₂ 0 (29B): C, 55.5; H, 6.17; N, 8.64. Found: C, 55.71; H, 5.92; N, 8.38.

Example XXXVI N-CBZ-g-Benzyl-L-aspartyl-V E-phenylalanine methyl ester (28)

A total of 0.86 g (2.4 mmoles) of N-CBZ-L- aspartic acid-g-benzyl ester dissolved in 30 ml of dry THF was cooled to 20° in a Dry Ice-carbon tetrachloride bath and 0.32 ml (2.9 mmoles) of N-methylmorpholine and 0.38 ml (2.9 mmoles) of isobutyl chloroformate were added. After 20 minutes a solution of 0.55 g (2.4 mmoles) of 26 in 10 ml of dioxane-water (7:3) containing 0.33 ml (2.4 mraoles) of triethylamine was

added. The mixture was allowed to stir at room temperature overnight, the THF was removed in vacuo, 30 ml of ether and 10 ml of H ₂ 0 were added and the solution was extracted. The aqueous portion was extracted with additional ether (2 x 10 ml), the ether layers combined, washed with 5% citric acid (2 x 20 ml), 5% NaHC0 ₃ (2 x 20 ml), water (1 x 20 ml), and dried over anhydrous Na ₂ 0 ₄ . The solution was filtered, concentrated in vacuo, and the resultant oil dried in vacuo over P ₂ Og to given 1.1 g (86%) of 28 as an amorphous solid. R _f (V)) = -.38; NMR (CDCI3) 6 7.5-7.4 (br s, 4H, PhCj^OCO-), 4.8-4.6 (m, IH, α-H) , 3.4 (br s, 3H, COOH3), 3.1-3.7 (ra, 3H, and COO-CH^), 2.4-2.1 and 1.6-1.4 (ra, 2H, cp-CH ₂ ).

Thus, compound is an intermediate for XXXVII.

Example XXXVII

L-Aspartyl-VE-phenylalanine methyl ester (30). .

A total of 1.5 g (2.8 mraoles) _28_ was dissolved in 50 ml of absolute methanol, 100 mg of 10% Pd/C was added, and hydrogen was bubbled through the solution for 4 hours. The solution was filtered through celite and the filtrate concentrated in vacuo. Partition chromatography of this material on Sephadex -10 using n-BuOH/HOAc/H ₂ 0 4:1:5 upper phase as eluant afforded pure 30 in fractions 94-107. These fractions were com¬ bined, the solvent removed in vacuo, and the resultant oil precipitated from methanol/ether to give 140 mg (16%) of ____- Rf(V) = 0.25; NMR (CD3OD) δ 7.6-7.3 (m, 5H, arH_), 4.5-4.1 (ra, lH, α-H) ,3.4 (s, 3H, COOCH3), 3.1-3.9 (m, IH, ), 2.5-2.3 (m, 2H, OOC-Cj^-), 2.3-1.7

OMPI _

This peptide is a potent long term sweetener for beverages and food products.

Example XXXVIII

Synthesis of Cyclopropyl Aspartyl Phenylalanine Methyl Ester

a. N-Boc-Dehydro-Aspartic Acid β-t-Butyl ct-Methyl Ester

A solution of 5 g of N-Boc-β-hydroxyaspartic acid ester in 25 mL of acetic anhydride should be heated to a temperature of app. 100° for 1 hr. and is evaporated to dryness in vacuo. The crude dehydro aspartic acid derivative should be precipitated from an appropriate solvent such as ethyl acetate using petroleum ether and used directly in the next step. The yield is quantitative. This is first intermediate.

b. N-Boc Cyclopropyl Aspartic Acid β-t-Butyl ct-Methyl Ester

The crude dehydro aspartic acid derivative should be dissolved in 50 mL of methylene chloride and gaseous diazomethane passed into the solution until excess reagent is present in the solution. After standing overnight, the solution is evaporated and the residue is dissolved in 10 mL of dry benzene and refluxed until no further ₂ is evolved. Evaporation of the solution gives the crude product which is purified by crystal¬ lization from an appropriate solvent or chromatographed on a silica column. If E- and Z-isomers are present these are to be separated by careful chromatography. The yield is 60-70%. This is the second intermediate.

c. N-Boc Cyclopropyl Aspartic Acid β-t-Butyl Ester

To a solution of 5 g of N-Boc cyclopropyl aspartic acid β-t-butyl ot-methyl ester in 50 mL of methanol should be added a 1.1 molar excess of 4N NaOH and the solution is allowed to stand at room temperature for four hours. The solution should then be evaporated to a very small volume in vacuo, diluted with 50 mL of water, and ten percent citric acid solution added until the pH is 5-6. The product precipitates and is collected on a filter. After washing with water, the product is dried in a desiccator and is crystallized from ethyl acetate. Yield quantitative.

d. N-Boc β-t-Butyl Cyclopropyl Aspartyl Phenylalanine Methyl Ester

To a solution of 5 g of N-Boc β-t-butyl cyclopropyl aspartic acid in 50 mL of methylene chloride should be added 1 molar equivalent each of dicyclohexyl carbodiimide, N-hydroxybenztriazole, and phenylalanine methyl ester. After 16 hr. at room temperature, the mixture should be filtered and the filtrate washed successively with 10% aHCθ , 10% HCl and brine and dried over anhyd. Na ₂ S0 ₄ . After evaporation to dryness, the residue should be crystallized from an appropriate solvent such as ethyl acetate. Yield is 70%.

Cyclopropyl Aspartyl Phenylalanine Methyl Ester

To a 1:1 solution of trifluoroacetic acid and methylene chloride (50 mL) should be added 5 g of N-Boc β-t-butyl cyclopropyl aspartyl phenylalanine methyl ester and the solution is allowed to stand at room temperature f or one hour and evaporated to

y

dryness. The crude peptide should be dissolved in 10% acetic acid and the solution should be passed through a column of Biogel P-2 and the ninhydrin reactive fractions pooled and lyophilized. Crystallization of the pure peptide from aqueous alcohol or some appropriate solvent mixture gives pure cyclopropyl aspartyl phenylalanine methyl ester, yield quantita¬ tive. This is the final product.

This peptide is very sweet to the taste and can be used to sweeten soft drinks, foods of all kinds, etc.

As noted above the new sweetner compounds have about 100 times to 200 times the sweetness power of ordinary sucrose. To sweeten an 8 ounce cup of coffee a 1 gram quantity of the new product can be employed for example depending on the taste of the user. One packet of 0.035 oz. or 1 gram of the products of Examples XXXV or XXXVIII is equivalent in sweetness power to 2 teaspoonfuls of sucrose.

A significant advantage of the present product of Examples XXXV and XXXVIII is that they do not hydrolyze in the intestine of the consumer when contacted by intestinal enzymes such as carboxypeptidase and chymotrypsin to generate methanol which leads to formation of formaldehyde. This means that a user can drink a cup of coffee, soda or other beverage sweetened with the cyclopropylized dipeptide VAsp-Phe-OCH ₃ or ASP-VPHE.OCH ₃ and while the sweetner performs its designated function it does not hydrolyze cleave or break down in the gastrointestinal tract or blood for at least 24 hours. This is sufficient time for the product in vivo to pass out of the body either with the feces or the urine.

OMPI

The significance of this lack of hydrolysis is that it has been alleged that metabolic fragments, i.e., hydrolysis products of normal L-Asp-Phe OCH ₃ do cause in some consumers side effects such as behavior modification, hyperactivity genetic changes, and brain tumors. The following comparative in vitro study of commercial ASPARTAME and the new food sweeteners of Example XXXV is presented to demonstrate the unexpected and beneficial utility in synthetic sweetners exhibited by the product of the invention.

COMPARATIVE PEPTIDE TESTS FOR STABILITY

₂ Aspartame and new stabilized Asp-V -Phe-OMe (29) of Example XXXV were comparison tested for stability to enzymatic hydrolysis by α-chymotrypsin. The product from hydrolysis of Aspartame would be Asp-Phe-OH ( 2 ) , and from _29_ Asp-V -Phe-OH. A ratio of 10:1 substrate:enzyme was used in a 0.5 M ammonium acetate solution which was adjusted to pH 8 with 10% ammonium hydroxide. After 15 minutes at room temperature aspartame was hydrolyzed to give the acid ( 2 ) and methanol. After 24 hours at room temperature, 29 failed to hydrolyze. Thus, response to hydrolysis by ct-chymotrypsin of compound 29 is completely different than the response of aspartame. The new food product of Example XXXV is hence demonstrated to have a sharp and distinct new property of resistance to peptide degradation by hydrolysis.

Example XXXIX

A powdered sweetener mix for use as the equivalent of sucrose can be prepared by mixing:

Ingredient Grams

Cyclopropyl Dipeptide of Example XXXV or 0.216

Example XXXVIII

Dextrose solids (50%) 1.70

Corn syrup solids (50%)

Tribasic Calcium Phosphate 0.01

Cellulose gum as a 0.11

Microcrystalline cellulose

This dry powderous material is free flowing and soluble instantly in aqueous hot or cold liquids. A single dose of 1 gram of this product will sweeten 300 c.c of a carbonated or non carbonated aqueous beverage. It is stable against all types of hydrolyz- ing agents, including food acids.

The new peptide sweetener prepared by the methods of the preceding examples can be advantageously employed in a new and unique beverage composition. This new food composition has the added merit over ordinary aspartame sweetened beverages in that despite the presence of an organic acid in the formulation the composition has long term stability of its sweetener ingredients. The following is an illustration of such a practical use although it is clear that a number of other food applications are feasible.

Example XL

A fruit flavored beverage mix can be prepared employing the following forraulation which includes the new peptide sweetener:

Ingredient Parts by Weight

Peptide Sweetener of

Example XXXV 0.89 citric Acid 5.53

Clouding agent as shown in U.S. Patent 3,023,106 2.28

Sodium carboxymethyl cellulose 0.90

Tricalcium phosphate 0.49

TRisodium citrate 0.70

Vitamin C 0.47

Tenfold orange oil 0.26

Vitamin A 0.04

Color (mixing 50/50 FDC No. 5 and FDC No. 6 - yellow) 0.01

6.5 grams of the above beverage mix can be reconstituted in a pint of water to produce a beverage exhibiting a highly acceptable mouth feel, cloud stability and natural fruit juice appearance.

Example XLI

A beverage can be manufactured and placed in 12 ounce cans which has a high margin of freedom from peptide sweetner decomposition and attendant problems of generation of methanol. The composition comprises:

Ingredient Grams Carbonated water. 299 Caramel color. 0 . .1 Phosphoric Acid, 0. .2 Artificial flavor, 0 , .12 Preservative-Sodium Benzoate, 0. .2

OMPI IΛ. WIPO , -

Ingredient Grams

Citric acid, 0.15

Caffeine, and 0.1

Peptide of Example XXXV 2.16

This makes 300 c.c of carbonated soda.

PREFERRED EMBODIMENT-PHARMACEUTICAL

APPLICATIONS-POLYPEPTIDES

The following Examples XLII and XLIII will serve to illustrate the preparation of several of our new peptides having enhanced stability against enzymatic cleavage as applied to the control of blood pressure. These peptides are renin inhibitors and are orally, intramuscularly or intravenously administered in dosage unit amounts comparable to those of known renin inhibitors. For instance, in a case where a peptide is formulated in a 50 cc vial it may contain a 50 milligram dose of peptide in glucose or isotonic saline solution for intravenous administration. The modified peptide of Examples XLII and XLIII will be substituted in like quantity and concentration whether the dose given is by oral or systemic administration to the subject.

SYNTHESIS OF RENIN INHIBITORS

Example XLII Boc-VLeu-Val-Tyr-OMe

To a solution of 2.43 g(10 mm) of Boc-VLeu-OH in 50 ml of CH ₂ C1 ₂ there is to be added 1.0 g (10 mm) of triethylamine and 1.4 g (10 mm) of isobutyl chloro- formate at 0°. After 15 min. a solution of 2.94 g

(10 mm) Val-Tyr-OMe in the same solvent is to be added and the mixture is stirred one hour at 0° and two hours at room temperature. After evaporation of the solution, the residue is to be dissolved in ethyl acetate and the solution should be washed successively with water (2 x 25 mL), 0.1 N HCl (2 x 25 mL), 5% NaHC0 ₃ (2 x 25 mL) and brine (2 x 25 mL). After drying over anhyd. Na ₂ Sθ3 the solution is evaporated to dryness and the crude peptide is crystallized from ethyl acetate or a suitable solvent; yield 89%. This compound is a useful intermediate VLeu-Val-Tyr-OMe

To 25 ml of a 1:1 mixture of CH ₂ Cl ₂ and trifluoro- acetic acid is to be added 5.19 g (10 mm) of Boc-VLeu-Val-Tyr-OMe and the solution should be allowed to stand one hour at room temperature. Evaporation of the solution gives the crude tripeptide as the TFA salt, VLeu-Val-Tyr-OMe-TFA, to be used directly in the next step. Boc-Pro-Phe-His-Leu-VLeu-Val-Tyr-OMe

To a solution of 6.26 g (10 mm) Boc-Pro-Phe-His- LeuNHNH ₂ in 50 mL of dry DMF should be added 1.17 g (10 mm) of AmONO while cooling in an ice bath. After one hour should be added a DMF solution containing 5.33 g (10 ram) of VLeu-Val-Tyr-OMe-TFA and 1.01 g (10 mm) of triethylamine over a period of fifteen min. After one hour at 0-5° and five hours at room temperature, the solution is to be partitioned between 250 mL of water and 500 mL of ethyl acetate in a separatory funnel. The organic layer is separated and washed successively with water (3 x 50 mL), 0.1 N HCl (2 x 50 mL), 5% NaHC0 ₃ (2 x 25 mL) and brine (2 x 25 mL) and dried over anhyd. Na ₂ S0 ₄ . Evaporation of the solution will give the crude blocked heptapeptide which is to be purified by crystallization from ethyl acetate

OMPI

or other suitable solvent. This is also an intermediate. Boc-Pro-Phe-His-Leu-VLeu-Val-Tyr-OH

To a solution fo 10.1 g (10 mm) of Boc-Pro-Phe- His-Leu- VLeu-Val-Tyr-OMe in 50 mL of methanol is to be added 5 mL of 2N NaOH at room temperature. After two hours, the solution is to be evaporated to dryness and the residue is to be dissolved in 25 mL of water. The solution is acidified to pH 4 with 10% citric acid and the precipitated peptide is filtered and washed with water. After drying the solid in vacuo, the solid is crystallized from water and ethanol or some other suitable solvent to give a 90% yield of Boc-Pro-Phe- His-Leu- VLeu-Val-Tyr-OH. . This is an intermediate. Pro-Phe-His-Leu-VLeu-Val-Tyr-OH

A solution of 9.99 g (10 mm) of Boc-Pro-Phe-His- Leu- VLeu-Val-Tyr-OH in 25 mL of 1:1 CH ₂ C1 ₂ /TFA is to be allowed to stand at room temperature for one hour. After evaporation to dryness, the residue is dissolved in 5% HOAc solution and passed through a Biogel P—2 column and the fractions reactive to ninhydrin are pooled and lyophilized; yield of pure Pro-Phe-His-Leu- VLei-Val-Tyr-OH is 90%.

This peptide should bind renin very tightly and be active as a renin inhibitor.

Example XLIII Boc-VLeu-Leu-Val-Tyr-OH

To a solution of 2.43 g (10 mm) of Boc-VLeu-OH in 50 mL of THF in a 250 mL round bottomed flask should be added 1.0 g (10 mm) of triethylamine and 1.4 g (10 mm) of isobutyl chloroforraate with ice cooling. After 15 min. , 4.07 g (10 mm) of leu-Val-Tyr-OMe (10 mm) is to

be added and the solution is stirred at 0-5° for one hour and room temperature overnight. After removal of the solvent in vacuo the residue is dissolved in 100 mL of ethyl acetate and washed successively with 0.1 N HCl (2 x 25 mL), 5% NaHC0 ₃ (2 x 25 mL) and saturated NaCl (2 x 25 mL). After drying over anhyd. NaS0 ₄ , the solution should be evaporated to dryness in vacuo. The crude product is recrystallized from a suitable solvent like ethyl acetate to give the pure tripeptide, Boc-VLeu-Leu-Val-Tyr-OMe, in 75% yield. VLeu-Leu-Val-Tyr-OMe.

To a 1:1 mixture of CH ₂ C1 and trifluoroacetic acid is to be added 6.32 g (10 mm) of Boc-VLeu-Leu-Val-Try-OMe and the solution should be allowed to stand one hour at room temperature. Evaporation of the solution will give the crude tetrapeptide TFA salt, VLeu-Leu-Val-Tyr-OMe-FTA, used without purification in the next step.

Example XLIV Boc-Pro-Phe-His- Leu-Leu-Val-Tyr-OMe

To a solution of 5.13 g (10 ram) of Boc-Pro-Phe- HisNHNH ₂ in 50 mL of dry DMF should be added 1.17 g (10 mm) of AmONO while cooling in an ice bath. After one hour is added a DMF solution containing 6.46 g (10 mm) of VLeu-Leu-Val-Tyr-OMe-TFA and 1.01 g (10 mm) of triethylamine over a period of fifteen rain. After one hour at 0-5° and five hours at room temperature, the solution is to be partitioned between 250 mL of water and 500 mL of ethyl acetate in a separatory funnel. The organic layer is separated and washed successively with water (3 x 50 mL), 0.1N HCl (2 x 50 mL), 5% NaHC0 ₃ (2 x 50 mL) and brine (2 x 25 mL) and dried over anhyd. Na ₂ S0 ₄ . Evaporation of the solution

will give the crude blocked heptapeptide which is to be purified by recrystallization from ethyl acetate or some other suitable solvent. This is an intermediate. Boc-Pro-Phe-His-VLeu-Leu-Val-Tyr-OH To a solution of 10.1 g (10 mm) of

Boc-Pro-His- Leu-Leu-Val-Tyr-OMe in 50 mL of methanol is to be added 5 mL of 2N NaOH at room temperature. After two hours, the solution is evaporated to dryness in vacuo and the residue is dissolved in 25 mL of water. The solution is acidified to pH 4 with 10% citric acid and the precipitated peptide is to be filtered and washed with water. After drying over anhyd. CaCL ₂ in vacuo the solid is to be recrystallized from ethanol and water to give a 90% yield of Boc-Pro- Phe-His- VLeu-Leu-Val-Tyr-OH. . This is an inter¬ mediate. Pro-Phe-His-VLeu-Leu-Val-Tyr-OH

A solution of 9.99 g (10 mm) Boc-Pro-Phe-His- VLeu-Leu-Val-Tyr-OH in 25 mL of 1:1 CH ₂ Cl ₂ /TFA is to be allowed to stand at room temperature for one hour.

After evaporation ofthe solution, the residue is passed through a Biogel P—2 column in 5% HOAc solution and the fractions reactive to ninhydrin are to be pooled and lyophilized; yield of pure Pro-Phe-His- VLeu-Leu-Val-Tyr is 90%.

This peptide should bind renin very tightly and be active as a renin inhibitor.

N-Boc-β-t-Butylaspartyl Cyclobutyl Phenylalanine Methyl Ester

To a solution of 5g of N-Boc β-t-butylaspartic acid in 50 mL of methylene chloride should be added 1 molar equivalent each of dicyclohexyl carbodiimide, hydroxybenztriazole, and cyclobutyl phenylalanine

raethyl ester. After 16 hr at room temperature, the mixture should be filtered and the filtrate washed successively with 10% NaHC0 ₃ , 10% HCl and brine and dried over anhyd. Na ₂ S0 ₄ . After evaporation to dryness, the residue should be crystallized from an appropriate solvent such as ethyl acetate. Yield is 70%.

N-Boc- -t-Butylaspartyl Cyclopentyl Phenylalanine Methyl Est

To a solution of 5 g of N-Boc β-t-butylaspartic acid in 50 mL of methylene chloride should be added 1 molar equivalent each of dicyclohexyl carbodiimide, hydroxybenztriazole, and cyclopentryl phenylalanine methyl ester. After 16 hr at room temperature, the mixture should be filtered and the filtrate washed successively with 10% NaHC0 ₃ , 10% HCl and brine and dried over anhyd. Na ₂ S0 ₄ . Afer evaporation to dryness, the residue should be crystallized from an appropriate solvent such as ethyl acetate. Yield is 70%.

Aspartyl Cyclobutyl Phenylalanine Methyl Ester.

To a 1:1 solution of trifluoroacetic acid and methylene chloride (50 mL) should be added 5 g of N-Boc-β-t-Butylaspartyl cyclobutylphenyl-alanine methyl ester and the solution is allowed to stand at room temperature of one hour and evaporated to dryness. The crude peptide should be dissolved in 10% acetic acid and the solution should be passed through a column of Biogel P-2 and the ninhydrin reactive fractions pooled and lyophilized. Crystallization of the pure peptide from aqueous alcohol or some appropriate solvent mixture gives pure aspartyl

cyclobutyl phenylalanine methyl ester, yield quantita¬ tive. This is the final product.

This peptide should be very sweet to the taste and can be used to sweeten soft drinks, foods of all kinds, etc

Aspartyl Cyclopentyl Phenylalanine Methyl Ester.

To a 1:1 solution of trifluoroacetic acid and methylene chloride (50 mL) should be added 5 g of N-boc-β-t-butylaspartyl cyclopentylphenylalanine methyl ester and the solution is allowed to stand at room temperature of one hour and evaporated to dryness. The crude peptide should be dissolved in 10% acetic acid and the solution should be passed through a column of Biogel P-2 and the ninhydrin reactive fractions pooled and lyophilized. Crystallization of the pure peptide from aqueous alcohol or some appropriate solvent mixture gives pure aspartyl cyclopentyl phenylalanine methyl ester, yield quantitative. This is the final product.

This peptide should be very sweet to the taste and can be used to sweeten soft drinks, foods of all kinds, etc.

Z-CyclopentylPhe-Leu-OMe

A solution of isobutyl chloroformate (546 mg, 4 mmol) in chloroform (10 ml) was added dropwise to a solution of Z-cyclopentylPhe OH (1) (4 mmol) and N-methylmorpholine (404 mg, 4 mmol) in chloroform (30 ml) at 0.5°C. After stirring for 20 min, a solution of Leu OMe HCl (1.45 g, 8 mmol) and

N-methylmorpholine (0.81 g, 8 mmol) in chloroform (20 ml) was added at 10°C. Adter stirring for 2 hr at 0.5°C and then overnight at room temperature, the reaction mixture was washed with H ₂ 0, 5% citric acid and 5% NaHC0 ₃ successively, and dried over Na ₂ S0 ₄ . The solvent was evaporated in vacuo and the residue was recrystallized from ethylacetate-hexane to give the product, (70%) as colorless needles.

Z-CyclobutylPhe-Leu OMe

A solution of isobutyl chloroformate (546 mg,

4 mmol) in chloroform (10 ml) was added dropwise to a solution of Z-cyclobutylPhe OH (1) (4 mmol) and N-methylmorpholine (404 mg, 4 mmol) in chloroform (30 ml) at 0.5°C. After stirring for 20 min, a solution of Leu OMe HCl (1.45 g, 8 mmol) and

N-methylmorpholine (0.81 g, 8 mmol) in chloroform (20 ml) was added at 10°C. After stirring for 2 hr at 0.5°C and then overnight at room temperature, the reaction mixture was washed with H ₂ 0, 5% citric acid and 5% NaHC0 ₃ successively, and dried over Na ₂ S0 ₄ . The solvent was evaporated in vacuo and the residue was recrystallized from ethylacetate-hexane to give the product, (70%) as colorless needles.

Cyclopentyl Phe-Leu OMe TFA

A solution of Z-cpPhe-Leu OMe (3 mmol) and thioanisole (2 ml) in trifluoroacetic acid (TFA) (20 ml) was stirred at 0° for 3 hr, and then room temperature overnight. The TFA was removed under reduced pressure, and the residue was triturated with ether (30 ml) and the precipitated crystals were collected by suction and washed with ether to give the

product (90% ) .

This compound has utility as an intermediate in synthesis of an enkephalin peptide which has opiate properties.

Z-Tyr-D-Ala-Gly-cbPhe-Leu OMe

To a solution of Z-Tyr-D-Ala-Gly OH (887 mg, 2 mmol) and cbPhe-Leu TFA (2 mraol) in THF (80 ml) was added N-methylmorpholine (202 mg, 2 mmol) HOBt (270 mg, 2 mmol) and EDC (l-ethyl-3(3-dimethylaminopropyl)- carbodimide HCl) (384 mg, 2 mmol) successively at

0°C. After stirring for 3 hr at 0°C and overnight at room temperature, the solvent was removed under reduced pressure, and the residue was extracted three with 100 ml of AcOEt. The combined AcOEt extracts were washed with 5% aHC0 , 0.2 N HCl, water, and dried over anhyd. Na ₂ S0 . The solvent was removed under reduced pressure, and the solid was recrystallized from ethylacetate-hexane to give Z-Tyr-D-Ala-Gly-cbPhe-Leu OMe as a colorless powder (90%).

This compound has utility as an intermediate in synthesis of an enkephalin peptide which inhibits muscle contraction.

Z-Tyr-D-Ala-Gly-cpPhe-Leu OMe

To a solution of Z-Tyr-D-Ala-Gly OH (887 mg, 2 mmol) and cpPhe-Leu TFA (2 mraol) in THF (80 ml) was added N-methylraorpholine (202 mg, 2 mmol) HOBt (270 mg, 2 mraol) and EDC (l-ethyl-3(3-dimethylaminopropyl)- carbodiraide'HCl) (384 mg, 2 mmol) successively at 0°C. After stirring for 3 hr at 0°C and overnight at

room temperature, the solvent was removed under reduced pressure, and the residue was extracted three with 100 ml of AcOEt. The combined AcOEt extracts were washed with 5% NaHC0 ₃ , 0.2 N HCl, water, and dried over anhyd. Na ₂ S0 ₄ . The solvent was removed under reduced pressure, and the solid was recrystallized from ethylacetate-hexane to give Z-Tyr-D-Ala-Gly-cpPhe- Leu'OMe as a colorless powder (90%).

This compound has utility as an intermediate in synthesis of an enkephalin peptide which inhibits muscle contraction.

Z-Tyr-D-Ala-Gly-cbPhe-Leu'OH

Z-Tyr-D-Ala-Gly-cbPhe-Leu ^* OMe (365 mg, 0.5 mmol) was dissolved in methanol (1 ml), the solution was cooled in an ice-water bath and IN NaOH (1 ml, 1 mmol) at 0°C was added. After stirring for 2 hr at 0°C, the solution was neutralized with IN HCl and diluted with water. The precipitated solid was collected by suction and dried under reduced pressure. The solid was purified by silica gel column chromatography using chloroforra-methanol (19:1) and chloroform-methanol- aceit acid (95:5:1) as eluants. After starting material was removed using CHC^/MeOH (19:1), Z-Tyr-D- Ala-Gly-cbPhe-Leu'OH (90%) .

Z-Tyr-D-Ala-Gly-cbPhe-Leu ^• OH

Z-Tyr-D-Ala-Gly-cbPhe-Leu*OMe (365 mg, 0.5 mmol) was dissolved in methanol (1 ml), the solution was cooled in an ice-water bath and IN NaOH (1 ml, 1 mmol) at 0°C was added. After stirring for 2 hr at 0°C, the solution was neutralized with IN HCl and diluted with

water. The precipitated solid was collected by suction and dried under reduced pressure. The solid was purified by silica gel column chromatography using chloroform-methanol (19:1) and chloroform-methanol- aceit acid (95:5:1) as eluants. After starting material was removed using CHC^/MeOH (19:1), Z-Tyr-D- Ala-Gly-cbPhe-Leu ^* OH (90%) .

Tyr-D-Ala-Gly-cbPhe-Leu

A solution of Z-Tyr-D-Ala-Gly-cbPhe-Leu*OH (143 mg, 0.2 mraol) and thioanisole (0.3 ml) in trifluoro- acetic acid (TFA) (3 ml) was stirred 1 hr at 0°C, and 4 hr at room temperature. The solvent was removed under reduced pressure and the residue was triturated with ether to give a TFA salt. This salt was dissolved in 5% AcOH and passed through a column containing a large excess of Amberlite CG400 (acetate form), followed by a column of BioGel P-2 (1.9 x 8.9 cm, 200- 400 mesh) (4 ml/20 min). The fractions containing product were pooled and lyophilized to give 75% of product.

This compound has utility as a stable opiate peptide and inhibits muscle contraction.

Tyr-D-Ala-Gly-cpPhe-Leu

A solution of Z-Tyr-D-Ala-Gly-cpPhe-Leu ^* OH (143 mg, 0.2 mmol) and thioanisole (0.3 ml) in trifluoro- acetic acid (TFA) (3 ml) was stirred 1 hr at 0°C, and 4 hr at room temperature. The solvent was removed under reduced pressure and the residue was triturated with ether to give a TFA salt. This salt was dissolved in 5% AcOH and passed through a column containing a

OMPI

large excess of Amberlite CG400 (acetate form), followed by a column of BioGel P-2 (1.9 x 8.9 cm, 200- 400 mesh) (4 ml/20 min). The fractions containing product were pooled and lyophilized to give 75% of product.

This compound has utility as a stable opiate peptide and inhibits muscle contraction.