WO1994018230A1

WO1994018230A1 - Inhibition of hiv replication by peptide-copper complexes

Info

Publication number: WO1994018230A1
Application number: PCT/US1994/000785
Authority: WO
Inventors: Andrew Branca
Original assignee: Procyte Corp
Current assignee: Procyte Corp
Priority date: 1993-02-02
Filing date: 1994-01-21
Publication date: 1994-08-18
Anticipated expiration: 1995-08-02
Also published as: ZA94707B

Abstract

There is disclosed methods for inhibiting HIV replication in an HIV-infected patient by administering a composition containing a peptide-copper complex in combination with a pharmaceutically acceptable carrier or diluent. The peptide-copper complexes of this invention are believed to inhibit HIV replication by inhibition or inactivation of HIV protease. In one embodiment, the peptide-copper complex has the structure [R1-R2-R3]:copper (II) wherein R1 is hydrogen, an amino acid or an amino acid derivative; R2 is histidine or a histidine derivative; and R3 is hydrogen or the remainder of the molecule. In a preferred embodiment, the peptide-copper complex is glycyl-histidyl-lysine:copper (II). Novel peptide-copper complexes are also disclosed.

Description

INHIBITION OF HIV REPLICATION BY PEPTIDE-COPPER COMPLEXES

Technical Field

The present invention relates generally to compositions and methods for inhibiting HIV replication utilizing peptide-copper complexes, and more specifically, to the administration of peptide-copper complexes to HIV-infected patients to inhibit HIV replication.

Background of the Invention

Human acquired immunodeficiency syndrome or "AIDS" is a fatal disease for which there is presently no cure. The disease is believed to be caused by a virus known, as human immunodeficiency virus, commonly referred to as "HIV." The virus is transmitted by HIV-infected individuals through the exchange of bodily fluids. HIV infection results most commonly from sexual contact with an infected partner and the sharing among intravenous drug users of hypodermic syringes previously used by an infected individual. A pregnant HIV-infected mother may infect her unborn child by trans-placental transmission, and HIV-contaminated blood is a possible source of infection for individuals subject to blood transfusion.

HIV infection causes a suppression of the immune system. The immune suppression renders the infected individual vulnerable to a variety of opportunistic infections and conditions that are otherwise kept in balance by a healthy immune system. Fatalities result from HIV infection due to the inability of AIDS patients to respond to treatment of the opportunistic infections and conditions as a consequence of their compromised immune systems. Because the virus may often remain dormant, the manifestation of AIDS from HIV infection may take as long as ten years.

One approach to the treatment of AIDS has targeted the opportunistic infections or conditions which result from HIV infection. The treatment of such infections or conditions, however, is ultimately ineffective and, while prolonging the life of the infected individual, does not treat the underlying HIV infection. A second approach to the treatment of AIDS targets the cause of the disease itself. Because AIDS results from viral infection, viral inactivation may ultimately provide a cure. Materials which are capable of viral inactivation or inhibition are referred to herein as "antiviral agents." To understand the mode of action of antiviral agents in the treatment of AIDS, an understanding of the process of HIV infection is necessary. HIV chronically infects To understand the mode of action of antiviral agents in the treatment of AIDS, an understanding of the process of HIV infection is necessary. HTV chronically infects specific immune cells known as T-helper cells, which are required for normal immune response. The HIV infected T-helper cells serve as hosts to the virus and facilitate the reproduction of the virus (the process of viral reproduction is commonly referred to as "replication"). After HIV infection, the infected host cell eventually dies, the replicated HIV virus is released, and the infection spreads to additional cells. This cycle continues unabated, depleting the population of T-helper cells and, in time, weakens the immune system to the onset of AIDS symptoms. Because T-helper cells are continuously produced by the body, the population of these cells may be reestablished in the absence of further HIV infection. Therefore, the progression of HIV infection (and the subsequent onset of AIDS) may be arrested by the prevention or inhibition of viral replication, and antiviral agents capable of inhibiting or preventing the replication of HIV should be effective in the treatment of AIDS. At the genetic level, HIV replication requires the insertion of viral deoxyribonucleic acid ("DNA") into the genome of the host cell. The genome of the host cell consists of the cell's own DNA, and is responsible for the synthesis of materials essential to the cell's own function and proliferation. Once the viral DNA is inserted into the host genome, the host facilitates replication of HIV. The inserted viral DNA is an enzymatic product derived from viral ribonucleic acid ("RNA") and the action of an enzyme known as HIV reverse transcriptase. Inhibition of HIV reverse transcriptase precludes the formation of viral DNA required for insertion into the genome of the host. Viral replication is prevented by the absence of viral DNA in the host cell genome. Antiviral agents which inhibit HIV reverse transcriptase are thus potential therapeutic drugs for treatment of AIDS.

Azidothymidine (AZT), dideoxyinosine (DDI), and dideoxycytosine (DDC) are examples of antiviral agents which inhibit HIV reverse transcriptase, and are currently used in the treatment of AIDS, and to prevent the subsequent onset of AIDS in an HIV- infected patient. While these therapeutic drugs have been successful in prolonging the lives of AIDS patients, their overall effectiveness has been limited by side-effects which arise from cross-reactivity which interferes with normal cell function.

Other antiviral agents which inhibit viral replication by targeting enzymes other than reverse transcriptase have also been proposed. For example, one of the earliest events in HIV replication is the formation of a single, large polypeptide chain known as a ftision protein. This fusion protein serves as the precursor to mature proteins required by the virus. In particular, the fusion protein consists of a series of connected individual proteins, and includes (among others) HIV reverse transcriptase. Cleavage of the fusion protein by a proteolytic enzyme (hereinafter referred to as "HIV protease") liberates the individual proteins which then mature and become fully functional. Therefore, inactivation or inhibition of HIV protease prevents fusion protein cleavage, thereby precluding the formation of HIV reverse transcriptase. Without HIV reverse transcriptase, viral DNA synthesis cannot occur, and replication of HIV is inhibited.

Accordingly, there is a need in the art for antiviral agents which inhibit HIV replication in HIV-infected patients, as well as a need for methods for inhibiting HIV replication by administration of an antiviral agent. Summary of the Invention

Briefly stated, the present invention discloses antiviral agents for inhibiting HIV replication, and methods relating to the administration thereof to an HIV-infected patient. The antiviral agents of this invention are peptide-copper complexes, and the methods include administration of a therapeutically effective amount of a composition which includes a peptide-copper complex in combination with a pharmaceutically acceptable carrier or diluent. Thus, compositions of this invention comprise a peptide- copper complex in a combination with a pharmaceutically acceptable carrier or diluent, and include medicaments for inhibiting HIV replication. The peptide-copper complexes of this invention are believed to enhance transport of copper into HIV infected cells which, in turn, inhibits or inactivates HIV protease and thus inhibits the replication of HIV. As used herein, the term "copper" is used to designate copper(II) (i.e., Cu⁺2), and the term "HIV" includes the various strains of the virus (such as HIV-1 and HIV-2).

Administration of the compositions of the present invention may be accomplished in any manner which will result in a systemic dose of a therapeutically effective amount of the peptide-copper complex to an HIV-infected animal or patient (including human patients). For example, such administration may be by injection (intramuscular, intravenous, subcutaneous or intradermal), oral, nasal, or suppository applications. Typically, compositions of the present invention include peptide-copper complexes in solution for various forms of injection, or in pharmaceutical preparations which are formulated for the sustained release of the peptide-copper complexes for oral, nasal, or suppository dosage application, and generally include one or more inert, physiological acceptable carriers. As used herein, the term "effective amount" means an amount of the peptide-copper complex which .inhibits HIV replication in the patient.

As used herein, the term "peptide-copper complex" means a peptide having least two amino acids (or amino acid derivatives), one of which is histidine (or a histidine derivative) or arginine, and which is capable of delivering copper by systemic appli^cation. Such peptide-copper complexes preferably have the following general structure. A:

A: [Rι-R2-R3]:copper(II) wherein:

R\ is hydrogen, an amino acid or an N-monoalkyl amino acid; R2 is histidine, a histidine derivative or arginine; and

R3 is hydrogen or remainder of the molecule, and wherein R\ and R2 are not both hydrogen.

As used in structure A above, the terms "amino acid", "N-monoalkyl amino acid", "histidine derivative" and "remainder of the molecule" are defined as follows. "Amino acid" means any carboxylic acid having an amino moiety, including, but not limited to, the naturally occuring α-amino acids: alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine. Further examples of amino acids include, but are not limited to, α-aminocaprolactam, - NHCH((CH2)_nNH3⁺)CO- where n = 1-20, and (X3)_n-L-tryptophan where X₃ is a - CH₂- or a -CH(OH)- moiety and n = 2-20. The term "N-monoalkyl amino acid" means any carboxylic acid having an alkyl- substituted amino moiety, wherein the alkyl moiety is a subsituted or unsubstituted, branched or unbranched, saturated or unsaturated carbon chain having from 1-50 carbon atoms, and preferably from 1-20 carbon atoms.

The term "histidine derivative" includes, but is not limited to, L-(3-W)-histidyl and L-(5-W)-histidyl, where W is an alkyl moiety containing from 1-12 carbon atoms or an aryl moiety containing from 6-12 carbon atoms. Preferred histidine derivatives include L-(3-methyl)-histidine and L-(5-methyl)-histidine.

The term "remainder of the molecule" means any chemical moiety linked to the R2 moiety including, but not limited to, one or more amino acids, one or mere basic amino moieties, one or more organic moieties, and any combination thereof. In this context, an amino acid is as defined above, and one or more amino acids includes all _ combinations of amino acids, including, but not limited to, L-valyl-L-phenylalanyl-L- valyl, L-phenylalanyl-L-phenylalanyl, (glycyl)y-L-tryptophan where y = 1-4, L-prolyl- X]-L-phenylalanyl-X2 or X]-L-phenylalanyl-X2 where X\ and X2 are selected from the group consisting of L-valine. L-alanine and glycine. As used herein, the term "basic amino moieties" include, but are not limited to, cadaverine, spermine and spermidine. Lastly, the term "organic moieties" means any chemical structure not derived from an amino acid, including, but not limited to, -NH2, an alkyl moiety containing from 1-18 carbon atoms, an aryl moiety containing from 6-18 carbon atoms, an alkoxy moiety containing from 1-18 carbon atoms, an aryloxy moiety containing from 6-18 carbon atoms, and an aminoalkyl moiety containing from 1 to 18 carbon atoms; as well as esters and amides, including, but not limited to, esters with sterol (such as cholesterol and estradiol), amides of aliphatic amines, and amides with τσ-amino fatty acids (i.e., H₂N(CH₂)_nCO2H where n = 1-19).

In a further preferred embodiment, the peptide-copper comlexes of the present invention are peptide-copper complexes having the following structures designated B through D:

B: [Rι-L-histidyl-R2]:copper(II) wherein: Rj is an amino acid; and

R2 is hydrogen or remainder of the molecule;

C: [L-histidyl-R₁-R2]:copper(II) wherein: R] is an amino acid; and

R2 is hydrogen or remainder of the molecule; and

D: [Ri-L-arginyl-R2J:copper(II) wherein: Rj is an amino acid;

R2 is hydrogen or remainder of the molecule.

As used in structures B through D above, the term "amino acid" is as defined above with regard to structure A, and includes, but is not limited to, alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine. The term "remainder of the molecule" means any_. chemical moiety, including, but not limited to, one or more amino acids, one or more basic amino moieties, one or more organic moieties, and any combination thereof. In this context, an amino acid is as defined above, and one or more amino acids includes all combinations of amino acids, including, but not limited to. L-valyl-L-phenylalanyl - L-valyl. L-phenylalanyl-L-phenylalanyl, (glycyl)y-L-tryptophan where y = 1-4, L-prolyl- Xl-L-phenylalanyl-X2 or Xι-L-phenylalanyl-X2_; where X\ and X2 are selected from the group consisting of L-valine, L-alrj ine and glycine. As used herein, the term "basic amino moieties" include, but are not limited to, cadaverine, spermine and spermidine. Lastly, the term "organic moieties" means any chemical structure not derived from an amino acid, including, but not limited to -NH2, an alkyl moiety containing from 1-18 carbon atoms, an aryl moiety containing from 6-18 carbon atoms, an alkoxy moiety containing from 1-18 carbon atoms, an aryloxy moiety containing from 6-18 carbon atoms, and an aminoalkyl moiety containing from 1-18 carbon atoms; as well as esters and amides, including, but not limited to, esters with sterol (such as cholesterol and estradiol), amides of aliphatic amines, and amides with τπ-amino fatty acids (i.e., H₂N(CH2)nCO₂H where n = 1-19).

In yet a further prefered embodiment, the peptide-copper complexes of the present invention are peptide-copper complexes having the following structures > designated E through G:

E: [Rι-L-histidyl-R2]:copper(II) wherein:

R is an amino acid; and R2 is hydrogen or at least one amino acid;

F: [L-histidyl-Rι-R2]:copper(II) wherein:

Rl is an amino acid; and R2 is hydrogen or at least one amino acid; and

G : [R 1 -L-arginyl-R2] : copper(II) wherein:

R\ is an amino acid; R2 is hydrogen or at least one amino acid. As used in structures E through G above, the term "amino acid" is as defined above with regard to structure A, and includes, but is not limited to, alanine, arginine, _. asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine. The term "at least one amino acid" means one or more amino acids, including, but not limited to, L-valyl-L-phenylalanyl-L-valyl, L- phenylalanyl-L-phenylalanyl, (glycyl)y-L-tryptophan where y = 1-4, L-prolyl-X_j-L- phenylalanyl-X2 or X]-L-phenylalanyl-X2 where X\ and X2 are selected from the group consisting of L-valine, L-alanine and glycine.

In atill a further embodiment of the present invention, an additional chelating agent may be added to the peptide-copper complexes disclosed above to form a ternary peptide-copper-chelating agent complex.

Other aspects of the present invention will become evident upon reference to the following detailed description.

Detailed Description of the Invention

The present invention discloses compositions and methods for inhibiting HIV replication utilizing a peptide-copper complex. The peptide-copper complexes of the present invention may be administered to an HIV-infected patient as a composition which contains the peptide-copper complex in combination with an pharmaceutically acceptable carrier or diluent. The peptide-copper complexes of this invention are.. believed to aid or enhance the transport of copper into HIV infected cells and, once in the cell, the copper is believed to inhibit or inactivate HIV protease, thus inhibiting the replication of HIV. The peptide-copper complexes of this invention preferably exhibit no adverse cross-reactivity with otherwise healthy cells.

As indicated above, peptide-copper complexes of the present invention have at least two amino acids (or amino acid derivatives), one of which is histidine (or a histidine derivative) or arginine. In one embodiment, the peptide-copper complexes of this invention have the following general structure:

[R₁-R₂-R₃]:copper(II) where R\ is hydrogen, an amino acid or an N-monoalkyl amino acid; R2 is histidine, a histidine derivative or arginine; R3 is hydrogen or remainder of the molecule, and wherein R\ and R2 are not both hydrogen. The terms "amino acid", "N-monoalkyl amino acid", "histidine derivative" and "remainder of the molecule" are as defined above. For example, when Rj is an amino acid, R2 is L-histidyl and R3 is hydrogen, the peptide-copper complex has the structure:

[(amino acid)-L-histidyl]:copper(II); when R\ is hydrogen, R2 is L-histidyl and R3 is an amino acid, the peptide-copper complex has the structure:

[L-histidyl-(amino acid)] xopper(II); and when Rj and R3 are amino acids and R2 is L-histidyl, the peptide copper complex has the structure: [(amino acid)-L-histidyl-(amino acid)]:copper(II).

More specifically, when R] is glycyl, R2 is L-histidyl and K.3 is L-lysyl, the peptide-copper complex ("GHK-Cu") has the structure:

[glycyl-L-histidyl-L-lysine]:copper(II); when R\ is glycyl, R2 is L-histidyl and R3 is L-lysyl -L-valyl-L-phenylalanyl-L-valyl, the peptide-copper complex has the structure:

[glycyl-L-histidyl-L-lysyl-L-valyl-L-phenylalanyl-L-valine]:copper(II); when R] is hydrogen, R₂ is L-histidyl and R3 is L-phenylalanyl-L-lysyl, the peptide- copper complex has the structure: [L-histidyl-L-phenylalanyl-L-lysine]:copper(II); and when R\ is glycyl, R2 is L-arginyl and R3 is L-lysyl, the peptide-copper complex has the structure:

[glycyl-L-arginyl-L-lysine]:copper(II).

In further preferred embodiments, the peptide-copper complexes of the present invention have the general formulas designated B through G above.

Within the present invention, the molar ratio of peptide to copper is greater than zero. The ratio will depend, in part, on the number of copper coordination sites that are occupied by the peptide. In the practice of the present invention, it is preferred that the molar ratio of peptide to copper be within the range of 1:1 to 3:1, and more preferably from 1 :1 to 2:1. For example, in the case of GHK-Cu, the preferred ratio of peptide to copper ranges from l .J to 2:1. with the GHK tripeptide occupying three coordination sites of the copper.

In another embodiment of the present invention, a chelating agent may be added to the peptide-copper complex to form a ternary peptide-copper-chelating agent complex. Suitable chelating agents include imidazole or imidazole-containing compounds, such as histidine, and sulfur containing amino acids, such as cysteine or methionine. Thus, if the peptide-copper complex is glycyl-L-histidyl-L-lysine:copper, histidine may be added to yield the ternary complex glycyl-L-histidyl-L- lysine:copper:histidine. However, to form such a ternary complex, the molar ratio of copper to peptide to chelating agent must be considered. For example, if the ratio of peptide to copper is 2:1, the addition of a chelating agent to the peptide-copper complex, although possible, is difficult due to site occupancy by the peptide. However, by maintaining the ratio of peptide to copper near 1 : 1 , a chelating group may readily be added to form the ternary complex. Preferably, the peptide to copper to chelating agent ratio is 1 :1 :1.

Furthermore, while the chiral arnino acids of the present invention have been designated as the L form, one skilled in the art would appreciate that the D forms of the amino acids (or amino acid derivatives) may be utilized in place of the L forms.

The peptides of the present invention may be synthesized by either solution or solid phase techniques. The general procedure involves the stepwise addition of protected amino acids to build up the desired peptide sequence. Such methodology is well known to those skilled in the art. Illustrative syntheses of complexes of the present invention are presented in the examples herein below.

The peptide-copper complexes of this invention may be screened for their ability to inhibit HIV replication using known techniques. For Example, HIV virus replication may be monitored using the Cytopathic Effect (CPE) assay disclosed by Bergeron et al. >

(J. Virol. 6^:5777-5787. 1992), and incorporated herein by reference in its entirety. In this assay, the degree of infection is monitored by the appearance of fused cellular membranes ("syncitium"). Alternatively, assays directed to activity of HIV protease may be employed. For example, the assays and techniques disclosed in the following references may be employed: Ashorn et al., Proc. Natl. Acad. Sci. U.S.A. 87:7472- 7476, 1990; Schramm et al., Biochem. Biophvs. Res. Commun. 179:847-851, 1991; Sham et al., Biochem. Biophvs. Res. Commun. 175:914-919, 1991; and Roberts et al., Science 248:358-361. 1990 (all of which is incorporated herein by reference in their entirety).

Administration of compositions of the present invention may be accomplished in any manner which will result in a systemic dose of the peptide-copper complex to the animal. For example, such administration may be by injection (intramuscular, intravenous, subcutaneous or intradermal), oral, nasal, or suppository applications. Typically, compositions of the present invention include peptide-copper complexes in solution for various forms for injection, or in pharmaceutical preparations which are formulated for the sustained release of the peptide-copper complexes for oral, nasal, or suppository dosage application. The balance of the composition or pharmaceutical preparation comprises an inert, physiological acceptable carrier. .

Methods for encapsulating compositions (such as in a coating of hard gelatin) for oral or suppository administration are well known in the art (Baker et al., Controlled Release of Biological Active Agents, John Wiley and Sons. 1986, incorporated herein by reference). Suitable pharmaceutically acceptable carriers for parenteral application, such as intravenous, subcutaneous or intramuscular injection, include sterile water. physiological saline, bacteriostatic saline (saline containing 0.9 mg/ml benzyl alcohol) and phosphate-buffered saline.

An effective dosage of the peptide-copper complexes of the present invention is an amount sufficient to inhibit HIV replication in an HIV-infected patient. Suitable dosages may range from approximately 0.01 to 20 mg of peptide-copper complex per kg body weight. The required dosage will vary according to the condition of the HIV- infected patient, as well as the severity of the AIDS or AIDS related conditions.

The following examples are offered by way of illustration, and not by way of limitation.

EXAMPLES The following examples present the preparation and characteristics of certain exemplary embodiments of the peptide-copper complexes of the present invention. To > summarize the examples that follow, Example 1 illustrates the preparation of peptide- copper complexes of the present invention by chelating the peptide to copper. Examples 2-19 illustrate representative peptides of this invention, and which may be chelated to copper to form the corresponding peptide-copper complex. Examples 20-23 illustrate the ability of a representative peptide-copper complex of the present invention to be transported to a cell, interact with HIV protease, and inhibit HIV replication. Specifically, Example 1 illustrates the preparation of peptide-copper complexes.

Example 2 illustrates the synthesis of glycyl-L-histidyl-L-lysine: copper (GHK-Cu). Example 3 illustrates the synthesis of glycyl-L-histidyl-L-lysine n-octyl amide. Example 4 illustrates the synthesis of glycyl-L-histidyl-L-lysyl-L-valyl-L-phenylalanyl- L-valine. Example 5 illustrates the synthesis of L-alanyl-L-histidyl-L-lysine. Example 6 illustrates the synthesis of L-lysyl-L-histidyl-glycine. Example 7 illustrates the synthesis of L-lysyl-L-histidyl-glycyl-L-valyl-L-phenylalanyl-L-valineL-lysyl-L- histidyl-glycyl-L-valyl-L-phenylalanyl-L-Valine. Example 8 illustrates the synthesis of glycyl-L-histidyl-L-caprolactam. Example 9 illustrates the synthesis of L-histidyl- glycyl-L-lysine. Example 10 illustrates the synthesis of L-alanyl-L-histidyl-L- phenylalanine. Example 11 illustrates the synthesis of glycyl -L-(3-methyl)histidyl-L- lysyl-L-tryptophan. Example 12 illustrates the synthesis of glycyl-L-histidyl-L-glutamic acid. Example 13 illustrates the synthesis of glycyl-L-histidyl-L-phenylalanine. Example 14 illustrates the synthesis of glycyl-L-histidyl-L-lysyl-L-phenylalanine. Example 15 illustrates the synthesis of glycyl -L-histidyl-L-lysyl-phenylalanyl-L- phenylalanine. Example 16 illustrates the synthesis of glycyl-L-arginyl-L-lysine. Example 17 illustrates the synthesis of L-histidyl-L-phenylalanyl-L-lysine. Example 18 illustrates the synthesis of L-histidyl-glycyl-L-lysyl-L-phenylalanine. Example 19 illustrates the synthesis of L-histidyl-glycyl-L-lysyl-tryptophan.

Example 20 illustrates transport of copper to human fibroblasts by GHK-Cu and copper-chloride. Example 21 illustrates binding of GHK-Cu to HIV protease. Example 22 illustrates inhibition of HIV replication by GHK-Cu.

Source of Chemicals

Chemicals and peptide intermediates utilized in the following examples may be purchased from a number of suppliers, including: Sigma Chemical So., St. Louis, Missouri; Peninsula Laboratories, San Carlos, California; Aldrich Chemical Company, Milwaukee, Wisconsin; Vega Biochemicals, Tucson, Arizona; Pierce Chemical Co., Rockford, Illinois; Research Biochemicals, Cleveland, Ohio; Van Waters and Rogers, South San Francisco, California; and Bachem, Inc., Torrance, California.

Example 1

Preparation of Peptide-Copper Complex The peptide-copper complexes of the present invention may be synthesized by dissolving the peptide in distilled water, followed by the addition of purified copper- chloride and then adjusting the pH of the solution. For example, copper complexes of glycyl-L-histidyl-L-lysine (GHK) with a molar ratio of peptide to copper of l .J, 2:1, or greater (e.g., 3.J), may be prepared by dissolving a given weight of GHK in distilled water (e.g., 50 mg/ml), and adding the desired molar amount of purified copper- chloride. The pH of the resulting peptide solution is then adjusted to about 7.0 by the addition of a sodium hydroxide solution. Alternatively, copper salts other than the copper-chloride may be utilized, such as copper-acetate or copper-sulfate.

Example 2 Synthesis of Glycyl-L-Histidyl-L-Lysine:Copper(ID Glycyl-L-histidyl-L-lysine (GHK) is commercially available as an acetate salt from Bachem Bioscience Inc. (Philadephia, PA) and Sigma Chemical Company (St. Louis. MO).

To prepare the peptide-copper complex: [glycyl-L-histidyl-L-lysine] opper(II) at a 1 : 1 molar ratio of peptide to copper, a solution of glycyl-L-histidyl-L-lysine was prepared by dissolving 0.01 mole (i.e., 4.3147 g) of GHK acetate in approximately 50 mL of distilled water. Separately, a solution of copper-chloride was prepared by dissolving 0.01 mole (i.e., 1.3212 g) of anhydrous copper(II)-chloride in approximately 10 mL of distilled water. The initial pH of the GHK solution was determined to be 7J5. The copper-chloride solution was slowly added to the rapidly stirring GHK solution, and the pH was constantly monitored with a pH meter. After all the copper- chloride solution was added, the combined solution pH was determined to be 3.44. The pH was then adjusted tp 7.00 by slow addition of a solution of 0.5 M NaOH, and the final volume was adjusted to 100 mL.

Example 3 Synthesis of Glycyl-L-Histidyl-L-Lysine n-Octyl Amide A solution of N-α-t-butyloxycarbonyl-N-ε-benzyloxycarbonyl-L-lysine in tetrahydrofuran was treated with N-methyl-morpholine, isobutyl chloroformate, and octylamine at -15°C. The resulting fully protected octyl amide was then treated with

50% trifluoroacetic acid in dichloromethane at room temperature, neutralized with > saturated aqueous potassium bicarbonate solution, and extracted into ethyl acetate. Evaporation gave the deblocked lysinamide which was added to a solution prepared from N-α-t-butyloxycarbonyl-N-im-benzyloxycarbonyl-L-histidine, N- methylmorpholine, and isobutyl chloroformate in dry tetrahydrofuran at -15°C.

The fully protected dipeptide formed above was deblocked by treatment with 50% trifluoroacetic acid in dichloromethane at room temperature followed by neutralization with saturated aqueous potassium bicarbonate. Extraction into ethyl acetate and evaporation gave the partially deblocked dipeptide, which was added to a solution prepared from benzyloxycarbonyl glycine, N-methylmorpholine, and isobutyl chloroformate in dry tetrahydrofuran at -15°C. The resulting protected tripeptide was deblocked by treatment with hydrogen in the presence of 10% palladium on carbon in glacial acetic acid. Filtration and lyophilization gave glycyl-L-histidyl-L-lysine n-octyl amide as its triacetate salt.

Example 4

Synthesis of Glycyl-L-Histidyl-L-Lysyl-L-Valyl-L-Phenylalanyl-L-Valine Glycyl-L-histidyl-L-lysyl-L-valyl-L-phenylalanyl-L-valine was synthesized by standard solution phase method using t-butyloxycarbonyl protecting group for the alpha nitrogen, benzyloxycarbonyl group for side-chain protection and mixed anhydride method for coupling. Briefly stated, L-valine benzyl ester p-toluenesulfonate salt was coupled with t-butyloxycarbonyl-L-phenylalanine using isobutyl chloroformate and N- methylmorpholine as a coupling agent (2 hours at -20°C, then 1 hour at ambient temperature). The t-butyloxycarbonyl protecting group of the dipeptide was then removed by 30% trifluoroacetic acid in dichloromethane at room temperature for 30 minutes. B'ocked amino acids (t-butyloxycarbonyl-L-valine, N-α-t-butyloxycarbonyl- N-ε-benzylυxycarbonyl-L-lysine, N-α-t-butyloxycarbonyl-N-im-benzyloxycarbonyl-L- histidine, benzyloxy-carbonyl-glycine) were added in sequential order, and t- butyloxycarbonyl protecting groups were removed to obtain the desired peptide. The final peptide was completely deprotected using hydrogen gas in acetic acid for 5 days in the presence of 10% Pd-C catalyst. The final peptide was lyophilized from water to obtain the tri-acetate salt.

Example 5

Synthesis of L-Alanyl-L-Histidyl-L-Lysine To a stirred solution of N-α-BOC-N-im-CBZ-L-histidine (9.74g, 25.0mmol) and N-methylmorpholine (5.8mL, 5.3g, 52.5mmol) in tetrahydrofuran (50mL) at -15°C was added isobutyl chloroformate (3.4mL, 3.6g, 26.3mmol). After 2min. (N-ε-CBZ)-L- lysine benzyl ester hydrochloride (10.2g, 25.0mmol) was added. The reaction mixture was stirred at -15°C for 1.5h and then allowed to warm to 0°C. At this time the reaction was quenched by the addition of 2M aqueous potassium bicarbonate. The products were extracted with ethyl acetate (3 x 150mL). The combined extracts were washed with 1M citric acid (3 x lOOmL), water, 2M KHCO3 (3 x lOOmL), water, and brine. The resulting solution was dried over sodium sulfate, filtered, and evaporated to give 17.2g (93%>) of the blocked dipeptide as a white semi-solid (Rf = 0.61, 10% methanol/dichloromethane), which was used in the following transformation without further purification.

A solution of the t-butyloxycarbonyl protected dipeptide (17.2g, 23.2mmol) in 35%) trifluoroacetic acid/dichloromethane (150mL) was stirred 1/2 h at room temperature. The resulting solution was concentrated in vacuo and neutralized with 2M aqueous potassium bicarbonate. The product was extracted into ethyl acetate (3 x 150mL). The combined extracts were dried over sodium sulfate, filtered, and evaporated to give 16.8g (ca. 100% + entrained solvent) of the free-amino compound as a white solid: Rf= 0.26 (10% methanol/dichloromethane).

To a stirred solution of N-CBZ-L-alanine (6.28g, 25.0mmol) and N- methylmorpholine (3.0mL, 2.8g, 27.5mmol) in tetrahydrofuran (50mL) at -15°C was added isobutyl chloroformate (3.4mL, 3.6g, 26.3mmol). After 2min. a solution of the above protected dipeptide (14.9g, 23.2mmol) in tetrahydrofuran (50mL) was added. The reaction mixture was stirred at -15°C for 1.5h and then allowed to warm to 0°C. At this time the reaction was quenched by the addition of 2M aqueous potassium bicarbonate. The products were extracted with ethyl acetate (3 x 150mL). The combined extracts were washed with 1M citric acid (3 x lOOmL), water, 2M KHCO3(3 x ICOmL), water, and brine. The resulting solution was dried over sodium sulfate, filtered, and evaporated to a syrup, from which the blocked tripeptide was precipitated by dilution with 95%) ethanol (300mL). The resulting material was collected on a filter, washed with 95% ethanol and dried to give a white solid: (R/= 0.49, 10% methanol/dichloromethane); mp l51-153°C.

To a suspension of the blocked tripeptide (21.5g, 21.9mmol) in ethanol (200mL) was added water (about 200mL). The resulting mixture was shaken with palladium chloride (4.25g, 24.0mmol) under an atmosphere of hydrogen (5 atm) for lh. The resulting mixture, in which the bulk of the material (other than the catalyst) became dissolved, was filtered and the filtrate was concentrated in vacuo to remove volatile organics. The remaining aqueous solution was lyophilized to give 10.88g of a white > solid. This material was dissolved in water, filtered through a 0.2m nylon membrane, and, again, lyophilized to give 10.50g (99%) of the desired tripeptide dihydrochloride as a white powder: [a]D -4.43°(c 3, H2O); ^]H NMR (500MHz, DMSO-dό) d 8.73 (IH, d, J = 7.8), 8.45 (IH, d, J = 7.5), 8.09 (IH, s), 7.08 (IH, s), 4.59 (IH, dd, J = 5.4, 7.5), 4.12 (IH, m), 3.88 (IH, q, J = 6.9), 3.03 (IH, dd, J = 15.0, 4.8), 2.96 (IH, dd, J = 15.0, 7.7), 2.74 (2H, t, J = 7.5), 1.76-1.68 (IH, m), 1.66-1.51 (3H, m), 1.41-1.21 (2H, m), 1.32 (3H, d, J = 7.0); ¹³C NMR (125MHz, DMSO-dό) d 174.0, 169.9, 169.5, 134.2, 130.5, 117.8, 52.6, 52.5, 48.0, 38.4, 30.3, 28.2, 26.5, 22.4, 17.2.

Example 6 Synthesis of L-Lysyl-L-Histidyl-Glycine N-α-t-butyloxycarbonyl-N-im-benzyloxycarbonyl-L-histidine was dissolved in tetrahydrofuran (THF) and neutralized with one equivalent of N-methylmorpholine. It was then coupled with benzyl glycinate p-toluenesulfonate salt using isobutyl chloroformate and N-methylmorpholine. After two hours at -20°C and an additional hour at ambient temperature, the reaction was quenched with 2 N aqueous potassium bicarbonate. The product was extracted into ethyl acetate, washed with 1 M aqueous citric acid, and saturated sodium bicarbonate. The organic phase was dried over anhydrous sodium sulfate. Filtration and evaporation gave benzyl N-oc-t- butyloxycarbonyl-N-im-benzyloxycarbonyl-L-histidyl-glycinate.

This product was dissolved in anhydrous methanolic hydrogen chloride (saturated at 0°C) for 5 minutes, followed by removal of solvent under reduced pressure, forming benzyl N-im-benzyloxycarbonyl-L-histidyl-glycinate. This was dissolved in tetrahydrofuran, and isobutyl chloroformate, N-methylmorpholine and N-α .N-ε-dibenzyloxycarbonyl-L-lysine were added to form benzyl N-α,N-ε- dibenzyloxycarbonyl-L-lysyl-N-im-benzyloxycai bonyl-L-histidyl-glycinate (3 hours at - 20°C, the 1 hour at ambient temperature). This product was then dissolved in methanol/acetic acid, 1 : 1 (v/v), and hydrogenated overnight in the presence of 10% Pd- C catalyst. The resultant L-lysyl-L-histidyl-glycine was lyophilized from water several times, then purified by liquid chromatography on a C- 18 reverse-phase column to yield the desired tripeptide triacetate salt as a foamy white solid.

Example 7

Synthesis of L-Lysyl-L-Histidyl-Glycyl-L-Valyl-L-Phenylalanyl-L-Valine L-lysyl-L-histidyl-glycyl-L-valyl-L-phenylalanyl-L-valine was synthesized by standard solution phase method using t-butyloxycarbonyl protecting group for the alpha > nitrogen, benzyloxycarbonyl group for side-chain protection and mixed anhydride method for coupling. Briefly stated, L-valine benzyl ester p-toluenesulfonate salt was coupled with t-butyloxycarbonyl-L-phenylalanine using isobutyl chloroformate and N- methylomorpholine as coupling agent (2 hours at -20°C, then 1 hour at ambient temperature). The t-butyloxycarbonyl protecting group of the dipeptide was then removed by 30%> trifluoroacetic acid in dichloromethane at room temperature for 30 minutes. Blocked amino acids (t-butyloxycarbonyl-L-valine, t- butyloxycarbonylglycine, N-α-t-butyloxycarbonyl-N-im-benzyloxycarbonyl-L-histidine,

N-α,N-ε-dibenzyloxycarbonyl-L-lysine) were added in sequential order and t- butyloxycarbonyl protecting groups were removed to obtain the desired peptide. The final peptide was completely deprotected using hydrogen gas in glacial acetic acid for five days in the presence of 10%) Pd-C catalyst. The final peptide was lyophilized from water and purified by liquid chromatography on a C-18 reverse phase column to produce the desired hexapeptide in multi-gram quantity.

Example 8 Synthesis of Glycyl-L-Histi.dyl-L-Caprolactam

L(-)-3-amino-ε-caprolactam was dissolved in tetrahydrofuran (THF) then coupled with Na-t-butyloxycarbonyl-Nim-benzyloxycarbonyl-L-histidine using isobutyl chloroformate and N-methylmorpholine in THF. After two hours at -20°C and an additional hour at ambient temperature, the reaction was quenched with 2 N aqueous potassium bicarbonate. This produce was extracted into ethyl acetate, washed with 1 M aqueous citric acid, and saturated sodium bicarbonate. The organic phase was dried over anhydrous sodium sulfate. Filtration and evaporation gave N-α-t- butyloxycarbonyl-N-im-benxyloxycarbonyl-L-histidyl-L-caprolactam.

The above protected dipeptide was dissolved in 30%» trifluoroacetic acid in dichloromethane for 30 minutes, then evaporated, forming N-im-benzyloxycarbonyl-L- histidyl-L-caprolactam. This was then dissolved in tetrahydrofuran, and isobutyl chloroformate, N-methylmorpholine and benzyloxycarbonylglycine were added to form benzyloxycarbonylglycyl-N-im-benzyloxycarbonyl-L-histidyl-L-caprolactam. This product was recrystallized once from ethyl acetate then dissolved in acetic acid and hydrogenated overnight in the presence of 10% Pd-C catalyst. The resultant glycyl-L- histidyl-L-caprolactam was lyophilized from water several times, then purified by liquid chromatography on a C-18 reverse-phase column to yield the desired tripeptide as a diacetate salt.

Example 9 Synthesis of L-Histidyl-Glycyl-L-Lysine N-ε-benxyloxycarbonyl-L-lysine benzyl ester hydrochloride salt was suspended in tetrahydrofuran (THF) and coupled with N-α-t-butyloxycarbonyl-glycine using isobutyl chloroformate and N-mefhylmorpholine in THF. After two hours at -20°C and an additional hour at ambient temperature, the reaction was quenched with 2 N aqueous potassium bicarbonate. The produce was extracted into ethyl acetate, washed with 1 M aqueous citric acid, and saturated sodium bicarbonate. The organic phase was dried over anhydrous sodium sulfate. Filtration and evaporation gave benzyl N-α-t- butyloxycarbonyl-glycyl-N-ε-benzyloxycarbonyl-L-lysinate.

The product was dissolved in 30% trifluoroacetic acid in dichloromethane for 30 minutes, then evaporated, forming benzyl glycyl-N-ε-benzyloxycarbonyl-L-lysinate.

This was dissolved in tetrahydrofuran, and isobutyl chloroformate, N-methylmorpholine and N- -benzyloxycarbonyl-N-im-benzyloxycarbonyl-L-histidine were added to form benzyl N-α-benzyloxycarbonyl-N-im-benzyloxycarbonyl-L-histidyl-glycyl-N-ε- benzyloxycarbonyl-L-lysinate. This product was then dissolved in acetic acid and hydrogenated overnight in the presence of 10%> Pd-C catalyst. The resultant L-histidyl- glycyl-L-lysine was lyophilized from water several times to yield the desired tripeptide as a diacetate salt: [a]rj-1.2° (c 2.0, water); - NMR (500MHz, D O) d 8.29 (IH, s), 7.30 (IH, s), 8.06 (IH, s), 4.29 (IH, t, J = 6.5), 4.20 (IH, dd, J = 5.3, 7.8), 4.04 (2H AB, J=17.0), 3.23 (2H, br t, J = 6), 3.01 (2H, t, J = 7.6), 1.99 (ca. 6H, s), 1.91-1.80 (IH, m), 1.70-1.67 (3H, m), 1.49-1.38 (2H, m); ¹³C NMR (125MHz, D2O) d 180.1, 178.4, 170.0, 134.9, 127.2, 118.0, 55.0, 52.3, 42.1, 39.0, 30.8, 26.9, 26.2, 22.7.

Example 10

Synthesis of L-Alanyl-L-Histidyl-L-Phenylalanine To a stirred solution of N-α-BOC-N-im-CBZ-histidine (9.74g, 25.0mmol) and N-methylmorpholine (5.8mL, 5.3g, 52.5mmol) in tetrahydrofuran (50mL) at -15°C was added isobutyl chloroformate (3.4mL, 3.6g, 26.3mmol). After 2 min. phenylalanine benzyl ester tosylate (10.7g, 25.0mmol) was added. The reaction mixture was stirred at -15°C for 1.5h and then allowed to warm to 0°C. At this time the reaction was quenched by the addition of 2M aqueous potassium bicarbonate. The products were extracted with ethyl acetate (3 x 150mL). The combined extracts were washed with 1M citric acid (3 x lOOmL), water, 2M KHCO3 (3 x lOOmL), water, and brine. The resulting solution was dried over sodium sulfate, filtered, and evaporated to give 13.7g (87%_>) of the blocked dipeptide as a white semi-solid (Ry = 0.75, 10% methanol/dichloromethane), which was used in the following transformation without further purification.

A solution of the t-butyloxycarbonyl protected dipeptide (12.9g, 20.6mmol) in 35%) trifluoroacetic acid/dichloromethane (150mL) was stirred l/2h at room temperature. The resulting solution was concentrated in vacuo and neutralized with 2M aqueous potassium bicarbonate. The product was extracted into ethyl acetate (3 x 150mL). The combined extracts were dried over sodium sulfate, filtered, and evaporated to give 13.3g (ca. 100% + entrained solvent) of the free-amino compound as a white solid: Rf= 0.49 (10% methanol/dichloromethane). To a stirred solution of N-CBZ-alanine (6.03g, 27.0mmol) and N- methylmorpholine (3.3mL, 3.0g, 29.7mmol) in tetrahydrofuran (50mL) at -15°C was added isobutyl chloroformate (3.7mL, 3.9g, 28.4mmol). After 2min. a solution of the suitably protected dipeptide (11.4g, 21.8mmol) in tetrahydrofuran (50mL) was added. The reaction mixture was stirred at -15°C for 1.5h and then allowed to warm to 0°C. At this time the reaction was quenched by the addition of 2M aqueous potassium bicarbonate. The products were extracted with ethyl acetate (3 x lOOmL). The combined extracts were washed with 1M citric acid (3 x lOOmL), water, 2M KHCO3(3 x 1 OOmL), water, and brine. The resulting solution was dried over sodium sulfate, filtered, and evaporated to give the blocked tripeptide as a white solid (Ry = 0.55, 10% methanol/dichloromethane), which was recrystallized from 95% ethanol to give 12.6g (79%>) of a free-flowing white powder: mp 147-147.5°C; Anal. Calcd. for C41H40N5O8: C, 67.39; H, 5.52; N, 9.58. Found: C, 66.78; H, 5.64; N, 9.24.

To a suspension of the blocked tripeptide (12.6g, 17.6mmol) in ethanol (150mL) was added water, until the mixture became very turbid (about 150mL). The resulting mixture was shaken with palladium chloride (1.56g, 8.8mmol) under an atmosphere of hydrogen (5 atm) for 16h. The catalyst was removed by filtration through a plug of Celite® and the filtrate was concentrated to remove volatile organic materials. The remainder was lyophilized to give 8.30g of white powder. This material was dissolved in water, filtered through a 0.2m nylon membrane and lyophilized to give 6.27g (87%>) of the desired tripeptide dihydrochloride as a free-flowing white powder: [a]D 5.1° (c 2.0, water); -Η NMR (500MHz, DMSO-d6) d 8.71 (IH, d, J = 7.9), 8.49 (IH, d, J = 7.8), 8.21 (IH, s), 7.30-7.22 (4H, m), 7.20-7.15 (IH, m), 7.12 (IH, s), 4.54 (IH, br q, J =^■= 7.1), 4.37 (IH, m), 3.86 (IH, q, J = 6.8), 3.12 (IH, dd, J=4.3, 13.8), 3.05-2.90 (2H, m), 2.88 (IH, dd, J=9.5, 13.8), 1.27 (3H, d, J = 6.8); ¹³C NMR (125MHz, DMSO-d6) d 173.5, 169.9, 169.5, 138.1, 134.2, 130.5, 129.2, 128.2. 126.4, 1 17.8. 54.4, 52.5, 48.0, 36.8, 28.5, 17.2. Example 11

Synthesis of Glycyl-L-G-methyDHistidyl-L-Lysyl-L-Tryptophan

To a stirred solution of N-α-BOC-N-ε-CBZ-lysine (7.0g, 18.5mmol) and N- methylmorpholine (4.3mL, 3.9g, 38.9mmol) in tetrahydrofuran (50mL) at -15°C was added isobutyl chloroformate (2.5mL, 2.7g, 19.4mmol). After 2min. a solution of tryptophan benzyl ester hydrochloride (6Jg, 18.5mmol) in dimethylformamide was added. The reaction mixture was stirred at -15°C for 1.5h and then allowed to warm to

0°C. At this time the reaction was quenched by the addition of 2M aqueous potassium bicarbonate. The products were extracted with ethyl acetate (3 x 150mL). The combined extracts were washed with 1M citric acid (3 x lOOmL), water, 2M KHCO3 (3 x lOOmL), water, and brine. The resulting solution was dried over sodium sulfate, filtered, and evaporated to give 12Jg (100% ) of the blocked dipeptide as a white solid (R/- 0.69, 10% methanol/dichloromethane). A solution of the t-butyloxycarbonyl protected dipeptide (12Jg, 18.5mmol) in

35% trifluoroacetic acid/dichloromethane (150mL) was stirred l/2h at room temperature. The resulting solution was concentrated in vacuo and neutralized with 2M aqueous potassium bicarbonate. The product was extracted into ethyl acetate (3 x lOOmL). The combined extracts were dried over sodium sulfate, filtered, and evaporated to give 13.3g (ca. 100% + entrained solvent) of the free-amino compound as a foaming white semi-solid: Ry= 0.43 (10% methanol/dichloromethane).

To a stirred solution of N-α-BOC-3 -methyl -L-histidine (4.98g, 18.5mmol) and N-methylmorpholine (2.2mL, 2Jg, 20.4mmol) in tetrahydrofuran (50mL) at -15°C was added isobutyl chloroformate (2.5mL, 2.7g, 19.4mmol). After 2min. a solution of the suitably protected dipeptide (10.3g, 18.5mmol) in dimethylformamide (40mL) was added. The reaction mixture was stirred at -15°C for 1.5h and then allowed to warm to 0°C. At this time the reaction was quenched by the addition of 2M aqueous potassium bicarbonate. The products were extracted with ethyl acetate (3 x 150mL). The combined extracts were washed with 1M citric acid (3 x lOOmL), water, 2M KHCO3 (3 x lOOmL), water, and brine. The resulting solution was dried over sodium sulfate, filtered, and evaporated to give 11.5g (77%) of the blocked tripeptide as a white semi-solid (Rf = 0.57, 10% methanol/dichloromethane).

A solution of the t-butyloxycarbonyl protected tripeptide (11.5g, 14.3mmol) in 35%o trifluoroacetic acid/dichloromethane (150mL) was stirred l/2h at room temperature. The resulting solution was concentrated in vacuo and neutralized with 2M aqueous potassium bicarbonate. The product was extracted into ethyl acetate (3 x 150mL). The combined extracts were dried over sodium sulfate, filtered, and evaporated to give 11.6g (ca. 100% + entrained solvent) of the free-amino compound as a white solid: Ry= 0.22 (10% methanol/dichloromethane).

To a stirred solution of N-CBZ-glycine (3J4g, 15.0mmol) and N- methylmorpholine (1.8mL, 1.7g, 16.5mmol) in tetrahydrofuran (50mL) at -15°C was added isobutyl chloroformate (2JmL, 2.2g, 15.8mmol). After 2min. a solution of the suitably protected tripeptide (9.0g, 12.8mmol) in tetrahydrofuran (50mL) was added. The reaction mixture was stirred at -15°C for 1.5h and then allowed to warm to 0°C. At this time the reaction was quenched by the addition of 2M aqueous potassium bicarbonate. The products were extracted with ethyl acetate (3 x 150mL). The combined extracts were washed with 1M citric acid (3 x lOOmL), water, 2M KHCO3(3 x lOOmL), water, and brine. The resulting solution was dried over sodium sulfate, filtered, and evaporated to give 11.99g (ca. 100%) of the blocked tetrapeptide as a white solid 0.64, 10% methanol/dichloromethane). Further purification could be effected by flash chromatography of silica (3»6% methanol/methylene chloride).

To a suspension of the blocked tetrapeptide (3Jg, 3.5mmol) in ethanol (150mL) was added water, until the mixture became very turbid (about 125mL). The resulting mixture was shaken with palladium chloride (0.61g, 3.5mmol) under an atmosphere of hydrogen (5 atm) for 16h. The catalyst was removed by filtration and the filtrate was evaporated to give 2.20g (ca. 100%) of pink solid: [a]D -18.0°(c 1.0, H2O); ^!H NMR (500MHz, D2O) d 8.92(1H, s), 8.88(1H, d, j=7.0), 8.50(1H, d, J=7.8), 8.36(1H, d, j=7.5), 8.60-8J0(3H, m), 7.54(1H, d, J=7.8), 7.34(1H, d, j=8.0), 7.27(1H, d, j=9.6), 7.05(1H, t, J=7.4), 6.98(1H, t, J=7.4). 4.70(1H, d, J=5J), 4.48(1H, d, j=5.2), 4.27(1H, d, J=5J), 3.90-3.60(2H, m), 3.69(3H, s), 3.30-3.00(6H, m), 1.80-1.20(6H, m).

Example 12

Synthesis of Glycyl-L-Histidyl L-Glutamic Acid

To a stirred solution of N- -BOC-N-im-CBZ-histidine (9.74g, 25.0mmol) and

N-methylmorpholine (5.8mL, 5.3g, 52.5mmol) in tetrahydrofuran (50mL) at -15°C was added isobutyl chloroformate (3.4mL, 3.6g, 26.3mmol). After 2 min. glutamic acid dibenzyl ester tosylate (12.5g, 25.0mmol) was added. The reaction mixture was stirred at -15°C for 1.5h and then allowed to warm to 0°C. At this time the reaction was quenched by the addition of 2M aqueous potassium bicarbonate. The products were extracted with ethyl acetate (3 x 150mL). The combined extracts were washed with 1M citric acid (3 x lOOmL), water, 2M KHCO3 (3 x lOOmL), water, and brine. The resulting solution was dried over sodium sulfate, filtered, and evaporated to give 15.2g

(87%) of the blocked dipeptide as a white semi-solid (Rf = 0.74, 10% methanol/dichloromethane), which was used in the following transformation without

> further purification. A solution of the t-butyloxycarbonyl protected dipeptide (15Jg, 21.6mmol) in

35%o trifluoroacetic acid dichloromethane (150mL) was stirred l/2h at room temperature. The resulting solution was concentrated in vacuo and neutralized with 2M aqueous potassium bicarbonate. The product was extracted into ethyl acetate (3 x 150mL). The combined extracts were dried over sodium sulfate, filtered, and evaporated to give 14.8g (ca. 100% + entrained solvent) of the free-amino compound as a white solid: Rf= 0.48 (10% methanol/dichloromethane).

To a stirred solution of N-CBZ-glycine (5.23g, 25.0mmol) and N- methylmorpholine (3.0mL, 2.8g, 27.5mmol) in tetrahydrofuran (50mL) at -15°C was added isobutyl chloroformate (3.4mL, 3.6g, 26.3mmol). After 2min. a solution of the suitably protected dipeptide (12.9g, 21.6mmol) in tetrahydrofuran (50mL) was added. The reaction mixture was stirred at -15°C for 1.5h and then allowed to warm to 0°C. At this time the reaction was quenched by the addition of 2M aqueous potassium bicarbonate. The products were extracted with ethyl acetate (3 x lOOmL). The combined extracts were washed with 1M citric acid (3 x lOOmL), water, 2M KHCO3 (3 x lOOmL), water, and brine. The resulting solution was dried over sodium sulfate, filtered, and concentrated to a syrup, which was diluted with absolute ethanol, and kept overnight at -20°C. The resulting precipitate was collected on a filter to afford 9.93g (58%) of the blocked tripeptide as a white solid (Rf =0.58, 10%) methanol/dichloromethane): mp 114-116°C. Anal. Calcd^'. for C43H43N5O10: C, 65.39; H, 5.49; N, 8.87. Found: C, 64.93; H, 5.56; N. 8.41. To a suspension of the blocked tripeptide (9.6g, 12.2mmol) in ethanol (150mL) was added water, unJl the mixture became very turbid (about 150mL). The resulting mixture was shaken -vitii palladium chloride (2.22g, 12.5mmol) under an atmosphere of hydrogen (5 atm) for 16h. The catalyst was removed by filtration through a plug of Celite® and the filtrate was concentrated to remove volatile organic materials. The remainder was lyophilized to give 4.72g of white powder. This material was dissolved in water, filtered through a 0.2m nylon membrane and lyophilized to give 4.64g (93%>) of the desired tripeptide dihydrochloride as a free-flowing white powder: [a]D -16.6° (c 2.0, water); ^]H NMR (500MHz, D2O) d 8.65 (IH, s), 7.35 (IH, s), 4.77 (IH, m), 4.46 (IH, m), 3.88 (2H, s), 3.28 (IH, dd, J=15.3, 6.1), 3.21 (IH, dd, J=15.3, 8.0), 2.47 (2H, m), 2.21 (2H, m), 2.00 (2H, m); ^C NMR (125MHz, D2O) d 179.9, 177.3, 174.3, 169.8, 136.5, 130.8, 120.4, 55.6, 54.9, 43.3, 32.8, 29.3, 28.5; Anal. Calcd for C13H21CI2N5O6: C, 37.69; H, 5.11; N, 16.91; Cl, 17.12. Found: C, 37.23; H, 5.07; N, 16.01; Cl, 17.95.

Example 13 Synthesis of Glycyl-L-Histidyl-L-Phenylalanine To a stirred solution of N-α-BOC-N-im-CBZ-histidine (9.74g, 25.0mmol) and N-methylmorpholine (5.8mL, 5.3g, 52.5mmol) in tetrahydrofuran (50mL) at -15°C was added isobutyl chloroformate (3.4mL, 3.6g, 26.3mmol). After 2 min. phenylalanine benzyl ester tosylate (10.7g, 25.0mmol) was added. The reaction mixture was stirred at -15°C for 1.5h and then allowed to warm to 0°C. At this time the reaction was quenched by the addition of 2M aqueous potassium bicarbonate. The products were extracted with ethyl acetate (3 x 150mL). The combined extracts were washed with 1M citric acid (3 x lOOmL), water, 2M KHCO3 (3 x lOOmL), water, and brine. The resulting solution was dried over sodium sulfate, filtered, and evaporated to give 13.0g (83%) of the blocked dipeptide as a white semi-solid (Rf = 0.79, 10% methanol/dichloromethane), which was used in the following transformation without further purification. A solution of the t-butyloxycarbonyl protected dipeptide (12.9g, 20.6mmol) in

35% trifluoroacetic acid/dichloromethane (150mL) was stirred l/2h at room temperature. The resulting solution was concentrated in vacuo and neutralized with 2M aqueous potassium bicarbonate. The product was extracted into ethyl acetate (3 x 150mL). The combined extracts were dried over sodium sulfate, filtered, and evaporated to give 12.3g (ca. 100% + entrained solvent) of the free-amino compound as a white solid: Rf= 0.50 (10% methanol/dichloromethane). To a stirred solution of N-CBZ-glycine (5.23g, 25.0mmol) and N- methylmo holine (3.0mL, 2.8g, 27.5mmol) in tetrahydrofuran (50mL) at -15°C was added isobutyl chloroformate (3.4mL, 3.6g, 26.3mmol). After 2min. a solution of ihe suitably protected dipeptide (10.8g, 20.6mmol) in tetrahydrofuran (50mL) was added. The reaction mixture was stirred at -15°C for 1.5h and then allowed to warm to 0°C. At this time the reaction was quenched by the addition of 2M aqueous potassium bicarbonate. The products were extracted with ethyl acetate (3 x 1 OOmL). The combined extracts were washed with IM citric acid (3 x lOOmL), water, 2M KHCO3(3 x lOOmL), water, and brine. The resulting solution was dried over sodium sulfate, filtered, and evaporated to give 14.0g (95%>) of the blocked tripeptide as a white solid (R/ = 0.64, 10% methanol/dichloromethane), which was recrystallized from absolute ethanol to give a free-flowing white powder.

To a suspension of the blocked tripeptide (6.0g, 8.3mmol) in ethanol (150mL) > was added water, until the mixture became very turbid (about 150mL). The resulting mixture was shaken with palladium chloride (1.47g, 8.3mmol) under an atmosphere of hydrogen (5 atm) for 16h. The catalyst was removed by filtration through a plug of

Celite® and the filtrate was concentrated to remove volatile organic materials. The remainder was lyophilized to give 1.46g of white powder. This material was dissolved in water, filtered through a 0.2m nylon membrane and lyophilized to give 1.38g (38%>) of the desired tripeptide dihydrochloride as a free-flowing white powder: [a]τj -7.5° (c

1.0, water); *H NMR (500MHz, D2O) d 8.59 (IH, s), 7.39-7.25 (5H, m), 7.21 (IH, s),

4.70 (IH, br t, J = 7), 3.80 (2H, s), 3.24 (IH, dd, J=14.0, 5.5). 3.16 (IH, dd, J=15.4,

6.9), 3.10 (IH, dd, J=15.4, 7.4), 3.03 (IH, dd, J=14.0, 9.1); ^C NMR (125MHz,

DMSO-d6) d 172.7, 169.5, 166.0, 137.6, 133.3, 129.2, 128.9. 128.3, 126.5, 116.8, 53.9, 51.8, 40.1, 36.4, 27.3.

Example 14

Synthesis of Glvcyl-L-Histidyl-L-Lysyl-L-Phenylalanine

To a stirred solution of N-α-BOC-N-ε-CBZ-lysine (9.5g, 25.0mmol) and N- methylmorpholine (5.8mL, 5.3g, 52.5mmol) in tetrahydrofuran (50mL) at -15°C was added isobutyl chloroformate (3.4mL, 3.6g, 26.7mmol). After 2min. phenylalanine benzyl ester tosylate (10.7g, 25.0mmol) was added. The reaction mixture was stirred at

-15°C for 1.5h and then allowed to warm to 0°C. At this time the reaction was quenched by the addition of 2M aqueous potassium bicarbonate. The products were extracted with ethyl acetate (3 x 150mL). The combined extracts were washed with IM citric acid (3 x lOOmL), water, 2M KHCO3 (3 x lOOmL), water, and brine. The resulting solution was dried over sodium sulfate, filtered, and evaporated to give 17.76g (ca. 100% + entrained solvent) of the blocked dipeptide as a white solid (R/ = 0.84, 10% methanol/dichloromethane), which was used in the following transformation without further purification. A solution of the t-butyloxycarbonyl protected dipeptide (15.4g, 25.0mmol) in

35% trifluoroacetic acid/dichloromethane (150mL) was stirred l/2h at room temperature. The resulting solution was concentrated in vacuo and neutralized with 2M aqueous potassium bicarbonate. The product was extracted into ethyl acetate (3 x lOOmL). The combined extracts were dried over sodium sulfate, filtered, and evaporated to give 15.8g (ca. 100% + entrained solvent) of the free-amino compound as a white semi-solid: Rf= 0.55 (10% methanol/dichloromethane).

To a stirred solution of N-α-BOC-N-im-CBZ-histidine (9.74g, 25.0mmol) and N-methylmorpholine (3.0mL, 2.8g, 27.5mmol) in tetrahydrofuran (50mL) at -15°C was added isobutyl chloroformate (3.4mL, 3.6g, 26.7mmol). After 2min. a solution of the suitably protected dipeptide (12.9g, 25.0mmol) in tetrahydrofuran (30mL) was added. The reaction mixture was stirred at -15°C for 1.5h and then allowed to warm to 0°C. At this time the reaction was quenched by the addition of 2M aqueous potassium bicarbonate. The products were extracted with ethyl acetate (3 x 150mL). The combined extracts were washed with IM citric acid (3 x lOOmL), water, 2M KHCO3 (3 x lOOmL), water, and brine. The resulting solution was dried over sodium sulfate, filtered, and evaporated to give 20.58g (93%) of the blocked tripeptide as a white semi-solid (Rf = 0.67, 10%) methanol/dichloromethane), which was used in the following transformation without further purification.

A solution of the t-butyloxycarbonyl protected tripeptide (20.5g, 23Jmmol) in 35%) trifluoroacetic acid/dichloromethane (150mL) was stirred l/2h at room temperature. The resulting solution was concentrated in vacuo and neutralized with 2M aqueous potassium bicarbonate. The product was extracted into ethyl acetate (3 x 150mL). The combined extracts were dried over sodium sulfate, filtered, and evaporated to give 20.5g (ca. 100% + entrained solvent) of the free-amino compound as a white solid: Ry= 0.51 (10% methanol/dichloromethane).

To a stirred solution of N-CBZ-glycine (7.24g, 34.6mmol) and N- methylmorpholine (4.2mL, 3.9g, 38Jmmol) in tetrahydrofuran (50mL) at -15°C was added isobutyl chloroformate (4.7mL, 5.0g, 36.3mmol). After 2min. a solution of the suitably protected tripeptide (18.2g, 23Jmmol) in 1 :1 tetrahydrofuran/dimethylformamide (50mL) was added. The reaction mixture was stirred at -15°C for 1.5h and then allowed to warm to 0°C. At this time the reaction was quenched by the addition of 2M aqueous potassium bicarbonate. The products were extracted with ethyl acetate (3 x 150mL). The combined extracts were washed with IM citric acid (3 x lOOmLy, water, 2M KHCO3(3 x lOOmL), water, and brine. The resulting solution was dried over sodium sulfate, filtered, and evaporated to give 21.6g (95%) of the blocked tetrapeptide as a white solid

0.85, 10%> methanol/dichloromethane), which was used in the following transformation without further purification.

To a suspension of the blocked tetrapeptide (21.5g, 21.9mmol) in ethanol (150mL) was added water, until the mixture became very turbid (about 125mL). The resulting mixture was shaken with palladium chloride (3.89g, 21.9mmol) under an atmosphere of hydrogen (5 atm) for 16h. The reaction mixture became clear within about l/2h, which may indicate completion of the reaction. The catalyst was removed by filtration and the filtrate was evaporated to give 13.7g of colorless semi-solid. This material was dissolved in water and lyophilized to give 11.5g (94%) of the desired tetrapeptide dihydrochloride as a free-flowing white powder: [a]rj -12.4°(c 2.0, H2O); IH NMR (500MHz, D2O) d 8.72 (IH, d, J=7.7), 8.40 (IH, d, J=7.8), 8.00 (IH, s), 7.30-7J9 (5H, m), 7.01 (IH, s), 4.62 (IH, br q, J=4.7), 4.44 (IH, m), 4.22 (IH, br q, J=4.9), 3.58 (2H, s), 3JO-2.90 (4H, m), 2.72 (2H, t, J=7.3), 1.65-1.20 (6H, m).

Example 15 Synthesis of Glycyl-L-Histidyl-L-Lysyl-Phenylalanyl-L-Phenylalanine

To a stirred solution of N-α-BOC-Phenylalanine (10.6g, 40.0mmol) and N- methylmorpholine (4.8mL, 4.5g, 44.0mmol) in tetrahydrofuran (50mL) at -15°C was added isobutyl chloroformate (5.5mL, 5.7g, 42.0mmol). After 2min. a solution prepared by mixing phenylalanine benzyl ester tosylate (17Jg, 40.0mmol), tetrahydrofuran (50mL), and N-methylmorpholine (4.4mL, 4.0g, 40.0mmol) was added. The reaction mixture was stirred at -15°C for 1.5h and then allowed to warm to 0°C. At this time the reaction was quenched by the addition of 2M aqueous potassium bicarbonate. The products were extracted with ethyl acetate (3 x 150mL). The combined extracts were washed with IM citric acid (3 x lOOmL), water, 2M KHCO3 (3 x lOOmL), water, and brine. The resulting solution was dried over sodium sulfate, filtered, and evaporated to give 19.8g (98%) of the blocked dipeptide as a white solid (Rf = 0.98, 10% methanol/dichloromethane).

A solution of the t-butyloxycarbonyl protected dipeptide (19.7g, 39.2mmol) in 35% trifluoroacetic acid/dichloromethane (150mL) was stirred l/2h at room temperature. The resulting solution was concentrated in vacuo and neutralized with 2M aqueous potassium bicarbonate. The product was extracted into ethyl acetate (3 x lOOmL). The combined extracts were dried over sodium sulfate, filtered, and evaporated to give 19.3g (ca. 100% + entrained solvent) of the free-amino compound : 0.65 (10%) methanol/dichloromethane).

To a stirred solution of N-α-BOC-N-ε-CBZ-lysine (15.2g, 40.0mmol) and N- methylmorpholine (4.8mL, 4.5g, 44.0mmol) in tetrahydrofuran (lOOmL) at -15°C was added isobutyl chloroformate (5.5mL, 5.7g, 42.0mmol). After 2min. the protected dipeptide (15.8g, 39.2mmol) was added. The reaction mixture was stirred at -15°C for 1.5h and then allowed to warm to 0°C. At this time the reaction was quenched by the addition of 2M aqueous potassium bicarbonate. The products were extracted with ethyl acetate (3 x 150mL). The combined extracts were washed with IM citric acid (3 x lOOmL), water, 2M KHCO3 (3 x lOOmL), water, and brine. The resulting solution was dried over sodium sulfate, filtered, and evaporated to give 29.9g (98%>) of the blocked tripeptide as a white solid (Rf= 0.84, 10%> methanol/dichloromethane).

A solution of the t-butyloxycarbonyl protected tripeptide (15.4g, 25.0mmol) in 35% trifluoroacetic acid dichloromethane (300mL) was stirred l/2h at room temperature. The resulting solution was concentrated in vacuo and neutralized with 2M aqueous potassium bicarbonate. The product was extracted into ethyl acetate (3 x lOOmL). The combined extracts were dried over sodium sulfate, filtered, and evaporated to give 28.7g (ca. 100% + entrained solvent) of the free-amino compound as a fluffy white solid: R/= 0.72 (10% methanol/dichloromethane).

To a stirred solution of N-α-BOC-N-im-CBZ-histidine (15.6g, 40.0mmol) and N-methylmorpholine (4.8mL, 4.5g, 44.0mmol) in tetrahydrofuran (80mL) at -15°C was added isobutyl chloroformate (5.5mL, 5.7g, 42.0mmol). After 2min. a solution of the suitably protected tripeptide (12.9g, 25.0mmol) in dimethylformamide (50mL) was added. The reaction mixture was stirred at -15°C for 1.5h and then allowed to warm to 0°C. At this time the reaction was quenched by the addition of 2M aqueous potassium bicarbonate. The products were extracted with ethyl acetate (3 x 150mL). The combined extracts were washed with IM citric acid (3 x lOOmL), water, 2M KHCO3 (3 x lOOmL), water, and brine. The resulting solution was dried over sodium sulfate, filtered, and evaporated to give 29Jg (72%) of the blocked tetrapeptide as a white solid

0.97, 10% methanol/dichloromethane).

A solution of the t-butyloxycarbonyl protected tetrapeptide (29Jg, 28.0mmol) in 35%) trifluoroacetic acid/dichloromethane (300mL) was stirred l/2h at room temperature. The resulting solution was concentrated in vacuo and neutralized with 2M aqueous potassium bicarbonate. The product was extracted into ethyl acetate (3 x 150mL). The combined extracts were dried over sodium sulfate, filtered, and evaporated to give 28.4g (ca. 100% + entrained solvent) of the free-amino compound as a white solid.

To a stirred solution of N-CBZ-glycine (7.32g, 35.0mmol) and N- methylmorpholine (4.2mL, 3.9g, 38Jmmol) in tetrahydrofuran (lOOmL) at -15°C was added isobutyl chloroformate (4.8mL, 5.0g, 36.7mmol). After 2min. a solution of the suitably protected tetrapeptide (26.3g, 28.0mmol) in 1 :1 tetrahydrofuran/dimethylformamide (50mL) was added. The reaction mixture was stirred at -15°C for 1.5h and then allowed to warm to 0°C. At this time the reaction was quenched by the addition of 2M aqueous potassium bicarbonate. The products were extracted with ethyl acetate (3 x 150mL). The combined extracts were washed with IM citric acid (3 x lOOmL), water, 2M KHCO3(3 x lOOmL), water, and brine. The resulting solution was dried over sodium sulfate, filtered, and evaporated to give 27.3g (87%o) of the blocked pentapeptide as a white solid

0.95, 10% methanol/dichloromethane)..

To a suspension of the blocked pentapeptide (27.3g, 24.2mmol) in ethanol (200mL) was added water, until the mixture became very turbid (about lOOmL). The resulting mixture was shaken with palladium chloride (4.3g, 24.4mmol) under an atmosphere of hydrogen (5 atm) for 16h. The reaction mixture became clear within about l/2h, which may indicate completion of the reaction. The catalyst was removed by filtration and the filtrate was evaporated to give 14.6g (82%) of the desired pentapeptide dihydrochloride as a free-flowing white powder: [a]_j) -12J°(c 2.0, methanol).

Example 16 Synthesis of Glycyl-L-Arginyl-L-Lysine To a stirred solution of N-α-BOC-Ng-Nitro-L-arginine (8.0g, 25.0mmol) and N- methylmorpholine (3.0mL, 2.8g, 27.5mmol) in tetrahydrofuran (50mL) at -15°C was added isobutyl chloroformate (3.4mL, 3.6g, 26.3mmol). After 2min. a solution of L-(N- e-CBZ)lysine benzyl ester hydrochloride (10.2g, 25.0mmol) and N-methylmorpholine (2.8mL, 2.5g, 25.0mmol) in tetrahydrofuran (30mL) was added. The reaction mixture was stirred at -15°C for 1.5h and then allowed to warm to 0°C. At this time the reaction was quenched by the addition of 2M aqueous potassium bicarbonate. The products were extracted with ethyl acetate (3 x 150mL). The combined extracts were washed with IM citric acid (3 x lOOmL), water, 2M KHCO3 (3 x lOOmL), water, and brine. The resulting solution was dried over sodium sulfate, filtered, and evaporated to give 16.3g (97%) of the blocked dipeptide as a white solid (Rf = 0.57, 10% methanol/dichloromethane) . A solution of the t-butyloxycarbonyl protected dipeptide (16.3g, 24.3mmol) in 35% trifluoroacetic acid/.-iichloromethane (150mL) was stirred for l/2h at room temperature. The resulting so tion was concentrated in vacuo and neutralized with 2M aqueous potassium bicarbonate. The product was extracted into ethyl acetate (3 x lOOmL). The combined extracts were dried over sodium sulfate, filtered, and evaporated to give 17.0g (ca. 100% + entrained solvent) of the free-amino compound as a white semi-solid: Ry= 0J2 (10% methanol/dichloromethane).

To a stirred solution of CBZ-glycine (7.32g, 35.0mmol) and N- methylmorpholine (4.2mL, 4.0g, 38.5mmol) in tetrahydrofuran (50mL) at -15°C was added isobutyl chloroformate (4.8mL, 5.0g, 36.8mmol). After 2min. a solution of the protected dipeptide (13.9g, 24.3mmol) in tetrahydrofuran (50mL) was added. The reaction mixture was stirred at -15°C for 1.5h and then allowed to warm to 0°C. At this time the reaction was quenched by the addition of 2M aqueous potassium bicarbonate, The products were extracted with ethyl acetate (3 x 150mL). The combined extracts were washed with IM citric acid (3 x lOOmL), water, 2M KHCO3(3 x lOOmL), water, and brine. The resulting solution was dried over sodium sulfate, filtered, and evaporated to give 17.7g (95%) of the blocked tripeptide as a white solid (Rf = 0.51, 10% methanol/dichloromethane) .

To a suspension of the blocked tripeptide (17.7g, 23.2mmol) in ethanol (250mL) was added water, until the mixture became very turbid (about lOOmL). The resulting mixture was shaken with palladium chloride (4.25g, 24.0mmol) under an atmosphere of hydrogen (5 atm) for 18h. The catalyst was removed by filtration and the filtrate was evaporated to give a white semi-solid. This material was dissolved in water, filtered through 0.45m nylon syringe filters, and lyophilized to give 10.2g (ca. 100%) of the desired tripeptide dihydrochloride as a white powder: [a]rj -14.6° (c 2, water); --H NMR (500MHz, D₂O) d 8.81(1H, br s), 8.30(1H, br s), 7.92(1H. br s), 4.37(1H, br s), 3.96(1H, d, J=4.8), 3.58(2H, d, J=8.8), 3J3(2H, br s), 2.74(2H, br s), 1.90-1.20(10H, m); ¹³C NMR (125MHz, D2O) d 175.2, 170.5, 166.9, 157.5, 1 15.0, 53.7, 52.6, 31.4, 29.2, 27.8, 26.8, 25.0, 22.5, 19.1.

Example 17 Synthesis of L-Histidyl-L-Phenylalanyl-L-Lysine To a stirred solution of N-α-BOC-L-phenylalanine (7.3g. 27.5mmol) and N- methylmorpholine (3.3mL, 3Jg, 30.2mmol) in tetrahydrofuran (50mL) at -15°C was added isobutyl chloroformate (3.8mL. 4.0g, 28.9mmol). After 2min. a solution of L-(N- ε-CBZ)lysine benzyl ester hydrochloride (10.2g, 25.0mmol) and N-methylmorpholine (2.9mL, 2.6g, 26.0mmol) in dimethylformamide (50mL) was added. The reaction mixture was stirred at -15°C for 1.5h and then allowed to warm to 0°C. At this time the reaction was quenched by the addition of 2M aqueous potassium bicarbonate. The products were extracted with ethyl acetate (3 x 150mL). The combined extracts were washed with IM citric acid (3 x lOOmL), water, 2M KHCO3 (3 x lOOmL), water, and brine. The resulting solution was dried over sodium sulfate, filtered, and evaporated to give 15.5g (100%) of the blocked dipeptide as a white solid (Rf = 0.92, 10% methanol/dichloromethane) .

A solution of the t-butyloxycarbonyl protected dipeptide (15.4g, 25.0mmol) in 35%o trifluoroacetic acid/dichloromethane (150mL) was stirred for l/2h at room temperature. The resulting solution was concentrated in vacuo and neutralized with 2M aqueous potassium bicarbonate. The product was extracted into ethyl acetate (3 x lOOmL). The combined extracts were dried over sodium sulfate, filtered, and

> evaporated to give 9.5g (73%) of the. free-amino compound as a white semi-solid: Rf= 0.55 (10%) methanol/dichloromethane).

To a stirred solution of L-(di-CBZ)histidine (8.47g, 20.0mmol) and N- methylmorpholine (2.4mL, 2.2g, 22.0mmol) in tetrahydrofuran (50mL) at -15°C was added isobutyl chloroformate (2.7mL, 2.9g, 21.0mmol). After 2min. a solution of the protected dipeptide (9.5g, 18.3mmol) in tetrahydrofuran (50mL) was added. The reaction mixture was stirred at -15°C for 1.5h and then allowed to warm to 0°C. At this time the reaction was quenched by the addition of 2M aqueous potassium bicarbonate. The products were extracted with ethyl acetate (3 x 150mL). The combined extracts were washed with IM citric acid (3 x lOOmL), water, 2M KHCO3(3 x lOOmL), water, and brine. The resulting solution was dried over sodium sulfate, filtered, and evaporated to give 15.7g (86%) of the blocked tripeptide as a white solid (Rf = 0.71, 10%) methanol/dichloromethane) .

To a suspension of the blocked tetrapeptide (15.0g, 16.3mmol) in isopropanol (175mL) was added water, until the mixture became very turbid (about 175mL). The resulting mixture was shaken with palladium chloride (2.93g, 16.3mmol) under an atmosphere of hydrogen (5 atm) for 18h. The catalyst was removed by filtration through Celite® and the filtrate was evaporated to give 7.54g of white solid. This material was dissolved in water, filtered through a 0.2m nylon filter, and lyophilized to give 7.84g (96%) of the desired tripeptide dihydrochloride as a white powder: [a]D 10.4° (c 2, water); IH NMR (500MHz, D O) d 8.69 (IH, s), 7.40-7.27 (6H, m), 4.31 (IH, T, J=6.8), 4.20 (IH, dd, J=5.8, 7.8), 3,39-3.35 (2H, m), 3J3-3.07 (2H, m). 2,98 (2H, t, j=7.6), 1.83-1.63 (4H, m), 1.37 (2H. pent, j=7.8). Example 18

Synthesis of L-Histidyl-Glycyl-L-Lysyl-L-Phcnylalanine

To a stirred solution of N-α-BOC-N-ε-CBZ-lysine (19.0g, 50.0mmol) and N- methylmoφholine (11.5mL, 10.6g, 105.0mmol) in tetrahydrofuran (lOOmL) at -15°C was added isobutyl chloroformate (6.8mL, 7.2g, 52.5mmol). After 2min. phenylalanine benzyl ester tosylate (21.3g, 50.0mmol) was added. The reaction mixture was stirred at

-15°C for 1.5h and then allowed to warm to 0°C. At this time the reaction was quenched by the addition of 2M aqueous potassium bicarbonate. The products were extracted with ethyl acetate (3 x 150mL). The combined extracts were washed with IM citric acid (3 x lOOmL), water, 2M KHCO3 (3 x lOOmL), water, and brine. The resulting solution was dried over sodium sulfate, filtered, and evaporated to give 28.25g (91%) of the blocked dipeptide as a white solid (Rf = 0.94, 10% methanol/dichloromethane), which was used in the following transformation without further purification.

A solution of the t-butyloxycarbonyl protected dipeptide (25.8g, 41.7mmol) in 35%) trifluoroacetic acid/dichloromethane (300mL) was stirred for l/2h at room temperature. The resulting solution was concentrated in vacuo and neutralized with 2M aqueous potassium bicarbonate. The product was extracted into ethyl acetate (3 x lOOmL). The combined extracts were dried over sodium sulfate, filtered, and evaporated to give 31. Og (ca. 100% + entrained solvent) of the free-amino compound as a white semi-solid: Rf= 0.45 (10% methanol/dichloromethane).

To a stirred solution of N-BOC-glycine (8.76g, 50.0mmol) and N- methylmorpholine (6.0mL, 5.6g, 55.0mmol) in tetrahydrofuran (lOOmL) at -15°C was added isobutyl chloroformate (6.8mL, 7.2g, 52.5mmol). After 2min. a solution of the protected dipeptide (21.6g, 41.7mmol) in tetrahydrofuran (lOOmL) was added. The reaction mixture was stirred at -15°C for 1.5h and then allowed to warm to 0°C. At this time the reaction was quenched by the addition of 2M aqueous potassium bicarbonate. The products were extracted with ethyl acetate (3 x 150mL). The combined extracts were washed with IM citric acid (3 x lOOmL), water, 2M KHCO3 (3 x lOOmL), water, and brine. The resulting solution was dried over sodium sulfate, filtered, and evaporated to give 31.40g (ca. 100%) of the blocked tripeptide as a white solid

0.66, 10% methanol/dichloromethane), which was used in the following transformation without further purification. A solution of the t-butyloxycarbonyl protected tripeptide (28Jg, 41.7mmol) in

35%) trifluoroacetic acid/dichloromethane (300mL) was stirred l/2h at room temperature. The resulting solution was concentrated in vacuo and neutralized with 2M aqueous potassium bicarbonrte. The product was extracted into ethyl acetate (3 x 150mL). The combined extracts were dried over sodium sulfate, filtered, and evaporated to give 43 Jg (ca. 100%) + entrained solvent) of the free-amino compound as a white solid: Ry= 0.26 (10% methanol/dichloromethane).

To a stirred solution of L-(di-CBZ)histidine (21.2g, 50.0mmol) and N- methylmorpholine (6.0mL, 5.6g, 55.0mmol) in tetrahydrofuran (lOOmL) at -15°C was added isobutyl chloroformate (6.8mL, 7.2g, 52.5mmol). After 2min. a solution of the protected tripeptide (23.9g, 41.7mmol) in 1 :1 tetrahydrofuran/dimethylformamide (50mL) was added. The reaction mixture was stirred at -15°C for 1.5h and then allowed to warm to 0°C. At this time the reaction was quenched by the addition of 2M aqueous potassium bicarbonate. The products were extracted with ethyl acetate (3 x 150mL). The combined extracts were washed with IM citric acid (3 x lOOmL), water, 2M KHCO3(3 x lOOmL), water, and brine. The resulting solution was dried over sodium sulfate, filtered, and evaporated to give 36.47g (89%>) of the blocked tetrapeptide as a white solid (Rf = 0.78, 10%) methanol/dichloromethane), which was used in the following transformation after flash chromatography on silica (3%> methanol/dichloromethane).

To a suspension of the blocked tetrapeptide (lOJg, 10.3mmol) in ethanol (250mL) was added water, until the mixture became very turbid (about 125mL). The resulting mixture was shaken with palladium chloride (1.83g, lOJmmol) under an atmosphere of hydrogen (5 atm) for lh. The reaction mixture became clear within about l/2h. The catalyst was removed by filtration and the filtrate was evaporated to give 4J2g of white semi-solid. This material was dissolved in water, filtered through a 0.2m nylon filter, and lyophilized to give 3.87g (67%>) of the desired tetrapeptide dihydrochloride: [a]o -19.4° (c 2.0, water); 1H NMR (500 MHz, D₂O) d 8.69 (IH, s), 7.49 (IH, s), 7.35-7.25 (5H, m), 4.72 (IH, dd, j=4.5, 8.5), 4.24 (IH, t, j=6.2), 4.40 (IH, d, J=16.9), 3.96 (IH, d, J=16.9), 3.45 (2H, d, J=6.2), 3.29 (IH, dd, J=12.9, 6.0), 3.08 (IH, dd, j=12.9, 7.7), 2.95 (2H, t, j=7.6), 1.75-1.60 (3H, m), 1.40-1.28 (3H, m).

Example 19 Synthesis of L-Histidyl-Glycyl-L-Lysyl-Trvptophan To a stirred solution of N-α-BOC-N-ε-CBZ-lysine (7.0g. 18.5mmol) and N- methylmorpholine (4.3mL, 3.9g, 38.9mmol) in tetrahydrofuran (50mL) at -15°C was added isobutyl chloroformate (2.5mL, 2.7g, 19.4mmol). After 2min. tryptophan benzyl ester hydrochloride (6Jg, 18.5mmol) was added. The reaction mixture was stirred at - 15°C for 1.5h and then allowed to warm to 0°C. At this time the reaction was quenched by the addition of 2M aqueous potassium bicarbonate. The products were extracted with ethyl acetate (3 x 150mL). The combined extracts were washed with IM citric acid (3 x lOOmL), water, 2M KHCO3 (3 x lOOmL), water, and brine. The resulting solution was dried over sodium sulfate, filtered, and evaporated to give 12.25g (100%) of the blocked dipeptide as a white solid

0.80, 10%) methanol/dichloromethane), which was used in the following transformation without further purification.

A solution of the t-butyloxycarbonyl protected dipeptide (25.8g, 41.7mmol) in 35% trifluoroacetic acid/dichloromethane (150mL) was stirred for l/2h at room temperature. The resulting solution was concentrated in vacuo and neutralized with 2M aqueous potassium bicarbonate. The product was extracted into ethyl acetate (3 x 150mL). The combined extracts were dried over sodium sulfate, filtered, and evaporated to give 11.75g (ca. 100% + entrained solvent) of the free-amino compound as a white semi-solid. To a stirred solution of N-BOC-glycine (3.24g, 18.5mmol) and N- methylmorpholine (2.2mL, 2.1g, 20.4mmol) in tetrahydrofuran (lOOmL) at -15°C was added isobutyl chloroformate (2.5mL, 2.7g, 19.4mmol). After 2min. a solution of the protected dipeptide (10.3g, 18.5mmol) in tetrahydrofuran (lOOmL) was added. The reaction mixture was stirred at -15°C for 1.5h and then allowed to warm to 0°C. At this time the reaction was quenched by the addition of 2M aqueous potassium bicarbonate. The products were extracted with ethyl acetate (3 x 150mL). The combined extracts were washed with IM citric acid (3 x lOOmL), water, 2M KHCO3 (3 x lOOmL), water, and brine. The resulting solution was dried over sodium sulfate, filtered, and evaporated to give 14J lg (ca. 100%) of the blocked tripeptide as a white solid

0.60, 10% methanol/dichloromethane), which was used in the following transformation without further purification.

A solution of the t-butyloxycarbonyl protected tripeptide (13Jg, 18.5mmol) in 35% trifluoroacetic acid dichloromethane (150mL) was stirred l/2h at room temperature. The resulting dark purple solution was concentrated in vacuo and neutralized with 2M aqueous potassium bicarbonate, fully discharging the color. The product was extracted into ethyl acetate (3 x 150mL). The combined extracts were dried over sodium sulfate, filtered, and evaporated to give 14.7g (ca. 100% + entrained solvent) of the free-amino compound as a foamy semi-solid: Rf = 0.20 (10% methanol/dichloromethane) . To a stirred solution of L-(di-CBZ)histidine (7.8g, 18.5mmol) and N- methylmorpholine (2.2mL, 2Jg, 20.4mmol) in tetrahydrofuran (50mL) at -15°C was added isobutyl chloroformate (2.5mL, 2.7g, 19.4mmol). After 2min. a solution of the protected tripeptide (11.3g, 18.5mmol) in THF (50mL) was added. The reaction mixture was stirred at -15°C for 1.5h and then allowed to warm to 0^CC. At this time the reaction was quenched by the addition of 2M aqueous potassium bicarbonate. The products were extracted with ethyl acetate (3 x 150mL). The combined extracts were washed with IM citric acid (3 x lOOmL), water, 2M KHCO3(3 x lOOmL), water, and brine. The resulting solution was dried over sodium sulfate, filtered, and evaporated to give 15.8g (84%>) of the blocked tetrapeptide as a yellow semi-solid (Rf = 0.85, 10%) methanol/dichloromethane), which was used in the following transformation after flash chromatography on silica (4% methanol/dichloromethane).

To a suspension of the blocked tetrapeptide (7.6g, 7.5mmol) in ethanol (120mL) was added water, until the mixture became very turbid. The resulting mixture was shaken with palladium chloride (1.33g, 7.5mmol) under an atmosphere of hydrogen (5 atm) for 18h. The catalyst was removed by filtration and the filtrate was evaporated to give a white semi-solid. This material was dissolved in water, filtered through a 0.2m nylon filter, and lyophilized to give 2.85g (64%>) of the desired tetrapeptide dihydrochloride as a white powder: [a] ) -21.9°(c 2,water).

Example 20 Transport of Copper to Human Fibroblasts bv GHK-Cu f2:l) and Copper-Chloride In this experiment, the ability of GHK-Cu (2:1) to transport copper to human fibroblast cells was compared to that of copper-chloride. Human neonatal foreskin fibroblast cells (Clonetics Coφ., San Diego, CA) were cultured in a medium (FGM, Clonetics Coφ., San Diego, CA) which contained neither serum or other factors (such as albumin and ceruloplasm) which may interfere with the delivery of copper to the cells. The cells were incubated with varying concentrations of GHK-Cu (2:1) or copper-chloride (i.e., 0, 0.022, 0.22 and 2.2 μM) for seven days at 37°C and under one atmosphere of 5%> (v/v) CO2. All cultures were prepared from the same aliquot of stock cells, thus all cultures began with an equal number of cells. Six cultures were used as the control group, and two cultures each of the above concentrations of GHK- Cu or copper-chloride were tested. At the end of the seven day incubation period, the viability of the cells was determined by a standard vital dye technique (i.e., the MTT assay) in which the conversion of the dye to a colorimetric product (measured at 570 nm) is directly proportional to the health of the cells (Mosman, J. Immunological Methods 65:55-63, 1983; incoφorated herein by reference in its entirety). The results of this experiment are expressed in Table 1 as percent increase relative to control cells which were not exposed to either GHK-Cu or copper-chloride.

TABLE 1 Viability of Human Fibroblasts

(Percent Increase Compared to Control Cells)

Copper-Chloride GHK-Cu

Cone. (μM): 0.022 0.22 2.2 0.022 0.22 2.2

Mean: -6.18 2.70 1.78 0.22 9.74 19.14 Std. Dev.: 1.29 7.20 2.21 1.85 0J8 2.40

As indicated by the data presented in Table 1 , copper-chloride had only a minor effect on the viability of the cells over the range of concentrations studied. However,- the cells treated with GHK-Cu exhibited a significant viability enhancement over the control group. Furthermore, the fibroblast cells exposed to GHK-Cu showed a viability enhancement that increased with increasing concentration of GHK-Cu. These results indicate that GHK-Cu is considerably more effective than copper-chloride in cell viability enhancement. This experiment also indicates that GHK is a suitable copper transport ligand, and is particularly well suited for both the delivery to cells, and uptake by cells, of copper. Furthermore, GHK-Cu does not adversely effect healthy cells, but rather the viability and overall health of normal cells is improved by exposure to GHK- Cu.

Example 21 Binding of GHK-Cu to HIV Protease

In this experiment, the direct interaction of GHK-Cu (1 :1) with HIV-1 protease is illustrated. GHK-Cu and HIV-1 protease were mixed, and their interaction was followed by changes in the UV absorbance at 300 nm. Specifically, to a solution of HIV-1 protease (Bachem Biosciences Inc., Philadelphia, PA) was added GHK-Cu (l .J) and the absorbance spectrum measured immediately after mixing (i.e., "0 minutes"), and at 1, 10, 20 and 30 minutes thereafter. The incubation conditions were as follows: 23°C, 0.435 milliliters total volume, pH 4.9, 1J4 x 10"⁷ M HIV protease, 47.7 x 10"³ M sodium acetate, 18.2 x 10^-3 M urea, 1J4 x 10^-7 M dithiothreitol, 0J91 M sodium chloride, and 5.7 x lO"*-⁵ M GHK-Cu. The GHK-Cu was added as 5 microliters of a 5 millimolar aqueous stock solution. A control sample without GHK-Cu was used to blank the spectrometer (Beckman Instruments, Model DU-65) and the absorbance meisurements were taken immediately following the addition of GHK-Cu.

TABLE 2 Interaction of GHK-Cu with HIV Protease

Minutes: 0 1 10 20 30

Absoφtion: 0 0.263 0.278 0.285 0.278

The increase in absorbance at 300 nm indicates a direct interaction between GHK-Cu and HIV protease, thus altering the physical properties HIV protease.

Example 22 Inhibition of HIV Replication bv GHK-Cu In this experiment, the inhibitory effect of GHK-Cu (1:1) on HIV replication in C8166 cells was demonstrated. C8166 is a cell line which is sensitive to infection by HIV, and has been described by Bergeron et al. (J. Virol. 66:5777-5787, 1992) and Warren et al. fAIDS Res. Hum. Retrovirus 6:1131-1137, 1990) (both of which references are incoφorated herein by reference in their entirety).

C8166 cells (Advanced Biotechnologies, Columbia, MD) were pre-incubated for 24 hours with varying concentrations of GHK-Cu (1 :1) (i.e., 0, 5, 50 and 500 nanomolar, "nM"), and then infected with HIV (i.e., HlVmg). The progress of infection was monitored by the appearance of syncitium (fused cellular membranes) in the cells, and is characteristic marker for HIV infected cells (see Bergeron et al., supra). The culture media was RPMI with 10% fetal bovine serum. Quadruplicate (4) cultures were used for each measurement. The cells were pre-treated with a concentration of GHK- Cu for 24 hours (15 ml at 200,000 cells/ml), and then concentrated to 500,000 cells/ml and incubated for 1 hour with six dilutions of the HIV virus preparation. Thus, there were quadruplicate cultures for each of the six vial dilutions for each concentration of GHK-Cu tested. After the virus absoφtion period (performed in serum free medium), the cells were washed with buffered saline and resuspended in media, with or without the indicated concentration of GHK-Cu, at concentration of 100,000 cells/ml. The cells were re-fed every 4 days, and the indicated concentrations of GHK-Cu were added back to the culture medium. The assay was controlled by cultures without GHK-Cu ("0 nm GHK-Cu"), as well as by cultures without the HIV virus. The latter control was used to determine any toxic effects of GHK-Cu on the cells in the absence of virus, and no toxic effects were observed.

The extent of viral replication was measured by the determination of the virus titer of the cells, and is reported in units of Tissue Culture Infective Dose 50 ("TCID50"). TCID50 is defined as the logarithm of the dilution of the HIV preparation necessary to infect 50% of the cells. Thus, if 50% of the cells were infected by a 10,000 fold dilution (1/10,000 or 1 x 10"⁴), the TCID₅₀ of the HIV preparation would be -4. If an agent is present in the preparation that inhibits HIV from infecting and cells, a greater concentration of HIV would be necessary to infect 50% of the cells. Thus, a culture that contains an HIV inhibitor may require a 100 fold dilution of the virus (1/100 or 1 x 10"-^***) to override the effect of the inhibitor and infect 50% of the cells. In such a case, the TCID50 would change from -4 to -2, and the difference between the two numbers (in this case 2) is referred to herein as the "log reduction" and represents inhibition of HIV infection (this technique for determining TCID5 is generally referred to as the Karber method).

TCID50 at days 4 and 14 alter infection and at various concentrations of GHK- Cu are presented in Table 3 below. At the concentrations indicated in Table 3, GHK- Cu inhibited HIV replication in C8166 cells.

TABLE 3 of HIV Re lication in C

In view of the data presented in Table 3 above, GHK-Cu resulted in a log reduction ranging from 0.25 to 0.75 over the concentrations reported (i.e., 0 to 500 nM). Moreover, no toxicity to the cells was observed by GHK-Cu in the absence of the HIV virus.

From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for puφoses of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not to be limited except as by the appended claims.

Claims

1. A method for inhibiting HIV replication in an HIV-infected patient, comprising administering to the patient an effective amount of a peptide-copper complex having the following structure:

[R₁-R₂-R₃]:copper(II) wherein:

R\ is hydrogen, an amino acid or an N-monoalkyl amino acid; R2 is histidine, a histidine derivative or arginine; and R3 is hydrogen or remainder of the molecule.

2. The method of claim 1 wherein the peptide-copper complex has the following structure:

[R 1 -L-histidyl-R2] xopper(II) wherein:

Rj is an amino acid; and

R2 is hydrogen or remainder of the molecule.

3. The method of claim 1 wherein the peptide-copper complex has the following structure:

[L-histidyl-Rj -R2] xopper(II) wherein:

Rj is an amino acid; and

R2 is hydrogen or remainder of the molecule.

4. The method of claim 1 wherein the peptide-copper complex is glycyl-histidyl- lysine: copper.

[R\ -L-arginyl-R2] xopper(II) wherein:

R\ is an amino acid; and

R2 is hydrogen or remainder of the molecule.

5. The method of claim 1 wherein the peptide-copper complex has the following structure:

[Rl -L-histidyl-R.2] xopper(II) wherein:

Ri is an amino acid; and

R is hydrogen or at least one amino acid.

6. The method of claim 1 wherein the peptide-copper complex has the following structure:

[L-histidyl-Rj -R₂] xopper(II) wherein:

Rj is an amino acid; and

R₂ is hydrogen or at least one amino acid.

7. The method of claim 1 wherein the peptide-copper complex is glycyl-histidyl- lysine: copper.

[R j -L-arginyl-R2] xopper(II) wherein:

R\ is an amino acid; and

R₂ is hydrogen or at least one amino acid.

8. The method of claim 1 wherein the peptide-copper complex is glycyl-histidyl- lysinexopper.

9. The method of claim 8 wherein peptide-copper complex has a molar ratio of peptide to copper ranging from 1 :1 to 2:1.

10. A composition for inhibiting HIV replication in an HIV-infected patient, the composition comprising an effective amount of a peptide-copper complex having the following structure:

[R₁-R₂-R3]:copper(II) wherein:

R\ is hydrogen, an amino acid or an N-monoalkyl amino acid;

R₂ is histidine, a histidine derivative or arginine; and

R3 is hydrogen or remainder of the molecule; and a pharmaceutically acceptable carrier or diluent.

1 1. The composition of claim 10 wherein the peptide-copper complex has the following structure:

[R j -L-histidyl-R2] xopper(II) wherein:

Rl is an amino acid; and

R2 is hydrogen or remainder of the molecule.

12. The composition of claim 10 wherein the peptide-copper complex has the following structure:

[L-histidyl-Ri -R2] xopper(II) wherein:

Rl is an amino acid; and

R2 is hydrogen or remainder of the molecule.

13. The composition of claim 10 wherein the peptide-copper complex is glycyl- histidyl-lysinexopper.

[Rl -L-arginyl-R2]xopper(II) wherein:

Rl is an amino acid; and

R2 is hydrogen or remainder of the molecule.

14. The composition of claim 10 wherein the peptide-copper complex has the following structure:

[Rl -L-histidyl-R2]:copper(II) wherein:

Rl is an amino acid; and

R2 is hydrogen or at least one amino acid.

15. The composition of claim 10 wherein the peptide-copper complex has the following structure:

[L-histidyl-R -R2] xopper(II) wherein:

Rl is an amino acid; and

R2 is hydrogen or at least one amino acid.

16. The composition of claim 10 wherein the peptide-copper complex is glycyl- histidyl-lysin xopper.

[R 1 -L-arginyl-R.2] xopper(II) wherein:

Rl is an amino acid; and

R2 is hydrogen or at least one amino acid.

17. The composition of claim 10 wherein the peptide-copper complex is glycyl- histidyl-lysinexopper.

18. The composition of claim 17 wherein peptide-copper complex has a molar ratio of peptide to copper ranging from 1 :1 to 2:1.