WO1998038990A1

WO1998038990A1 - Method of treating trauma associated with brain, spinal cord or peripheral nerve injury using carbobenzyloxy n-protected di- and tripeptide phenylalaninals

Info

Publication number: WO1998038990A1
Application number: PCT/US1998/001414
Authority: WO
Inventors: Matthew D. Linnik
Original assignee: Hoechst Marion Roussel, Inc.
Priority date: 1997-03-07
Filing date: 1998-01-27
Publication date: 1998-09-11
Also published as: ZA981741B; AU6134498A

Abstract

The present invention relates to a method of treating trauma associated with brain or spinal cord injury in a patient in need thereof with a therapeutically effective amount of carbobenzyloxy n-protected di- and tripeptide phenylalaninals.

Description

METHOD OF TREATING TRAUMA ASSOCIATED WITH BRAIN. SPINAL CORD

OR PERIPHERAL NERVE INJURY USING CARBOBENZYLOXY N-PROTECTED DI- AND TRIPEPTIDE PHENYLALANINALS

The present invention provides a method of treating trauma associated with brain, spinal cord or peripheral nerve injury in a patient in need thereof comprising administering to said patient a therapeutically effective amount of a compound of formula (1)

wherein R is hydrogen, OH, C1-C4 alkyl, C1-C4 alkoxy, NO2, NH₂ or halogen; R₁ and R₂ are each independently C₁-C4 alkyl; and n is the integer zero or one; or a stereoisomer or a pharmaceutically acceptable salt thereof.

The present invention also provides a method of treating traumatic injury to the central nervous system in a patient in need thereof comprising administering tc said patient a therapeutically effective amount of a compound of formula (1). The present invention further provides a method of reducing contusion volume in neuronal tissue in a patient in need thereof comprising administering to said patient a therapeutically effective amount of a compound of formula (1).

As used in this application:

a) the designation " ^— " refers to a bond that protrudes forward out of the plane of the page;

b) the designation " " refers to a bond that protrudes backward out of the plane of the page;

c) the designation " — " refers to a bond between achiral molecules or a bond between chiral molecules for which the stereochemistry is not designated. For the purposes of this invention, it is contemplated that either L- or D- isomers or mixtures of the two are encompassed by this designation.

As used herein, the term "C₁-C₄ alkyl" refers to a saturated straight or branched chain hydrocarbon radical of one to four carbon atoms. Included within the scope of this term are methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl and the like. The term "C₁-C₄ alkoxy" refers to an alkoxy radical made up of an oxygne radical bearing a saturated straight or branched chain hydrocarbon radical of one to four carbon atoms. Included within the scope of this term are methoxy, ehtoxy, propoxy, n-butoxy, isobutoxy, sec-butoxy, t-butoxy and the like. The terms "halo", "halogen" or "halide" refer to a fluorine, chlorine, bromine or iodine atom.

The terms "Cbz" or "carbobenzyloxy" refer to a carbobenzyloxy functionality of the formula:

The terms "BOC" or '1-butyloxycarbonyl" refer to a t-butyloxycarbonyl functionality of the formula:

The term "stereoisomer" is a general term for all isomers of individual molecules that differ only in the orientation of their atoms in space. It includes mirror image isomers (enantiomers), geometric (cis/trans) isomers and isomers of compounds with more than one chiral center that are not mirror images of one another (diastereomers). For amino acids, the designations L. D, or R/S can be used as described in IUPAC-IUB Joint Commission on Biochemical Nomenclature, Eur. J. Biochem. 138, 9-37 (1984).

The term "pharmaceutically acceptable salt" refers to those salts that are not substantially toxic at the dosage administered to achieve the desired effect and do not independently possess significant pharmacological activity. The salts included within the scope of this term are hydrobromic, hydrochloric, sulfuric, phosphoric, nitric, formic, acetic, propionic, succinic, glycolic, lactic, malic, tartaric, citric, ascorbic, α-ketoglutaric, glutamic, aspartic, maleic, hydroxymaleic, pyruvic, phenylacetic, benzoic, p-aminobenzoic, anthranilic, p-hydroxybenzoic, salicyclic, hydroxyethanesulfonic, ethylenesulfonic, halobenzenesulfonic, toluenesulfonic, naphthalenesulfonic, methanesulfonic, sulfanilic, and the like.

As used herein the term "trauma" refers to an injury caused by contact with a physical object, as defined by Stedman's Medical Dictionary, 23rd Edition, copyright 1976, Williams & Wilkins, Baltimore, MD.

As used herein, the term "contusion" refers to bruising of the brain, spinal cord or peripheral nerve with extravasation of blood and the secondary mass lesion accompanying that hemorrhage. As used herein, the term "patient" refers to a warm blooded animal such as a mammal which has either suffered trauma associated with injury to the central nervous system or to a peripheral nerve, particularly the brain or spinal cord. It is understood that guinea pigs, gerbils, dogs, cats, rats, mice, horses, cattle, sheep and humans are examples of patients within the scope of the meaning of the term.

Diagnosis of patients suffering from trauma associated with brain or spinal cord injury is well within the ability and knowledge of one skilled in the art. For example, individuals who have symptoms of overt external injury, loss of consciousness, acute confusion and memory impairment, or any neurological deficit associated with a traumatic injury are generally considered within the diagnosis of trauma. A clinician skilled in the art can readily identify, by use of clinical tests and/or physical examination, those patients who are suffering from trauma associated with brain or spinal cord injury.

The term "therapeutically effective amount" refers to an amount which is effective, upon continuous infusion or upon single or multiple dose administration to the patient, in providing a reduction in the extent of damage associated with trauma associated with brain or spinal cord injury, leading to an improved outcome and/or delay or prevention in damage progression as compared to outcomes expected in the absence of treatment. The term "therapeutically effective amount" does not necessarily indicate a total elimination or prevention of damage related to trauma associated with injury to the brain or spinal cord. In determining the therapeutically effective amount or dose, a number of factors are considered by the attending diagnostician, including, but not limited to: the species of the animal; its size, age, and general health; the specific disease involved; the degree of or involvement or the severity of the disease or trauma; the response of the individual patient; the particular compound administered; the mode of administration; the bioavailability characteristics of the preparation administered; the dose regimen selected; the use of concomitant medication; and other relevant circumstances.

A therapeutically effective amount of a compound of formula (1) is expected to vary from about 0.1 milligram per kilogram of body weight per day (mg/kg/day) to about 100 mg/kg/day. Preferred amounts are expected to vary from about 0.5 to about 30 mg/kg/day.

In effecting treatment of a patient afflicted with a disease state described above, a compound of formula (1) can be administered in any form or mode which makes the compound bioavailable in effective amounts, including oral and .parenteral routes. For example, compounds of formula (1) can be administered orally, subcutaneousiy, intramuscularly, intravenously, transdermally, intranasally, rectally, topically, and the like. Oral or intravenous administration is generally preferred. One skilled in the art of preparing formulations can readily select the proper form and mode of administration depending upon the particular characteristics of the compound selected for the disease state to be treated, the stage of the disease, and other relevant circumstances. Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Co. (1990).

The compounds can be administered alone or in the form of a pharmaceutical composition in combination with pharmaceutically acceptable carriers or excipients, the proportion and nature of which are determined by the solubility and chemical properties of the compound selected, the chosen route of administration, and standard pharmaceutical practice. The compounds of the invention, while effective themselves, may be formulated and administered in the form of their pharmaceutically acceptable salts, such as for example, acid addition salts, for purposes of stability, convenience of crystallization, increased solubility and the like.

The pharmaceutical compositions are prepared in a manner well known in the pharmaceutical art. The carrier or excipient may be a solid, semi-solid, or liquid material which can serve as a vehicle or medium for the active ingredient. Suitable carriers or excipients are well known in the art. The pharmaceutical composition may be adapted for oral, parenteral, or topical use and may be administered to the patient in the form of tablets, capsules, suppositories, solution, suspensions, or the like.

The compounds of the present invention may be administered orally, for example, with an inert diluent or with an edible carrier. They may be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the compounds may be incorporated with excipients and used in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, chewing gums and the like. These preparations should contain at least 4% of the compound of the invention, the active ingredient, but may be varied depending upon the particular form and may conveniently be between 4% to about 70% of the weight of the unit. The amount of the compound present in compositions is such that a suitable dosage will be obtained. Preferred compositions and preparations according to the present invention are prepared so that an oral dosage unit form contains between 5.0-300 milligrams of a compound of the invention.

The tablets, pills, capsules, troches and the like may also contain one or more of the following adjuvants: binders such as microcrystalline cellulose, gum tragacanth or gelatin; excipients such as starch or lactose, disintegrating agents such as alginic acid, Primogel, corn starch and the like; lubricants such as magnesium stearate or Sterotex; glidants such as colloidal silicon dioxide; and sweetening agents such as sucrose or saccharin may be added or a flavoring agent such as peppermint, methyl salicylate or orange flavoring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol or a fatty oil. Other dosage unit forms may contain other various materials which modify the physical form of the dosage unit, for example, as coatings. Thus, tablets or pills may be coated with sugar, shellac, or other enteric coating agents. A syrup may contain, in addition to the present compounds, sucrose as a sweetening agent and certain preservatives, dyes and colorings and flavors. Materials used in preparing these various compositions should be pharmaceutically pure and non-toxic in the amounts used.

For the purpose of parenteral therapeutic administration, the compounds of the present invention may be incorporated into a solution or suspension. These preparations should contain at least 0.1% of a compound of the invention, but may be varied to be between 0.1 and about 50% of the weight thereof. The amount of the inventive compound present in such compositions is such that a suitable dosage will be obtained. Preferred compositions and preparations according to the present invention are prepared so that a parenteral dosage unit contains between 5.0 to 100 milligrams of the compound of the invention.

The solutions or suspensions may also include one or more of the following adjuvants: sterile diluents such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylene diaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parenteral preparation can be enclosed in ampules, disposable syringes or multiple dose vials made of glass or plastic.

The compound of formula (1) where R is hydrogen, Ri is isopropyl, n is zero and the benzyl moiety at bond "a" is a mixture of D- and L- isomers or is the L- isomer is preferred in the method of use according to the present invention. It is further preferred that the Ri moiety at bond "b" is in the L-configuation. In the end use application provided by the present invention, the preferred compound of formula (1) is carbamic acid, [1-[[(1-formyl-2-phenylethyl)amino]carbonyl]-2-methylpropyl]-, phenylmethyl ester, better known in its shorthand designation Cbz-Val-Phe-H, or MDL 28,170. Compounds of the present invention, including carbamic acid, [1-[[(1- formyl-2-phenylethyl)amino]carbonyl]-2-methylpropyl]-, phenylmethyl ester have been disclosed by Bey, P. et al., European Patent Application OPI No. 0 363 284, with a publication date of April 11 , 1990 as being effective as calpain inhibitors, particularly in the treatment of stroke and by Cordell, B. et al., PCT Int. Publ. No. WO 95/09838, with a publication date of April 13, 1995 as being inhibitors of β-amyloid protein production.

The recognized abbreviations for the α-amino acids are set forth in Table 1. TABLE 1

The following list illustrates some of the compounds according to the present invention:

Cbz-Val-Phe-H;

Cbz-lle-Phe-H;

Cbz-Ala-Phe-H;

Cbz-Abu-Phe-H;

Cbz-Leu-Phe-H;

Cbz-Nva-Phe-H;

Cbz-Nie-Phe-H; Cbz-Val-Tyr-H;

Cbz-lle-Tyr-H;

Cbz-Ala-Tyr-H;

Cbz-Abu-Tyr-H;

Cbz-Leu-Tyr-H;

Cbz-Nva-Tyr-H;

Cbz-Nle-Tyr-H;

Cbz-Val-Tyr(OMe)-H;

Cbz-lle-Tyr(OMe)-H;

Cbz-Ala-Tyr(OMe)-H;

Cbz-Abu-Tyr(OMe)-H;

Cbz-Leu-Tyr(OMe)-H;

Cbz-Nva-Tyr(OMe)-H;

Cbz-Nle-Tyr(OMe)-H;

Cbz-Val-Phe(4-CI)-H;

Cbz-lle-Phe(4-CI)-H;

Cbz-Ala-Phe(4-Br)-H;

Cbz-Abu-Phe(4-Br)-H;

Cbz-Leu-Phe(4-CI)-H;

Cbz-Nva-Phe(4-F)-H;

Cbz-Nle-Phe(4-I)-H;

Cbz-Val-Phe(N0₂)-H;

Cbz-lle-Phe(NH₂)-H;

Cbz-Ala-Phe(N0₂)-H;

Cbz-Abu-Phe(NH₂)-H

Cbz-Leu-Phe(N0₂)-H

Cbz-Nva-Phe(NH₂)-H

Cbz-Nle-Phe(NH₂)-H;

Cbz-lle-Val-Phe-H;

Cbz-Val-lle-Phe-H;

Cbz-Leu-Ala-Phe-H;

Cbz-Leu-Abu-Phe-H;

Cbz-Ala-Leu-Phe-H; Cbz-Leu-Nva-Phe-H;

Cbz-Val-Nle-Phe-H;

Cbz-Leu-Val-Tyr-H;

Cbz-lle-lle-Tyr-H; Cbz-Ala-Ala-Tyr-H;

Cbz-Val-Abu-Tyr-H;

Cbz-Ala-Leu-Tyr-H;

Cbz-lle-Val-Tyr(OMe)-H;

Cbz-Val-lle-Tyr(OMe)-H; Cbz-Leu-Ala-Tyr(OMe)-H

Cbz-Leu-Val-Phe(4-CI)-H;

Cbz-Ala-lle-Phe(4-CI)-H;

Cbz-lle-Ala-Phe(4-Br)-H;

Cbz-Leu-Leu-Phe(N0₂)-H; Cbz-Ala-Nva-Phe(NH₂)-H;

Cbz-Nva-N le-Phe(N H₂)-H ;

Starting material for Scheme 3 for the preparation of compounds of formula (1) can be prepared as described in Scheme 1. All the substituents, unless otherwise indicated, are previously defined. The reagents and starting materials are readily available to one of ordinary skill in the art.

SCHEME 1

(6)

Pg = Protecting Group

X = suitable carboxylic acid protecting group or resin ln Scheme 1 , step A, compounds of structure (2) are coupled with compounds of structure (3a) using standard reactions analogously known in the art, such as those used in peptide synthesis. For example, in an ordinary peptide synthesis, peptides are elongated by deprotecting the α-amine of the N-terminal residue and coupling the next suitably N-protected amino acid through a peptide linkage using the methods described. This deprotection and coupling procedure is repeated until the desired sequence is obtained. This coupling can be performed with the constituent amino acids in stepwise fashion, as depicted in Scheme 1 , or by condensation of fragments or a combination of both processes, or by solid phase peptide synthesis according to the method originally described by Merrifield, J. Am. Chem. Soc, 1963, 85, 2149-2154. When a solid phase synthetic approach is employed, the C-terminal carboxylic acid is attached to an insoluble carrier (usually polystyrene). These insoluble carriers form a bond which is stable to the elongation conditions but readily cleaved later. Examples of such carriers are: chloro- or bromomethyl resin, hydroxymethyl resin, and aminomethyl resin. Many of these resins are commercially available with the desired C-terminal amino acid already incorporated.

In addition to the foregoing, peptide synthesis are described in Stewart and Young, "Solid Phase Peptide Synthesis", 2nd ed., Pierce Chemical Co., Rockford, IL (1984); Gross, Meienhofer, Udenfriend, Eds., "The Peptides: Analysis, Synthesis, Biology", Vol 1 , 2, 3, 5 and 9, Academic Press, New York, 1980-1987; Bodanszky, "Peptide Chemistry: A Practical Textbook", Springer- Verlag, New York (1988); and Bodanszky, et al. "The Practice of Peptide Synthesis", Springer- Verlag, New York (1984).

Coupling between two amino acids, an amino acid and a peptide, or two peptide fragments can be carried out using standard coupling procedures such as the azide method, mixed carbonic-carboxylic acid anhydride (isobutyl chloroformate) method, carbodiimide (dicyclohexylcarbodiimide, diisopropylcarbodiimide, or water- soluble carbodiimide) method, active ester (p-nitrophenyl ester, N-hydroxy-succinic imido ester) method, Woodward reagent K method, carbonyldiimidazole method, phosphorus reagents such as BOP-CI, or oxidation-reduction methods. Some of these methods (especially the carbodiimide method) can be enhanced by adding 1- hydroxybenzotriazole. These coupling reactions can be performed in either solution (liquid phase) or solid phase.

The functional groups of the constituent amino acids generally must be protected during the coupling reactions to avoid formation of undesired bonds. The protecting groups that can be used are listed in Greene, "Protective Groups. in Organic Chemistry", John Wiley & Sons, New York (1981) and "The Peptides: Analysis, Synthesis, Biology", Vol. 3, Academic Press, New York (1981), the disclosure of which is hereby incorporated by reference.

The α-carboxyl group of the C-terminal residue is usually protected by an ester that can be cleaved to give the carboxylic acid. Protecting groups which can be used include: 1) alkyl esters such as methyl and t-butyl, 2) aryl esters such as benzyl and substituted benzyl, or 3) esters which can be cleaved by mild base treatment or mild reductive means such as trichloroethyl and phenacyl esters.

The α-amino group of each amino acid to be coupled to the growing peptide chain must be protected. Any protecting group known in the art can be used. Examples of these protecting groups include: 1) acyl types such as formyl, trifluoroacetyl, phthaloyi, and p-toluenesulfonyl; 2) aromatic carbamate types such as benzyloxycarbonyl (Cbz or Z) and substituted benzyloxycarbonxyls, 1-(p-biphenyl)-1- methylethoxy-carbonyl, and 9-fluorenylmethyloxycarbonyl (Fmoc); 3) aliphatic carbamate types such as tert-butyloxycarbonyl (Boc), ethoxycarbonyl, diisopropyl- methoxycarbonyl, and allyloxycarbonyl; 4) cyclic alkyl carbamate types such as cyclopentyloxycarbonyl and adamantyloxycaronbyl; 5) alkyl types such as triphenylmethyl and benzyl; 6) trialkylsilanes such as trimethylsilane; and 7) thiol containing types such as phenylthiocarbonyi and dithiasuccinoyl. The preferred α-amino protecting group is either Boc, Cbz or Fmoc, preferably Boc. Many amino acid derivatives suitably protected for peptide synthesis are commercially available.

The α-amino group protecting group of the newly added amino acid residue is cleaved prior to the coupling of the next amino acid. Conditions for cleavage of such protecting groups are described in Greene, "Protective Groups in Organic Chemistry", Chapter 7, John Wiley & Sons, New York (1981). When the Boc group is used, the methods of choice are trifluoroacetic acid, neat or in dichloromethane, or HCI in dioxane or ethyl acetate. The resulting ammonium salt is then neutralized either prior to the coupling or in situ with basic solutions such as aqueous buffers, or tertiary amines in dichloromethane or dimethylformamide. When the Fmoc group is used, the reagents of choice are piperidine or substituted piperidine in dimethylformamide, but any secondary amine or aqueous basic solutions can be used. The deprotection is carried out at a temperature between 0°C and room temperature.

Any of the amino acids bearing side chain functionalities must be protected during the preparation of the peptide using any of the above-described groups. Those skilled in the art will appreciate that the selection and use of appropriate protecting groups for these side chain functionalities depends upon the amino acid and presence of other protecting groups in the peptide. The selection of such protecting groups is important in that it must not be removed during the deprotection and coupling of the α-amino group.

For example, when Boc is used as the α-amino protecting group, a benzyl (Bn) ether can be used to protect the hydroxy containing side chains of amino acids such as Tyr, Ser or Thr.

When a solid phase synthesis is used, the peptide is cleaved from the resin usually simultaneously with the protecting group removal. When the Boc protection scheme is used in the synthesis, treatment with anhydrous HF containing additives such as dimethyl sulfide, anisole, thioanisole, or p-cresol at 0°C is the preferred method for cleaving the peptide from the resin. The cleavage of the peptide can also be accomplished by other acid reagents such as trifluoromethanesulfonic acid/trifluoroacetic acid mixtures. If the Fmoc protection scheme is used the N- terminal Fmoc group is cleaved with reagents described earlier. The other protecting groups and the peptide are cleaved from the resin using a solution of trifluoroacetic acid and various additives such as anisole, etc. More specifically, in Scheme 1 , step A an α-amino acid of structure (2) wherein X is a suitable α-carboxyl protecting group, such as a methyl ester, is dissolved in a suitable anhydrous organic solvent, such as anhydrous DMF or anhydrous methylene chloride under an inert atmosphere, such as nitrogen. To this solution is added an equivalent of N-hydroxybenzotriazole hydrate, an equivalent of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and an equivalent of the protected α-amino acid of structure (3a) dissolved in a suitable anhydrous organic solvent, such as anhydrous DMF or anhydrous methylene chloride. The reaction is then allowed to stir for about 1 to 15 hours. The coupled product of structure (4) is then isolated and purified by techniques well known in the art, such as extractive techniques and flash chromatography. For example, the reaction is diluted with a suitable organic solvent such as ethyl acetate, rinsed with water, dried over anhydrous magnesium sulfate, filtered and concentrated under vacuum. The residue is purified by flash chromatography on silica gel with a suitable eluent, such as ethyl acetate/hexane to provide the coupled product (4).

Alternatively, in Scheme 1 , step A a suitably protected α-amino acid of structure (3a) is dissolved in a suitable organic solvent under an inert atmosphere, such as nitrogen. Examples of suitable organic solvents are petroleum ethers, a chlorinated hydrocarbon such as carbon tetrachloride, ethylene chloride, methylene chloride, or chloroform; a chlorinated aromatic such as 1 ,2,4-trichlorobenzene, or o- dichlorobenzene; carbon disulfide; an ethereal solvent such as diethyl ether, tetrahydrofuran, or 1 ,4-dioxane, or an aromatic solvent such as benzene, toluene, or xylene. Methylene chloride is the preferred solvent for this coupling reaction. The solution is then treated with one to four equivalents of a suitable amine. Examples of suitable amines are tertiary organic amines such as tri-(lower alkyl)amines, for example, triethylamine; or aromatic amines such as picolines, collidines, and pyridine. When pyridines, picolines, or collidines are employed, they can be used in high excess and act therefore also as the reaction solvent. Particularly suitable for the coupling reaction is N-methylmorpholine (NMM). The solution is then cooled to about -20°C and one equivalent of isobutyl chloroformate is added. The reaction is allowed to stir for about 10 to 30 minutes and 1 to 4 equivalents of the amino acid ester of structure (2) (X is an ester group, such as methyl or ethyl and the amino acid can be an acid addition salt or a free base), is added to the reaction. The reaction is stirred for 30 minutes to 2 hours at about -20°C and then it is allowed to warm to room temperature and stirred for 1 to 3 hours. The coupled product (4) is then isolated and purified by techniques well known in the art, such as extractive techniques and flash chromatography. For example, the reaction is diluted with a suitable organic solvent such as methylene chloride, rinsed with water, dried over anhydrous magnesium sulfate, filtered and concentrated under vacuum. The residue is purified by flash chromatography on silica gel with a suitable eluent, such as ethyl acetate/hexane to provide the coupled product (4).

In Scheme 1 , step A1 , the protecting group (Pg) on the coupled product (4) is removed under conditions well known in the art, as described by T.W. Green, "Protective Groups in Organic Synthesis", Chapter 7, 1981 , John Wiley & Sons, Inc and the primary amine is coupled with carbobenzyloxy ("Cbz") to provide the coupled product of structure (5). For example, when Pg is a tert-butyl carbamate (BOC) on the coupled product (4), the compound is dissolved in methanolic hydrochloric acid, stirred for several hours and then concentrated under vacuum. The residue is then dissolved in water, neutralized with saturated sodium bicarbonate and extracted with ethyl acetate. The organic extracts are dried over anhydrous magnesium sulfate, filtered and concentrated under vacuum. The residue is purified by flash chromatography on silica gel with a suitable eluent, such as ethyl acetate/hexane to provide the primary amine.

Alternatively, when Pg is a tert-butyl carbamate (BOC) on the coupled product (4), the compound can be dissolved in trifluoroacetic acid and stirred at room temperature for 1 to 12 hours. The reaction is then poured carefully into water, neutralized with sodium bicarbonate and extracted with ethyl acetate. The combined organic extracts are dried over anhydrous magnesium sulfate, filtered and concentrated under vacuum. The residue can be purified by flash chromatography on silica gel with a suitable eluent, such as ethyl acetate/hexane to provide the primary amine. ln Scheme 1 , step A2, the above prepared primary amine is coupled to Cbz to provide the coupled product (5) under conditions analogous to the procedures described in Scheme 1 , step A above, or by those well known in the art. For example, dibenzyl pyrocarbonate ((PhCH OCO)₂0) is reacted with the above prepared primary amine at basic pH in NaOH/H₂θ, dioxane/ H₂0 (1/1) EtsN, or dioxane/H₂0 (4/1) NaOH according to the method taught by Sennyey, G. et al., Tetrahedron Lett., 27, 5375-5376 (1986), which also teaches how to make the dibenzyl pyrocarbonate reagent. The residue is purified by flash chromatography on silica gel with a suitable eluent to provide the coupled product (5).

The coupled product (5) can also be prepared directly in Scheme 1 , step B by a coupling reaction of the α-amino acid of structure (2) wherein X is a suitable α- carboxyl protecting group, such as a methyl ester, with the α-amino acid of structure (3b). The α-amino acid (3b) is readily prepared by coupling the Cbz substituent to the amino acid of structure (9b')

wherein X is a suitable α-carboxyl protecting group, such as a methyl ester, under conditions well known to one of ordinary skill in the art, such as the procedures described in Scheme 1 , step A. The α-carboxyl protecting group of this coupled product is then removed under conditions well known in the art to provide the α- amino acid of structure (3b). For example, wherein X is a methyl or ethyl group, the compound is dissolved in ethanol, treated with an equal volume of water and an equivalent of lithium hydroxide. The reaction is allowed to stir for 1 to 6 hours. The resulting acid is then isolated by techniques well known in the art. For example, the organic solvent is removed under vacuum and the remaining aqueous solution is acidified with dilute hydrochloric acid. The aqueous is then extracted with a suitable organic solvent, such as ethyl acetate, and the combined organic extracts are dried over anhydrous magnesium sulfate, filtered and concentrated under vacuum to provide the α-amino acid (3b). ln Scheme 1 , step C, the coupled product (5) is then deprotected or cleaved from the solid phase under conditions well known in the art to provide the acid of structure (6). For example, wherein X is a methyl or ethyl group on structure (5) , the compound is dissolved in a suitable organic solvent, such as ethanol and treated with approximately an equal volume of water. To this solution, with stirring is added 1 to 2 equivalents of lithium hydroxide and the reaction is allowed to stir for 1 to 6 hours. The resulting acid is then isolated and purified by techniques well known in the art. For example, the organic solvent is removed under vacuum and the remaining aqueous solution is acidified with dilute hydrochloric acid. The aqueous phase is then extracted with a suitable organic solvent, such as ethyl acetate, and the combined organic extracts are dried over anhydrous magnesium sulfate, filtered and concentrated under vacuum. The residue can then be purified by flash chromatography on silica gel with a suitable eluent, such as methanol/chloroform to provide the acid (6).

Additional starting material for Scheme 3 for the preparation of compounds of formula (1) can be prepared as described in Scheme 2. All the substituents, unless otherwise indicated, are previously defined. The reagents and starting materials are readily available to one of ordinary skill in the art.

SCHEME 2

(8)

Pg = protecting group

X = suitable carboxyl protecting group or resin SCHEME 2 (cont.)

(9)

Pg = Protecting group

X = sutiable carboxyl protecting group or a resin

In Scheme 2, step A1 the coupled product (4) (prepared in Scheme 1) is deprotected to produce the primary amine under conditions analogous to the procedure described in Scheme 1 , step A1. The resulting primary amine is then subjected to a coupling reaction with the protected α-amino acid of structure (3a") in a manner analogous to the procedures described previously in Scheme 1 , step A to provide the coupled product (7).

In Scheme 2, step C1 the above prepared coupled product (7) is deprotected to produce the primary amine under conditions analogous to the procedure described in Scheme 1 , step A1. The resulting primary amine is then subjected to a coupling reaction with carboxybenzoyi in a manner analogous to the procedures described previously in Scheme 1 , step A or Scheme 1 , step A2 to provide the coupled product (8). Alternatively, the above coupled product (8) may be prepared directly as described in Scheme 2, steps B1 and B2. The coupled product (4) is deprotected to produce the primary amine under conditions analogous to the procedure described in Scheme 1 , step A1. The resulting primary amine is then subjected to a coupling reaction with the α-amino acid of structure (3b") (as prepared in Scheme 1, wherein R₂ is substituted for R^ in a manner analogous to the procedures described previously in Scheme 1 , step A to provide the coupled product (8).

In Scheme 2, step C the above prepared coupled product (8) is deprotected or cleaved from the solid phase under conditions well known in the art, such as that described previously in Scheme 1 , step C to provide the acid of structure (9).

The compounds of formula (1) can be made according to techniques and procedures described in the prior art and according to Scheme 3 below. All of the substituents are as previously defined unless otherwise indicated. The reagents and starting materials are readily available to one of ordinary skill in the art.

SCHEME 3

(6) or (9)

ion

(10)

(1)

In Scheme 3, step A, either the acid of structure (6) or the acid of structure (9) is transformed into the Weinreb amide (10). This amidation can be performed utilizing a coupling reaction as between two amino acids using either the acid (6) or the acid (9) and the N-alkyl-O-alkylhydroxylamine. The standard coupling reaction can be carried out using standard coupling procedures as described above in Scheme 1 , step A to provide the amide (10). ln Scheme 3, step B, the peptide Weinreb amide (10) is reduced to provide the desired aldehyde of formula (1).

For example, the peptide Weinreb amide (10) is dissolved in a suitable organic solvent, such as tetrahydrofuran and cooled to 0°C under an atmosphere of nitrogen. An excess of a suitable reducing agent is added to the solution. Examples of suitable reducing agents are lithium aluminum hydride, diisobutylaluminum hydrides, tri-tert-butyloxyaluminum hydrides, sodium aluminum hydrides, diaminoaluminum hydrides and the like. The preferred reducing agent is lithium aluminum hydride. The reaction is stirred for 20 minutes to 2 hours at a temperature of about 0°C to 20°C. The reaction is then quenched and the product isolated by techniques well known in the art. For example, the reaction is quenched with 10% potassium hydrogen sulfate followed by addition of 10% hydrochloric acid. The aqueous mixture is then extracted with a suitable organic solvent such as ethyl acetate. The organic extract is washed with water, dried over magnesium sulfate, filtered and concentrated under vacuum to provide the aldehyde of formula (1).

Alternatively, the compounds of formula (1) may be made according to the methods set forth in Scheme 4. All substituents, unless otherwise indicated, are previously defined. The reagents and starting materials are readily available to one of ordinary skill in the art.

SCHEME 4

StepB

Deprotection

(11) (12)

StepC

HCI- Coupling

(13)

StepD

Reduction

(10)

(1) The required starting material defined by compound (11) is readily available either commercially or by applying known prior art principles and techniques. The term "Pg" refers to a suitable amine protecting group. The selection, use and removal of protecting groups utilizing suitable protecting groups such as those described in Protecting Groups in Organic Synthesis by T. Greene, Wiley-

Interscience (1981) is well known and appreciated in the art. An example αf such a compound is the suitably protected amino acid L-phenylalanine.

In Scheme 4, step A, the protected amino acid (11) is transformed into the amide (12) using techniques and procedures previously set forth above in Scheme 3, step A.

In Scheme 4, step B, the amide (12) is deprotected under conditions previously set forth in Scheme 1 , step A1 to provide the deprotected amide (13).

In Scheme 4, step C, the deprotected amide (13) is elongated by coupling the next suitably protected amino acid through a peptide linkage using standard coupling procedures, or by condensation of fragments, or by combination of both processes to provide the elongated peptide (10). Standard coupling procedures are described above, for example, in Scheme 1 , step A.

In Scheme 4, step D, the peptide Weinreb amide (10) is reduced to provide the desired aldehyde of formula (1) according to procedures described above in Scheme 3, step B.

Alternatively, the compounds of formula (1) may be made according to the methods set forth in Scheme 5. All substituents, unless otherwise indicated, are previously defined. The reagents and starting materials are readily available to one of ordinary skill in the art. SCHEME 5

SCHEME 5 (cont.)

In Scheme 5, step A, the Cbz-N-protected amino acid or dipeptide of structure

(14) is coupled with an amino alcohol of structure (15) in a manner analogous to the procedures described previously in Scheme 1 , step A to provide the Cbz-N-protected peptide alcohol of structure (16).

In Scheme 5, step B, the Cbz-N-protected peptide alcohol of structure (16) is oxidized to provide the Cbz-N-protected peptide aldehyde of structure (17). For example, the Cbz-N-protected peptide alcohol of structure (16) is dissolved in a buffered solvent mixture comprising a nonoxidizable, polar, water-immiscible solvent, for example, ethyl acetate, and a small amount of water. A stoichiometric amount of sodium bromide is then added to the mixture and the Cbz-N-protected peptide alcohol of structure (16) is oxidized, while cooling (about -5°C to about 5°C), using a catalytic amount of 2,2,6,6-tetramethylpiperidin-N-oxyl (TEMPO), or a derivative thereof. The reaction mixture is then treated with an aqueous solution of sodium hypochlorite and the reaction is terminated by adding a reducing agent. The reaction mixture is worked up by separating the two phases and extracting the aqueous phase with a nonoxidizable, polar and water-immiscible solvent and finally obtaining the Cbz-N-protected peptide aldehyde of structure (17) from the pooled organic phases.

It is preferred that the content of water in the solvent mixture be kept relatively low in comparison to the nonoxidizable, polar, water-immiscible solvent. Preferably, the water component of the solvent mixture should be < 10%. Most preferably, the water component of the solvent mixture should be < 5%. Preferably, either sodium hydrogen carbonate or potassium hydrogen carbonate is used as a buffer for the solvent mixture. Particular preference is given to sodium hydrogen carbonate as a 1-10% strength, preferably 3-5% strength solution.

Preferably, either 2,2,6,6-tetramethylpiperidin-N-oxyl (TEMPO), or one of its derivatives, is employed as the catalyst for the oxidation reaction for producing the Cbz-N-protected peptide aldehyde of structure (17). Suitable derivatives of TEMPO are the 4-hydroxy derivative or the 4-acetamide derivative. However, TEMPO is a particularly preferred catalyst.

The sodium hypochlorite solution is 10-20% strength, preferably 12-15% strength.

Preferred reducing agents are those which are soluble in water. Particularly preferred are dithionites, sulfite, dimethyl sulfide and/or thiosulfates.

The two phases of the reaction mixture which form may be separated and the aqueous phase of these two phases is extracted preferably with ethyl acetate. The combined organic phases are back-extracted with water. The Cbz-N-protected peptide aldehyde of structure (17) may be isolated by distilling off the water/ethyl acetate mixture. An additional purification can be achieved by subsequently precipitating the product from a nonpolar solvent, preferably n-propane or π-hexane.

In Scheme 5, step C, the Cbz-N-protected peptide aldehyde of structure (17) is racemized to form the Cbz-N-protected peptide aldehyde racemate of structure (18). For example, the Cbz-N-protected peptide aldehyde of structure (17) is dissolved in a mixture of silica gel, methylene chloride and ethyl acetate. The silica gel is then filtered and washed with ethyl acetate. The filtrate is evaporated to give a residue, which is slurried in a nonpolar solvent, preferably n-heptane. Crude product of structure (18) is then filtered off, washed with nonpolar solvent, then dried. The Cbz-N-protected peptide aldehyde racemate of structure (18) may be purified according to techniques and procedures well known in the art, such as extraction and evaporation.

The following examples illustrate the preparation and use of the compounds of formula (1). Melting points are obtained on a Thomas Hoover melting point apparatus. NMR spectra are recorded on a Varian XL 400 at 400 Mhz for .¹H, and 100 Mhz for ¹³C and/or Varian GEMINI-300 spectrometers at 300 Mhz for ¹H, and 75 Mhz for ¹³C. All chemical shifts are reported in parts per million (ppm, δ) relative to TMS standard. Mass spectra are obtained on a MAT TSQ 700 spectrometer at 120 eV. The diastereomeric ratio of the compounds is determined by capillary zone electrophoresis on a Beckman P/ACE 5000 CE system equipped with a 57 cm x 75 μm i.d. x 365 μm o.d. column, 50 cm to detector. As used herein, the following terms have the indicated meanings: "g" refers to grams; "mmol" refers to millimoles; "mL" refers to milliliters; "L" refers to liters; "mol" refers to moles; "bp" refers to boiling point; "mp" refers to melting point; "°C" refers to degrees Celsius; "mm Hg" refers to millimeters of mercury; "μL" refers to microliters; "μg" refers to micrograms; "μM" refers to micromolar; "Cbz" refers to carbobenzyloxy; "DMF" refers to dimethylformamide; "THF" refers to tetrahydrofuran; "TBAF" means tetrabutylammonium fluoride; "NMM" means N-methylmorpholine; "DMSO" means dimethylsulfoxide; "HOBT" means hydroxybenzotriazole; "EDC" means 1-(3- dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride. These examples are illustrative only and are not intended to limit the scope of the invention in any way.

EXAMPLE 1

Preparation of Carbamic Acid. ri-[ (1-formyl-2-phenylethyl)amino1carbonvn-2- methylpropylK phenylmethyl ester (Cbz-.L.-Val-.D.L.-Phe-H. MDL 28,170)

a) Cbz-(L)-Val-(L)-Phe-Λ/-methoxy-N-methylamide

Scheme 3, step A; Cool a mixture of Cbz-(L)-Val-(L)-Phe-OH (40.0 g, 100.4 mmol) in CH₂CI₂ (400 mL) to -25°C (nitromethane/dry ice) with stirring under a N₂ atmosphere. Add N-methylmorpholine (22.6 mL, 20.8 g, 205.8 mmol, 2.05 eq.) dropwise over 5 min and allow the suspension to stir for 20 min. Add isobutyl chloroformate (13.6 mL, 14.3 g, 104.4 mmol, 1.04 eq) dropwise via syringe over 5 min such that the temperature does not exceed -23°C. Stir the mixture for 20 min. Add NOdimethylhydroxyamine • HCI (10.4 g, 106.4 mmol, 1.06 eq) to the reaction mixture in 2 x 5.2 g portions over 5 min, and allow the mixture to stir at -25°C for 1 h. Allow the reaction mixture to warm to room temperature over 1.5 h and stirr at room temperature for 1 h. Quencht the reaction by adding NH CI (125 mL) with stirring for 15 min. Separate the layers and extract the organic layer with half-sat. NH CI (1 x 75 mL), sat. NaHC0₃ (1 x 100 mL), and brine (1 x 75 mL). Dry the organic solution over MgS0₄, filter to remove inorganics, and concentrate in vacuo (42°C / 35 torr) to give the title Weinreb amide as a crude oil (45 g, 101.9 mmol, 102% yield). TLC: (75% EtOAc / 25% hexane) R_f = 0.50. Dissolve the oil in Et₂0 and a white solid precipitates upon stirring. Collect the white solid by filtration and allow to dry overnight to provide the title Weinreb amide (26.0 g, 58.9 mmol, 59% yield). ¹H NMR (CDCI₃): δ 7.42-7.08 (m, 10H, Ar), 6.66 (d, J=7.5 Hz, 1 H, NH), 5.33 (d, J=10 Hz, 1 H, NH), 5.25 (m, 1 H, NCH), 5.11 (d, J=4.5 Hz, 2H, ArCH₂0), 4.06 (m, 1 H, NCH), 3.65 (s, 3H, OCH₃), 3.16 (s, 3H, NCH₃), 3.08 (m, 1H), 2.93 (m, 1H), 2.06 (sept, 1H, J=6.5 Hz, CH(CH₃)₂, 0.91 (d, 3H, J=6.5 Hz, CH₃), 0.84 (d, 3H, J=6.5 Hz, CH₃);

¹³C NMR (CDCI₃): δ 171.4, 170.7, 156.2, 136.3, 136.1 , 129.4, 128.5, 128.4, 128.1 , 128.0, 126.9, 66.9, 61.6, 60.1 , 50.3, 38.3, 31.2, 19.1 , 17.5;

MS (Cl, NH₃), M/Z (rel. intensity), 460 (21), 459 (M+NH₄, 77), 442 (M+H, 100), 398 (4), 381 (11), 351 (2), 334 (15), 308 (12), 278 (2), 234 (2), 210 (2), 162 (2), 125 (5), 108 (9), 91 (3);

Calcd. for C₂4H3iN₃05, M.W. 441.53: C, 65.29; H, 7.08; N, 9.52 Found: C, 65.40; H, 7.03; N, 9.56.

b) Carbamic Acid, ri-rf(1-formyl-2-phenylethyl)aminolcarbonyll-2- methylpropyn-. phenylmethyl ester (Cbz-(L)-Val-,D,L)-Phe-H, MDL 28.170)

Scheme 3, step B; Dissolve the Weinreb amide (17.25 g, 39.1 mmol) of Example 1 , step (a) in tetrahydrofuran (200 mL) and cool the solution to -15°C with stirring under a N₂ atmosphere. Add a solution of LAH in tetrahydrofuran (1M, 50.0 mL, 50.0 mmol, 1.25 eq) dropwise with stirring such that the temperature does not exceed -10°C. CAUTION: exotherm and foaming occurs upon addition of the first portion of the solution! After the addition is complete, stir the mixture at -10°C for 0.5 h. Decant the mixture into a separator funnel and wash the solution with sat.

NaHCOβ (3 x 80 mL), and brine (1 x 100 mL). Dry the organic layer over MgS0₄, filter through filter aid to remove the inorganics and remove the solvent in vacuo (45°C / 35 torr) to give a yellow solid. Dissolve the solid in CH₂CI₂ and purify by plug filtration on silica gel (33% EtOAc / 67% hexane as eluent) to provide the title aldehyde (8.6 g, 22.5 mmol, 58% yield). TLC: (75% EtOAc / 25% hexane) R_f = 0.64.

¹H NMR (CDCI₃): δ 9.60 (2-s, 1 H, CHO), 7.42-7.08 (m, 10H, Ar), 6.39 (2-d, 1 H, NH), 5.25 (d, J=8 Hz, 1 H, NH), 5.11 and 5.10 (2-s, 2H, ArCH₂0), 4.73 (2-dd, 1 H, NCH), 4.02 (m, 1 H, NCH), 3.13 (2-d, 2H, ArCH₂CHCHO), 2.09 (sept, 1 H, J=6.5 Hz, CH(CH₃)₂), 0.88 (m, 6H, CH(CH₃)₂);

¹³C NMR (CDCI₃): δ 198.5, 198.3, 171.4, 171.3, 156.3, 136.1 , 135.3, 135.2, 129.3, 129.2, 128.8, 128.6, 128.3, 128.1 , 127.3, 67.2, 60.3, 59.6, 59.5, 35.2, 35.1 , 30.8, 19.2, 17.6, 17.4;

MS (Cl, NH₃), M/Z (rel. intensity), 400 (M+NH₄, 19), 383 (M+H, 100), 355 (7), 249 (5), 206 (6), 162 (5), 120 (5), 108 (9), 91 (9), 72 (2);

Calcd. for C₂₂H₂₆N₂0₄, M.W. 382.46: C, 69.09; H, 6.85; N, 7.32 Found: C, 69.04; H, 6.91 ; N, 7.37.

EXAMPLE 2

Preparation of Carbamic Acid. ri-rr(1-formyl-2-phenylethyl)amino1carbonyri-3- methylbutyll-, phenylmethyl ester (Cbz-.L)-Leu-(D.L)-Phe-H)

a) Cbz-,L)-Leu-(L)-Phe-0-tert-butyl

Scheme 1, step B; Suspend HCkPhe-O-tert-butyl (4.65 g, 18 mmol) is in DMF (40 mL). Cool the suspension to 0°C and add triethylamine (5.6 mL, 40 mmol). After stirring for 10 minutes, add THF (50 mL), followed by the addition of Cbz-Leu-OH (4.77 g, 18 mmol, in 100 mL THF), HOBt (2.6 g, 19 mmol) and EDC (3.63 g, 19 mmol). Stir the reaction at 0°C for 3 hours and then at room temperature overnight. Then concentrate the reaction under vacuum. Dissolve the residue in 1 N HCI (100 mL) and extract the aqueous with ethyl acetate (4 x 100 mL). Combine the organic extracts, rinse with saturated sodium carbonate (100 mL), brine (100 mL), dry over anhydrous magnesium sulfate, pass through a short pad of silica gel and concentrate the filtrate under vacuum to provide the title coupled product (9.93 g) as an oil.

b) Cbz-,L)-Leu-(L)-Phe-OH

Scheme 1 , step C; Dissolve the above coupled product (9.93 g) in methylene chloride (20 mL) and treat with trifluoroacetic acid (10 mL). Stir the reaction overnight at room temperature and then concentrate under vacuum to provide the acid as a sticky oil. c) Cbz-(L)-Leu-(L)-Phe-Λ/-methoxy-Λ/-methylamide

Scheme 3, step A; Prepare by the method of Example 1 (a) using Cbz-(L)- Leu-(L)-Phe-OH (100.4 mmol). Purify by chromatography to give the title compound.

d) Carbamic Acid, f 1 -IT(1 -formyl-2-phenylethyl)amino1carbonvπ-3-methylbutylK phenylmethyl ester (Cbz-(L)-Leu-fD.L)-Phe-H)

Scheme 3, step B; Prepare by the method of Example 1 (b) using Cbz-(L)-

Leu-(L)-Phe-Λ/-methoxy-Λ/-methylamide (39.1 mmol). Purify by chromatography to give the title compound.

EXAMPLE 3

Preparation of Carbamic Acid. f1-frri-formyl-2-(4-hvdroxyphenylethyl)- aminolcarbonvn-2-methylpropyn-, phenylmethyl ester

■Cbz-,L)-Val-,D,L)-Tyr-H)

a) Cbz-(L)-Val-,L)-Tyr-Q-tert-butyl

Scheme 1 , step B; Suspend HCI*Tyr-0-tert-butyl (18 mmol) in DMF (40 mL). Cool the suspension to 0°C and add triethylamine (5.6 mL, 40 mmol). After stirring for 10 minutes, add THF (50 mL), followed by addition of Cbz-Val-OH (18 mmol, in 100 mL THF), HOBt (2.6 g, 19 mmol) and EDC (3.63 g, 19 mmol). Stir the reaction at 0°C for 3 hours and then at room temperature overnight. Then concentrate the reaction under vacuum. Dissolve the residue in 1 N HCI (100 mL) and extract the aqueous with ethyl acetate (4 x 100 mL). Combine the organic extracts, rinse with saturated sodium carbonate (100 mL), brine (100 mL), dry over anhydrous magnesium sulfate, filter and concentrate under vacuum to provide the coupled product.

b) Cbz-.L)-Val-(L)-Tyr-OH

Scheme 1 , step C; Dissolve the above coupled product in methylene chloride (20 mL) and treat with trifluoroacetic acid (10 mL). Stir the reaction overnight at room temperature and then concentrate under vacuum to provide the acid as a sticky oil.

c) Cbz-(L)-Val-(L)-Tyr-Λ/-methoxy-/V-methylamide

Scheme 3, step A; Prepare by the method of Example 1 (a) using Cbz-(L)- Val-(L)-Tyr-OH (100.4 mmol). Purify by Ghromatography to give the title compound.

d) Carbamic Acid, f 1 -NT 1 -formyl-2-,4-hvdroxyphenylethyl)-aminolcarbonyl1-2- methylpropyπ-. phenylmethyl ester (Cbz-(L)-Val-(D.D-Tyr-H)

Scheme 3, step B; Prepare by the method of Example 1 (b) using Cbz-(L)-

Val-(L)-Tyr-Λ/-methoxy-Λ/-methylamide (39.1 mmol). Purify by chromatography to give the title compound.

EXAMPLE 4

Preparation of Carbamic Acid, ri-rrH-formyl-2-(4-methoxyphenylethyl)- aminolcarbonvn-2-methylpropyn-, phenylmethyl ester

(Cbz-.L)-Val-(D,L)-Tyr(OMe)-H)

a) Cbz-(L)-Val-(L)-Tyr(OMe)-OBz

Scheme 1 , step B; To a solution of N-benzyloxycarbonyl-L-valine anhydride (0.339 g, 0.7 mmol) in anhydrous dichloromethane (15 ml) is added O-methyl-L- tyrosine, benzyl ester, toluene-4-sulfonate (0.330 g, 0.7 mmol) and N-methyl morpholine (0.081 g, 0.8 mmol). The reaction is stirred at room temperature overnight. The reaction is concentrated under vacuum and the residue is purified by flash chromatography (silica gel: 2:8 ethyl acetate/cyclohexane) to provide the coupled compound.

b) Cbz-(L)-Val-,L)-Tyr(OMe)-OH

Scheme 1 , step C; Dissolve the above prepared coupled product (14.3 mmol) in THF (100 mL) and water (100 mL). Add lithium hydroxide monohydrate (670 mg, 16 mmol) and stir the reaction at room temperature for 2 hours. Then wash the reaction with diethyl ether (100 mL) and acidify the aqueous layer with 6N HCI to approximately pH 2. Extract the acidified aqueous layer with ethyl acetate (3 x 100 mL). Combine the organic extracts, dry over anhydrous magnesium sulfate, filter and concentrate the filtrate under vacuum to provide the acid.

c) Cbz-(L)-Val-(L)-Tyr(OMe)-Λ/-methoxy- /-methylamide Scheme 3, step A; Prepare by the method of Example 1 (a) using Cbz-(L)- Val-(L)-Tyr(OMe)-OH (100.4 mmol). Purify by chromatography to give the title compound.

d) Carbamic Acid, H-ffH-formyl-2-(4-methoxyphenylethyl)-amino.carbonvH-2- methylpropyll-. phenylmethyl ester (Cbz-(L)-Val-(D,L)-Tyr,OMe)-H)

Scheme 3, step B; Prepare by the method of Example 1 (b) using Cbz-(L)- Val-(L)-Tyr(OMe)-Λ/-methoxy-Λ/-methylamide (39.1 mmol). Purify by chromatography to give the title compound.

EXAMPLE 5

Preparation of Carbamic Acid. ri-rrri-formyl-2-(4-nitrophenylethyl)-aminolcarbonyll-2- methylpropyll-, phenylmethyl ester ,Cbz-(L)-Val-(D,L)-Phe.4-NQp)-H)

a) N-benzyloxycarbonyl-L-valyl-4-nitro-L-phenylalanine methyl ester

Scheme 1 , step B; To a solution of N-benzyloxycarbonyl-L-valine anhydride (4.80 g, 10 mmol) in anhydrous dichloromethane (50 ml) is added 4-nitro-L- phenylalanine methyl ester (2.24 g, 10 mmol). Stir the mixture at room temperature overnight. Concentrate the reaction under vacuum and purify the residue by flash chromatography (silica gel: 4:6 ethyl acetate/cyclohexane) to provide N- benzyloxycarbonyl-L-valyl-4-nitro-L-phenylalanine methyl ester. Rf ~ 0.32 (ethyl acetate/cyclohexane 1 :1).

b) N-benzyloxycarbonyl-L-valyl-4-nitro-L-phenylalanine

Scheme 1 , step C; Dissolve N-benzyloxycarbonyl-L-valyl-4-nitro-L- . phenylalanine methyl ester (14.3 mmol, prepared above) in THF (100 mL) and water (100 mL). Add lithium hydroxide monohydrate (670 mg, 16 mmol) and stir the reaction at room temperature for 2 hours. Then wash the reaction with diethyl ether (100 mL) and acidify the aqueous layer with 6N HCI to approximately pH 2. Extract the acidified aqueous layer with ethyl acetate (3 x 100 mL). Combine the organic extracts, dry over anhydrous magnesium sulfate, filter and concentrate the filtrate under vacuum to provide the acid.

c) Cbz-.L)-Val-(L)-Phe(4-NOg)-Λ/-methoxy-Λ/-methylamide

Scheme 3, step A; Prepare by the method of Example 1 (a) using N- benzyloxycarbonyl-L-valyl-4-nitro-L-phenylalanine (100.4 mmol). Purify by chromatography to give the title compound.

d) Carbamic Acid. H-rrπ-formyl-2-(4-nitrophenylethyl)-amino1carbonvπ-2- methylpropyll-. phenylmethyl ester ,Cbz-(L)-Val-(D,L)-Phe(4-NOp)-H)

Scheme 3, step B; Prepare by the method of Example 1 (b) using Cbz-(L)- Val-(L)-Phe(4-N0₂)-N-methoxy-N-methylamide (39.1 mmol). Purify by chromatography to give the title compound. EXAMPLE 6

Preparation of Carbamic Acid, ri-ffri-formyl-2-(4-aminophenylethyl)amino1carbonvn-

2-methylpropyπ-, phenylmethyl ester (Cbz-(L)-Val-(D.L)-Phe(4-NHp)-H)

a) N-benzyloxycarbonyl-L-valyl-4-amino-L-phenylalanine methyl ester

A solution of N-benzyloxycarbonyl-L-valyl-4-nitro-L-phenylalanine methyl ester (0.91 g, 2 mmol, prepared in example 5) and Tin (II) chloride dihydrate (1.56 g, 7 mmol) in absolute ethanol (50 ml) and N,N-dimethylformamide (5 ml) is heated under reflux for 4 hours. The mixture is cooled, diluted with water, neutralized with sodium hydrogenocarbonate and extracted with ethyl acetate (3 x 50 ml). The organic extracts are combined, dried over anhydrous magnesium sulfate, filtered and concentrated under vacuum to provide the amine compound.

b) N-benzyloxycarbonyl-L-valyl-4-amino-L-phenylalanine

Scheme 1 , step C; The above prepared amino compound (14.3 mmol) is deprotected in manner analogous to the procedure described in example 5, step (b) with lithium hydroxide monohydrate (16 mmol) in water (100 mL) and THF (100 mL) to provide the acid.

c) Cbz-(L)-Val-(L)-(4-amino)Phe-Λ/-methoxy-/V-methylamide Scheme 3, step A; Prepare by the method of Example 1 (a) using N- benzyloxycarbonyl-L-valyl-4-amino-L-phenylalanine (100.4 mmol). Purify by chromatography to give the title compound.

d) Carbamic Acid. ri-rr[1-formyl-2-.4-aminophenylethyl)amino1carbonvn-2- methylpropyll-. phenylmethyl ester .Cbz-(L)-Val-,D,L)-Phe.4-NHp)-H)

Scheme 3, step B; Prepare by the method of Example 1 (b) using Cbz-(L)- Val-(L)-(4-amino)Phe-Λ/-methoxy-Λ/-methylamide (39.1 mmol). Purify by chromatography to give the title compound.

EXAMPLE 7

Preparation of Cbz-,L)-lle-(L)-Val-,D,L)-Phe-H

a) Boc-(L)-Val-,L)-Phe-OCHg

Scheme 1 , step A; HCI»Phe-OCH₃ (4.75 g, 22 mmol) is dissolved in DMF (30 mL). The solution is cooled to 0°C and triethylamine (6.2 mL, 44 mmol) is added. After 10 minutes, a solution of N-t-butoxycarbonyl-Val (22 mmol dissolved in 130 mL of DMF) is added to the solution, followed by addition of HOBt (2.97 g, 22 mmol) and EDC (4.2 g, 22 mmol). The reaction is stirred at 0°C for 3 hours and then allowed to warm to room temperature overnight. The reaction is then concentrated under vacuum, the residue taken up in ethyl acetate (100 mL) and rinsed with 1 N HCI (100 mL). The aqueous rinse is extracted with ethyl acetate (2 x 100 mL). The organic layer and the organic extracts are combined, rinsed with saturated sodium bicarbonate (100 mL), brine (100 mL), dried over anhydrous magnesium sulfate, passed through a short pad of silica gel and the filtrate concentrated under vacuum to provide the coupled product.

b) TFA«(L)-Val-(L)-Phe-OCHg

Scheme 2, step C1 ; The above coupled product is dissolved in methylene chloride (20 mL) and treated with trifluoroacetic acid (10 mL). The reaction is stirred overnight at room temperature and then concentrated under vacuum to provide deprotected amine.

c) Cbz-.D-lle-fϋ-Val-.D-Phe-OCH-.

Scheme 2, step C2; The above prepared deprotected amine (22 mmol) is dissolved in DMF (30 mL). The solution is cooled to 0°C and triethylamine (6.2 mL, 44 mmol) is added. After 10 minutes, a solution of Cbz-lle (22 mmol dissolved in 130 mL of DMF) is added to the solution, followed by addition of HOBt (2.97 g, 22 mmol) and EDC (4.2 g, 22 mmol). The reaction is stirred at 0°C for 3 hours and then allowed to warm to room temperature overnight. The reaction is then concentrated under vacuum, the residue taken up in ethyl acetate (100 mL) and rinsed with 1 N HCI (100 mL). The aqueous rinse is extracted with ethyl acetate (2 x 100 mL). The organic layer and the organic extracts are combined, rinsed with saturated sodium bicarbonate (100 mL), brine (100 mL), dried over anhydrous magnesium sulfate, passed through a short pad of silica gel and the filtrate concentrated under vacuum to provide the coupled product.

d) Cbz-,L)-lle-,L)-Val-(L)-Phe-OH

Scheme 2, step D; The above prepared coupled product (14.3 mmol) is deprotected in manner analogous to the procedure described in example 5, step B with lithium hydroxide monohydrate (16 mmol) in water (100 mL) and THF (100 mL) to provide the acid.

e) Cbz-(L)-lle-(L)-Val-.L)-Phe-Λ/-methoxy-Λ/-methylamide Scheme 3, step A; Prepare by the method of Example 1 (a) using Cbz-(L)-lle- (L)-Val-(L)-Phe-QH (100.4 mmol). Purify by chromatography to give the title compound.

f) Cbz-(L)-lle-(L)-Val-(D.L)-Phe-H

Scheme 3, step B; Prepare by the method of Example 1 (b) using Cbz-(L)-lle- (L)-Val-(L)-Phe-Λ/-methoxy-Λ/-methylamide (39.1 mmol). Purify by chromatography to give the title compound.

EXAMPLE 8

Preparation of Cbz-(L)-Val-(L)-VaKD.L)-Phe-H

a) Cbz-(L)-Val-(L)-Val-(L)-Phe-OCHr.

Scheme 2, step C2; The deprotected amine prepared in example 7, step B (22 mmol) is dissolved in DMF (30 mL). The solution is cooled to 0°C and triethylamine (6.2 mL, 44 mmol) is added. After 10 minutes, a solution of Cbz- Val (22 mmol dissolved in 130 mL of DMF) is added to the solution, followed by addition of HOBt (2.97 g, 22 mmol) and EDC (4.2 g, 22 mmol). The reaction is stirred at 0°C for 3 hours and then allowed to warm to room temperature overnight. The reaction is then concentrated under vacuum, the residue taken up in ethyl acetate (100 mL) and rinsed with 1 N HCI (100 mL). The aqueous rinse is extracted with ethyl acetate (2 x 100 mL). The organic layer and the organic extracts are combined, rinsed with saturated sodium bicarbonate (100 mL), brine (100 mL), dried over anhydrous magnesium sulfate, passed through a short pad of silica gel and the filtrate concentrated under vacuum to provide the coupled product.

b) Cbz-(L)-Val-(L)-Val-(L)-Phe-OH

Scheme 2, step D; The above prepared coupled product (14.3 mmol) is deprotected in a manner analogous to the procedure described in example 5, step B with lithium hydroxide monohydrate (16 mmol) in water (100 mL) and THF (100 mL) to provide the acid.

c) Cbz-(L)-Val-(L)-Val-(L)-Phe-Λ/-methoxy-Λ/-methylamide

Scheme 3, step A; Prepare by the method of Example 1 (a) using Cbz-(L)- Val-(L)-Val-(L)-Phe-OH (100.4 mmol). Purify by chromatography to give the title compound.

d) Cbz-,L)-Val-(L)-Val-(L)-Phe-H

Scheme 3, step B; Prepare by the method of Example 1 (b) using Cbz-(L)-

Val-(L)-Val-(L)-Phe-Λ/-methoxy-Λ/-methylamide (39.1 mmol). Purify by chromatography to give the title compound.

EXAMPLE 9

Preparation of Carbamic Acid. ri-rff1-formyl-2-(4-chlorophenylethyl)-amino1carbonvn-

2-methylpropyll-. phenylmethyl ester (Cbz-(L)-Val-(D.L)-Phe(4-CI)-H)

a) N-BOC-p-chloro-L-Phe-OCH..

N-BOC-p-chloro-L-Phe (20 mmol, commercially available from Sigma Chemical Company, St. Louis, MO 63178) is dissolved in diethyl ether (400 mL), cooled to 0°C and treated with a slight excess of diazomethane (faint yellow color persists). Several drops of dilute acetic acid are added to quench the excess diazomethane. The reaction is then rinsed with brine (200 mL), dried over anhydrous magnesium sulfate, filtered and concentrated to provide the methyl ester (N-BOC-p- chloro-L-Phe-OCH₃).

b) p-Chloro-L-Phe-OCHg

Scheme 1 , step A1 ; The above prepared methyl ester is dissolved in methylene chloride (20 mL) and treated with trifluoroacetic acid (10 mL). The reaction is stirred overnight at room temperature and then concentrated under vacuum to provide the deprotected compound, (p-chloro-L-Phe-OCH₃).

c) Cbz-Val-Phe(4-CI)-OCH_?

Scheme 1 , step A2; The above prepared deprotected compound (22 mmol) is coupled with CBz-Val (22 mmol dissolved in 130 mL DMF) in a manner analogous to the procedure described in example 7, step (c) with triethylamine (44 mmol), HOBt (22 mmol) and EDC (22 mmol) to provide the coupled compound.

d) Cbz-Val-Phe,4-CI)-OH

Scheme 1 , step C; Dissolve the above prepared coupled product (14.3 mmol) in THF (100 mL) and water (100 mL). Add lithium hydroxide monhydrate (670 mg, 16 mmol) and stir the raction at room temperature for 2 hours. Then wash the reaction with diethyl ether (100 mL) and acidify the aqueous layer with 6N HCI to approximately pH 2. Then extract the acidified aqueous layer with ethyl acetate (3 x 100 mL). Combine the organic extracts, dry over anhydrous magnesium sulfate, pass through a short pad of silica gel and concentrate the filtrate under vacuum to provide the acid.

e) Cbz-Val-Phe(4-CI)-Λ/-methoxy-Λ/-methylamide

Scheme 3, step A; Prepare by the method of Example 1 (a) using Cbz-Val- Phe(4-CI)-OH (100.4 mmol). Purify by chromatography to give the title compound.

f) Carbamic Acid. .1 -\W\ -foιmyl-2-.4-chlorophenylethyl)-aminolcarbonvπ-2- methylpropyll-. phenylmethyl ester (Cbz-(L)-Val-(D.L)-Phe(4-CI)-H)

Scheme 3, step B; Prepare by the method of Example 1 (b) using Cbz-Val- Phe(4-CI)-Λ/-methoxy-Λ/-methylamide (39.1 mmol). Purify by chromatography to give the title compound.

EXAMPLE 10

Preparation of Carbamic Acid. ri-Fr(1-formyl-2-phenylethyl)amino1carbonyll-ethvn-. phenylmethyl ester (Cbz-(L)-Ala-ω.L)-Phe-H)

a) Cbz-(L)-Ala-(L)-Phe-Λ/-methoxy-/V-methylamide

Scheme 3, step A; Prepare by the method of Example 1 (a) using N-Cbz-Ala- Phe-OH (100.4 mmol, available from Sigma Chemical Company, St. Louis, MO 63178). Purify by chromatography to give the title compound.

b) Carbamic Acid. M -fK1 -formyl-2-phenylethyl)amino1carbonyl1-ethyll-, phenylmethyl ester (Cbz-(L)-Ala-(D.D-Phe-H)

Scheme 3, step B; Prepare by the method of Example 1 (b) using Cbz-(L)- Ala-(L)-Phe-Λ/-methoxy-Λ/-methylamide (39.1 mmol). Purify by chromatography to give the title compound.

EXAMPLE 11

Preparation of Carbamic Acid. ri-ff(1-formyl-2-phenylethyl)amino1carbonvn-2- methylbutvn-. phenylmethyl ester .Cbz-.L)-lle-(D.L)-Phe-H)

a) Cbz-(L)-lle-(L)-Phe-N-methoxy-N-methylamide

Scheme 3, step A; Prepare by the method of Example 1 (a) using N-Cbz-lle- Phe-OH (100.4 mmol, available from Sigma Chemical Company, St. Louis, MO 63178). Purify by chromatography to give the title compound.

b) Carbamic Acid, H-rr(1-formyl-2-phenylethyl)amino1carbonvπ-2-methylbutyl1-. phenylmethyl ester (Cbz-(L)-lle-(D.D-Phe-H)

Scheme 3, step B; Prepare by the method of Example 1 (b) using Cbz-(L)-lle- (L)-Phe-Λ/-methoxy-Λ/-methylamide (39.1 mmol). Purify by chromatography to give the title compound.

EXAMPLE 12

Preparation of Carbamic Acid. M-πT1-formyl-2-(4- hvdroxyphenylethyl)laminolcarbonyl1-3-methylbutvn-, phenylmethyl ester

,Cbz-.L)-Leu-.D.L)-Tyr-H)

a) Cbz-(L)-Leu-(L)-Tyr-Λ/-methoxy-Λ/-methylamide

Scheme 3, step A; Prepare by the method of Example 1 (a) using N-Cbz-Leu- Tyr-OH (100.4 mmol, available from Sigma Chemical Company, St. Louis, MO 63178). Purify by chromatography to give the title compound.

b) Carbamic Acid, f 1 -KM -formyl-2-(4-hvdroχyphenylethyl)1amino1carbonvn-3- methylbutyll-. phenylmethyl ester .Cbz-.L)-Leu-(D.L)-Tyr-H)

Scheme 3, step B; Prepare by the method of Example 1 (b) using Cbz-(L)- Leu-(L)-Tyr-Λ/-methoxy-Λ/-methylamide (39.1 mmol). Purify by chromatography to give the title compound.

EXAMPLE 13

Preparation of Cbz-(L)-Ala-.L)-Abu-(D,L)-Phe,4-CI)-H

a) Cbz-(L)-Ala-(L)-Abu-(L)-Phe-OCH3

Scheme 1 , step A2; The deprotected compound of Example 9, step (b) (22 mmol) is coupled with CBz-Ala-Abu-OH (22 mmol dissolved in 130 mL DMF, available from Sigma Chemical Company, St. Louis, MO 63178) in a manner analogous to the procedure described in example 7, step (c) with triethylamine (44 mmol), HOBt (22 mmol) and EDC (22 mmol) to provide the coupled compound.

b) Cbz-(L)-Ala-.L)-Abu-(L)-Phe.4-CI)-OH

e) Cbz-(L)-Ala-(L)-Abu-(L)-Phe(4-CI)-Λ/-methoxy-Λ/-methylamide Scheme 3, step A; Prepare by the method of Example 1 (a) using Cbz-(L)- Ala-(L)-Abu-(L)-Phe(4-CI)-OH (100.4 mmol). Purify by chromatography to give the title compound.

f) Cbz-.L)-Ala-(L)-Abu-(D,L)-Phe,4-CI)-H

Scheme 3, step B; Prepare by the method of Example 1 (b) using Cbz-(L)- Ala-(L)-Abu-(L)-Phe(4-CI)-Λ/-methoxy-Λ/-methylamide (39.1 mmol). Purify by chromatography to give the title compound.

EXAMPLE 14

Preparation of Cbz-(L)-Val-.L)-Phe-H

a) Cbz-,L)-Val-(L)-Phenylalaninol

Scheme 5, step A; Charge a mixture of Cbz-(L)-valine (551 g, 2.19 mol) and anhydrous THF (6.0 L) to a 12-L, three-necked flask fitted with a stirrer, thermometer, dropping funnel and continuous nitrogen purge. Stir the solution and cool to -20°C, then add Λ/-methylmorpholine (241 mL, 222.9 g, 2.20 mol) over a period of 15 min while maintaining the reaction temperature of -20°C. After stirring for an additional 15 min, add isobutylchlorofomate (300 g, 2.20 mol) over a period of 30 min while maintaining a reaction temperature of from -20°C to -15°C. After stirring for a period of 30 min at -20°C, add Λ/-methylmorpholine (241 mL, 222.9 g, 2.20 mol), then add (S)-(-)-2-amino-3-phenyl-1-propanol (331.8 g, 2.19 mol) in ca. 65 g portions over 15 min while maintaining a reaction temprature of -15°C to -20°C. Stir the reaction mixture for 30 min at -15°C to -20°C, then remove the cooling bath and stir the reaction mixture at ambient temperature for 2 hr (reaction temperature now 10°C). Dilute the reaction mixture with ethyl acetate, then wash with water (5 L), 0.5 N HCI (4.8 L), and finally a solution of NaHC0₃ (480 g diluted to 5 L with water). Dry the organic phase (500 g of MgS0₄), filter to remove drying agent which is washed with ethyl acetate (2 x 1 L), then evaporate the filtrate at 40°C/50 torr. Slurry the obtained solid in 4 L of heptane. Filter off Cbz-(L)-Val-(L)-phenylalaninol, wash with* heptane (4 x 0.5 L), then air dry at ambient temperature to give Cbz-(L)-Val-(L)-phenylalaninol (786 g, 93% yield).

b) Cbz-(L)-Val-(L)-Phe-H

Scheme 5, step B; Charge a solution of Cbz-(L)-Val-(L)-phenyialaninol (250 g, 0.65 mol) in ethyl acetate (25 L) to a 20 gallon glass-lined reactor. Cool the stirred solution to 0°C, then add a solution of NaBr (82.5 g, 0.80 mol) in 1.5 L of water, followed by the addition of KHC0₃ (440 g, 4.39 mol) and TEMPO (2.5 g). Maintain the reaction temperature at -2°C to +2°C while adding a solutionn of 12.5% NaOCI (463 g, 0.78 mol) in 1.5 L of water over 35 min. After an additional 20 min, analyze the reaction mixture using tic (silica gel, 9/1 CH₂CI₂/EtOAc, R_f of Cbz-(L)-Val-(L)- phenylalaninol = 0.22, R_f of Cbz-(L)-Val-(L)-Phe-H = 0.3, vanillin stain). Add a solution of 12.5% NaOCI (28 g, 0.06 mol) in 150 mL of water. After stirring for 20 min, conduct tic analysis of the reaction mixture to determine if reaction is complete. Add Me₂S (60 mL) and stir the mixture for 15 min. Filter off the insolubleson dicalite and wash with ethyl acetate (4 x 1 L). Separate the organic phase, wash with saturated NaCl (5 L), then dry (500 g of MgSθ₄). Filter off the drying agentand wash with ethyl acetate (4 x 1 L). Evaporate the filtrate at 35°C/50 torr. Dilute the obtained solid with heptane (2 L), filter off, wash with heptane (2 x 0.5 L), then air dry to give the title compound (218.7 g, 88.0% yield). EXAMPLE 15

Alternative Preparation of Cbz-(L)-Val-(P,L)-Phe-H

a) Cbz-(L)-Val-,D.L)-Phe-H

Scheme 5, step C; Charge a mixture of Cbz-(L)-Val-(L)-Phe-H (500 g, Example 14), silica gel (10.5 kg, 230-400 mesh), CH₂CI₂ (45 L) and ethyl acetate (5.0 L) to a 20 gallon glass-lined reactor and vigorously stir for 4.5 h at 22°C. Filter off silica gel and wash with ethyl acetate (5 x 5 L). Evaporate the filtrate at 25-45°C/50 torr to give a residue, which is slurried in heptane (2 L). Filter off crude aldehyde, wash with heptane (2 x 1 L), then air dry at ambient temperature to give the title compound (487 g, 97% recovery).

b) Purification of Cbz-.L)-Val-(D,L)-Phe-H

Dissolve Cbz-(L)-Val-(D,L)-Phe-H (941 g) in ethyl acetate (6.6 L) by heating the stirred mixture to reflux. Gravity filter the hot solution in a 22 L flask and rinse the filter funnel with hot ethyl acetate (400 mL). Dilute the stirred filtrate with hot (50°C) heptane. Cool the stirred mixture to -12°C over a perioof 5.5 h. Filter off the solid, wahs with 2 x 3 L of 9/1 heptane/ethyl acetate, air dry at ambient temperature, then dry for 24 h at 45°C/50 torr to give pure Cbz-(L)-Val-(D,L)-Phe-H (880 g, 94% recovery).

Calcd. for C2₂H₂₆N₂0₄, M.W. 382.45: C, 69.09; H, 6.85; N, 7.32 Found: C, 68.63, 68.93; H, 6.80; N, 7.12,7.18. EXAMPLE 16

Effects of Carbamic Acid. ri-rf(1-formyl-2-phenylethyl)aminolcarbonyl1-2- methylpropylK phenylmethyl ester Infusion on Segualea of Traumatic Brain Injury in Rats

a) Methods

Surgical Preparation: Male Sprague-Dawley rats weighing an average of 290.0 ± 2.4 g were used as subjects. Traumatic Brain Injury ('TBI") was carried out using procedures described by Dixon et al., J. Neurosci. Methods 39, 253-262 (1991). Briefly, the animals were anesthetized with isofluorane in 0₂/N₂0 and an indwelling catheter was placed in the right jugular vein for administration of compound or vehicle. The animal was then prepared with a craniectomy over the right parietal cortex of concussive head injury and a craniectomy over the left cortex for the countrecoup area. Injury level was adjusted to be of moderate severity (2 mm deformation of the cortex on the right side). Following injury, animals were restrained in vests and harnesses designed to prevent the i.v. line from becoming tangled or dislodged.

Drug Administration: Carbamic Acid, [1-[[(1-formyl-2-phenylethyl)amino]carbonyl]-2- methylpropyl]-, phenylmethyl ester (MDL 28,170) was dissolved in a vehicle of 10% EtOH/90% PEG 300 at 10 mg/ ml. Doses were administered according to Table 2 below, as a bolus of 2 ml/kg and infusion of 7.2 ml over 24 h via the venous catheter in the jugular vein. Drug and vehicle treatments were randomized each day and run in a double-blind fashion so that the surgeon did not know the group assignments. TABLE 2

Table 2. Doses of MDL 28.170 administered to rats 5 min after concussive head injury and the outcome measures used in the present study. In this table "n" stands for the number of rats in a particular group.

Histological Outcome: Twenty-four h after injury, each rat was sacrificed with an overdose of sodium pentathol and perfused transcardially, with saline followed by 10% formalin. The brain was removed and embedded in paraffin for sectioning. Slices (5 μm in thickness) were taken through the extent of the injury and stained with hemotoxylin and eosin (H&E). Area of injury was determined for each of 10 sections per brain, using an image analysis system that projected an image of each slide onto a computer monitor for analysis of injured vs. non-injured tissue. A single experimenter carried out the histological analysis and remained blinded to the sample's group assignment. Contusion volumes were derived mathematically by integration of area under the curve. Mean contusion volumes were expressed for each treatment group.

Statistical Analysis: One-way ANOVA was used to analyze the data, with post hoc pairwise comparisons using the Neuman-Keuls method. b) Results

Completion Percentages: Table 3 shows the completion percentages for each group. The column labeled "Excluded/Died-Surgery" includes animals that died during the surgical procedure and those that were lost to technical mishaps (e.g., i.v. line lost or pump failure). No vehicle treated animals died during the study-. The distribution of animals that died in the three drug groups shows no difference in death rates (chi squared test:p>.05), so there was no dose-dependent death rate.

TABLE 3

Table 3. Completion percentages for each group. Although more drug treated animals died than vehicle treated animals, there were no differences between dose groups in the death rate.

Contusion Volumes: Contusion volumes measured at 24 h post-injury showed a clear effect of drug treatment compared to the vehicle group. Table 4 shows the mean volume of damage for each group and indicates the percent reduction achieved by MDL 28,170 treatment. A one-way ANOVA revealed a significant effect of group (F93,38)=5.4; p=003). Post hoc comparisons using Neuman-Keuls procedure showed that the low dose group, medium dose group and high dose group each differed from the vehicle treated group, but did not differ from one another. TABLE 4

Table 4. Mean contusion volumes for each dose group in the dose-response. Treatment with MDL 28,170 significantly reduced contusion volume in all dose groups ^*p<.05.

MDL 28,170 administered to rats 5 min following head injury of moderate severity was effective in reducing contusion volume evaluated 24 h post-injury. All the doses used in the present study were equally efficacious, indicating that a minimally effective dose (M.E.D.) has not been achieved in this model.

One method of demonstrating the utility of the compounds of formula (1) in the treatment of trauma associated with spinal cord injury involves the following test protocol:

Adult rats are anesthetized, for example, with a mixture of ketamine (77.5 mg/kg) and xylazine (12.5 mg/kg). Under aseptic conditions, an incision is made to expose the thoracic spinal cord and a laminectomy is performed at the T8 level. Animals are then placed in a stereotaxic apparatus designed for spinal cord injury in the rat based on the design of Wrathall, J.R., J. Neurotrauma 9:S129-S143 (1992). A stainless steel cylinder weighing 15 g with a flat tip of 2 mm diameter is dropped from a height of 8 cm through a guided tube onto the exposed dura. The aponeurotic plane and the skin are separated sutured with nylon thread and postoperative care is provided as appropriate. Outcome is determined by evaluation of motor function using one more of several established motor function tests, the measurement of lymphocyte accumulation as assessed by myeloperoxidase activity, the evaluation of trauma- induced edema, the amplitude of motor evoked potentials, the histological assessment of the spinal cord after sacrifice of the animal and any other measures deemed appropriate for evaluating the sequale of damage associated with traumatic injury.

Any other method for testing the compounds of formula (1) for the treatment of trauma associated with spinal cord or peripheral nerve injury may be used. For example, adult rats are anesthetized, for example, with sodium pentobarbital (2.1 mg/100 g body weight for adult male rats). Under aseptic conditions, an incision is made to expose the spinal cord and a laminectomy is performed at the T11-12 level. Static compression injury is induced by placing a weight of 25 to 40 g extradurally on the spinal cord at T12 for 5 min. The weight has a round concave lower surface of 6 mm diameter and a square top with an area of 4 mm² which exactly fits the spinal cord; Iwasa, K. et al., Free Rad. Biol. Med. 6, 599-606 (1989), Nakaguchi, K. et al., J. Neurotrauma 13, 573-582 (1996). Outcome is determined by evaluation of motor function using one or more of several established motor function tests, the measurement of lymphocyte accumulation as assessed by myeloperoxidase activity, the evaluation of trauma-induced edema, the amplitude of motor evoked potentials, the histological assessment of the spinal cord after sacrifice of the animal and any other measures deemed appropriate for evaluating the sequale of damage associated with traumatic injury.

Claims

WHAT IS CLAIMED IS:

1. A method of treating trauma associated with brain, spinal cord or peripheral nerve injury in a patient in need thereof comprising administering to said patient a therapeutically effective amount of a compound of formula (1 )

wherein R is hydrogen, OH, C₁-C₄ alkyl, C₁-C₄ alkoxy, N0₂, NH₂ or halogen; Ri and R₂ are each independently C₁-C₄ alkyl; and n is the integer zero or one; or a stereoisomer or a pharmaceutically acceptable salt thereof.

2. A method according to claim 1 wherein n is zero.

3. A method according to claim 1 wherein R is hydrogen.

4. A method according to claim 2 wherein R is hydrogen.

5. A method according to claim 1 wherein said compound is carbamic acid, [1-[[(1-formyl-2-phenylethyl)amino]carbonyl]-2-methylpropyl]-, phenylmethyl ester.

6. A method according to claim 1 wherein said trauma is associated with brain injury.

7. A method according to claim 6 wherein said compound is carbamic acid,

[1 -[[(1 -formyl-2-phenylethyl)amino]carbonyl]-2-methylpropyl]-, phenylmethyl ester.

8. A method of treating traumatic injury to the central nervous system in a patient in need thereof comprising administering to said patient a therapeutically effective amount of a compound of formula (1)

wherein R is hydrogen, OH, C1-C4 alkyl, C1-C4 alkoxy, N0₂, NH₂ or halogen; Ri and R₂ are each independently C₁-C₄ alkyl; and n is the integer zero or one; or a stereoisomer or a pharmaceutically acceptable salt thereof.

9. A method according to claim 8 wherein n is zero.

10. A method according to claim 8 wherein R is hydrogen.

11. A method according to claim 9 wherein R is hydrogen.

12. A method according to claim 1 wherein said compound is carbamic acid, [1 -[[(1 -formyl-2-phenylethyl)amino]carbonyl]-2-methylpropyl]-, phenylmethyl ester.

13. A method of reducing contusion volume in neuronal tissue in a patient in need thereof comprising administering to said patient a therapeutically effective amount of a compound of formula (1 )

wherein R is hydrogen, OH, C₁-C₄ alkyl, C C alkoxy, N0₂, NH₂ or halogen; Ri and R₂ are each independently C₁-C₄ alkyl; and n is the integer zero or one; or a stereoisomer or a pharmaceutically acceptable salt thereof.

14. A method according to claim 13 wherein n is zero.

15. A method according to claim 13 wherein R is hydrogen.

16. A method according to claim 14 wherein R is hydrogen.

17. A method according to claim 13 wherein said compound is carbamic acid, [1 -[[(1 -formyl-2-phenylethyl)amino]carbonyl]-2-methylpropyl]-, phenylmethyl ester.

AMENDED CLAIMS

[received by the International Bureau on 18 August 1998 (18.08.98); original claims 1-17 amended (3 pages)]

1. Use in the manufacture of a medicament for treating trauma associated with brain, spinal cord or peripheral nerve injury of compounds according to formula 0)

wherein R is hydrogen, OH, C C₄ alkyl, d-C alkoxy, N0₂, NH₂ or halogen; Ri and R₂ are each independently C1-C4 alkyl; and n is the integer zero or one; or a stereoisomer or a pharmaceutically acceptable salt thereof.

2. Use as claimed in claim 1 wherein n is zero.

3. Use as claimed in claim 1 wherein R is hydrogen.

4. Use as claimed in claim 2 wherein R is hydrogen.

5. Use as claimed in claim 1 wherein said compound is carbamic acid, [1-[[(1 - formyl-2-phenylethyl)amino]carbonyl]-2-methylpropyl]-, phenylmethyl ester.

6. Use as claimed in claim 1 wherein said trauma is associated with brain injury.

7. Use as claimed in claim 6 wherein said compound is carbamic acid, [1 -[[(1 - formyl-2-phenylethyl)amino]carbonyl]-2-methylpropyl]-, phenylmethyl ester.

8. Use in the manufacture of a medicament for treating traumatic injury to the central nervous system of compounds according to formula (1)

wherein R is hydrogen, OH, C C alkyl, C C₄ alkoxy, N0₂, NH₂ or halogen; Ri and R₂ are each independently C C₄ alkyl; and n is the integer zero or one; or a stereoisomer or a pharmaceutically acceptable salt thereof.

9. Use as claimed in claim 8 wherein n is zero.

10. Use as claimed in claim 8 wherein R is hydrogen.

11. Use as claimed in claim 9 wherein R is hydrogen.

12. Use as claimed in claim 1 wherein said compound is carbamic acid, [1-[[(1- formyl-2-phenylethyl)amino]carbonyl]-2-methylpropyl]-, phenylmethyl ester.

13. Use in the manufacture of a medicament for reducing contusion volume in neuronal tissue of compounds of formula (1)

wherein R is hydrogen, OH, C C₄ alkyl, Cι-C alkoxy, N0₂, NH or halogen; R-i and R₂ are each independently C C₄ alkyl; and n is the integer zero or one; or a stereoisomer or a pharmaceutically acceptable salt thereof.

14. Use as claimed in claim 13 wherein n is zero.

15. Use as claimed in claim 13 wherein R is hydrogen.

16. Use as claimed in claim 14 wherein R is hydrogen.

17. Use as claimed in claim 13 wherein said compound is carbamic acid, [1- [[(1 -formyl-2-phenylethyl)amino]carbonyl]-2-methylpropyl]-, phenylmethyl ester.