WO1997016410A1 - Nouveaux inhibiteurs de la liaison de peptides aux proteines de classe ii du cmh - Google Patents

Nouveaux inhibiteurs de la liaison de peptides aux proteines de classe ii du cmh Download PDF

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Publication number
WO1997016410A1
WO1997016410A1 PCT/US1996/017333 US9617333W WO9716410A1 WO 1997016410 A1 WO1997016410 A1 WO 1997016410A1 US 9617333 W US9617333 W US 9617333W WO 9716410 A1 WO9716410 A1 WO 9716410A1
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Prior art keywords
nva
lys
leu
methyl
boc
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PCT/US1996/017333
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English (en)
Inventor
Alan D. Adams
A. Brian Jones
Victoria K. Lombardo
Richard L. Tolman
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Merck & Co., Inc.
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Priority claimed from GBGB9602976.4A external-priority patent/GB9602976D0/en
Application filed by Merck & Co., Inc. filed Critical Merck & Co., Inc.
Priority to AU75257/96A priority Critical patent/AU7525796A/en
Publication of WO1997016410A1 publication Critical patent/WO1997016410A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0821—Tripeptides with the first amino acid being heterocyclic, e.g. His, Pro, Trp
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
    • C07K5/0207—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the structure -NH-(X)4-C(=0), e.g. 'isosters', replacing two amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/06034—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms
    • C07K5/06052—Val-amino acid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
    • C07K5/0808—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0812—Tripeptides with the first amino acid being neutral and aromatic or cycloaliphatic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0815—Tripeptides with the first amino acid being basic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1016—Tetrapeptides with the first amino acid being neutral and aromatic or cycloaliphatic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention provides novel compounds, novel compositions, methods of their use and methods of their manufacture, where such compounds may be pharmacologically useful as agents in therapies whose mechanism of action rely on the inhibition of peptide binding to major histocompatibility complex (MHC) class II molecules, and more particularly useful in therapies for the treatment and prevention of autoimmune diseases.
  • MHC major histocompatibility complex
  • a basic function of the immune system is to distinguish self from non-self, an activity carried out primarily by T cells. Failure of mechanisms which control the tolerance of T cells to self antigens and the prevention of activation of T cells by self antigens may lead to autoimmunity. In individuals afflicted with autoimmune diseases, an increased frequency of alleles for specific human leukocyte antigens (HLAs) are found, and it is believed that the disease-associated HLA molecules have the ability to bind the autoantigen and present it to T cells, thereby inducing and/or maintaining the autoimmune process.
  • HLAs human leukocyte antigens
  • Currently available immunosuppressive drugs are inadequate because of limited efficacy, lack of selectivity and considerable toxicity.
  • the present invention is directed to compounds which inhibit the binding of peptides to the major histocompatibility complex class II, a more selective target for therapeutic treatment and prevention of autoimmune diseases.
  • Major histocompatibility complex class II molecules are cell-surface glycoproteins that bind antigenic peptide fragments and display them at the cell surface to CD4- positive helper T-cells. The action of these molecules forms part of a pathway of the immune system for identifying and responding to foreign antigens.
  • antigen presenting cells internalize foreign proteins. Once internalized, the proteins are proteolytically degraded and short sequences of the degraded proteins are bound to MHC class II molecules in an endosomal compartment. These complexes of the short sequences bound to the MHC Class II molecule are then exposed on the cell surface where they initiate a series of immunogenic events.
  • MHC Class II proteins are synthesized and assembled in the endoplasmic reticulum as trimers composed of highly polymorphic and ⁇ -chain polypeptides and a non-polymorphic invariant chain polypeptide.
  • the invariant chain prevents the premature binding of peptides to MHC class II.
  • the invariant chain contains a sequence that targets the ⁇ / ⁇ heterodimer into the low pH, protease-rich endosomal compartment. In this compartment, the invariant chain is removed, leaving the MHC class II ⁇ / ⁇ heterodimers free to bind antigenic peptides.
  • class I and class LI histocompatibility proteins have different domain organizations but similar structures, with two membrane-proximal immunoglobulin-like domains and a membrane- distal peptide-binding site formed by an eight stranded ⁇ -sheet and two oc -helical regions. Polymorphic residues in both class I and II proteins are clustered in the peptide-binding region and are responsible for the different peptide specificities observed for different histocompatibility proteins.
  • Class I histocompatibility proteins are specific for peptides of defined length, usually 8-10 residues and have allele-specific binding motifs characterized by strong preferences for a few side chains at some positions in the peptide, and wide tolerance for many side chains at the other positions.
  • Class II histocompatibility proteins bind longer peptides with no apparent restriction on peptide length. Class II proteins also have allele specific motifs, which have been more difficult to characterize because of the difficulty in aligning peptide sequences of different lengths.
  • Antigen presenting cells expressing MHC class II molecules capture proteins from extracellular fluids.
  • APCs can take up antigens through surface immunoglobulin receptors, through Fc receptor-mediated intemalization of antibody/antigen complexes, or through bulk-phase endocytosis. Internalized antigens are then transported to endosomal compartments where they are digested into peptide fragments. A subset of these peptides can associate with a specific binding groove at the interface of MHC class II ⁇ and ⁇ -chain heterodimers.
  • MHC class II/peptide complexes are then transported to the cell surface where they are recognized by T-cell receptors (TCRs) on CD4-positive T-cells. This process is pivotal for the generation of both humoral and cellular immune responses.
  • HLA-DP Three genetic loci within the human MHC encode class U antigen-presenting molecules: HLA-DP, HLA-DQ, and HLA-DR. These loci are highly polymorphic. For instance, there are over 30 DR ⁇ alleles in the human population. Since each individual expresses only a small number of different histocompatibility proteins, each histocompatibility protein must be able to bind a large number of different peptides in order to ensure an immune response against many possible pathogens. The extensive polymorphism of histocompatibility genes may be the result of selection of alleles that can present peptides from particular pathogens.
  • MHC class II alleles are linked to susceptibility to many autoimmune diseases.
  • a prominent example of this is susceptibility to rheumatoid arthritis (RA) which is genetically associated with a small subset of related DR alleles (DR4Dw4,
  • DR4Dwl4, and DRl See, Skinner et al., Annals of the Rheumatic Diseases 53:171-177 (1994). Over 90% of RA patients possess at least one of these 3 DR alleles compared to 27% in an age-matched control group. Autoimmune conditions are thought to involve the T-cell recognition of self-components by MHC Class U proteins, a situation which is normally avoided. This presentation generates an undesirable immune response to self.
  • the present invention is concerned with compounds which interfere with the binding of peptides to MHC class II molecules and a method of treating and preventing autoimmune diseases employing such compounds which interfere with the binding of peptides to MHC class II molecules associated with disease.
  • a method of treating and preventing autoimmune diseases employing such compounds which interfere with the binding of peptides to MHC class II molecules associated with disease.
  • Specifically blocking the formation of the MHC Class LJ/self- peptide complex is a manner of disrupting the aberrant process of the autoimmune disorder without globally depressing immune function.
  • Hurtenbach et al., J. Exp. Med. 177: 1499-1504 (1993) demonstrated that treatment with MHC class II complex -blocking peptide prevented autoimmune diabetes in non-obese diabetic mice. Further, Guery et al., J.
  • the binding inhibitors of the present invention may prevent the presentation of self-peptides to autoreactive T-cells that drive the disease process.
  • An advantage of the immunotherapy and immunotherapeutic agents of the present invention is that they are very selective agents, targeting only certain alleles of MHC Class II, which may minimize the risk of opportunistic infections during long term treatment.
  • competition for MHC binding among peptides is known, no non-peptide (or pseudopeptide) inhibitor of MHC Class LI binding has been known. Due to the inherent pharmacological limitations of peptides, particularly within a system that involves proteolytic degradation of proteins, the compounds of the present invention having less peptidic character may present a useful avenue for therapy.
  • novel compounds of the present invention are those of structural formula I:
  • the compounds of the present invention may be used in the treatment and prevention of autoimmune diseases, including rheumatoid arthritis, Type I diabetes, multiple sclerosis, lupus erythematosis, Graves disease and pemphigus.
  • an object of this invention to provide compounds that have activity in the inhibition of peptide binding to MHC Class proteins. It is an additional object of this invention to provide methods of using the compounds of formula I for the treatment of autoimmune conditions such as rheumatoid arthritis, Type I diabetes, multiple sclerosis, lupus erythematosis, Graves disease and pemphigus. It is a further object of this invention to provide pharmaceutical compositions for the compounds of formula I. Still another object of the present invention is to provide a method for in vitro inhibition of peptide binding of MHC Class II proteins.
  • novel compounds of this invention have the structural formula I:
  • R 1 is selected from
  • Ci-io alkyl unsubstituted or substituted with one to three substituents selected from:
  • R2 is selected from
  • R3 and R4 are each independently selected from:
  • R5 is selected from: (a) Ci -5 alkyl, unsubstituted or substituted with one to three substituents independently selected from:
  • R6 is C 1 -5 alkyl, unsubstituted or substituted with one to three substituents selected from:
  • R7 is selected from:
  • each R 8 is independently selected from
  • cycloalkyl is independently selected at each occurrence from (a) C3-8 saturated cycloalkyl unsubstituted or substituted with one to three substituents selected from:
  • hydroxy, halogen is independently selected at each occurrence from: (a) F, (b) Cl,
  • a heterocyclic ring is independently selected at each occurrence from: (a) C3-8 cycloalkyl wherein one or two of the carbon atoms are replaced with a heteroatom selected from oxygen, nitrogen, and sulfur, unsubstituted or substituted with one to three substituents selected from
  • alkyl is intended to include both branched- and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms, e.g, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl and isomers thereof such as isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, isohexyl, etc.
  • Alkoxy represents an alkyl group having the indicated number of carbon atoms attached through an oxygen bridge, e.g. methoxy, ethoxy, propyloxy, isopropoxy, etc.
  • Alkoxycarbonyl represents alkyl-O-C(O)- wherein the indicated number of carbons refers to those of the alkyl residue.
  • Acyl represents an alkyl group having the indicated number of carbon atoms attached through a -C(O)- bridge.
  • Sulfonyl represents an alkyl group having the indicated number of carbon atoms attached through a -S0 - bridge.
  • halogen and halo refer to F, Cl, Br and I.
  • the heterocyclic or aryl ring may be attached to the structural formula I at any nitrogen atom (in the case of heterocyclic) or carbon atom (in either case) in the ring which results in the creation of a stable, uncharged structure.
  • X is CHR 8 ;
  • Z is selected from: (a) CH, and (b) N;
  • Rl is selected from:
  • R3 and R4 are each independently selected from: (a) -CONR 8 R 8 , (b) -COOR 8 , and
  • R5 is C 1-4 alkyl, unsubstituted or substituted with one to three substituents selected from: (a) aryl, (b) NH2, and
  • a class of compounds within this embodiment further limited to those wherein: X is CH 2; Z is N; R 1 is selected from:
  • R 3 and R 4 are each independently selected from:
  • R 5 is Ci-4 alkyl, unsubstituted or substituted with one to three substituents selected from:
  • aryl is selected from: (a) phenyl,
  • Examples of compounds illustrating the present invention include, but are not limited to, the following:
  • N- ⁇ -((3-Cyclohexyl)propyl))-pyAla-Nva-cLys N- ⁇ -((3-Cyclohexyl)propyl))-Nva-Nva-Leu-NH 2
  • N- ⁇ -Ethylcarbamoyl-Cha-Val-Nva-NH-NH 2 ( 8 ) ( ⁇ -CBZ)-( ⁇ -BOC)Lys-Nva-Leu-NH2, (9) ( ⁇ -CBZ)-Lys-Nva-Leu-NH2, (10 ⁇ -[Cinnamoyl]-( ⁇ -BOC)Lys-Nva-Leu-NH2,
  • N-ethylcarbamoyl-phenylalanine-valine-alanine-leucin- hydrazide The compounds of the present invention are named by reference to a tetrapeptide of the general format: cap-Pl-P2-P3-P4 where "PX" represents the amino acid in the "xth" position in the tetrapeptide starting from PI at the N-terminus.
  • the 'cap' is a non- amino acid group attached to the N-terminus.
  • P4 is the carboxy terminal residue.
  • the compounds of the present invention are of substantially non-peptide character, yet inhibit peptide binding MHC Class proteins. Because the compounds of the present invention have substantially reduced peptide character relative to known inhibitors, the compounds of the present invention will be more likely to penetrate cellular membranes to access the Class II loading compartment within the cell, where competition for peptide binding is thought to occur. They are also likely to be more stable than peptides in the proteolytic environment of the endosomal compartment and hence better able to compete with the endogenous peptides. Based on knowledge within the art regarding peptide versus nonpeptide pharmacology, the compounds of the present invention are expected to have better oral bioavailability and longer in vivo half life than intact peptides.
  • pharmaceutically acceptable salts of the compounds of formula I where a basic or acidic group is present on the structure.
  • the compounds of the present invention may be administered in the form of pharmaceutically acceptable salts.
  • pharmaceutically acceptable salt is intended to include all acceptable salts such as acetate, lactobionate, benzenesulfonate, laurate, benzoate, malate, bicarbonate, maleate, bisulfate, mandelate, bitartrate, mesylate, borate, methylbromide, bromide, methylnitrate, calcium edetate, methylsulfate, camsylate, mucate, carbonate, napsylate, chloride, nitrate, clavulanate, N-methylglucamine, citrate, ammonium salt, dihydrochloride, oleate, edetate, oxalate, edisylate, pamoate (embonate), estolate, palmitate, e
  • salts of the compounds of this invention include those formed from cations such as sodium, potassium, aluminum, calcium, lithium, magnesium, zinc, and from bases such as ammonia, ethylenediamine, N-methyl-glutamine, lysine, arginine, ornithine, choline, N,N'-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, diethylamine, piperazine, tris(hydroxymethyl)aminomethane, and tetramethylammonium hydroxide.
  • bases such as ammonia, ethylenediamine, N-methyl-glutamine, lysine, arginine, ornithine, choline, N,N'-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, diethylamine, piperazine, tris(hydroxymethyl)aminomethan
  • a free acid by reacting a free acid with a suitable organic or inorganic base.
  • a suitable organic or inorganic base such as amino, an acidic salt, i.e. hydrochloride, hydrobromide, acetate, pamoate, and the like, can be used as the dosage form.
  • esters can be employed, e.g. acetate, maleate, pivaloyloxymethyl, and the like, and those esters known in the art for modifying solubility or hydrolysis characteristics for use as sustained release or prodrug formulations.
  • variable e.g., Rl , R6, etc.
  • its definition on each occurrence is independent of its definition at every other occurrence. Also, combinations of substituents and/or variables are permissible only if such combinations result in stable compounds.
  • the compounds of the present invention may have chiral centers other than those centers whose stereochemistry is depicted in formula I, and therefore may occur as diastereomers, with all such isomeric forms being included in the present invention as well as mixtures thereof. Furthermore, some of the crystalline forms for compounds of the present invention may exist as polymorphs and as such are intended to be included in the present invention. In addition, some of the compounds of the instant invention may form solvates with water or common organic solvents. Such solvates are encompassed within the scope of this invention.
  • terapéuticaally effective amount means that amount of a drug or pharmaceutical agent that will elicit the biological or medical response of a tissue, system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other clinician, which includes alleviation of the symptoms of the disorder being treated.
  • the novel methods of treatment of this invention are for disorders known to those skilled in the art.
  • the term “mammal” includes humans.
  • the present invention has the objective of providing methods of treating and preventing autoimmune diseases including: rheumatoid arthritis, Type I diabetes, multiple sclerosis, lupus erythematosis, Graves disease and pemphigus by oral, systemic, parenteral or topical administration of the novel compounds of formula I either alone or in combination with other agents useful in treating autoimmune diseases.
  • such agents which may be used in combination with the novel compounds of structural formula (I) include, but are not limited to: aspirin; NSAIDs including fenoprofen, tolmetin, sulindac, meclofenamate, indomethacin, ibuprofen, naproxen, ketoprofen, piroxicam, flurbiprofen, and diclofenac; gold sodium thiomalate; aurothioglucose; auranofin; penicillamine; hydroxychloroquine; sulfasalazine, corticosteroids; methotrexate; azathioprine; and cyclophosphamide.
  • aspirin include, but are not limited to: aspirin; NSAIDs including fenoprofen, tolmetin, sulindac, meclofenamate, indomethacin, ibuprofen, naproxen, ketoprofen, piroxicam,
  • agents which may be used in combination with the novel compounds of structural formula (I) include, but are not limited to: insulin therapy.
  • agents which may be used in combination with the novel compounds of structural formula (I) include, but are not limited to: prednisone, dexamethazone, azathioprine, copolymer 1 , cyclophosphamide, interferon, plasmapheresis, and baclofen.
  • agents which may be used in combination with the novel compounds of structural formula (I) include, but are not limited to: antimalarials such as hydroxychloroquinine, chloroquine, and quinacrine; prednisone and methyl prenisolone; and cyclophosphamide.
  • antimalarials such as hydroxychloroquinine, chloroquine, and quinacrine
  • prednisone and methyl prenisolone include cyclophosphamide.
  • agents which may be used in combination with the novel compounds of structural formula (I) include, but are not limited to: systemic corticosteroids, prednisone, methotrexate, cyclophosphamide and azathioprine.
  • the present invention also has the objective of providing suitable systemic including oral and parenteral including topical pharmaceutical formulations for use in the novel methods of treatment and prevention of the present invention.
  • treatment is intended to include ameliorating the autoimmune symptoms and/or arresting the progression of an autoimmune disease in an individual known to be, or believed to be suffering from an autoimmune disease.
  • prevention is intended to include ameliorating the underlying cause of an autoimmune condition in an individual who may not have begun to experience recognizable symptoms of an autoimmune condition, and arresting the progress of an autoimmune disease in a patient who has not begun to experience recognizable symptoms of an autoimmune condition.
  • compositions containing the present compounds as the active ingredient for use in the treatment of the above-noted conditions can be administered in a wide variety of therapeutic dosage forms in conventional vehicles for systemic administration.
  • the compounds can be administered in such oral dosage forms as tablets, capsules (each including timed release and sustained release formulations), pills, powders, granules, elixirs, tinctures, solutions, suspensions, syrups and emulsions, or by injection.
  • they may also be administered in intravenous (both bolus and infusion), intraperitoneal, subcutaneous, topical with or without occlusion, or intramuscular form, all using forms well known to those of ordinary skill in the pharmaceutical arts.
  • the daily dosage of the products may be varied over a range from 0.01 to 1 ,000 mg per adult human/per day.
  • the compositions are preferably provided in the form of tablets containing 0.01 to 1 ,000 mg, and particularly dosages of 0.01 , 0.05, 0.1 , 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, and 50.0 milligrams of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated.
  • An effective amount of the drug is ordinarily supplied at a dosage level of from about 0.0002 mg/kg to about 50 mg/kg of body weight per day.
  • the range is more particularly from about 0.001 mg/kg to 7 mg/kg of body weight per day.
  • compounds of the present invention may be administered in a single daily dose, or the total daily dosage may be administered in divided doses of two, three or four times daily.
  • compounds for the present invention can be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art.
  • the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen.
  • the compounds of the present invention may also be used in the preparation of a medicament or an agent useful in the treatment of autoimmune diseases, including rheumatoid arthritis,
  • Type I diabetes multiple sclerosis, lupus erythematosis, Graves disease and pemphigus.
  • the compounds of the present invention may be administered in a pharmaceutical composition comprising the active compound in combination with a pharmaceutically acceptable carrier adapted for topical administration.
  • Topical pharmaceutical compositions may be, e.g., in the form of a solution, cream, ointment, gel, lotion, shampoo or aerosol formulation adapted for application to the skin.
  • These topical pharmaceutical compositions containing the compounds of the present invention ordinarily include about 0.005% to 5% by weight of the active compound in admixture with a pharmaceutically acceptable vehicle.
  • the compounds of the present invention may be used together with agents known to be useful in treating autoimmune disease, discussed previously.
  • the active agents can be administered concurrently, or they each can be administered at separately staggered times.
  • the dosage regimen utilizing the compounds of the present invention is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular compound thereof employed.
  • a physician or veterinarian of ordinary skill can readily determine and prescribe the effective amount of the drug required to prevent, counter or arrest the progress of the condition.
  • Optimal precision in achieving concentration of drug within the range that yields efficacy without toxicity requires a regimen based on the kinetics of the drug's availability to target sites. This involves a consideration of the distribution, equilibrium, and elimination of a drug.
  • the compounds herein described in detail can form the active ingredient, and are typically administered in admixture with suitable pharmaceutical diluents, excipients or carriers (collectively referred to herein as "carrier” materials) suitably selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, syrups and the like, and consistent with conventional pharmaceutical practices.
  • carrier suitable pharmaceutical diluents, excipients or carriers
  • the active drug component can be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like.
  • suitable binders, lubricants, disintegrating agents and coloring agents can also be incorporated into the mixture.
  • suitable binders include, without limitation, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes and the like.
  • Lubricants used in these dosage forms include, without limitation, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like.
  • Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum and the like.
  • the liquid forms in suitably flavored suspending or dispersing agents such as the synthetic and natural gums, for example, tragacanth, acacia, methyl-cellulose and the like.
  • Other dispersing agents which may be employed include glycerin and the like.
  • sterile suspensions and solutions are desired. Isotonic preparations which generally contain suitable preservatives are employed when intravenous administration is desired.
  • Topical preparations containing the active drug component can be admixed with a variety of carrier materials well known in the art, such as, e.g., alcohols, aloe vera gel, allantoin, glycerine, vitamin A and E oils, mineral oil, PPG2 myristyl propionate, and the like, to form, e.g., alcoholic solutions, topical cleansers, cleansing creams, skin gels, skin lotions, and shampoos in cream or gel formulations. See, e.g., EP 0 285 382.
  • the compounds of the present invention can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles.
  • Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholines.
  • the compounds of the present invention may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydro-pyrans, polycyanoacrylates and cross-linked or amphipathic block copolymers of hydrogels.
  • biodegradable polymers useful in achieving controlled release of a drug
  • a drug for example, polylactic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydro-pyrans, polycyanoacrylates and cross-linked or amphipathic block copolymers of hydrogels.
  • the compounds of the present invention can be prepared readily according to the following Schemes and Examples or modifications thereof using readily available starting materials, reagents and conventional synthesis procedures. In these reactions, it is also possible to make use of variants which are themselves known to those of
  • Boc- is t-butoxycarbonyl
  • BOP is benzotriazole- 1-y l-oxy-tris-(dimethylamino)- phosphonium hexafluorophosphate
  • DCC is N,N'- dicyclohexylcarbodiimide
  • DCM is dichloromethane
  • DIEA is N, N'- diisopropylethyl amine
  • DMF is N,N'-dimethyl formamide
  • EDC is 1 - (3-dimethylaminopropyl)-3-ethyl carbodimide hydrochloride
  • EtOAc is ethyl acetate
  • ESI electrospray ionization
  • ESI-MS electrospray ionization mass spectroscopy
  • Examples 1 through 3 were prepared by solid-phase synthesis using well documented procedures. Standard Fmoc chemistry on RLNK-MBHA resin was used to link the first three C-terminal amino acids. Couplings were performed with DCC/HOBT in NMP for 70 min. Fmoc cleavage was achieved with 20% piperidine in NMP for 20min. Following cleavage of the N-terminal Fmoc group of the tripeptide, the resins were treated with -8 equivalents of cyclohexylpropionaldehyde, ⁇ 9 equivalents sodium cyanoborohydride in NMP for 120 min. Cleavage from the resin was achieved using 100% TFA. The crude materials were purified by reversed phase (C18) HPLC, using a mixed water/acetonitrile eluant containing 0.1% trifluoroacetic acid, and lyophilized. Materials were validated by mass spectral characterization.
  • Standard Fmoc chemistry on RLNK-MBHA resin was used to link the first three C-terminal amino acids. Couplings were performed with DCC/HOBT in NMP for 70 min. Fmoc cleavage was achieved with 20% piperidine in NMP for 20 min. Following cleavage of the N-terminal Fmoc group, the tripeptide was cleaved from the resin by treatment with 100% TFA. The tripeptide (5.1 mg) was dissolved in MeOH (lOO ⁇ L). Cyclohexylpropionaldehyde (1.6 mg) was added followed by sodium cyanoborohydride (2.0 mg). After lh NaHC ⁇ 3 solution was added and the mixture extracted with EtOAc, dried over magnesium sulfate, filtered and concentrated.
  • the title Hydrazide was prepared by solid-phase synthesis using well documented procedures. Standard Fmoc chemistry on Wang resin was used to link the three amino acids. Couplings were performed with DCC/HOBT in NMP for 70 min. Fmoc cleavage was achieved with 20% piperidine in NMP for 20min. Following cleavage of the N- terminal Fmoc group of the tripeptide the resin was treated with ethyl chloroformate (leq) and diisopropylethylamine (leq) in DMF for 120 min. The material was cleaved from the resin and the C-terminal hydrazide generated by treatment with hydrazine (30 eq) in DMF for 2 days. The crude material was purified by reversed phase (C18) HPLC using a mixed water/acetonitrile eluant containing 0.1 % trifluoroacetic acid, lyophilized and validated by mass spectral characterization.
  • C18 reversed phase
  • Step 1 Preparation of ( ⁇ -CBZW ⁇ -BOOLvs-Nva-Leu-NH?
  • Step 2 Preparation of ( ⁇ -CBZ)-( ⁇ -BOC)Lys-Nva-Leu-NH2: Typical CBZ Removal
  • the BOC amine (21.7 mg, 0.036 mmol, the product of Example 10) was dissolved in CH3OH (0.5 mL) at 0°C. Methanolic HCI 1.3N (1.5 mL) was added dropwise. The mixture was diluted with methanol (2 mL) after 1/4 Hr and concentrated . vac. The crude product was purified by preparatory reverse phase HPLC on an E. Merck LiChroprep RP-18 (31 Ox 25 mm) column with 0.1 % TFA(aq) : CH3CN (65 : 35) as eluent. The product was recovered as an oil.
  • the BOC amine (20.7 mg, 0.034 mmol, product of Example 12) was dissolved in glacial acetic acid (2 ml) at room temperature. Trifluoroacetic acid (2 ml) was added dropwise. The reaction mixture was then cooled in an ice bath and stirred at 0°C for lhr 15 minutes. The reaction mixture was diluted with methanol and concentrated . vac. The crude product was purified by preparatory reverse phase HPLC on an E. Merck LiChroprep RP-1 column with 0.1 % TFA(aq) : CH3CN (62 : 38). The product was recovered as an oil.
  • Example 14 The deprotection of the BOC amine of Example 14 (36.6 mg, 0.061 mmol) was carried out as described in Example 9.
  • the crude product was purified by preparatory reverse phase HPLC on an E. Merck LiChroprep RP-18 column with 0.1 % TFA(aq) : CH3CN (38 : 62).
  • the product was recovered as an oil.
  • iH NMR [400MHz, d4 Methanol] Selected peaks: 7.5 (m, 2H), 7.37 (m, 3H), 6.28 (s, IH), 4.39 (m, 2H), 2.93 (t, 2H), 2.51 (s 3H), 0.94 (m, 9H).
  • Example 16 The deprotection of the BOC amine of Example 16 (34.0 mg, 0.057 mmol) was carried out as described in Example 13. Residual acetic acid was removed by azeotropic evaporation with toluene. The crude product was purified by preparatory reverse phase HPLC on an E. Merck LiChroprep RP-18 column with 0.1 % TFA(aq) : CH3CN (62 : 38). Two diastereomers were separated. The absolute configurations are unknown. The early eluting product was recovered as an oil.
  • Example 13 The deprotection of the BOC amine of Example 1 (32.6 mg, 0.053 mmol) was carried out as described in Example 13.
  • the crude product was purified by preparatory reverse phase HPLC on an E. Merck LiChroprep RP- 18 (310 x 25 mm) column with 0.1 % TFA(aq) : CH3CN (57 : 43) as eluent.
  • the product was recovered as an amo ⁇ hous solid.
  • iH NMR [400MHz, d4 Methanol] Selected peaks: 7.23 (m, 5H), 4.34 (m, IH), 4.27 (m, 2H), 2.9 (m, 2H), 1.1 1 (h, I H), 0.92 (m, 9H), 0.81 (m, IH).
  • ( ⁇ -CBZ)-Lys-Nva-Leu-NH2 is prepared by the general procedures typified by Examples 8 and 9 starting from ( ⁇ -BOC)-( ⁇ - CBZ)-Lysine.
  • the acylation of ( ⁇ -CBZ)Lys-Nva-Leu-NH2 (1 10.6 mg, 0.226mmol.) by 1 -phenyl- 1 -cyclopropyl carboxylic acid (44.3 mg, by 1 -phenyl- 1 -cyclopropyl carboxylic acid (44.3 mg, 0.271 mmol) was carried out as described in Example 10. .
  • the CBZ protected amine of Example 20 (48.3 mg, 0.076 mmol) was dissolved in glacial acetic acid (6 mL) with Pd/C 10 % (11.0 mg, -20% w/w).
  • the reaction flask was purged three times vacuum versus H2 and stirred vigorously under one atm. of H2 at room temperature. The reaction was typically complete after 1-2 hours as judged by TLC.
  • the reaction mixture was filtered through a pad of CeliteTM diatomaceous earth to remove the catalyst and concentrated . vac. Residual acetic acid is removed as the toluene azeotrope /. vac.
  • the product was recovered as an oil.
  • Example 21 The deprotection of the CBZ protected amine prepared according to the procedures of Example 23 (40.5 mg, 0.066 mmol) was carried out as described in Example 21.
  • the crude product was purified by preparatory reverse phase HPLC on an E. Merck LiChroprep RP-18 (310 x 25 mm) column with 0.1 % TFA(aq): CH3CN (62:38) as eluent.
  • the product was obtained as an amo ⁇ hous solid.
  • Example 25 The deprotection of the CBZ amine produced in Example 25 (50.0 mg, 0.078 mmol) was carried out as described in Example 21.
  • the crude product was purified by preparatory reverse phase chromatography on an E. Merck LiChroprep RP-18 (310 x 25 mm) column with 0.1 % TFA (aq) : CH3CN (65 : 35) as eluent. Two diastereomers were separated. The absolute configurations are unknown.
  • Example 27 The deprotection of the BOC amine of Example 27 (30.1 mg, 0.042 mmol) was carried out as described in Example 9.
  • the crude product was purified by preparatory reverse phase HPLC on an E. Merck LiChroprep RP-18 (310 x 25 mm) column with 0.1 % TFA(aq) CH3CN (62 : 38) as eluent.
  • the product was recovered as an oil.
  • iH NMR [400MHz, d4 Methanol] Selected peaks: 7.28 (m, 5H), 4.3 (m, 3H), 3.05 (m, 2H), 2.91 (t, 2H), 2.76 ( dd,lH), 1.29 (s, 9H), 0.95 (m, 6H), 0.9 (d, 3H).
  • Example 9 The deprotection of the BOC amine prepared according to the procedures of Example 29 (31.9 mg, 0.052 mmol) was carried out as described in Example 9.
  • the crude product was purified by preparatory reverse phase HPLC on an E. Merck LiChroprep RP-18 (310 x 25 mm) column with 0.1 % TFA(aq) : CH3CN (62 : 38) as eluent.
  • the product was recovered as an oil.
  • Step 2 Preparation of ⁇ -[3R-methyl-3-cyclohexylpropionyl]( ⁇ - BOOLvs Nva Leu-NH_9
  • Example 9 The deprotection of the BOC amine produced according to the procedures of Example 37 (15.4 mg, 0.027 mmol) was performed as for Example 9.
  • the crude product was purified by preparatory reverse phase chromatography on an E. Merck LiChroprep RP-18 (310 x 25 mm) column with 0.1 % TFA (aq) : CH3CN (68 : 32) as eluent.
  • the product was recovered as an amo ⁇ hous solid.
  • Example 39 The deprotection of the BOC amine produced according to the procedures of Example 39 (15 mg, 0.026 mmol) was performed as for Example 9.
  • the crude product was purified by preparatory reverse phase chromatography on an E. Merck LiChroprep RP-18 (310 x 25 mm) column with 0.1% TFA (aq) : CH3CN (60 : 40) as eluent.
  • the product was recovered as an amo ⁇ hous solid.
  • ⁇ -aza-peptide isosteres or N-alkylcarbazates and carbamates have been inco ⁇ orated to protect the tetrapeptide mimic from protease degradation.
  • Figure 1 shows the structure and nomenclature of the ⁇ -
  • ⁇ -aza-amino acid isostere function may be inco ⁇ orated at any of the potential protease cleavage sites, terminal substitutions are most likely to provide maximum utility for protection from enzymatic cleavage, based on published investigation. See ref. below for enzyme stability studies.
  • the protected hydrazine derivative either the BOC hydrazine [t-butylcarbazate] as shown in Scheme I below, or the analogous CBZ hydrazine [benzylcarbazate] is typically dissolved in methanol at room temperature or below with one equivalent of the desired aldehyde or ketone. Glacial acetic acid is added in excess to facilitate the formation of the imminium intermediate and its subsequent reduction. A slight excess of a 1 molar solution of sodium cyanoborohydride is added to reduce the imminium intermediate to the desired alkylated carbazate. Minor quantities of dialkylation products are removed by chromatography.
  • the carbazate may be treated with a slight excess of the alkyl chloroformate either in two phase mixture of methylene chloride and an aqueous base, or in homogeneous solution of dioxane and an aqueous base.
  • Treatment of the alkylated carbazate with potassium cyanate in the presence of a protic acid, for instance in dioxane solution with an excess of 2 N hydrochloric acid leads to the semicarbazide analog, or ⁇ -aza-amino acid amide isostere.
  • Treatment with other acylating reagents such as acid chlorides under similar conditions lead to analogs with less exact relationships to natural amino acids.
  • the peptide hydrazide may be prepared by hydrazine resin cleavage from the usual solid phase synthesis, or hydrazides may be prepared by the solution phase coupling of either t-Butyl or benzylcarbazate under the usual carbodiimide HOBT peptide coupling conditions followed by deprotection.
  • the steps required for the conversion of the resulting peptide hydrazide analogs produced to the desired final products are substantially the same as those described above for the simpler carbazate derivatives.
  • Some dialkylation product is also typically obtained by this route, and must be removed chromatographically.
  • Benzyl carbazate (1.0 Eq, 1.0 g, 6 mmol) was dissolved CH3OH (12 mL) and cooled to OOC. Isobutyraldehyde (1.0 Eq, 434 mg, 6 mmol) and acetic acid (2.0 Eq, 0.7 mL, 12 mmol) were added and the reaction mixture stirred briefly. A IM THF solution of NaCNBH3 was added and the mixture was allowed to come to RT and stirred one hour. The mixture was poured into H2O and CH2CI2 and the aqueous phase extracted three times CH2CI2. The combined organic phases were washed with H2O, pH 2.3 citrate buffer satd. aq. sodium bicarbonate and brine. The extracts were dried over Na2S ⁇ 4 and concentrated . vac. The product was recovered as an oil (used without purification).
  • EDC hydrochloride (2.4 Eq, 260 mg, 1.36 mmol) was added and the mixture stirred overnight at 0°C followed by 1 Hr at RT. The mixture was poured into H2O and EtOAc and the aqueous phase extracted three times with EtOAc. Combined EtOAc extracts were washed with satd. aq. sodium bicarbonate, washed with brine, dried over Na2S04 and concentrated . vac. The crude product was purified by chromatography on Si ⁇ 2 (40-63 ⁇ , 10 g) eluting with 8 : 1 : 1 toluene : EtOAc : iPrOH. The product was obtained as amo ⁇ hous solid.
  • Example 9 The deprotection of the BOC amine from Example 42 (40 mg, 0.058 mmol) was performed as for Example 9.
  • the crude product was purified by preparatory reverse phase chromatography on an E. Merck LiChroprep RP-18 (310 x 25 mm) column with 0.1% TFA (aq) : CH3CN (57 : 43) as eluent.
  • the product was recovered as an amo ⁇ hous solid.
  • the crude amine (1.0 Eq, 20 mg, 0.052 mmol) was dissolved THF (1.25 mL) with commercial ( ⁇ -BOC)( ⁇ -CBZ)Lysine N- hydroxysuccinimide ester (1.5 Eq, 37 mg, 0.078 mmol) at RT.
  • Diisopropylethyl amine ( 1.0 Eq, 9 ⁇ L, 0.052 mmol) was added and the solution stined at RT for 1 Hr.
  • the mixture was diluted EtOAc and H2O and the aqueous phase extracted three times EtOAc. The EtOAc extracts were washed with H2O, washed with satd. aq.
  • Step 1 Preparation of ( ⁇ -CBZ Lvs-Nva- ⁇ -AzaNle-OEt
  • the deprotection of the BOC amine obtained following the procedures of Example 445 (30 mg) was carried out as described in Example 9.
  • the product was purified by elution from a Sephadex LH- 20-100 column (780 x 12.7 mm) with methanol.
  • the product was obtained as amo ⁇ hous solid.
  • Step 2 Preparation of ⁇ -[3-Cyclohexyl ⁇ ropyl]-( ⁇ -CBZ)Lys-Nle- ⁇ -
  • the crude product was purified by chromatograph on Si ⁇ 2 ( 40-63 ⁇ , 50: 1 Si02 : crude mass ratio) eluting with toluene : isopropyl alcohol (75 : 25). The product was recovered as an amo ⁇ hous solid.
  • the CBZ protected amine obtained following the procedures of Example 49 (22.0 mg, 0.034 mmol) was dissolved in absolute ethanol (2 mL) with Pd/C 10 % (7.0 mg, 30% w/w).
  • the reaction flask was purged three times vacuum versus H2 and stirred vigorously under one atm. of H2 at room temperature. The reaction was typically complete after 2.5 hours as judged by TLC.
  • the reaction mixture was filtered through a pad of CeliteTM diatomaceous earth to remove the catalyst and concentrated . vac.
  • the crude product was purified by preparatory reverse phase chromatography on an E.
  • Example 51 Deprotection of the CBZ amine prepared according to the procedures of Example 51 ( 12.5 mg, 0.016 mmol ) was carried out as described in Example 50.
  • the product was purified by preparatory reverse phase chromatography on an E. Merck LiChroprep RP-8 (310 x 25 mm) column with 0.1 % TFA (aq) : CH3CN (40 : 60) as eluent.
  • the product was recovered as an amo ⁇ hous solid.
  • iH NMR [400MHz, d4 Methanol] Selected peaks: 7.07 (s, 4H), 4.34 (broad, IH), 4.1 1 (broad s, 2H), 3.89 (q, IH).
  • Step 1 Preparation of Val-Ala-Leu-® fMerrifield Standard Boc deprotection protocol for resin: Boc-V-A-L-
  • Standard reductive cleavage protocol The resin was then transferred to a dry flask and a solution of lithium borohydride (67 mg, 3 mmol) in dry THF (3 mL) was added followed by DIEA (50 ⁇ L, 0.3 mmol). The reaction was stirred at room temperature (1-5 hr) under an atmosphere of N2. The reaction was concentrated in vacuo and the resin taken up in water, filtered, and the filtrate lyophilized.
  • Standard purification protocol The resulting product was purified by reverse phase high performance liquid chromatography (RP-HPLC) (Waters Delta Pak Cis, RCM 25X10, 10 mL/min., 20- >80% CH3CN gradient, 30 min). The fractions containing the desired product were combined and lyophilized, providing the peptide (5_) as a white powder.
  • RP-HPLC reverse phase high performance liquid chromatography
  • Example 53 Starting with N-ethylcarbamoyl-phenylalanine-valine- norvalanine-leucine-® Pam resin (80 mg, Preparation 4) the standard reductive cleavage protocol of Example 53, Step 4 was used to provide the crude product which was taken up in water, filtered, and the filtrate lyophilized. The product was purified using the standard purification protocol Example 53, Step 4, providing the desired product as a white powder.
  • Boc-V-A-L-® (Merrifield) resin (91 mg, Preparation 1 ) was deprotected by the standard Boc deprotection protocol of Example 53, Step 1.
  • Step 2 Standard solid phase reductive amination procedure: The N-deprotected resin was suspended in DMF (2 mL) and cyclohexylpropionaldehyde (9.5 mg, 69 ⁇ mol) added. The reaction was agitated briefly (5-15 min. typical reaction time) and sodium cyanoborohydride (4.5 mg, 69 ⁇ mol) added in one portion. The reaction was agitated (5-24 hours typical reaction times) and the resin was rinsed with DMF (2 x 2 mL) and MeOH (2 x 2 mL). Kaiser test on the resin was negative.
  • Example 53 The product was liberated from the resin using the standard reductive cleavage protocol of Example 53, Step 4, and the resulting product purified by the standard peptide purification protocol of Example 53, Step 4. This afforded the desired material as a white powder.
  • Boc-V-A-L-® (Merrifield) resin (93 mg, Preparation 1) was deprotected by the standard Boc deprotection protocol of Example 53, Step 1 and alkylated by using the standard solid phase reductive amination procedure of Example 55, Step 2 with ⁇ -methyl-?ra/u -cinnamaldehyde (8 ⁇ L, 55 ⁇ mol).
  • the product was liberated from the resin using the standard reductive cleavage protocol of Example 53, Step 4, and the resulting product purified by the standard peptide purification protocol of Example 53, Step 4. This afforded the desired material as a white powder.
  • Step 1 Standard resin cleavage procedure for amines: N-ethylcarbamoyl-phenylalanine-valine-alanine-® Pam resin (180 mg, Preparation 5) was swelled with DMF (200 ⁇ L) and 3,3- dimethylbutyl amine (800 ⁇ L) was added. The reaction was stirred at RT (4 h to 24 h typical reaction times). The reaction mixture was concentrated in vacuo and taken up into EtOH and the resin filtered off affording a solution of the desired product.
  • the resin was suspended in hot EtOH/H2 ⁇ , filtered, and lyophilized.
  • the material was purified using the standard peptide purification procedure of Example 53, Step 4, affording the desired hydrazide as a white powder.
  • Example 60 From the synthesis of Example 60, a second component was also isolated during purification and identified as the alanine deletion product.
  • Boc-norvaline (2.0 g, 9.2 mmol) and leucinamide (1.53 g, 9.2 mmol) were dissolved in dry DMF (90 mL).
  • the reaction was cooled to 0 °C and DCC (2.3 g, 1 1 mmol), HOBt (1.48 g, 1 1 mmol), and DIEA (1.9 mL, 1 1 mmol) added.
  • the mixture was stirred for 20 min at 0 °C and allowed to slowly warm to room temperature ovemight ( 17 h). The mixture was concentrated to 1/3 its original volume, cooled, and filtered.
  • Boc-valine-norvaline-leucinamide (21.4 mg, 50 ⁇ mol, product of Example 63) was dissolved in 50% TFA/DCM (5.0 mL) and the solution stirred for 30 min. at RT. The solution was concentrated in vacuo affording a white powder that was used directly in the subsequent reaction.
  • Step 2 Standard solution phase reductive amination procedure
  • the N-deprotected material was dissolved in MeOH (280 ⁇ L) and 3 -cyclohexylpropionaldehyde (7.6 ⁇ L, 50 ⁇ mol) added. The reaction was stirred briefly ( 1-5 min.) and a solution of sodium cyanoborohydride (3.2 mg, 50 ⁇ mol) in MeOH (200 ⁇ L) was added. The pH of the reaction mixture was adjusted to 7 by the addition of small aliquots ( 1 ⁇ L) of DIEA and the solution was stirred ovemight at RT (5-24 hr). 50% HOAc/MeOH (1 mL) was added to the reaction mixture and the mixture concentrated in vacuo.
  • the product was purified by the standard peptide purification protocol of Example 53, Step 4, affording the desired material as a white powder.
  • Step 1 N-9-fluorenylmethoxycarbonyl-valine-alanine-leucine-®
  • Rink Amide MBHA resin (92 mg, Preparation 7) was deprotected using the standard Fmoc deprotection protocol for resin described in Preparation 6. The deprotected resin was then alkylated using the standard solid phase reductive amination procedure of Example 55 with a-methy]-trans -cinnamaldehyde (60 ⁇ L, 0.4 mmol).
  • Step 2 Standard acidic cleavage for Fmoc resin protocol A
  • the peptide was cleaved from the resin using TFA/H2O (24: 1 , 2 mL) for 2 hr at RT.
  • the resin was filtered off and the filtrate concentrated in vacuo , taken up into 25% aq. CH3CN, and lyophilized.
  • the peptide was purified using the standard peptide purification protocol of Example 53, Step 4, affording the desired material as a white powder.
  • Step 1
  • Lysine-norvaline-leucine-Rink Amide MBHA resin (102 mg, Preparation 8) was rinsed with DMF (2x1.5 mL) and suspended in DMF (1.5 mL) and DIEA added (20 ⁇ L). 3-phenylpropanoic acid (14 mg, 85 ⁇ mol), and PyBOP (44.1 mg, 85 ⁇ mol) was added. The reaction was agitated at RT (1-18 h typical reaction time) and the resin filtered and rinsed with MeOH (3x1.5 mL). Step 2: Standard acidic cleavage for Fmoc resin protocol B
  • the peptide was cleaved from the resin by treatment with TFA/anisole/water (98: 1 :1 ) (3 mL) for 3 h at RT.
  • the resin was filtered off and the filtrate concentrated in vacuo, taken up into 25% aq. CH3CN, and lyophilized.
  • the product was purified using the standard peptide purification protocol of Example 53, Step 4 with a 25-55% aq. CH3CN gradient over 30 min. This afforded the desired material as a white powder.
  • EXAMPLE 68 Oral Composition
  • 5 mg of a compound of structural formula I is formulated with sufficient finely divided lactose to provide a total amount of 580 to 590 mg to fill a size 0 hard gelatin capsule.
  • Boc-V-A-L-® (Merrifield resin): 1 mmol of Boc-
  • Leu-® (Merrifield) resin (Bachem California, 0.6 mmol/g) was swelled in DCM in a fritted vessel equipped for manual peptide synthesis.
  • Boc-V-A-L-® (PAM resin): The resin-bound tripeptide was prepared from 1.5 mmol of Boc-Leu-® (PAM) resin (Bachem California, 0.6 mmol/g), using the synthesis protocol outlined in Preparation 1. NMP was used as the reaction solvent in place of DCM.
  • EtOCO-F-V-A-L-® PAM resin
  • the resin-bound tripeptide was prepared from 1.0 mmol of Boc-Leu-® (PAM) resin (NovaBiochem, 0.56 mmol/g), using the synthesis protocol outlined in Preparation 1. NMP was used as the reaction solvent in place of DCM. The N-terminal Boc protecting group was removed by treatment with 50% TFA/DCM (30 min., RT) and the tripeptide was capped by the addition of ethylchloroformate (382 ⁇ L, 4.0 mmol). The resin was finally rinsed with NMP (3x20 mL), then DCM (3x20 mL), and dried affording the loaded resin.
  • EtOCO-F-V-Nva-L-® (PAM resin): The resin-bound tripeptide was prepared from 1.0 mmol of Boc-Leu-® (PAM) resin (Bachem California, 0.6 mmol/g), using the synthesis procedure outlined in Preparation 3.
  • EtOCO-F-R(N ⁇ 2)-Nva-L-® PAM resin
  • the resin-bound tetrapeptide was prepared from 1.0 mmol of Boc-Leu-® (PAM) resin (Bachem California, 0.6 mmol/g), using the synthesis procedure outlined in Preparation 3.
  • the Arg(N ⁇ 2) residue was introduced as the N-Fmoc-protected amino acid using the standard Fmoc-amino acid coupling protocol illustrated below.
  • Standard Fmoc-amino acid coupling protocol The resin bound, deprotected dipeptide (422 mg, Nva-Leu-® PAM) was suspended in DMF (10 mL) and Fmoc-Arg(N ⁇ 2) (333 mg, 0.76 mmol), BOP (672 mg, 1.5 mmol), HOBt (206 mg, 1.5 mmol), and DIEA (132 ⁇ L, 0.76 mmol) added. The reaction was agitated at RT (1 to 24 h typical reaction time). The resin was filtered and rinsed with DMF (3 x 15 mL) and DCM (3 x 15 mL).
  • Standard Fmoc deprotection protocol for resin The resin was suspended in 20% piperidine/DMF (10 mL) and agitated at room temperature (10 to 60 min typical reaction time). The resin was filtered and rinsed with DMF (5 x 15 mL) and DCM (3 x 15 mL). A Kaiser test on the resin was positive for the presence of a free amino group. 12) Kaiser, E.; Colescott, R. L.; Bossinger, C. D.; Cook, P.
  • Fmoc-V-A-L-® (Rink amide MBHA resin): 1.5 mmol of Rink amide MBHA resin (NovaBiochem, 0.47 mmol/g) was swelled in DMF in a fritted vessel equipped for manual peptide synthesis. Nitrogen sparging was used as a source of agitation. The resin was deprotected using the standard Fmoc deprotection protocol for resin described in Preparation 6, and the Leu, Ala, and Val residues sequentially coupled to the resin using standard Fmoc-amino acid coupling protocol (see reference 13 below). DCC activation was used with DMF as the reaction solvent. Following the synthesis of the resin- bound tripeptide, the material was rinsed with DMF (2x20 mL), DCM (2x20 mL), MeOH (2x20 mL), and dried (N2 stream) affording the loaded resin.
  • K-Nva-L-® (Rink amide MBHA resin): The resin was prepared on an ABI 431 A peptide synthesizer (Applied Biosystems, Inc.) using the standard scale ABI 431 A Fmoc peptide synthesis protocol (reference 14 below) on a 0.25 mmol scale starting with unloaded Rink amide MBHA resin (0.47 mmol/g).
  • the conditions of the assays are shown to be in ligand excess, because twofold reduction of these class II concentrations does not change the measured ED50 values.
  • the DR-peptide complexes (50 ⁇ L) are transferred to wells of a 96-well ELA plate precoated with LB 3.1 , the monoclonal antibody which recognizes the DR alleles of MHC Class II, and blocked with PBS with fetal calf serum (FCS).
  • FCS fetal calf serum
  • An additional 50 ⁇ L of 50 mM Tris, pH 7.0, containing 0.75% octyl glucoside is added to each well and the mixture incubated ovemight at 4°C.
  • Excess peptide is removed by washing with PBS containing 0.05% Tween 20 (Polyoxyethylene sorbitan monolaurate) and 0.01 % NaN3.
  • Europium- labeled streptavidin (Wallac Inc.) is added and incubated ovemight.
  • complexes are measured by the addition of EnhanceTM buffer, the tradename for 0.1 M acetate phthalate buffer, pH 3.2, containing 0.1 % Triton X-100, tradename for polyoxyethylene ethers and other surface active compounds of Union Carbide Chemicals and Plastics Co., Inc.
  • LB3.1 ability of LB3.1 to bind DRIDwl and DR4Dw4 is shown to be equivalent by measuring the capacity of Ab-coated plates to bind serial dilutions of biotinylated DR molecules.
  • Europium streptavidin is used to measure the number of DR molecules bound as described for the peptide binding assay.
  • the inhibition assay format is identical to the procedure described above with the exception that the unlabeled antagonist is serially diluted and incubated with constant concentrations of biotinylated RMBP 90-102 (0.3 nM for DRIDwl or 0.9 nM for DR4Dw4) and the MHC class II proteins.
  • the concentration of unlabeled compound that prevents 50% of the labeled peptide from binding is the IC50 value.
  • the concentration of the biotinylated RMBP 90-102 in each assay is experimentally determined to be at least one- sixth of its measured ED50 to assure the inhibition was primarily measuring the binding characteristics of the competitor. This was confirmed by demonstrating that a two- or four fold reduction in the biotinylated agonist peptide did not alter the IC50 values obtained with unlabeled competitor proving that the receptor concentration was not limiting.

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Abstract

Composés répondant à la formule (I), servant d'inhibiteurs de la liaison de peptides aux protéines de type II du complexe majeur d'histocompatibilité, et trouvant application dans le traitement et la prévention des maladies auto-immunes telles que la polyarthrite rhumatoïde, le diabète insulino-dépendant, la sclérose en plaques, le lupus érythémateux, la maladie de Graves et le pemphigus. On a également prévu de nouvelles compositions, des procédés de traitement faisant intervenir lesdits composés, et des procédés de fabrication des composés répondant à la formule développée (I).
PCT/US1996/017333 1995-10-30 1996-10-25 Nouveaux inhibiteurs de la liaison de peptides aux proteines de classe ii du cmh WO1997016410A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU75257/96A AU7525796A (en) 1995-10-30 1996-10-25 Novel inhibitors of peptide binding to mhc class ii proteins

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US805995P 1995-10-30 1995-10-30
US60/008,059 1995-10-30
GB9602976.4 1996-02-13
GBGB9602976.4A GB9602976D0 (en) 1996-02-13 1996-02-13 Novel inhibitors of peptide binding to mhc class II proteins

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0958829A1 (fr) * 1998-05-21 1999-11-24 Tecnogen S.C.P.A. Utilisation d'un composé peptidique pour le traitement de SLE
WO1999067221A1 (fr) * 1998-06-22 1999-12-29 Elan Pharmaceuticals, Inc. COMPOSES D'INHIBITION DE LA LIBERATION DU PEPTIDE β-AMYLOIDE ET/OU DE SA SYNTHESE
WO1999067219A1 (fr) * 1998-06-22 1999-12-29 Elan Pharmaceuticals, Inc. Composes destines a inhiber la liberation et/ou la synthese du peptide beta-amyloide
US6552013B1 (en) 1998-06-22 2003-04-22 Elan Pharmaceuticals, Inc. Deoxyamino acid compounds, pharmaceutical compositions comprising same, and methods for inhibiting β-amyloid peptide release and/or its synthesis by use of such compounds
US6569851B1 (en) 1998-06-22 2003-05-27 Elan Pharmaceutials, Inc. Cycloalkyl, lactam, lactone and related compounds, pharmaceutical compositions comprising same, and methods for inhibiting β-amyloid peptide release and/or its synthesis by use of such compounds
US6635632B1 (en) 1996-12-23 2003-10-21 Athena Neurosciences, Inc. Cycloalkyl, lactam, lactone and related compounds, pharmaceutical compositions comprising same, and methods for inhibiting β-amyloid peptide release and/or its synthesis by use of such compounds
US6958330B1 (en) 1998-06-22 2005-10-25 Elan Pharmaceuticals, Inc. Polycyclic α-amino-ε-caprolactams and related compounds
US11021514B2 (en) 2016-06-01 2021-06-01 Athira Pharma, Inc. Compounds

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J. MED. CHEM., Volume 36, No. 26, issued 1993, KEVIN T. CHAPMAN et al., "Inhibition of Matrix Metalloproteinases by N-Carboxyalkyl Peptides", pages 4293-4301. *
J. MED. CHEM., Volume 37, No. 19, issued 1994, DIETER SCHOLZ et al., "Inhibitors of HIV-1 Proteinase Containing 2-Heterosubstituted 4-Amino-3-hydroxy-5 Phenylpentanoic Acid: Synthesis, Enzyme Inhibition and Antiviral Activity", pages 3079-3089. *
J. MED. CHEM., Volume 38, No. 25, issued 1995, "3D-Quantitative Structure-Activity Relationship of Human Immunodeficiency Virus Type-1 Proteinase Inhibitors: Comparative Molecular Field Analysis of 2-Heterosubstituted Statine Derivatives- Implications for the Design of Novel Inhibitors", pages 4917-4928. *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6635632B1 (en) 1996-12-23 2003-10-21 Athena Neurosciences, Inc. Cycloalkyl, lactam, lactone and related compounds, pharmaceutical compositions comprising same, and methods for inhibiting β-amyloid peptide release and/or its synthesis by use of such compounds
EP0958829A1 (fr) * 1998-05-21 1999-11-24 Tecnogen S.C.P.A. Utilisation d'un composé peptidique pour le traitement de SLE
US6303577B1 (en) 1998-05-21 2001-10-16 Tecnogen S.C.P.A. Use of a peptide compound in the treatment of systemic lupus erythematosus
WO1999067221A1 (fr) * 1998-06-22 1999-12-29 Elan Pharmaceuticals, Inc. COMPOSES D'INHIBITION DE LA LIBERATION DU PEPTIDE β-AMYLOIDE ET/OU DE SA SYNTHESE
WO1999067219A1 (fr) * 1998-06-22 1999-12-29 Elan Pharmaceuticals, Inc. Composes destines a inhiber la liberation et/ou la synthese du peptide beta-amyloide
US6552013B1 (en) 1998-06-22 2003-04-22 Elan Pharmaceuticals, Inc. Deoxyamino acid compounds, pharmaceutical compositions comprising same, and methods for inhibiting β-amyloid peptide release and/or its synthesis by use of such compounds
US6569851B1 (en) 1998-06-22 2003-05-27 Elan Pharmaceutials, Inc. Cycloalkyl, lactam, lactone and related compounds, pharmaceutical compositions comprising same, and methods for inhibiting β-amyloid peptide release and/or its synthesis by use of such compounds
US6958330B1 (en) 1998-06-22 2005-10-25 Elan Pharmaceuticals, Inc. Polycyclic α-amino-ε-caprolactams and related compounds
US11021514B2 (en) 2016-06-01 2021-06-01 Athira Pharma, Inc. Compounds

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