CA2793683A1 - Peptidomimetics for modulating interleukin-1 receptor - Google Patents

Abstract

The present invention provides, among other things, improved Allosteramers for modulating IL-I receptor activity. In particular, the present invention provides peptidomimetics containing lactams and/or Bab residues. Pharmaceutical compositions of the peptidomimetic compounds of the present invention and methods of using these compositions in the treatment of various disorders are also provided.

Description

REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Patent Application No.
61/162,070, filed March 20, 2009, the contents of which are hereby incorporated by reference in their entireties.

BACKGROUND OF THE INVENTION

[0002] The Interleukin-1 (IL-1) family of polypeptide hormones represents an important class of cytokines which are expressed by a variety of cell types including monocytes (which are the predominant source of IL-1), fibroblasts, endothelial cells, smooth muscle cells, osteoclasts, astrocytes, epithelial cells, T-cells, B-cells and numerous cancer cells. This family of cytokines includes more than 7 distinct but structurally related molecules including IL-la and IL-1(3. Receptors for IL-1 recognize both a and (3 forms and both forms have similar biological properties. The biological properties of IL-1 are numerous and include mediating many immunological and inflammatory responses to infection and injury.

[0003] Two distinct receptor proteins of IL-1 have been cloned and characterized: IL-1RI (Sims, et at. 1989), which generates the biological effects of IL-1; and IL-1RII. In addition, a receptor accessory protein (IL-1RAcP), which is the putative signal-transducing subunit of the receptor complex, has been identified. Generally, one of the first events in signal transduction, following IL-1 binding, is the formation of an IL-1R/IL-1RacP complex which leads to IRAK (IL-1 receptor associated kinase) recruitment to the complex and to a cascade of phosphorylation by kinases, causing the activation of transcriptional factors including NFKB and AP- 1. The IL-1 R/IL-1 RacP complex can also recruit and activate kinases like P13K and Akt and can also lead to the activation of the PLC/PKC
pathway of signalization (Daun and Fenton, 2000).

[0004] Despite its normally beneficial effects on an organism response to infection and injury, actions of IL-1 can be harmful in some instances. For example, inappropriate production or response to IL-1 have been shown in many acute and chronic inflammatory diseases such as rheumatoid arthritis, inflammatory bowel disease (IBD), osteoarthritis, psoriasis, septic shock, encephalitis and respiratory distress syndrome. IL-1 has also been shown to play a role in several other illnesses including Alzheimer's disease, periventricular leukomalacia, meningitis, stroke, and a number of autoimmune diseases.

[0005] Generally, Interleukin-1 (IL-1) plays a role in the regulation of inflammation by stimulating generation of inflammatory mediators like IL-6, prostaglandin E2 (PGE2; via the induction the COX-2 and PGE synthase (mPGES) expression) and itself, therefore enhancing the process of inflammation. Another biological activity of IL-1 is to induce proliferation and activation of numerous cell types like T-cells (Cullinan, et at. 1998; Dunne and O'Neill 2003). IL-1 may also increase the level of collagenase in an arthritic joint and has been implicated in the acute and chronic stages of immunopathology in rheumatoid arthritis. IL-1 may be responsible for altering endothelial cell function, directing the chemotaxis of lymphocytes and leucocytes into synovial tissue and inducing the secretion of latent collagenase by chondrocytes and fibroblasts. IL-1 is considered, along with TNF, as the prototype of inflammatory cytokines. However, the effects of IL-1 are not limited to inflammation and this cytokine also plays a role in bone formation and remodeling, insulin secretion and fever induction.

[0006] As a major pro-inflammatory cytokine, IL-1 is a potentially powerful target for therapeutic intervention in diseases like articular cartilage injury such as in arthritis.
Osteoarthritis and rheumatoid arthritis are only second to heart disease for causing work disabilities in North America and their prevalence increase dramatically with age (Hallegua and Weisman 2002).

[0007] Current approaches for treating IL-1 related diseases include the development of soluble receptors, monoclonal antibodies, mimetics of cytokines, antisense techniques and kinase inhibitors. Recently, short peptides known as AllosteramersTM that specifically target the IL-1 receptor activity have been developed. See, U.S. Patent No.
7,432,341, entitled "Cytokine receptor modulators and method of modulating cytokine receptor activity," and U.S. Pub. No. 20060094663 entitled "Interleukin-1 Receptor Antagonists, Compositions, and Methods of Treatment," the entire disclosure of both of which are hereby incorporated by reference.

SUMMARY OF THE INVENTION

[0008] The present invention encompasses the discovery that IL-1R modulatory peptides (e.g., IL-1R AllosteramersTM) can be optimized by incorporating one or more lactams and/or Bab residues into the peptide. Thus, the present invention provides, among other things, peptidomimetics that have improved ability to modulate (e.g., inhibit or activate) IL-1 receptor activity.

[0009] In one aspect, the present invention provides an IL-1R modulatory peptidomimetic comprising one or more lactams and/or Bab residues. In some embodiments, the present invention provides a peptidomimetic comprising one or more lactams and/or Bab residues and at least three (e.g., at least four, five, or six) out of seven contiguous amino acids that appear in an extracellular region of the IL-1 receptor (SEQ ID NO: 1) or IL-1 receptor accessory protein (IL-1RacP) (SEQ ID NO:2), wherein the at least three (e.g., at least four, five, or six) amino acids maintain their relative positions as they appear in the extracellular region of the IL-1 receptor or IL-1 receptor accessory protein (IL-1RacP). In other embodiments, the present invention provides a peptidomimetic comprising one or more lactams and/or Bab residues and at least three (e.g., at least four, five, or six) out of seven contiguous amino acids that appear in an extracellular region of the IL-1 receptor (SEQ ID
NO: l) or IL-1 receptor accessory protein (IL-1RacP) (SEQ ID NO:2), wherein the at least three (e.g., at least four, five, or six) amino acids maintain their relative positions, but in the inverse configuration, as they appear in the extracellular region of the IL-1 receptor or IL-1 receptor accessory protein (IL-1RacP).

[0010] In some embodiments, the present invention provides a peptidomimetic comprising one or more lactams and/or Bab residues and at least three (e.g., at least four, five, or six) amino acids from any one peptide selected from SEQ ID NO:3-40, wherein the at least three (e.g., at least four, five, or six) amino acids maintain their relative positions as they appear in said peptide sequence. In some embodiments, the present invention provides a peptidomimetic comprising one or more lactams and/or Bab residues and at least three (e.g., at least four, five, or six) amino acids from RYTVELA (SEQ ID NO: 12) wherein the at least three (e.g., at least four, five, or six) amino acids maintain their relative positions as they appear in RYTVELA (SEQ ID NO:12).

[0011] In some embodiments, lactams suitable for the present invention have the structure of formula (I) or (II), as defined and described herein. In some embodiments, lactams suitable for the present invention are selected from the group consisting of alpha-amino-gamma-lactam (Agl), beta-amino-gamma-lactam (Bgl), beta-hydroxy-alpha-amino-gamma-lactam (Agl(4-OH)), and combination thereof In some embodiments, lactams suitable for the present invention contain haa, I9aa, or Qaa.

[0012] In some embodiments, the present invention provides a peptidomimetic modified from any one of SEQ ID NO:3-40, wherein the peptidomimetic comprises at least three (e.g., at least four, five or six) amino acids from the corresponding peptide sequence and a lactam and/or Bab residue replacement or insertion. In some embodiments, the at least three (e.g., at least four, five or six) amino acids maintain their relative positions as they appear in the corresponding peptide sequence. In some embodiments, the invention provides peptidomimetics modified from any one of peptide as shown in Tables 1 and 2 (e.g., SEQ ID
NOs:3-60), wherein the modification comprises a lactam replacement or insertion. In some embodiments, a lactam suitable for the present invention is selected from the group consisting of alpha-amino-gamma-lactam (Agl), beta-amino-gamma-lactam (Bgl), beta-hydroxy-alpha-amino-gamma-lactam (Agl(4-OH)), and combination thereof In some embodiments, a lactam suitable for the present invention contains haa, I9aa, or Qaa.

[0013] In some embodiments, a peptidomimetic of the present invention comprises at least one D-amino acid. In some embodiments, all of the amino acids present in a peptidomimetic of the present invention are D-amino acids.

[0014] In some embodiments, a peptidomimetic of the present invention comprises one or more modifications to increase protease resistance, serum stability and/or bioavailability, such as N- and/or C-terminal acetylation, glycosylation, biotinylation, amidation, substitution with D-amino acid or unnatural amino acid, and/or cyclization of the peptide.

[0015] In another aspect, the present invention provides a method of modifying an IL-1R modulatory peptide (e.g., AllosteramerTM). In some embodiments, a method according to the invention includes steps of. (a) providing a peptide that modulates the IL-1R activity; (b) modifying the peptide by introducing one or more lactams and/or Bab residues into the peptide; and (c) testing the IL-1R modulatory activity of the modified peptide. In some embodiments, the peptide modulates the IL-1R activity non-competitively. In some embodiments, the peptide is a Negative Allosteric Modulator (NAM) of the IL-1R
activity.
In some embodiments, the peptide is a Positive Allosteric Modulator (PAM) of the IL-1R
activity. In some embodiments, the peptide is both an NAM and a PAM of the IL-activity. In some embodiments, a method according to the invention further includes a step of identifying a modified peptide having improved ability to modulate (e.g., inhibit or activate) the IL-1R activity as compared to a control (e.g., the corresponding unmodified parent peptide).

[0016] The present invention also encompasses any IL-1R modulatory peptide and modified IL-1R modulatory peptide according to various methods described herein. In some embodiments, the invention provides a peptidomimetic having a sequence of any one of SEQ
ID NO:61-105.

[0017] The invention further relates to pharmaceutical compositions containing various peptides or peptidomimetics described herein and their uses. For example, the present invention provides a method of inhibiting the activity of an IL-1 receptor in a cell by contacting the cell with a peptide or peptidomimetic described herein. The present invention further provides a method of treating an IL-1 related disease, disorder or condition (e.g., an inflammatory disease, disorder or condition) by administering to a subject in need of treatment a peptide or peptidomimetic described herein.

[0018] In this application, the use of "or" means "and/or" unless stated otherwise. As used in this application, the term "comprise" and variations of the term, such as "comprising"
and "comprises," are not intended to exclude other additives, components, integers or steps.

[0019] Other features, objects, and advantages of the present invention are apparent in the detailed description, drawings and claims that follow. It should be understood, however, that the detailed description, the drawings, and the claims, while indicating embodiments of the present invention, are given by way of illustration only, not limitation.
Various changes and modifications within the scope of the invention will become apparent to those skilled in the art.

DEFINITIONS

[0020] In order to facilitate the understanding of certain terms used in the specification and claims, a number of definitions are first provided herein below. Additional definitions of these terms and other terms are provided throughout the specification.

[0021] Unless defined otherwise, the scientific and technological terms and nomenclature used herein have the same meaning as commonly understood by a person of ordinary skill to which this invention pertains. Generally, the procedures of cell cultures, infection, molecular biology methods and the like are common methods used in the art. Such standard techniques can be found in reference manuals such as, for example, Ausubel et at., Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001;
and Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, N.Y., 2001.

[0022] In order for the present invention to be more readily understood, certain terms are first defined. Additional definitions for the following terms and other terms are set forth throughout the specification.

[0023] About or approximately: As used herein, the term "approximately" or "about," as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value). The terms "about" and "approximately" are used as equivalents.

[0024] Agonist: As used herein, the term "agonist" refers to a compound that directly or indirectly stimulates, activates or enhances a biological activity of a target protein. For example, an IL-1 agonist is a compound that directly or indirectly stimulates, activates or enhances a biological activity of IL-1 or IL-1R/IL-1RacP. The term "agonist"
also includes potentiators of known compounds with agonist properties. In some embodiments, an agonist can be a peptide or peptidomimetic. In some embodiments, an agonist is a peptide or peptidomimetic that stimulates, activates or enhances an activity of IL-1 or IL-1R/IL-1RacP
without competing with a natural ligand of the IL-1 receptor. Such an agonist is also referred to as an allosteric agonist or a Positive Allosteric Modulator (PAM). In some embodiments, an IL-1 agonist binds to IL-1 or IL-1 R/IL-1 RacP without competing with the binding of a natural ligand.

[0025] Antagonist: As used herein, the term "antagonist" refers to any molecule that directly or indirectly inhibits, suppresses, inactivates and/or decreases an activity of a target protein. For example, an IL-1 antagonist is a compound that directly or indirectly stimulates, activates or enhances an activity of IL-1 or IL-1R/IL-lRacP. The term "antagonist" also includes potentiators of known compounds with antagonistic properties. In some embodiments, an antagonist can be a peptide or peptidomimetic. In some embodiments, an antagonist is a peptide or peptidomimetic that inhibits, suppresses, inactivates and/or decreases an activity of IL-1 or IL-1R/IL-lRacP without competing with a natural ligand of the IL-1 receptor. Such an antagonist is also referred to as an allosteric antagonist or a Negative Allosteric Modulator (NAM). In some embodiments, an IL-1 antagonist binds to IL-1 or IL-1R/IL-1RacP without competing with the binding of a natural ligand.

[0026] Amino acid: As used herein, the term "amino acid," in its broadest sense, refers to any compound and/or substance that can be incorporated into a polypeptide chain.
In some embodiments, "amino acid" refers to a molecule containing both an amino group and a carboxyl group. In certain embodiments, an amino acid is an alpha amino acid. Suitable amino acids include, without limitation, natural alpha-amino acids such as L-isomers of the 20 common naturally occurring alpha-amino acids, D-isomers of naturally occurring alpha-amino acids, unnatural alpha-amino acids, natural beta-amino acids (e.g., beta-alanine), and unnatural beta-amino acids. a,a-Disubstituted amino acids, N-alkyl amino acids, lactic acid and other unconventional amino acids may also be suitable components for the peptides or peptidomimetics of the present invention. Examples of unconventional amino acids include but are not limited to citrulline, ornithine, norvaline, 4-(E)-butenyl-4(R) -methyl-N-methylthreonine (MeBmt), N-methyl-leucine (MeLeu), aminoisobutyric acid, statine, N-methyl-alanine (MeAla). As used herein, the twenty natural amino acids and their abbreviations follow conventional usage. However, unless specifically noted, an amino acid abbreviation should cover both L- and D-isomers.

[0027] Animal: As used herein, the term "animal" refers to any member of the animal kingdom. In some embodiments, "animal" refers to humans, at any stage of development. In some embodiments, "animal" refers to non-human animals, at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and/or worms. In some embodiments, an animal may be a transgenic animal, genetically-engineered animal, and/or a clone.

[0028] Biological activity: As used herein, the term "biological activity,"
when used in the context of IL-1 receptor, or "IL-1R/IL-lRacP activity" or "receptor activity," refers to any detectable biological activity of IL-1 or IL-1R/IL-lRacP gene or protein.
It can include specific biological activity of IL-1R/IL-lRacP proteins in cell signaling. A
suitable biological activity may be measured by PGE2 production, proliferation assays and changes in down stream gene and protein expression (e.g., IL-6, IL-1, COX enzymes). A
suitable biological activity may also include for example, binding ability to, e.g., substrates, interacting proteins and the like. For example, measuring the effect of a test compound on its ability to inhibit or increase (e.g., modulate) IL-1 response or IL-1R binding or interaction, is considered herein as measuring a biological activity of IL-1R according to the present invention. Broadly intra-or inter-molecular binding of the receptor subunits (e.g., IL-1R and IL-1RacP) in the absence vs the presence of the peptide, peptide derivative or peptidomimetic of the invention is yet another example of a biological activity according to the invention.
IL-1R/IL-lRacP biological activity also includes any biochemical measurement of this receptor, conformational changes, phosphorylation status, any downstream effect of the receptor's signaling such as protein phosphorylation (or any other posttranslational modification e.g. ubiquitination, sumolylation, palmytoylation, prenylation etc), kinase effect or any other feature of the protein that can be measured with techniques known in the art.
Finally, IL-1R/IL-1RacP biological activity includes a detectable change in cell architecture, cell proliferation or other cell phenotype that is modulated by the action of a ligand (i.e., 1L-1) on the predetermined receptor.

[0029] Comprising: As used in this specification and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having"
(and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, un-recited elements or method steps. The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one" but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one."

[0030] Control: As used herein, the term "control" has its art-understood meaning of being a standard against which results are compared. Typically, controls are used to augment integrity in experiments by isolating variables in order to make a conclusion about such variables. In some embodiments, a control is a reaction or assay that is performed simultaneously with a test reaction or assay to provide a comparator. In one experiment, the "test" (i.e., the variable being tested) is applied. In the second experiment, the "control," the variable being tested is not applied. In some embodiments, a control is a historical control (i.e., of a test or assay performed previously, or an amount or result that is previously known). In some embodiments, a control is or comprises a printed or otherwise saved record.
A control may be a positive control or a negative control.

[0031] Functional: As used herein, a "functional" biological molecule is a biological molecule in a form in which it exhibits a property and/or activity by which it is characterized.

[0032] Functional derivative: As used herein, the term "functional derivative"
denotes, when used in the context of a functional derivative of an amino acid sequence, refers to a molecule that shares structural similarity and retains a biological activity that is substantially similar to that of the original sequence. A functional derivative or equivalent may be a natural derivative or may be prepared synthetically. Such derivatives include amino acid sequences having substitutions, deletions, or additions of one or more amino acids, provided that the biological activity of the protein is conserved (e.g., it acts as a non-competitive antagonist of the IL-1 receptor). The substituting amino acid generally has chemico-physical properties, which are similar to that of the substituted amino acid. The similar chemico-physical properties include, similarities in charge, bulkiness, hydrophobicity, hydrophylicity and the like. The term "functional derivatives" is intended to include "segments", "variants", "analogs" or "chemical derivatives" of the subject matter of the present invention. In some embodiments, a functional derivative shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the original amino acid sequence.

[0033] IL-1: Unless otherwise noted, "IL-1" refers to either or both IL-l a and IL-1(3.
The term "IL" refers to the broad family of interleukins.

[0034] Inhibiting: The terms "inhibiting," "reducing" or "prevention," or any variation of these terms, when used in the claims and/or the specification include any measurable decrease as compared to a baseline control or complete inhibition of the receptor activity to achieve a desired result. For example, a peptide is said to be inhibiting IL-I
activity when a decrease in PGE2 production is measured following a treatment with the peptides, peptide derivatives or peptidomimetics of the present invention as compared to in the absence of these peptides.

[0035] In vitro: As used herein, the term "in vitro" refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within a multi-cellular organism.

[0036] In vivo: As used herein, the term "in vivo" refers to events that occur within a multi-cellular organism such as a non-human animal.

[0037] Inflammatory disease, disorder or condition: As used herein, the term an "inflammatory disease, disorder or condition" refers to any disease, disorder, or condition in which the immune system abnormally activated or suppressed. In some embodiments, an inflammatory disease, disorder, or condition that can be treated according to the invention is inflammation of the upper and lower respiratory tract, for example, bronchial asthma, allergic asthma, non-allergic asthma, lymphomatous tracheobronchitis, allergic hypersensitivity or a hypersecretion condition, such as chronic bronchitis and cystic fibrosis;
pulmonary fibrosis of various aetiologies (e.g., idiopathic pulmonary fibrosis), chronic obstructive pulmonary disease (COPD), sarcoidosis, allergic and non-allergic rhinitis; allergic or non-allergic urticaria; a skin-related diseases characterized by deregulated inflammation, tissue remodeling, angiogenesis, and neoplasm, a disease of the gastrointestinal tract, such as Crohn's disease, Hirschsprung's disease, diarrhea, malabsorption conditions, and inflammatory conditions; a disorder of the central and peripheral nervous system, such as depression, anxiety states, Parkinson's disease, migraine and other forms of cranial pain, strokes, emesis; a disease of the immune system, such as in the splenic and lymphatic tissues, an autoimmune disease or other immune-related diseases; a disease of the cardiovascular system, such as pulmonary edema, hypertension, atherosclerosis, pre-eclampsia, complex regional pain syndrome type 2, stroke and chronic inflammatory diseases such as arthritis, a bone-related diseases such as rheumatoid arthritis, as well as pain, chronic pain such as fibromyalgia, and other disorders in which the action of neurokinins, tachykinins or other related substances (e.g., hemokinins) are involved in the pathogenesis, pathology, and aetiology.

[0038] Additional examples of inflammatory disorders include acne vulgaris;
acute respiratory distress syndrome; Addison's disease; allergic intraocular inflammatory diseases, ANCA-associated small-vessel vasculitis; ankylosing spondylitis; atopic dermatitis;
autoimmune hemolytic anemia; autoimmune hepatitis; Behcet's disease; Bell's palsy; bullous pemphigoid; cerebral ischaemia; cirrhosis; Cogan's syndrome; contact dermatitis; Cushing's syndrome; dermatomyositis; diabetes mellitus; discoid lupus erythematosus;
lupus nephritis;
eosinophilic fasciitis; erythema nodosum; exfoliative dermatitis; focal glomerulosclerosis;
focal segmental glomerulosclerosis; segmental glomerulosclerosis; giant cell arteritis; gout;
gouty arthritis; graft-versus-host disease; hand eczema; Henoch-Schonlein purpura; herpes gestationis; hirsutism; idiopathic cerato- scleritis; idiopathic thrombocytopenic purpura;
immune thrombocytopenic purpura inflammatory bowel or gastrointestinal disorders, inflammatory dermatoses; lichen planus; lymphomatous tracheobronchitis;
macular edema;
multiple sclerosis; myasthenia gravis; myositis; nonspecific fibrosing lung disease;
osteoarthritis; pancreatitis; pemphigoid gestationis; pemphigus vulgaris;
periodontitis;
polyarteritis nodosa; polymyalgia rheumatica; pruritus scroti;
pruritis/inflammation, psoriasis;
psoriatic arthritis; pulmonary histoplasmosis; relapsing polychondritis;
rosacea caused by sarcoidosis; rosacea caused by scleroderma; rosacea caused by Sweet's syndrome; rosacea caused by systemic lupus erythematosus; rosacea caused by urticaria; rosacea caused by zoster-associated pain; sarcoidosis; scleroderma; septic shock syndrome;
shoulder tendinitis or bursitis; Sjogren's syndrome; Still's disease; Sweet's disease; systemic lupus erythematosus; systemic sclerosis; Takayasu's arteritis; temporal arteritis;
toxic epidermal necrolysis; transplant-rejection and transplant-rejection-related syndromes;
tuberculosis;
type-1 diabetes; ulcerative colitis; uveitis; vasculitis; and Wegener's granulomatosis.
Desirably the autoimmune disorder is inflammatory bowel disease, an inflammatory skin disorder such as psoriasis, or multiple sclerosis.

[0039] Isolated: As used herein, the term "isolated" refers to a substance and/or entity that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature and/or in an experimental setting), and/or (2) produced, prepared, and/or manufactured by the hand of man.
Isolated substances and/or entities may be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, substantially 100%, or 100% of the other components with which they were initially associated. In some embodiments, isolated agents are more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, substantially 100%, or 100% pure. As used herein, a substance is "pure" if it is substantially free of other components. As used herein, the term "isolated cell" refers to a cell not contained in a multi-cellular organism.

[0040] Modulator: As used herein, the term "modulator" refers to a compound that alters or elicits an activity. For example, the presence of a modulator may result in an increase or decrease in the magnitude of a certain activity compared to the magnitude of the activity in the absence of the modulator. In certain embodiments, a modulator is an inhibitor or antagonist, which decreases the magnitude of one or more activities. In certain embodiments, an inhibitor completely prevents one or more biological activities. In certain embodiments, a modulator is an activator or agonist, which increases the magnitude of at least one activity. In certain embodiments the presence of a modulator results in an activity that does not occur in the absence of the modulator. As used herein, the terms "inhibiting,"
"reducing," "preventing," or "antagonizing," or any variations of these terms as used herein, refer to a measurable decrease of a biological activity. In some embodiments, the decrease is a 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% reduction in the biological activity relative to a control. As used herein, the terms "stimulating," "increasing,"
or "agonizing," or any variations of these terms as used herein, refer to a measurable increase of a biological activity. In some embodiments, the increase is a 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% increase in the biological activity relative to a control.

[0041] Molecule: As used herein, the terms "molecule", "compound", "agent" or "ligand" are used interchangeably and broadly to refer to natural, synthetic or semi-synthetic molecules or compounds. The term "molecule" therefore denotes for example chemicals, macromolecules, cell or tissue extracts (from plants or animals) and the like.
Non-limiting examples of molecules include peptides, antibodies, carbohydrates and pharmaceutical agents. The agents can be selected and screened by a variety of means including random screening, rational selection and by rational design using for example protein or ligand modeling methods such as computer modeling. The terms "rationally selected" or "rationally designed" are meant to define compounds which have been chosen based on the configuration of interacting domains of the present invention or on the configuration of antagonist peptides and/or peptidomimetics of the present invention. As will be understood by the person of ordinary skill, macromolecules having non-naturally occurring modifications are also within the scope of the term "molecule." For example, peptidomimetics, well known in the pharmaceutical industry and generally referred to as peptide analogs can be generated by modeling as mentioned above. Similarly, in a preferred embodiment, the polypeptides of the present invention are modified to enhance their stability. In some cases this modification enhances the biological activity of the peptide. The molecules identified in accordance with the teachings of the present invention have a therapeutic value in diseases or conditions in which the physiology or homeostasis of the cell and/or tissue is compromised by a defect in IL-1 production or response. Non-limiting examples of such diseases or conditions include acute and chronic inflammatory diseases such as rheumatoid arthritis, inflammatory bowel disease (IBD), osteoarthritis, psoriasis, septic shock, encephalitis and respiratory distress syndrome, Alzheimer's disease, periventricular leukomalacia, meningitis, stroke, and a number of autoimmune diseases. It will be understood that the compounds are herein described interchangeably as "API-X" "TTI-X" or simply by the number of the compound (for example: "101.10", "API-101.10" or "TTI-101.10").

[0042] Peptides: As used herein, the term "peptides" refers to macromolecules which comprise a multiplicity of amino or imino acids (or their equivalents) in peptide linkage. In the polypeptide or peptide notation used herein, the left-hand direction is the amino-terminal direction and the right-hand direction is the carboxy-terminal direction, in accordance with standard usage and convention. Peptides may include moieties other than amino acids (e.g., may be glycoproteins, proteoglycans, etc.) and/or may be otherwise processed or modified.
Peptides may contain L-amino acids, D-amino acids, or both and may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, e.g., terminal acetylation, amidation, glycosylation, biotinylation, substitution with D-amino acid or unnatural amino acid, and/or cyclization of the peptide. In some embodiments, peptides may comprise natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof. As used herein, a "short peptide" refers to any peptide containing up to 25 amino acids (e.g., up to 20, 15, 12, 10, 9, 8, 7, 6, 5, 4, or 3). In some embodiments, a short peptide contains 5-25 amino acids. As used in this application, peptides also include peptidomimetics unless indicated otherwise.

[0043] Peptidomimetic: Herein the terminologies "mimic," "mimetic,"
peptidomimetic" and the like are used herein interchangeably.

[0044] Pharmaceutically acceptable carrier: As used herein, the term "pharmaceutically acceptable carrier" refers to a non-toxic carrier medium that does not destroy the pharmacological activity of the compound with which it is formulated.

[0045] Purified: As used herein, the term "purified" refers to a molecule (e.g. IL-1 receptor, peptides, peptide derivatives, peptidomimetics, nucleic acids, proteins etc.) having been separated from a component of the composition in which it was originally present.
Thus, for example, a "purified IL-1 receptor" has been purified to a level not found in nature.
A "substantially pure" molecule is a molecule that is lacking in most other components (e.g., 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 100% free of contaminants). By opposition, the term "crude" means molecules that have not been separated from the components of the original composition in which it was present. Therefore, the term "separating" or "purifying" refers to methods by which one or more components of the biological sample are removed from one or more other components of the sample.
Sample components include nucleic acids in a generally aqueous solution that may include other components, such as proteins, carbohydrates, or lipids. A separating or purifying step preferably removes at least about 70% (e.g., 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 100%), more preferably at least about 90% (e.g., 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%) and, even more preferably, at least about 95% (e.g., 95, 96, 97, 98, 99, 100%) of the other components present in the sample from the desired component. For the sake of brevity, the units (e.g. 66, 67...81, 82,...91, 92%....) have not systematically been recited but are considered, nevertheless, within the scope of the present invention.

[0046] Reverse peptide: As used herein, the term "reverse peptide" refers to peptides arranged in a reverse sequence relative to a corresponding region of the IL-1R
or IL-lRacP
protein. Likewise, the term "reverse-D peptide" refers herein to peptides containing D-amino acids, arranged in a reverse sequence relative to a corresponding peptide containing L-amino acids. For example, the C-terminal residue of an L-peptide becomes N-terminal for the reverse D-peptide, and so forth. Without wishing to be bound by theory, it is contemplated that reverse D-peptides may often retain the same tertiary conformation and therefore the same activity, as the L-amino acid peptides. It is also contemplated that a D-peptide may be more stable and resistant to enzymatic degradation in vitro and in vivo.
Therefore, a D-peptide may have greater therapeutic efficacy than the corresponding L-peptide.

[0047] Subject: As used herein, the term "subject" or "patient" refers to an animal, preferably a mammal, most preferably a human who is the object of treatment, observation or experiment.

[0048] Substantially: As used herein, the term "substantially" refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result. The term "substantially" is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0049] Suffering from: An individual who is "suffering from" a disease, disorder, and/or condition has been diagnosed with or displays one or more symptoms of the disease, disorder, and/or condition.

[0050] Susceptible to: An individual who is "susceptible to" a disease, disorder, and/or condition has not been diagnosed with the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition may not exhibit symptoms of the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition will develop the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition will not develop the disease, disorder, and/or condition.

[0051] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" of a therapeutic agent means an amount that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and/or condition, to treat, diagnose, prevent, and/or delay the onset of the symptom(s) of the disease, disorder, and/or condition. It will be appreciated by those of ordinary skill in the art that a therapeutically effective amount is typically administered via a dosing regimen comprising at least one unit dose.

[0052] Therapeutic agent: As used herein, the phrase "therapeutic agent"
refers to any agent that, when administered to a subject, has a therapeutic effect and/or elicits a desired biological and/or pharmacological effect. In some embodiments, a therapeutic agent of the invention refers to a peptide inhibitor or derivatives thereof according to the invention.

[0053] Treating: As used herein, the term "treat," "treatment," or "treating"
refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of and/or reduce incidence of one or more symptoms or features of a particular disease, disorder, and/or condition. Treatment may be administered to a subject who does not exhibit signs of a disease and/or exhibits only early signs of the disease for the purpose of decreasing the risk of developing pathology associated with the disease.

[0054] Definitions of specific functional groups and chemical terms are described in more detail below. For purposes of this invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March March's Advanced Organic Chemistry, 5th Edition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; Carruthers, Some Modern Methods of Organic Synthesis, 3rd Edition, Cambridge University Press, Cambridge, 1987;
the entire contents of each of which are incorporated herein by reference.

[0055] Unless otherwise stated, structures depicted herein are also meant to include all isomeric (e.g., enantiomeric, diastereomeric, and geometric (or conformational)) forms of the structure; for example, the R and S configurations for each asymmetric center, Z and E
double bond isomers, and Z and E conformational isomers. Therefore, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the present compounds are within the scope of the invention. Unless otherwise stated, all tautomeric forms of the compounds of the invention are within the scope of the invention.
Additionally, unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures including the replacement of hydrogen by deuterium or tritium, or the replacement of a carbon by a 13C- or 14C-enriched carbon are within the scope of this invention. Such compounds are useful, for example, as analytical tools, as probes in biological assays, or as therapeutic agents in accordance with the present invention.

[0056] Where a particular enantiomer is preferred, it may, in some embodiments be provided substantially free of the corresponding enantiomer, and may also be referred to as "optically enriched." "Optically-enriched," as used herein, means that the compound is made up of a significantly greater proportion of one enantiomer. In certain embodiments the compound is made up of at least about 90% by weight of a preferred enantiomer.
In other embodiments the compound is made up of at least about 95%, 98%, or 99% by weight of a preferred enantiomer. Preferred enantiomers may be isolated from racemic mixtures by any method known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts or prepared by asymmetric syntheses. See, for example, Jacques et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen, et al., Tetrahedron 33:2725 (1977); Eliel, E.L. Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); Wilen, S.H. Tables of Resolving Agents and Optical Resolutions p. 268 (E.L. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN 1972).

[0057] Alkenyl: The term "alkenyl," as used herein, denotes a monovalent group derived from a straight- or branched-chain aliphatic moiety having at least one carbon-carbon double bond by the removal of a single hydrogen atom. In certain embodiments, alkenyl contains 2-6 carbon atoms. In certain embodiments, alkenyl contains 2-5 carbon atoms. In some embodiments, alkenyl contains 2-4 carbon atoms. In another embodiment, alkenyl contains 2-3 carbon atoms. Alkenyl groups include, for example, ethenyl ("vinyl"), propenyl ("allyl"), butenyl, 1-methyl-2-buten-1-yl, and the like.

[0058] Alkyl: The term "alkyl," as used herein, refers to a monovalent saturated, straight- or branched-chain hydrocarbon radical derived from an aliphatic moiety containing between one and six carbon atoms by removal of a single hydrogen atom. In some embodiments, alkyl contains 1-5 carbon atoms. In another embodiment, alkyl contains 1-4 carbon atoms. In still other embodiments, alkyl contains 1-3 carbon atoms. In yet another embodiment, alkyl contains 1-2 carbons. Examples of alkyl radicals include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, iso-butyl, sec-butyl, sec-pentyl, iso-pentyl, tert-butyl, n-pentyl, neopentyl, n-hexyl, sec-hexyl, n-heptyl, n-octyl, n-decyl, n-undecyl, dodecyl, and the like.

[0059] Alkylene: The term "alkylene" refers to a bivalent alkyl group. An "alkylene chain" is a polymethylene group, i.e., -(CH2)õ-, wherein n is a positive integer, preferably from 1 to 6, from 1 to 4, from 1 to 3, from 1 to 2, or from 2 to 3. A
substituted alkylene chain is a polymethylene group in which one or more methylene hydrogen atoms are replaced with a substituent. Suitable substituents include those described below for a substituted aliphatic group.

[0060] Alkynyl: The term "alkynyl," as used herein, refers to a monovalent group derived from a straight- or branched-chain aliphatic moiety having at least one carbon-carbon triple bond by the removal of a single hydrogen atom. In certain embodiments, alkynyl contains 2-6 carbon atoms. In certain embodiments, alkynyl contains 2-5 carbon atoms. In some embodiments, alkynyl contains 2-4 carbon atoms. In another embodiment, alkynyl contains 2-3 carbon atoms. Representative alkynyl groups include, but are not limited to, ethynyl, 2-propynyl ("propargyl"), 1-propynyl, and the like.

[0061] Aliphatic: The term "aliphatic" or "aliphatic group", as used herein, denotes a hydrocarbon moiety that may be straight-chain (i.e., unbranched), branched, or cyclic (including fused, bridging, and spiro-fused polycyclic) and may be completely saturated or may contain one or more units of unsaturation, but which is not aromatic.
Unless otherwise specified, aliphatic groups contain 1-6 carbon atoms. In some embodiments, aliphatic groups contain 1-4 carbon atoms, and in yet other embodiments aliphatic groups contain 1-3 carbon atoms. Suitable aliphatic groups include, but are not limited to, linear or branched, alkyl, alkenyl, and alkynyl groups, and hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.

[0062] Aryl: The term "aryl" used alone or as part of a larger moiety as in "aralkyl", "aralkoxy", or "aryloxyalkyl", refers to monocyclic and bicyclic ring systems having a total of five to 10 ring members, wherein at least one ring in the system is aromatic and wherein each ring in the system contains three to seven ring members. The term "aryl"
may be used interchangeably with the term "aryl ring". In certain embodiments of the present invention, "aryl" refers to an aromatic ring system which includes, but not limited to, phenyl, biphenyl, naphthyl, anthracyl and the like, which may bear one or more substituents.
Also included within the scope of the term "aryl", as it is used herein, is a group in which an aromatic ring is fused to one or more non-aromatic rings, such as indanyl, phthalimidyl, naphthimidyl, phenantriidinyl, or tetrahydronaphthyl, and the like.

[0063] Cycloalkylene: As used herein, the term "cycloalkylene" refers to a bivalent cycloalkyl group. In certain embodiments, a cycloalkylene group is a 1,1-cycloalkylene group (i.e., a spiro-fused ring). Exemplary 1,1-cycloalkylene groups include .
In other embodiments, a cycloalkylene group is a 1,2-cycloalkylene group or a 1,3-X, d cycloalkylene group. Exemplary 1,2-cycloalkylene groups include and [0064] Cycloaliphatic: The terms "cycloaliphatic", "carbocycle", "carbocyclyl", "carbocyclo", or "carbocyclic", used alone or as part of a larger moiety, refer to a saturated or partially unsaturated cyclic aliphatic monocyclic or bicyclic ring systems, as described herein, having from 3 to 10 members, wherein the aliphatic ring system is optionally substituted as defined above and described herein. Cycloaliphatic groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl, and cyclooctadienyl. In some embodiments, the cycloalkyl has 3-6 carbons. The terms "cycloaliphatic", "carbocycle", "carbocyclyl", "carbocyclo", or "carbocyclic" also include aliphatic rings that are fused to one or more aromatic or nonaromatic rings, such as decahydronaphthyl, tetrahydronaphthyl, decalin, or bicyclo[2.2.2]octane, where the radical or point of attachment is on an aliphatic ring.

[0065] Halogen: The terms "halo" and "halogen" as used herein refer to an atom selected from fluorine (fluoro, -F), chlorine (chloro, -Cl), bromine (bromo, -Br), and iodine (iodo, -I).

[0066] Heteroaryl: The terms "heteroaryl" and "heteroar-", used alone or as part of a larger moiety, e.g., "heteroaralkyl", or "heteroaralkoxy", refer to groups having 5 to 10 ring atoms, preferably 5, 6, or 9 ring atoms; having 6, 10, or 14 it electrons shared in a cyclic array; and having, in addition to carbon atoms, from one to five heteroatoms.
The term "heteroatom" refers to nitrogen, oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur, and any quaternized form of a basic nitrogen. Heteroaryl groups include, without limitation, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, and pteridinyl.
The terms "heteroaryl" and "heteroar-", as used herein, also include groups in which a heteroaromatic ring is fused to one or more aryl, cycloaliphatic, or heterocyclyl rings, where the radical or point of attachment is on the heteroaromatic ring. Nonlimiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and pyrido[2,3-b]-1,4-oxazin-3(4H)-one. A
heteroaryl group may be mono- or bicyclic. The term "heteroaryl" may be used interchangeably with the terms "heteroaryl ring", "heteroaryl group", or "heteroaromatic", any of which terms include rings that are optionally substituted. The term "heteroaralkyl"
refers to an alkyl group substituted by a heteroaryl, wherein the alkyl and heteroaryl portions independently are optionally substituted.

[0067] Heteroatom: The term "heteroatom" means one or more of oxygen, sulfur, nitrogen, phosphorus, or silicon (including, any oxidized form of nitrogen, sulfur, phosphorus, or silicon; the quaternized form of any basic nitrogen or; a substitutable nitrogen of a heterocyclic ring, for example N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) or NR-'- (as in N-substituted pyrrolidinyl)).

[0068] Heterocycle: As used herein, the terms "heterocycle", "heterocyclyl", "heterocyclic radical", and "heterocyclic ring" are used interchangeably and refer to a stable 4- to 7-membered monocyclic or 7-10-membered bicyclic heterocyclic moiety that is either saturated or partially unsaturated, and having, in addition to carbon atoms, one or more, preferably one to four, heteroatoms, as defined above. When used in reference to a ring atom of a heterocycle, the term "nitrogen" includes a substituted nitrogen. As an example, in a saturated or partially unsaturated ring having 0-3 heteroatoms selected from oxygen, sulfur or nitrogen, the nitrogen may be N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl), or +NR (as in N-substituted pyrrolidinyl).

[0069] A heterocyclic ring can be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure and any of the ring atoms can be optionally substituted. Examples of such saturated or partially unsaturated heterocyclic radicals include, without limitation, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, pyrrolidonyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl. The terms "heterocycle", "heterocyclyl", "heterocyclyl ring", "heterocyclic group", "heterocyclic moiety", and "heterocyclic radical", are used interchangeably herein, and also include groups in which a heterocyclyl ring is fused to one or more aryl, heteroaryl, or cycloaliphatic rings, such as indolinyl, 3H-indolyl, chromanyl, phenanthridinyl, 2-azabicyclo [2.2. 1 ]heptanyl, octahydroindolyl, or tetrahydroquinolinyl, where the radical or point of attachment is on the heterocyclyl ring. A
heterocyclyl group may be mono- or bicyclic. The term "heterocyclylalkyl" refers to an alkyl group substituted by a heterocyclyl, wherein the alkyl and heterocyclyl portions independently are optionally substituted.

[0070] Optionally substituted: As described herein, compounds of the invention may contain "optionally substituted" moieties. In general, the term "substituted", whether preceded by the term "optionally" or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. Unless otherwise indicated, an "optionally substituted" group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at each position. Combinations of substituents envisioned under this invention are preferably those that result in the formation of stable or chemically feasible compounds. The term "stable", as used herein, refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein.

[0071] Suitable monovalent substituents on a substitutable carbon atom of an "optionally substituted" group are independently halogen; -(CH2)o-4R ; -(CH2)0-40R ; -0-(CH2)0_4C(O)OR ; -(CH2)0-4CHOR )2; -(CH2)0_4SR ; -(CH2)0-4Ph, which may be substituted with R ; -(CH2)0 40(CH2)0_1Ph which may be substituted with R ; -CH=CHPh, which may be substituted with R ; -NO2; -CN; -N3; -(CH2)o-4N(R )2; -(CH2)o-4N(R )C(O)R ;
-N(R )C(S)R ; -(CH2)o-4N(R )C(O)NR 2; -N(R )C(S)NR 2; -(CH2)o-4N(R )C(O)OR ;
-N(R )N(R )C(O)R ; -N(R )N(R )C(O)NR 2; -N(R )N(R )C(O)OR ; -(CH2)0 4C(O)R ;
-C(S)R ; -(CH2)0_4C(O)OR ; -(CH2)0C(O)SR ; -(CH2)0C(O)OSiR 3; -(CH2)0_40C(O)R
; -OC(O)(CH2)0_4SR-, SC(S)SR ; -(CH2)0SC(O)R ; -(CH2)0_4C(O)NR 2; -C(S)NR 2; -C(S)SR ; -SC(S)SR , -(CH2)0_40C(O)NR 2; -C(O)N(OR )R ; -C(O)C(O)R ;
-C(O)CH2C(O)R ; -C(NOR )R ; -(CH2)0SSR ; -(CH2)0S(0)2R ; -(CH2)0S(0)20R ;
-(CH2)00S(0)2R ; -S(0)2NR 2; -(CH2)0_4S(O)R ; -N(R )S(0)2NR 2; -N(R )S(0)2R ;
-N(OR )R ; -C(NH)NR 2; -P(0)2R ; -P(O)R 2; -OP(O)R 2; -OP(O)(OR )2; -SiR 3; -(C1_4 straight or branched alkylene)O-N(R )2; or -(C1_4 straight or branched alkylene)C(O)O-N(R )2, wherein each R may be substituted as defined below and is independently hydrogen, C1-6 aliphatic, -CH2Ph, -O(CH2)o-IPh, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of R , taken together with their intervening atom(s), form a 3-12-membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, which may be substituted as defined below.

[0072] Suitable monovalent substituents on R (or the ring formed by taking two independent occurrences of R together with their intervening atoms), are independently halogen, -(CH2)0-2R', -(haloR'), -(CH2)0 20H, -(CH2)0_20R', -(CH2)0-2CH(OR')2;

-O(haloR'), -CN, -N3, -(CH2)02C(O)R -(CH2)02C(O)OH, -(CH2)02C(O)OR', -(CH2)0_ 2SR', -(CH2)0_2SH, -(CH2)02NH2, -(CH2)0_2NHR', -(CH2)02NR'2, -NO2, -SiR'3, -OSiR'3, -C(O)SR', -(C1.4 straight or branched alkylene)C(O)OR', or -SSR' wherein each R' is unsubstituted or where preceded by "halo" is substituted only with one or more halogens, and is independently selected from C1-4 aliphatic, -CH2Ph, -O(CH2)o-IPh, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents on a saturated carbon atom of R include =0 and =S.

[0073] Suitable divalent substituents on a saturated carbon atom of an "optionally substituted" group include the following: =O, =S, =NNR*2, =NNHC(O)R*, =NNHC(O)OR*, =NNHS(0)2R*, =NR*, =NOR*, -O(C(R*2))2_30-, or -S(C(R*2))2_3S-, wherein each independent occurrence of R* is selected from hydrogen, Ci_6 aliphatic which may be substituted as defined below, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents that are bound to vicinal substitutable carbons of an "optionally substituted" group include: -O(CR*2)2_30-, wherein each independent occurrence of R* is selected from hydrogen, Ci_6 aliphatic which may be substituted as defined below, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0074] Suitable substituents on the aliphatic group of R* include halogen, -R', -(haloR'), -OH, -OR', -O(haloR'), -CN, -C(O)OH, -C(O)OR', -NH2, -NHR', -NR'2, or -NO2, each R' is unsubstituted or where preceded by "halo" is substituted only with one or more halogens, and is independently Ci_4 aliphatic, -CH2Ph, -O(CH2)o_1Ph, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0075] Suitable substituents on a substitutable nitrogen of an "optionally substituted"
group include -Rt, -NRt2, -C(O)Rt, -C(O)ORt, -C(O)C(O)Rt, -C(O)CH2C(O)Rt, -S(O)2Rt, -S(O)2NRt2, -C(S)NRt2, -C(NH)NRt2, or -N(R)S(O)2Rt; wherein each Rt is independently hydrogen, Ci_6 aliphatic which may be substituted as defined below, unsubstituted -OPh, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of Rt, taken together with their intervening atom(s) form an unsubstituted 3-12-membered saturated, partially unsaturated, or aryl mono-or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0076] Suitable substituents on the aliphatic group of Rt are independently halogen, -R', -(haloR'), -OH, -OR', -O(haloR'), -CN, -C(O)OH, -C(O)OR', -NH2, -NHR', -NR'2, or -NO2, each R' is unsubstituted or where preceded by "halo" is substituted only with one or more halogens, and is independently Ci_4aliphatic, -CH2Ph, -O(CH2)o_1Ph, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0077] Partially unsaturated: As used herein, the term "partially unsaturated"
refers to a ring moiety that includes at least one double or triple bond between ring atoms but is not aromatic. The term "partially unsaturated" is intended to encompass rings having multiple sites of unsaturation, but is not intended to include aryl or heteroaryl moieties, as herein defined.

[0078] Unsaturated: The term "unsaturated", as used herein, means that a moiety has one or more units of unsaturation.

BRIEF DESCRIPTION OF THE DRAWINGS

[0079] The drawings are for illustration purposes only, not for limitation.

[0080] Figure 1 is an exemplary graph showing the inhibition of IL-1 induced cell proliferation by peptides 54-59.

[0081] Figure 2 is an exemplary graph showing the inhibition of IL-1 induced cell proliferation by compounds 75-78.

[0082] Figure 3 illustrates activity of exemplary peptidomimetics 101.164-101.167.
DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0083] The present invention provides, among other things, peptidomimetics containing one or more lactams and/or Bab residues that can effectively modulate (e.g., inhibit or activate) the biological activity of IL-1 R. In some embodiments, peptidomimetics of the present invention are modified from IL-1R modulatory peptides (such as AllosteramersTM) by lactam and/or Bab replacement or insertion.
Peptidomimetics according to the present invention can be used to treat various IL-1 related diseases, disorders or conditions.

[0084] Various aspects of the invention are described in detail in the following sections. The use of sections is not meant to limit the invention. Each section can apply to any aspect of the invention. In this application, the use of "or" means "and/or" unless stated otherwise.

IL-IR Modulatory Peptides [0085] Various IL-1R modulatory peptides may be optimized according to the present invention. Suitable IL-1R modulatory peptides include antagonistic peptides or agonistic peptides. As used herein, an IL-1R antagonistic peptide refers to any peptide that can directly or indirectly inhibit, suppress, inactivate and/or decrease the receptor activity of IL-1R and/or IL-lRacP. As used herein, an IL-1R agonistic peptide refers to any peptide that can directly or indirectly activates, enhance, stimulate and/or increase the receptor activity of IL-1R
and/or IL-lRacP. In particular, suitable IL-1R modulatory peptides may be AllosteramersTM
(short peptides composed of 5-25 amino acids derived from body's own proteins that can interact with the protein from which it is derived, or with its associated proteins, without competing with a natural ligand) that interact with a region of the extracellular domain of the IL-1R/IL-lRacP receptor complex. Typically, suitable peptides for the invention modulate the IL-1R receptor activity non-competitively (i.e., without competing with a natural ligand of the receptor). In some embodiments, a suitable peptides for the invention is a Negative Allosteric Modulator (NAM) of the IL-1R activity. In some embodiments, a suitable peptide for the invention is a Positive Allosteric Modulator (PAM) of the IL-1R
activity. In some embodiments, a suitable peptide is both an NAM and a PAM of the IL-1R
activity.

[0086] In some embodiments, suitable IL-1R modulatory peptides may be strategically designed to interact with at least one of an extracellular flexible region of the IL-1R/IL-1RacP complex that are responsible for oligomerization, and that are important for the appropriate conformation of the IL-1 receptor which enables signaling. Such flexible regions include, but are not limited to, juxtamembranous regions, regions containing a helix, R sheet, loops and/or R turns, regions between domains, regions between two R chains, and combinations thereof. Thus, in some embodiments, IL-1R modulatory peptides are designed based on amino acid sequences that appear within such flexible regions. This approach is known as Module X technology and is further described in U.S. Patent No.
7,432,341, entitled "Cytokine receptor modulators and method of modulating cytokine receptor activity," and in U.S. Pub. No. 20060094663 entitled "Interleukin-1 Receptor Antagonists, Compositions, and Methods of Treatment," the entire disclosure of both of which are hereby incorporated by reference.

[0087] In some embodiments, suitable IL-1R modulatory peptides may be designed simply based on the primary linear amino acid sequence of extracellular or transmembrane regions of the IL-1R and IL-1RacP protein without first characterizing any tertiary or secondary structure of the receptor. This method is also referred to as the Module X
"walking" method and is further described in U.S. Provisional Application Serial No.
61/172,533, entitled "TNF receptor antagonists and uses thereof," the entire disclosure of which is hereby incorporated by reference.

[0088] For example, suitable IL-1R modulatory peptides may be designed based on contiguous amino acids that appear in the extracellular and/or transmembrane regions of the IL-1 receptor. The amino acid sequence of the IL-1R is shown below. The projected transmembrane domain is underlined and bolded. The extracellular region is N-terminal to the transmembrane domain.

MKVLLRLICFIALLISSLEADKCKEREEKIILVSSANEIDVRPCPLNPNEHKGTITWYKD
DSKTPVSTEQASRIHQHKEKLWFVPAKVEDSGHYYCVVRNSSYCLRIKISAKFVENE
PNLCYNAQAIFKQKLPVAGDGGLV CPYMEFFKNENNELPKLQ WYKDCKPLLLDNIH
FSGVKDRLIVMNVAEKHRGNYTCHASYTYLGKQYPITRVIEFITLEENKPTRPVIVSP
ANETMEVDLGSQIQLICNVTGQLSDIAYWKWNGSVIDEDDPVLGEDYYSVENPANK
RRSTLITVLNI SEIE SRFYKHPFTCFAKNTHGIDAAYIQLIYPVTNFQKHMIGIC VTLT
VIIVCSVFIYKIFKIDIVLWYRDSCYDFLPIKASDGKTYDAYILYPKTVGEGSTSDCDIF
VFKVLPEVLEKQCGYKLFIYGRDDYVGEDIVEVINENVKKSRRLIIILVRETSGFSWLG
GSSEEQIAMYNALVQDGIKVVLLELEKIQDYEKMPESIKFIKQKHGAIRWSGDFTQGP
QSAKTRFWKNVRYHMPVQRRSPSSKHQLLSPATKEKLQREAHVPLG (SEQ ID NO: 1) [0089] In some embodiments, suitable IL-1R modulatory peptides may be designed based on contiguous amino acids that appear in the extracellular regions of the IL-1RacP.
The amino acid sequence of an exemplary isoform of the IL-1RacP protein is shown below.
The sequences of other isoforms are known in the art. The projected transmembrane domain is underlined and bolded. The extracellular region is N-terminal to the transmembrane domain.

MTLLWCVV SLYFYGILQ SDASERCDDWGLDTMRQIQVFEDEPARIKCPLFEHFLKFN
YSTAHSAGLTLIWYWTRQDRDLEEPINFRLPENRISKEKDVLWFRPTLLNDTGNYTC
MLRNTTYCSKVAFPLEVVQKDSCFNSPMKLPVHKLYIEYGIQRITCPNVDGYFPSSV
KPTITWYMGCYKIQNFNNVIPEGMNLSFLIALISNNGNYTCVVTYPENGRTFHLTRTL
TVKVVGSPKNAVPPVIHSPNDHVVYEKEPGEELLIPCTVYFSFLMDSRNEVWWTIDG
KKPDDITIDVTINESISHSRTEDETRTQILSIKKVTSEDLKRSYVCHARSAKGEVAKAA
KVKQKVPAPRYTVELACGFGATVLLVVILIVVYHVYWLEMVLFYRAHFGTDETIL
DGKEYDIYVSYARNAEEEEFVLLTLRGVLENEFGYKLCIFDRDSLPGGIVTDETLSFIQ
KSRRLLV VLSPNYVLQGTQALLELKAGLENMASRGNINVILV QYKAVKETKVKELK
RAKTVLTVIKWKGEKSKYPQGRFWKQLQVAMPVKKSPRRSSSDEQGLSYSSLKN
(SEQ ID NO:2) [0090] Typically, a suitable peptide is a short peptide. As used herein, a short peptide includes any peptide that contains up to 25 amino acids (e.g., up to 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, or 25 amino acids) or equivalents thereof. In some embodiments, a suitable peptide contains 5-25 amino acids (e.g., 5-20, 5-15, 5-12, 5-10, 6-25, 6-20, 6-15, 6-12, 6-10, 7-25, 7-20, 7-15, 7-12, or 7-10 amino acids) or equivalents thereof. In some embodiments, a suitable peptide is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, or 25 amino acids long.

[0091] In some embodiments, candidate peptides are designed to contain a sequence corresponding to at least 5 (e.g., at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 18, 20, or 25) contiguous amino acids that appear in an extracellular or transmembrane region of the IL-l RacP or IL-1 R protein.

[0092] In some embodiments, a sequence corresponding to at least 5 (e.g., at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 18, 20, or 25) contiguous amino acids that appear in an extracellular or transmembrane region of a receptor includes any sequence having at least 70% (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identical to the sequence of at least 5 (e.g., at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 18, 20, or 25) contiguous amino acids. Percentage of amino acid sequence identity can be determined by alignment of amino acid sequences. Alignment of amino acid sequences can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. Preferably, the WU-BLAST-2 software is used to determine amino acid sequence identity (Altschul et at., Methods in Enzymology 266, 460-480 (1996);
http://hg.wustl.edu/blast/README.html). WU-BLAST-2 uses several search parameters, most of which are set to the default values. The adjustable parameters are set with the following values: overlap span=1, overlap fraction=0.125, word threshold (T)=11. HSP score (S) and HSP S2 parameters are dynamic values and are established by the program itself, depending upon the composition of the particular sequence, however, the minimum values may be adjusted and are set as indicated above.

[0093] In some embodiments, a sequence corresponding to at least 5 (e.g., at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 18, 20, or 25) contiguous amino acids that appear in an extracellular or transmembrane region of a receptor includes any sequence otherwise identical to the sequence of the at least 5 (e.g., at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 18, 20, or 25) contiguous amino acids but incorporating one or more D-amino acid substitutions for corresponding L-amino acids. Peptides containing such a sequence are also known as D-isomers. In some embodiments, a sequence corresponding to at least 5 (e.g., at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 18, 20, or 25) contiguous amino acids that appear in an extracellular or transmembrane region of a receptor includes any sequence that is an inverse of the at least (e.g., at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 18, 20, or 25) contiguous amino acids.
Peptides containing such a sequence are also known as reversed L-peptides. In some embodiments, a sequence corresponding to at least 5 (e.g., at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 18, 20, or 25) contiguous amino acids that appear in an extracellular or transmembrane region of a receptor includes any sequence that is otherwise an inverse of the at least 5 (e.g., at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 18, 20, or 25) contiguous amino acids but incorporating one or more D-amino acid substitutions for corresponding L-amino acids.
Peptides containing such a sequence are also known as reversed D-peptides.

[0094] In some embodiments, a peptide suitable for the invention contains a sequence that includes at least 4 (e.g., at least 5, 6, or 7) from at least 7 (e.g., at least 8, 9, 10, 11, 12) contiguous amino acids that appear in an extracellular or transmembrane region of the IL-lRacP or IL-1R protein, wherein the at least 4 (e.g., at least 5, 6, or 7) amino acids maintain their relative positions and/or spacing as they appear in the extracellular or transmembrane region of the IL-lRacP or IL-1R protein. In some embodiments, a peptide suitable for the invention contains a sequence that includes at least 4 (e.g., at least 5, 6, or 7) from at least 7 (e.g., at least 8, 9, 10, 11, 12) contiguous amino acids that appear in an extracellular or transmembrane region of the IL-lRacP or IL-1R protein, the at least 4 (e.g., at least 5, 6, or 7) amino acids maintain their relative positions and/or spacing, but in the inverse configuration, as they appear in the extracellular or transmembrane region of the IL-1RacP or IL-1R protein.

[0095] Exemplary IL-1R antagonistic peptides suitable for the present invention are shown in Table 1.

Table 1: Exemplary Peptides LIST OF SEQUENCE NUMBERS
SEQ ID NO:3 API-101 APRYTVELA
SEQ ID NO:4 API-101.1 AARYTVELA
SEQ ID NO:5 API-101.2 APAYTVELA
SEQ ID NO:6 API-101.3 APRATVELA
SEQ ID NO:7 API-101.4 APRYAVELA
SEQ ID NO:8 API-101.5 APRYTAELA
SEQ ID NO:9 API-101.6 APRYTVALA
SEQ ID NO:10 API-101.7 APRYTVELA
SEQ ID NO:11 API-101.9 PRYTVELA
SEQ ID NO: 12 API-101.10 RYTVELA
SEQ ID NO: 13 API-101.11 YTVELA
SEQ ID NO: 14 API-101.12 TVELA
SEQ ID NO:15 API-101.101 XYTVELA (X = Citrulline) SEQ ID NO:16 API-101.102 XYTVQLA (X = Citrulline) SEQ ID NO: 17 API-101.103 RYTVQLA
SEQ ID NO: 18 API-101.104 RFTVELA
SEQ ID NO: 19 API-101.105 RYSVELA
SEQ ID NO:20 API-101.106 RYVVELA
SEQ ID NO:21 API-101.107 RYTPELA
SEQ ID NO:22 API-101.108 RYTVEL
SEQ ID NO:23 API-101.113 RYTPEL
SEQ ID NO:24 API-101.114 KYTPELA
SEQ ID NO:25 API-101.115 XYTPELA (X = Omithine SEQ ID NO:26 API-101.116 RWTPELA
SEQ ID NO:27 API-101.117 RYTPDLA
SEQ ID NO:28 API-101.118 RYTPQLA
SEQ ID NO:29 API-101.119 RYTPEFA
SEQ ID NO:30 API-101.120 RYTPEMA
SEQ ID NO:31 API-101.121 XRYTPELA (X = Acetyl) SEQ ID NO:32 API-101.122 RYTPEPA
SEQ ID NO:33 API-101.123 RYTPALA
SEQ ID NO:34 API-101.126 XYTPEL (X = Ornithine SEQ ID NO:35 API-101.127 RFVPELA
SEQ ID NO:36 API-101.128 RWTPEL

SEQ ID NO:37 API-101.129 RYTPEV
SEQ ID NO:38 API-101.132 RFTPEL
SEQ ID NO:39 API-101.133 KYTPEL
SEQ ID NO:40 API-101.134 XYTPEL (X = Citrulline) [0096] Suitable peptides may include naturally-occurring amino acids and/or unnatural amino acids. For example, suitable peptides may be composed of all L-amino acids, all D-amino acids, a combination of L- and D-amino acids, or a combination of naturally-occurring and unnatural amino acids. Exemplary peptides containing a combination of L-amino acids, D-amino acids and unnatural amino acids are shown in Table 2.

Table 2: Additional Exemplary Peptides and Peptidomimetics Unless otherwise noted, lower cases represent D-amino acids and upper cases represent L-amino acids.

LIST OF SEQUENCE NUMBERS
SEQ ID NO:41 101.135 Rytpel SEQ ID NO:42 101.145 Tel SEQ ID NO:43 101.146 rytPel SEQ ID NO:44 101.147 rytpEl SEQ ID NO:45 101.148 rytpeL
SEQ ID NO:46 101.149 rtppel SEQ ID NO:47 101.150 ryPpel SEQ ID NO:48 101.151 rypPel SEQ ID NO:49 101.152 ryPPel SEQ ID NO:50 101.153 ryxpel x=D- i ecolate SEQ ID NO:51 101.154 ryXpel X=L- i ecolate SEQ ID NO:52 101.155 rytxel x=D- i ecolate SEQ ID NO:53 101.156 rytXel X=L- i ecolate SEQ ID NO:54 101.157 rhtpel (d-amino acids SEQ ID NO:55 101.158 rztpel z=D-Do a SEQ ID NO:56 101.159 r(cha)tpel cha=D-c clohex alanine SEQ ID NO:57 101.160 rytp('D-alpha-aminoadiis acid )l SEQ ID NO:58 101.161 rytpe(cha) cha=D-c clohex alanine SEQ ID NO:59 101.162 HN=C H2 NH CH2 4CO- el SEQ ID NO:60 101.163 Rytphl [0097] Additional examples of IL-1R antagonistic peptides are disclosed in U.S.
Application Publication No. US 2006/0094663, entitled "Interleukin-1 Receptor Antagonists, Compositions, and Methods of Treatment," the entire disclosure of which is hereby incorporated by reference.

Peptidomimetics containing lactam and/or Bab residues [0098] One useful modification in optimizing a peptide structure is the addition of conformational constraints. Without wishing to be bound by theory, it is contemplated that conformational constraints can lock the secondary structure of a peptide in a bioactive conformation, thus enhancing biological potency by reducing the entropic cost of binding. If the conformation constraint stabilizes the bioactive conformation, the stabilization often results in a significant increase in biological activity. For example, alpha-amino-gamma-lactam (Agl) and beta-amino-gamma-lactam (Bgl) can be utilized to constrain the backbone conformation of linear peptides to give beta-turn mimics. In Agl, the gamma-lactam may force the C-terminal amide into the trans-orientation and restricts the psi-torsion angle to the range -125 100, favoring a type II' beta-turn geometry contingent on configuration.
Similarly, Bgl too can stabilize a type II' beta-turn conformation contingent on configuration.

HZN, 4,q NH NH NH NH
HZN HZN, (S)-Agl (R)-Agl (R)-Bgl (S)-Bgl [0099] In some embodiments, the present invention provides a peptidomimetic comprising one or more lactams of formula (I):

~ Rx ) n m j N f H

(I) wherein:

each RX is independently -R, halogen, -OR, -SR, -N(R)2, -CN, -NO2, -C(O)R, -CO2R, -C(O)N(R)2, -C(O)C(O)R, -C(O)CH2C(O)R, -S(O)R, -SO2R, -SO2N(R)2, -NRC(O)R, -NRC(O)N(R)2, -NRSO2R, -NRSO2N(R)2, -N(R)N(R)2, -C=NN(R)2, -C=NOR, -OC(O)R, or -OC(O)N(R)2;

each R is independently hydrogen or an optionally substituted group selected from C1-6 aliphatic; phenyl; a 3- to 7-membered saturated or partially unsaturated carbocyclic ring;
a 5- to 6-membered monocyclic heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; or a 4- to 7-membered saturated or partially unsaturated heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; or two R groups attached to the same nitrogen atom may be taken together with their intervening atoms to form a 4- to 7- membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independent selected from nitrogen, oxygen, and sulfur;

m is 0, 1, or 2; and nis0, 1, 2, or 3.

[0100] In some embodiments, m of formula (I) is 0. In other embodiments, m is 1 or 2.

[0101] In some embodiments, n of formula (I) is 1. In other embodiments, n is 0 or 2.

[0102] In some embodiments, a lactam of formula (I) is alpha-amino-gamma-lactam (Agl), beta-amino-gamma-lactam (Bgl), or beta-hydroxy-alpha-amino-gamma-lactam (Agl(4-OH)).

[0103] In some embodiments, a lactam of formula (I) will take up the space of approximately one amino acid residue in a peptidomimetic.

[0104] In some embodiments, the present invention provides a peptidomimetic comprising one or more lactams of formula (II):

H
q HN

N TOP_ O (I
I) wherein:

each R3' is independently -R, halogen, -OR, -SR, -N(R)2, -CN, -NO2, -C(O)R, -CO2R, -C(O)N(R)2, -C(O)C(O)R, -C(O)CH2C(O)R, -S(O)R, -SO2R, -SO2N(R)2, -NRC(O)R, -NRC(O)N(R)2, -NRSO2R, -NRSO2N(R)2, -N(R)N(R)2, -C=NN(R)2, -C=NOR, -OC(O)R, or -OC(O)N(R)2;

each R is independently hydrogen or an optionally substituted group selected from C1-6 aliphatic; phenyl; a 3- to 7-membered saturated or partially unsaturated carbocyclic ring;
a 5- to 6-membered monocyclic heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; or a 4- to 7-membered saturated or partially unsaturated heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; or two R groups attached to the same nitrogen atom may be taken together with their intervening atoms to form a 4- to 7- membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independent selected from nitrogen, oxygen, and sulfur;

p is 0, 1, or 2; and each q is independently 0, 1, 2, or 3.

[0105] In some embodiments, p of formula (II) is 0. In other embodiments, p is 1 or 2.

[0106] In some embodiments, each q of formula (II) is 1 or 2.

[0107] In some embodiments, a lactam of formula (II) is indolizidin-2-one amino acid (haa), indolizidin-9-one amino acid (I9aa), or quinolizidinone amino acid (Qaa).

[0108] In some embodiments, a lactam of formula (II) will take up upto two amino acid residue space in a peptidomimetic.

[0109] In some embodiments, lactams suitable for the invention are (3-, y- or lactams. In some embodiments, lactams suitable for the invention are four-, five-, six-, seven- or eight-membered lactams.

[0110] In addition, benzylaminobutyryl "Bab" residue may be used to optimize a peptide according to the present invention. In some embodiments, a peptidomimetic according to the present invention includes one ore more Bab residues as shown below:

H
Benzylaminobutyryl "Bab" residue [0111] In some embodiments, the present invention provides derivatives containing lactams and/or Bab residues of various peptides described herein (e.g., in Tables 1 and 2).
Suitable derivatives may contain one or more (e.g., one, two or three) lactam and/or Bab replacements or insertions. In some embodiments, derivatives according to the invention comprise a lactam and/or Bab insertion or replacement at position 1, 2, 3, 4, 5, 6, 7, 8 or 9 from the N-terminus of any one of the peptides described herein, for example as shown in Tables 1 (SEQ ID NO:3-40) and 2 (SEQ ID NO:41-60). In particular embodiments, derivatives according to the invention comprise a lactam and/or Bab insertion or replacement at position 1, 2, 3, 4, 5, 6, or 7 from the N-terminus of peptide 101.10 RYTVELA (SEQ ID
NO:12).

[0112] In some embodiments, the present invention provides a peptidomimetic comprising one or more lactams and/or Bab residues and at least three (e.g., at least four, at least five, or at least six) out of seven contiguous amino acids that appear in an extracellular or transmembrane region of the IL-1 receptor (SEQ ID NO: 1) or IL-1 receptor accessory protein (IL-1RacP) (SEQ ID NO:2), wherein the at least three (e.g., at least four, at least five, or at least six) amino acids maintain their relative positions as they appear in the extracellular or transmembrane region of the IL-lR or IL-lRacP protein. In other embodiments, the present invention provides a peptidomimetic comprising one or more lactams and/or Bab residues and at least three (e.g., at least four, at least five, or at least six) out of seven contiguous amino acids that appear in an extracellular or transmembrane region of the IL-1R
or IL-lRacP protein, wherein the at least three (e.g., at least four, at least five, or at least six) amino acids maintain their relative positions, but in the inverse configuration, as they appear in the extracellular or transmembrane region of the IL-1R or IL-1 RacP
protein. In some embodiments, the one or more lactams and/or Bab residues replace one or more amino acids of the seven contiguous amino acids. In some embodiments, the one or more lactams and/or Bab residues are inserted at the N-terminus, C-terminus, or internally.

[0113] In some embodiments, the present invention provides a peptidomimetic comprising one or more lactams and/or Bab residues and at least three (e.g., at least four, at least five, at least six) amino acids from any one of SEQ ID NOS:3-40, wherein the at least three (e.g., at least four, at least five, or at least six) amino acids maintain their relevant positions as they appear in the corresponding sequence. In some embodiments, the present invention provides a peptidomimetic comprising one or more lactams and/or Bab residues and at least three (e.g., at least four, at least five, or at least six) amino acids from RYTVELA
(SEQ ID NO: 12), wherein the at least three (e.g., at least four, at least five, or at least six) amino aicds maintain their relevant positions as they appear in RYTVELA (SEQ
ID NO: 12).
In some embodiments, one or more lactams and/or Bab residues replace one or more amino acids of RYTVELA (SEQ ID NO:12). In some embodiments, the one or more lactams are inserted at the N-terminus, C-terminus, or internally.

[0114] Various methods can be used to introduce lactam and/or Bab residue into the peptides described herein. In some embodiments, systematic scans of a peptide described herein with lactam and/or Bab can be used. Various synthetic strategies for the synthesis of Agl peptide mimics are known in the art and can be used for the present invention. See, for example, Toniolo, C. Int. J. Pept. Protein Res. 1990, 35, 287, and references therein; Wolf, J-P.; Rapoport, H. J. Org. Chem. 1989, 54, 3164. Schuster, M.; Blechert, S.
Angew. Chem. Int.
Ed. 1997, 36, 2036. Wolfe, M. S.; Dutta, D.; Aube, J. J. Org. Chem. 1997, 62, 654. (b) Noth, J.; Frankowski, K. J.; Neuenwander, B.; Aube, J. J. Comb. Chem. 2008,10,456;
Piscopio, A.
P. D.; Miller J. F.; Koch K. Tetrahedron Letters. 1998, 39, 2667; Armstrong, S.K. J. Chem.
Soc., Perkin Trans. 1 1998, 1, 371; Piscopio, A. D.; Miller, J. F.; Koch K.
Tetrahedron 1999, 55, 8189; Galaud, F.; Lubell, W. D. Biopolymers (Peptide Science) 2005, 80, 665; Bhooma, R.; Rodney, J. J. Org. Chem. 2006, 71, 2151, and references therein, the teachings of all of which are incorporated herein by reference.

[0115] Exemplary methods are described in the Examples section. Additional methods are described in co-owned U.S. Patent Application entitled "Processes for Preparing Amino-substituted gamma-lactams" filed on even date, the entire disclosure of which is hereby incorporated by reference. In addition, various methods have been used to incorporate various lactams to other proteins and peptides. Those methods and lactams are suitable for the present invention and can be found in the following:
Freidinger et al, Science 210:656-8 (1980) (alpha-amino-gamma-lactam incorporated into luteinizing hormone-releasing hormone (LRHH); Mishra et al, Prog, Neuropsychopharmacol. Biol.
Psych. 14:821-827 (1990) (amino-gamma-lactam analogs of the Pro-Gly-Leu-NHe sequence for D2 dopamine receptor modulation); United States Publication No. 2006/0229240 (parathyroid hormone (PTH) derivatives containing lactam bridges); United States Publication No.
2009/0209491 (lactam-containing inhibitors of post-proline cleaving enzymes (PPCE));
Gomez et al, Bioorg. Med. Chem. Lett. 19:1733-1736 (2009) (lactam-containing peptidomimetic inhibitors of STAT3).

[0116] Although exemplary methods described in the Example sections are based on IL-1R antagonistic peptide 101.10 RYTVELA (SEQ ID NO: 12), similar methods can be used to introduce various lactams and/or Bab residues into any one of the peptides described herein (e.g., peptides having an amino acid sequence as shown in Tables 1 and 2) or variants thereof. As used herein, variants of a peptide include peptides that have an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, identical to the sequence of the parent peptide. In some embodiments, variants of peptides as shown in Tables 1 and 2 (e.g., SEQ ID NOs:3-60) include peptides having at least three, four, five, six, or seven amino acids from any one of peptides as shown in Tables 1 and 2 (e.g., SEQ ID NOs:3-60), wherein the at least three, four, five, six, or seven amino acids maintain their relative positions and/or spacing as they appear in the corresponding parent peptide.

[0117] The biological activity of peptidomimetics synthesized according to the invention can be tested using various functional assays known in the art and as described herein. For example, the lactam peptidomimetics synthesized according to the invention can be tested for its ability to inhibit or enhance the activity of the IL-1 receptor using various IL-1 functional assays (e.g., thymocyte TF-1 proliferation assay). Exemplary methods are described in the Examples section. Additional assays are described in U.S.
Application Publication No. US 2006/0094663, entitled "Interleukin-1 Receptor Antagonists, Compositions, and Methods of Treatment," the entire disclosure of which is hereby incorporated by reference.

[0118] The present invention further relates to methods of improving the biological activity of a peptide by imposing conformational constraints. In some embodiments, the methods according to the invention include introducing a lactam and/or Bab residue replacement or insertion into a peptide such that the lactam/Bab structure locks the secondary structure of a peptide in a bioactive conformation. In some embodiments, a lactam/Bab residue is introduced at N-terminus (also referred to as position 1 from the N-terminus) of a peptide. In some embodiments, a lactam/Bab residue is introduced at position 2, 3, 4, 5, 6, 7, 8 or 9, from the N-terminus of the peptide. In some embodiments, the methods include a step of testing the activity of modified peptide as compared to a control to identify modified peptides that have improved biological activity (e.g., the IL-1R antagonistic or agonistic activity). In some embodiments, the unmodified parent peptide is used as the control. In some embodiments, a modified peptide according to the invention has 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, or 10-fold increased IL-1R modulatory (e.g., antagonistic or agonistic) activity as compared to a control (e.g., an unmodified parent peptide).

[0119] Exemplary lactam peptidomimetics are shown in Tables 3, 4, and 6-8, and in the Examples.

Table 3. Exemplary Compounds and TF-1 Cells Proliferation Assay SEQ ID NO Compound Compound Compound Structure Percentage Number Name Control (/o) - - Control - 100 (Proliferation) 12 101.10 101.10 vela-NHz 69 90 94 LRM 17 ryt-(S)-Agl-ela-NH2 132 89 93 LRM 18 ry- -A l-vela-NHz 150 Amino pentanoic-y-99 3 WC125 (3R,6R,9R)-I2aa-ql- 88 NHZ
100 4 WC126 Omega amino 165 hexanoic-y-(3R,6R,9R)-I2aa-gl-101 5 WC127 oy-(3R,6R,9R)-I2 aa-ql- 95 96 6 WC115 oy-(3R,6R,9R)-I2 aa-el- 94 97 7 WC116 Amino pentanoic-y-3R,6R,9R -I as-el-NH2 98 8 WC117 Amino hexanoic-y-3R,6R,9R -I as-el-NHz 66 59 AJ-303-A ve- R -A l-a-NH2 93 65 58 AJ-350-A rytv-(R)-Agl-la-NH2 76 64 57 AJ-328-A ryt-(R)-Agl-ela-NH2 78 63 56 AJ-351-A ry- R -A l-vela-NH2 122 62 55 AJ-366-A r- R -A l-tvela-NH2 74 61 54 AJ-333-A R -A l- vela-NH2 44 70 74 AJ-328-B ryt-(R)-Bab-ela-NH2 41 69 73 AJ-351-B ry- R -Bab-vela-NH2 42 68 72 AJ-366-B r- R -Bab-tvela-NH2 59 67 71 AJ-333-B R -Bab- vela-NH2 56 *Results are shown as percentage of control.

Table 4. Exemplary peptidomimetics Crude Purity Yield HRMS
Peptide' Purity % b %C %a m/z m/z (calcd) (obsd) ...............................................................................
..........................
(R)-Agl-D-Tyr-D-Thr-D-Val-D-Glu-D-Leu-D-Ala-NH2 (SEQ ID NO:61) 55 >99 19 777.4141 777.4138 54 .......................................... .....
....... ....... .......
D-Arg-(R)-Agl-D-Thr-D-Val-D-Glu-D-Leu-D-Ala-NH2 (SEQ ID NO:62) 41 >99 15 770.4519 770.4514 55 .......................
...... .................. ....... ..........
D-Arg-D-Tyr-(R)-Agl-D-Val-D-Glu-D-Leu-D-Ala-NH2 (SEQ ID NO:63) 58 >99 7 832.4675 832.4676 56 ............................~.................. .~.........................
D-Arg-D `Tyr-D-Thr-(R) `Agl-D `Glu-D-Leu-D-Ala-NH2 (SEQ ID NO:64) 50 >99 11 833.4468 833.4458 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
D-Arg-D-Tyr-D-Thr-D-Val-(R)-Agl-D-Leu-D-Ala-NH2 (SEQ ID NO:65) 36 >99 21 803.4726 803.4724 ......................................
...............................................................................
...................................................
D-Arg-D-Tyr-D-Thr-D-Val-D-Glu-(R)-Agl-D-Ala-NH2 (SEQ ID
N0:66) 81 ; >99 7 820.4311 820.4306 'Bold lettering indicates lactam residue. bRP-HPLC purity at 214 nm of the crude peptide.
'RP-HPLC purity at 214 nm of the purified peptide dYields after purification by RP-HPLC
are based on Fmoc Table 5. Exemplary peptidomimetics HRMS
Crude Purity Yield :.............................
%C %d m/z m/z Peptide' Purity y (calcd) obsd) (R)-Bab-D-Tyr-D-Thr-D-Val-D-Glu-D-Leu-D-Ala-NH2 (SEQ ID NO:67) 28 >99 10 885.4714 885.4716 D-Arg-(R)-Bab-D-Thr-D-Val-D-Glu-D-Leu-D-Ala-NH2 (SEQ ID 45 >99 15 878.5092 878.5094 NO:68) D-Arg-D-Tyr-(R)-Bab-D-Val-D-Glu-D-Leu-D-Ala-NH2 (SEQ ID 27 >99 3 940.5240 940.5251 NO:69) D-Arg-D-Tyr-D-Thr-(R)-Bab-D-Glu-D-Leu-D-Ala-NH2 (SEQ ID 6 >99 4 942.5027 942.5043 NO:70) 'Bold lettering indicates lactam residues. ARP-HPLC % at 214 nm of the crude mixture of the parent lactam peptide. ARP-HPLC purity at 214 nm of the purified peptide dYields after purification by RP-HPLC are based on Fmoc loading test for Rink resin.

Table 6. Exemplary peptidomimetics Crude HRMS
Yiel Purity %b Punt Peptide' (lactamizati Y%' %d m/z m/z (calcd) (obsd) on time (R)-Bgl-D-Tyr-D-Thr-D-Val-D-Glu- 777,414 D-Leu-D-Ala-NH2 (SEQ ID NO:71) 71(4 h) >99 22 1 777.4145 D-Arg-(R)-Bgl-D-Thr-D-Val-D-Glu- 770.451 D-Leu-D-Ala-NH2 (SEQ ID NO:72) 87(6 h) >99 24 770.4521 D-Arg-D-Tyr-(R)-B gl-D-Val-D-Glu-832.467 D-Leu-D-Ala-NH2 (SEQ ID NO:73) 86(4 h) >99 23 6 832.4677 D-Arg-D-Tyr-D-Thr-(R)-Bgl-D-Glu- 833.446 79(7 h) 4~ 8 833.4470 D-Leu-D-Ala-NH2(SEQIDNO`7 >99 17 D-Arg-D-Tyr-D-Thr-D-Val-(R)-Bgl- 826.454 826.4544 D-Leu-D-Ala-NH2 (SEQ ID NO:75) 87(4 h) >99 7 6e e D-Arg-D-Tyr-D-Thr-D-Val-D-Glu- 820.431 (R)-Bgl-D-Ala-NH2 (SEQ ID NO:76) 50(4 h) >99 14 2 820.4313 (S)-BgI-D-Tyr-D-Thr-D-Val-D-Glu- 777,414 D-Leu-D-Ala-NH2 (SEQ ID NO:77) 70(10 h) >99 13 1 777.4138 D-Arg-(S)-Bgl-D-Thr-D-Val-D-Glu- 770.451 D-Leu-D-Ala-NH2 (SEQ ID NO:78) 82(10 h) >99 16 9 770.4518 D-Arg-D-Tyr-(S)-Bgl-D-Val-D -Glu-832.467 D-Leu-D-Ala-NH2 (SEQ ID NO:79) 60(8 h) >99 17 6 832.4674 D-Arg-D-Tyr-D-Thr-(S)-Bgl-D-Glu- 833.446 D-Leu-D-Ala-NH2 (SEQ ID NO:80) 87(7 h) >99 14 8 833.4469 D-Arg-D-Tyr-D-Thr-D-Val-(S)-Bgl- 803.472 D-Leu-D-Ala-NH2 (SEQ ID NO:81) 90(4 h) >99 13 6 803.4724 D-Arg-D-Tyr-D-Thr-D-Val-D-Glu- 820.413 (S)-Bgl-D-Ala-NH2 (SEQ ID NO:82) 96(4 h) >99 11 1 820.4136 ....... 6 ...............................................................................
......................................
'Bold lettering indicates lactam residues. RP-HPLC purity at 214 nm of the crude peptide.
CRP-HPLC purity at 214 nm of the purified peptide dYields after purification by RP-HPLC
are based on Fmoc loading test for Rink resin. eThe cation [M+Na]+ was observed, theoretical mass was calculated accordingly.

Table 7. Exemplary peptidomimetics Crude HRMS
Purity Yield \`
Peptide' Purity o e 0/ d m/z m/z o b ~o o ~o calcd) (obsd) D-Arg-(S)-Bgl-D-Tyr-D-Thr-D-Val-D-Glu-D-Leu-D-Ala-NH2 (SEQ ID 56 >99 18 933.5152 933.5152 NO:83) D-Arg- D-Tyr-(S)-Bgl-D-Thr-D-Val-D-Glu-D-Leu-D-Ala-NH2 (SEQ ID
N0:84) 55 >99 6 933.5152 933.5137 D-Arg-D-Tyr-D-Thr-(S)-Bgl-D-Val-71 >99 10 933.5152 933.5152 D-Glu-D-Leu-D-Ala-NH2 (SEQ ID

NO:85) D-Arg `D-Tyr `D-Thr-D-Val-(S)-Bgl-D-Glu-D-Leu-D-Ala-NH2 (SEQ ID 68 >99 6 933.5152 933.5147 NO:86) D-Arg-D `Tyr-D-Thr `D-Val-D-Glu-(S)-Bgl-D-Leu-D-Ala-NH2 (SEQ ID
N0:87) 37 >99 3 933.5152 933.5154 D-Arg-D-Tyr-D-Thr-D-Val-D-Glu-D-Leu-(S)-BgI-D-Ala-NH2 (SEQ ID
N0:88) 97 >99 18 933.5152 933.5150 Bold lettering indicates lactam residues. RP-HPLC purity at 214 nm of the crude peptide.
'RP-HPLC purity at 214 nm of the purified peptide dYields after purification by RP-HPLC
are based on Fmoc loading test for Rink resin. eThe cation [M+Na]+ was observed, theoretical mass was calculated accordingly.

Table 8. Exemplary peptidomimetics Purity Yield HRMS
Peptide Purity a (%)b m/z calcd m/z obsd 93 D-Arg-D-Tyr-(S)-Agl-D-Val-D-Glu-D-Leu-D-Ala-NH2 (SEQ ID NO:89) 32 >99 15 832.4676 832.4678 94 D-Arg-D-Tyr-D-Thr-(S)-Agl-D-Glu-D-Leu-D-Ala-NH2 (SEQ ID NO:90) 18 >99 5 834.4468 834.4459 (R)-Agl-D-Tyr-D-Thr-D-Pro-D-Glu-D-Leu-D-Ala-NH2 (SEQ ID NO:91) 55 >95 10 775.3985 775.3986 96 (R)-Agl-D-Tyr-D-Thr-(R)-Agl -D-Glu-D-Leu-D-Ala-NH2 (SEQ ID
NO:92) 30 >99 5 761.3828 761.3832 97 (R)-Agl-(R)-Agl -D-Thr-D-Val -D-Glu-D-Leu-D-Ala-NH2 (SEQ ID
NO:93) 45 >65 5 697.3880 697.3901 aRP-HPLC purity at 214 nm of the crude peptide. RP-HPLC purity at 214 nm of the purified peptide 'Yields after purification by RP-HPLC are based on resin and lantern loading. Bold lettering indicates lactam residues.

Peptide preparation [0120] Peptides (including the peptide portions of peptidomimetics) of the present invention are obtained by any method of peptide synthesis known to those skilled in the art, including synthetic (e.g., exclusive solid phase synthesis, partial solid phase synthesis, fragment condensation, classical solution synthesis) and recombinant techniques. For example, the peptides or peptide derivatives can be obtained by solid phase peptide synthesis, which in brief, consists of coupling the carboxyl group of the C-terminal amino acid to a resin (e.g., benzhydrylamine resin, chloromethylated resin, hydroxymethyl resin) and successively adding N-alpha protected amino acids. The protecting groups maybe any such groups known in the art. Before each new amino acid is added to the growing chain, the protecting group of the previous amino acid added to the chain is removed.
Such solid phase synthesis has been described, for example, by Merrifield, 1964, J. Am. Chem.
Soc. 85: 2149;
Vale et al., 1981, Science, 213: 1394-1397, in US Patent Nos. 4,305,872 and 4,316,891, Bodonsky et at., 1966, Chem. Ind. (London), 38:1597; Pietta and Marshall, 1970, Chem.
Comm. 650. The coupling of amino acids to appropriate resins is also well known in the art and has been described in US Patent No. 4,244,946. (Reviewed in Houver-Weyl, Methods of Organic Chemistry. Vol E22a. Synthesis of Peptides and peptidomimetics, Murray Goodman, Editor-in-Chief, Thieme. Stuttgart. New York 2002).

[0121] During any process of the preparation of a compound of the present invention, it may desirable to protect sensitive reactive groups on any of the molecule concerned. This may be achieved by means of conventional protecting groups such as those described in Protective Groups In Organic Synthesis by T.W. Greene & P.G.M. Wuts, 1991, John Wiley and Sons, New-York; and Peptides: chemistry and Biology by Sewald and Jakubke, 2002, Wiley-VCH, Wheinheim p.142. For example, alpha amino protecting groups include acyl type protecting groups (e.g., trifluoroacetyl, formyl, acetyl), aliphatic urethane protecting groups (e.g., t-butyloxycarbonyl (BOC), cyclohexyloxycarbonyl), aromatic urethane type protecting groups (e.g., fluorenyl-9-methoxy-carbonyl (Fmoc), benzyloxycarbonyl (Cbz), Cbz derivatives) and alkyl type protecting groups (e.g., triphenyl methyl, benzyl). The amino acids side chain protecting groups include benzyl (For Thr and Ser), Cbz (Tyr, Thr, Ser, Arg, Lys), methyl ethyl, cyclohexyl (Asp, His), Boc (Arg, His, Cys) etc. The protecting groups may be removed at a convenient subsequent stage using methods known in the art.

[0122] In one embodiment, the peptides of this invention, including the analogs and other modified variants, may generally be synthesized according to the FMOC
protocol in an organic phase with protective groups. They can be purified with a yield of 70%
with HPLC
on a C18 column and eluted with an acetonitrile gradient of 10-60%. Their molecular weight can then be verified by mass spectrometry (Reviewed in Fields, G.B. "Solid-Phase Peptide Synthesis". Methods in Enzymology. Vol. 289, Academic Press, 1997).

[0123] Alternatively, peptides of this invention that consist of genetically encoded amino acids may be prepared in recombinant systems using polynucleotide sequences encoding the peptides. It is understood that a peptide of this invention may contain more than one of the above-described modifications within the same peptide.

[0124] Purification of the synthesized peptide or peptide derivatives is carried out by standard methods, including chromatography (e.g., ion exchange, size exclusion, affinity), centrifugation, precipitation or any standard technique for the purification of peptides and peptide derivatives. In one embodiment, thin-layered chromatography is employed. In another embodiment, reverse phase HPLC is employed. Other purification techniques well known in the art and suitable for peptide isolation and purification may be used in the present invention.

[0125] Where the processes for the preparation of the compounds according to the present invention give rise to mixtures of stereoisomers, these isomers may be separated by conventional techniques such as preparative chromatography. The compounds may be prepared in racemic form, or individual enantiomers may be prepared either by enantiospecific synthesis or by resolution. The compounds may, for example, be resolved into their components enantiomers by standard techniques such as the formation of diastereoisomeric pairs by salt formation with an optically active acid followed by fractional crystallization and regeneration of the free base. The compounds may also be resolved by formation of diastereomeric esters or amides, followed by removal of the chiral auxiliary.
Alternatively, the compounds may be resolved using chiral HPLC column.

Further peptidomimetic modifications [0126] Peptide mimetics that are structurally related to therapeutically useful peptides may be used to produce an equivalent or enhanced therapeutic or prophylactic effect.
Generally, peptidomimetics are structurally similar to paradigm polypeptide (i.e., a polypeptide that has a biological or pharmacological activity) such as naturally occurring receptor-binding polypeptides but have one or more peptide linkages optionally replaced by linkages like -CH2NH-, -CH2S-, -CH2-CH2-, -CH=CH- (cis and trans), -CH2SO-, -CH(OH)CH2-, -COCH2- etc., by methods well known in the art (Spatola A.F., Peptide Backbone Modifications, Vega Data, March 1983, 1(3): 267; Spatola et at., Life Sci., 1986, 38:1243-1249; Hudson D. et at., Int. J. Pept. Res. 1979., 14: 177-185;
Weinstein. B., 1983, Chemistry and Biochemistry, of Amino Acids, Peptides and Proteins, Weinstein Eds, Marcel Dekker, New-York). Such peptide mimetics may have significant advantages over natural polypeptides including more economical production, greater chemical stability, enhanced pharmacological properties (e.g., half-life, absorption, potency, efficiency etc), reduced antigenicity and others.

[0127] While peptides are effective in inhibiting wild-type IL-1 in vitro, their effectiveness in vivo might be compromised by the presence of proteases. Serum proteases have specific substrate requirements. The substrate must have both L-amino acids and peptide bonds for cleavage. Furthermore, exopeptidases, which represent the most prominent component of the protease activity in serum, usually act on the first peptide bond of the peptide and require a free N-terminus (Powell et at. 1993). In light of this, it is often advantageous to utilize modified versions of peptides also termed peptide analogs or derivatives. The modified peptides retain the structural characteristics of the original L-amino acid peptides that confer biological activity with regard to IL-1, but are advantageously not readily susceptible to cleavage by protease and/or exopeptidases.

[0128] Systematic substitution of one or more amino acids of a consensus sequence with D-amino acid of the same type (e.g., D-lysine in place of L-lysine) may be used to generate more stable peptides. Thus, a peptide derivative or peptidomimetic of the present invention may be all L, all D or mixed D, L peptide. In some embodiments, peptides or peptidomimetics contain all D-amino acids. In some embodiments, peptides or peptidomimetics contain a D-amino acid at N-terminus or C-terminus. Reverse-D
peptides are peptides containing D-amino acids, arranged in a reverse sequence relative to a peptide containing L-amino acids. Thus, the C-terminal residue of an L-amino acid peptide becomes N-terminal for the D-amino acid peptide, and so forth. It is thought that a reverse D-peptide retains the same tertiary conformation and therefore the same activity, as the corresponding L-amino acid peptide, but is more stable to enzymatic degradation in vitro and in vivo, and thus have greater therapeutic efficacy than the original peptide. In addition to reverse-D-peptide, constrained peptides comprising a consensus sequence or a substantially identical consensus sequence variation may be generated by methods well known in the art (Rizo et Gierasch, Ann. Rev. Biochem., 1992., 61: 387), for example, by adding cysteine residues capable of forming disulfide bridges which cyclize the peptide. Cyclic peptides have no free N- or C-termini.

[0129] A cyclic derivative containing intramolecular disulfide bond may be prepared by conventional solid phase synthesis while incorporating suitable S-protected cysteine or homocysteine residues at the positions selected for cyclization such as the amino and carboxy termini (SahM et at., 1996., J. Pharm. Pharmacol. 48: 197). Following completion of the chain assembly, cyclization can be performed either (1) by selective removal of the S-protecting group with a consequent on-support oxidation of the corresponding two free SH-functions, to form a S-S bonds, followed by conventional removal of the product from the support and appropriate purification procedure; or (2) by removal of the peptide from the support along with complete side chain deprotection, followed by oxidation of the free SH-functions in highly dilute aqueous solution.

[0130] A cyclic derivative containing an intramolecular amide bond may be prepared by conventional solid phase synthesis while incorporating suitable amino and carboxyl side chain protected amino acid derivatives, at the position selected for cyclization. The cyclic derivatives containing intramolecular -S-alkyl bonds can be prepared by conventional solid phases while incorporating an amino acid residue with a suitable amino-protected side chain, and a suitable S-protected cysteine or homocysteine residue at the position selected for cyclization.

[0131] Substitution of unnatural amino acids for natural amino acids in a subsequence of the peptides can also confer resistance to proteolysis. Such a substitution can, for instance, confer resistance to proteolysis by exopeptidases acting on the N-terminus.
Such substitutions have been described and these substitutions do not affect biological activity.
Examples of non-naturally occurring amino acids include a,a -disubstituted amino acids, N-alkyl amino acids, lactic acids, C-a-methyl amino acids, and (3-methyl amino acids. Amino acids analogs useful in the present invention may include but are not limited to (3-alanine, norvaline, norleucine, 4-aminobutyric acid, orithine, hydroxyproline, sarcosine, citrulline, cysteic acid, cyclohexylalanine, 2-aminoisobutyric acid, 6-aminohexanoic acid, t-butylglycine, phenylglycine, o-phosphoserine, N-acetyl serine, N-formylmethionine, 3-methylhistidine and other unconventional amino acids. Furthermore, the synthesis of peptides with unnatural amino acids is routine and known in the art.

[0132] One other effective approach to confer resistance to peptidases acting on the N-terminal or C-terminal residues of a peptide is to add chemical groups at the peptide termini, such that the modified peptide is no longer a substrate for the peptidase. One such chemical modification is glycosylation of the peptides at either or both termini. Certain chemical modifications, in particular N-terminal glycosylation, have been shown to increase the stability of peptides in human serum (Powell et at. 1993). Other chemical modifications which enhance serum stability include, but are not limited to, the addition of an N-terminal alkyl group, consisting of a lower alkyl of from 1 to 20 carbons, such as an acetyl group, and/or the addition of a C-terminal amide or substituted amide group. In particular the present invention includes modified peptides consisting of peptides bearing an N-terminal acetyl group and/or a C-terminal amide group.

[0133] Also included by the present invention are other types of peptide derivatives containing additional chemical moieties not normally part of the peptide, provided that the derivative retains the desired functional activity of the peptide. Examples of such derivatives include (i) N-acyl derivatives of the amino terminal or of another free amino group, wherein the acyl group may be an alkanoyl group (e.g., acetyl, hexanoyl, octanoyl), an aroyl group (e.g., benzoyl) or a blocking group such as Fmoc (fluorenylmethyl-O-CO-); (ii) esters of the carboxy terminal or of another free carboxy or hydroxyl group; (iii) amide of the carboxyterminal or of another free carboxyl group produced by reaction with ammonia or with a suitable amine; (iv) phosphorylated derivatives; (v) derivatives conjugated to an antibody or other biological ligand and other types of derivatives.

[0134] Longer peptide sequences which result from the addition of extra amino acid residues to the peptides of the invention are encompassed by the present invention.

[0135] Other derivatives included in the present invention are dual peptides consisting of two of the same, or two different peptides of the present invention covalently linked to one another either directly or through a spacer, such as by a short stretch of alanine residues or by a putative site for proteolysis (e.g., by cathepsin, see US Patent No.
5,126,249 and European Patent No. 495,049). Multimers of the peptides of the present invention consist of polymer of molecules formed from the same or different peptides or derivatives thereof.

[0136] In another embodiment, the peptide derivatives of the present invention are chimeric or fusion proteins comprising a peptide of the present invention or fragment thereof linked at its amino or carboxy terminal end, or both, to an amino acid sequence of a different protein. Such a chimeric or fusion protein may be produced by recombinant expression of a nucleic acid encoding the protein. In one embodiment such a chimeric or fusion protein contains at least 6 amino acids of a peptide of the present invention and has a functional activity equivalent or greater than that of a peptide of the invention.

[0137] Peptide derivatives of the present invention can be made by altering the amino acid sequences by substitutions, additions or deletions that provide for functionally equivalent molecules, or functionally enhanced or diminished molecules, as desired. The derivative of the present invention include, but are not limited to those containing, as primary amino acid sequence, all or part of the amino acid sequence of the peptides of the present invention including altered sequences in which functionally equivalent amino acid residues are substituted for an equivalent in the sequence. For example, one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity, which act as a functional equivalent, resulting in a silent alteration. Substitution for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs. For example, the positively charged (basic) amino acids include, arginine, lysine and histidine. The nonpolar (hydrophobic) amino acids include, leucine, isoleucine, alanine, phenylalanine, valine, proline, tryptophan and methionine. The uncharged polar amino acids include serine, threonine, cysteine, tyrosine, asparagine and glutamine. The negatively charged (acid) amino acids include glutamic acid and aspartic acid. The amino acid glycine is sometimes included in the nonpolar amino acids family and sometimes in the uncharged (neutral) polar amino acids family. Substitutions that are done within a family of amino acids are generally understood to be conservative substitutions.

[0138] Once a peptidomimetic of the present invention is identified, it may be isolated and purified by any number of standard methods including but not limited to differential solubility (i.e., precipitation), centrifugation, chromatography (affinity, ion exchange, size exclusion and the like) or by any other standard techniques used for the purification of peptides, peptidomimetics or proteins. The functional properties of an identified peptide of interest may be evaluated using any functional assay known in the art. In one embodiment assays for evaluating downstream receptor function in intracellular signaling are used (e.g., PGE2 synthesis).

[0139] In one embodiment, peptidomimetic compounds of the present invention are obtained with the following three-phase process: 1) scanning the peptides of the present invention to identify regions of secondary structure involved in the binding of the IL-1 receptor; 2) using conformationally constrained dipeptide surrogates to refine the backbone geometry and provide organic platforms corresponding to these surrogates; and 3) using the best organic platforms to display organic pharmocophores in libraries of candidates designed to mimic the desired activity of the native peptide. In more details the three phases are as follows. In phase 1, the peptide leads are scanned and their structure abridged to identify the requirements for their activity. A series of peptide analogs of the original are synthesized. In phase 2, the best peptide analogs are investigated using the conformationally constrained dipeptide surrogates. Indolizidin-2-one, indolizidin-9-one and quinolizidinone amino acids (haa, I9aa and Qaa respectively) are used as platforms for studying backbone geometry of the best peptide candidates. These and related platforms (Reviewed in Halab, Li;
Gosselin, F;
Lubell, WD; Biopolymers (Peptide Science) Vol 55, 101-122. 2000; Hanessian, S.J.
McNaughton-Smith G; Lombart, H-G.; Lubell,W.D. Tetrahedron Vol. 53, 12789-12854, 1997) may be introduced at specific regions of the peptide in order to orient the pharmacophores in different directions. Biological evaluation of these analogs identifies improved leads that mimic the geometric requirements for activity. In phase 3, the platforms from the most active leads are used to display organic surrogates of the pharmacophores responsible for activity of the native peptide. The pharmacophores and scaffolds are combined in a parallel synthesis format. Derivation of peptides and the different phases can be done by other means and methods known in the art.

[0140] Structure function relationships determined from peptidomimetics of the present invention may be used to refine and prepare analogous molecular structures having similar or better properties. Thus, also within the scope of the present invention are molecules, in addition to those specifically disclosed, that share the structure, polarity, charge characteristics and side chain properties of the specific embodiments exemplified herein.

[0141] Peptidomimetics of the present invention are functionally active (i.e., capable of exhibiting one or more of the identified functional activities associated with a peptide of the present invention). For example, such peptides, peptide derivatives, peptidomimetics or analogs that inhibit a desired property (e.g., binding of the IL-lRacP to a protein partner or ligand) can be used as inhibitors of such property and its physiological correlates. Peptides, derivatives, peptidomimetics or analogs of the peptides of the present invention can be tested for the inhibition of cell signaling through the IL-1 R/IL-1 RacP receptor by any functional assay known in the art (e.g., PGE2 synthesis).

Assays [0142] Methods for testing the ability of candidate compounds to inhibit or activate IL-1 receptor activity are presented herein. It will be understood that the invention is not so limited. Indeed, other assays well known in the art can be used in order to identify non-competitive, extracellular agonists or antagonists of the present invention.

[0143] Generally, screens of an IL-1R/IL-lRacP antagonist or agonist may be based on assays that measure a biological activity of IL-1R/IL-lRacP. Assays of the present invention employ either a natural or recombinant IL-1 receptor. A cell fraction or cell free screening assays for antagonists of IL-1 activity can use in situ purified, or purified recombinant IL-1 receptor. Cell-based assays can employ cells which express IL-1 receptor naturally, or which contain recombinant IL-1 receptor. In all cases, the biological activity of IL-1 receptor can be directly or indirectly measured; thus inhibitors or activators of IL-1 receptor activity can be identified.

[0144] In some embodiments, an assay is a cell-based assay in which a cell which expresses a IL-1R/IL-lRacP receptor complex or biologically active portion thereof, either natural or recombinant in origin, is contacted with a test compound, and the ability of the test compound to modulate IL-1R/IL-lRacP receptor biological activity, e.g., modulation of PGEz production, proliferation assays, binding of IL-1R to a binding partner (IL-1RacP) or any other measurable biological activity of the IL-1 receptor is determined.

[0145] In some embodiments, determining the ability of the test compound to modulate the activity of IL-1R/IL-1RacP receptor complex can be accomplished by determining the ability of the test compound to modulate the activity of a downstream effector of an IL-1R/IL-1RacP receptor target molecule. For example, the activity of the test compound on the effector molecule can be determined. Non-limiting examples of such downstream effector, include interleukin receptor activated kinase (IRAK);
TRAF, activation of NI-KB (e.g. p65), mutagenic activated protein kinases (MAPK). Other examples of effector molecules which could be assayed to define the modulatory (agonist or antagonist) activity of the compounds of the present invention are described in Sims et at. 2002; and Kashiwamura et at. 2002.

[0146] In some embodiments, it may be desirable to immobilize either IL-1, IL-1R, IL-1RacP or an interacting peptide or peptidomimetic of the present invention to facilitate separation of complexed from uncomplexed forms of one or both of the interacting proteins, as well as to accommodate automation of the assay. Binding of a test compound to IL-1R
protein or interaction of IL-1R protein with a target molecule (e.g., IL-1RacP) in the presence and absence of a candidate compound can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes and micro-centrifuge tubes. In one embodiment a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix. For example, glutathione-S-transferase/IL-1R fusion proteins or glutathione-S-transferase/IL-1RacP fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St.
Louis, MO), or glutathione derivatized microtiter plates, which are then combined with the test compound or the test compound and either the non-adsorbed target protein or IL-1R
protein and the mixture incubated under conditions conducive to complex formation (e.g. at physiological conditions for salt and pH). Following incubation the beads or microtiter plate wells are washed to remove any unbound components, and complex formation determined either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of IL-1R binding or activity determined using standard techniques.

[0147] It will be understood that the in vivo experimental models such as described and exemplified herein can also be used to carry out an in vitro assay.

In vitro assays [0148] In some embodiments, candidate peptides or peptidometics are tested for their ability to activate or inhibit ability of an IL-1 receptor to modulate cellular proliferation with the incorporated tritiated thymidine method. In some embodiments, candidate peptides are tested for their ability to inhibit ability of an IL-1 receptor to modulate cellular proliferation, using for example, the assays described in (Baker et at. 1995; Cheviron, Grillon et at. 1996);
(Elliott et at. 1999; Hu et at. 1999).

[0149] In some embodiments, candidate peptides or peptidomimetics are tested for their ability to modulate the phosphorylation state of IL-1R or portion thereof, or an upstream or downstream target protein in the IL-1R/IL-1RacP pathway, using for example an in vitro kinase assay.

[0150] In some embodiments, candidate peptides targeting IL-1R are tested for levels, IL-6 or collagenase expression or any other molecule having a level which is modified following IL-1 stimulation in IL-1R/IL1RacP expressing cells, such as chondrocytes and fibroblasts.

[0151] Additonal assays are known in the art and examples are described in In vivo [0152] The assays described above may be used as initial or primary screens to detect promising lead compounds for further development. Lead peptides will be further assessed in additional, different screens. Therefore, this invention also includes secondary IL-1R screens that may involve various assays utilizing mammalian cell lines expressing these receptors or other assays.

[0153] Tertiary screens may involve the study of the identified inhibitors in animal models for clinical symptoms. Accordingly, it is within the scope of this invention to further use an agent (peptide or peptidomimetic) identified as described herein in an appropriate animal model such as a rat or a mouse. For example, a peptide can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent.
Alternatively, an agent identified as described herein can be used in an animal model to determine the mechanism of action of such an agent. Furthermore, this invention pertains to uses of novel agents identified by the above-described screening assays for treatment (e.g.
treatments of different types of disorders associated with a deregulation or malfunction of IL-1 receptor), as described herein. Non-limiting animal models which can be used in such assays include: collagen-induced arthritis in rat, animal model of acute IBD, tumor growth in immunosuppressed mouse, sensitization of the airways in newborn mice and any other known animal model including transgenic animals.

[0154] Additional assays for testing and identifying IL-1R antagonists are described in U.S. Application Publication No. US 2006/0094663, entitled "Interleukin-1 Receptor Antagonists, Compositions, and Methods of Treatment," the disclosure of which is incorporated by reference.

Pharmaceutical compositions [0155] The present invention relates to a method for modulating (e.g., inhibiting or activating) IL-1 receptor activity through its interaction with the peptides, peptide derivatives and peptidomimetics of the present invention. In view of the importance of IL-1 and or IL-1 R/IL I RacP receptor function in numerous pathways and conditions in animals, the peptides, peptide derivatives and peptidomimetics of the present invention are useful in the treatment of IL-1 related diseases, disorders or conditions.

[0156] Therefore, methods of the present invention comprise administering to a subject in need thereof or at risk of being in need thereof an effective amount of a peptide, peptide derivative or peptidomimetic, or a composition comprising a peptide, peptide derivative or peptidomimetic to a subject, to modulate (e.g., inhibit) IL-1R/RacP biological activity. In one embodiment, an effective amount of a therapeutic composition comprising a peptide or peptide derivative thereof and a suitable pharmaceutical carrier is administered to a subject to inhibit IL-1 R/IL-1 RacP biological activity to prevent, ameliorate symptoms or treat a disorder, disease or condition related to abnormal signaling through IL-1R/IL-lRacP (e.g., overstimulation of the IL-1/IL-1RacP receptor via an overproduction of IL-1/IL-1RacP ligand or via a constitutively active receptor or any other defect). In one embodiment, the subject is an animal. In another embodiment, the subject is a mammal, and preferably a human.

[0157] The peptides, peptide derivatives and peptidomimetics of the present invention are used in the treatment, prophylaxis or amelioration of symptoms in any disease condition or disorder where the inhibition of IL-1R/IL-lRacP biological activity might be beneficial.
Diseases, conditions or disorders to which the peptides, peptide derivatives or peptidomimetics of the present invention may be beneficial include, but are not limited to the following examples: chronic and acute inflammation diseases like rheumatoid arthritis, inflammatory bowel disease, septic shock, osteoarthritis, psoriasis, encephalitis, glomerulonephritis, respiratory distress syndrome and Reiter's syndrome. Other conditions include, systemic lupus erythematosus, scleroderma, Crohn's disease, ulcerative colitis, inflammatory joint disease, cachexia in certain leukemias, Alzheimer's disease, numerous types of cancers, diabetes mellitus (type I), pulmonary hypertension, stroke, periventricular leucopenia and meningitis.

[0158] The present invention can also be used to treat other inflammatory diseases, disorders and conditions including, but not limited to, CNS demyelinating diseases, multiple sclerosis, acute disseminated encephalomyelitis (ADEM), idiopathic inflammatory demyelinating disease, transverse myelitis, Devic's disease, progressive multifocal leukoencephaly, Guillain-Barre syndrome, chronic inflammatory demyelinating polyneuropathy, anti-MAG neuropathy, inflammatory bowel disease, sepsis, septic shock, adult respiratory distress syndrome, pancreatitis, trauma-induced shock, asthma, bronchial asthma, allergic rhinitis, cystic fibrosis, stroke, acute bronchitis, chronic bronchitis, acute bronchiolitis, chronic bronchiolitis, gout, spondylarthropathris, ankylosing spondylitis, Reiter's syndrome, psoriatic arthropathy, enterapathric spondylitis, juvenile arthropathy or juvenile ankylosing spondylitis, reactive arthropathy, infectious or post-infectious arthritis, gonoccocal arthritis, tuberculous arthritis, viral arthritis, fungal arthritis, syphilitic arthritis, Lyme disease, arthritis associated with "vasculitic syndromes," polyarteritis nodosa, hypersensitivity vasculitis, Luegenec's granulomatosis, polymyalgin rheumatica, joint cell arteritis, calcium crystal deposition arthropathris, pseudo gout, non-articular rheumatism, bursitis, tenosynomitis, epicondylitis (tennis elbow), carpal tunnel syndrome, repetitive use injury (typing), miscellaneous forms of arthritis, neuropathic joint disease (charco and joint), hemarthrosis (hemarthrosic), Henoch-Schonlein purpura, hypertrophic osteoarthropathy, multicentric reticulohistiocytosis, arthritis associated with certain diseases, surcoilosis, hemochromatosis, sickle cell disease and other hemoglobinopathries, hyperlipoproteineimia, hypogammaglobulinemia, hyperparathyroidism, acromegaly, familial Mediterranean fever, Behat's Disease, systemic lupus erythrematosis, and relapsing polychondritis, inflammatory conditions resulting from harmful stimuli, such as pathogens, damaged cells, or irritants, sarcoidosis, disseminated intravascular coagulation, atherosclerosis, Kawasaki's disease, macrophage activation syndrome (MAS), HIV, graft-versus-host disease, Sjogren's syndrome, vasculitis, autoimmune thyroiditis, dermatitis, atopic dermatitis, myasthenia gravis, inflammatory conditions of the skin, cardiovascular system, nervous system, liver, kidney and pancreas, cirrhosis, eosinophilic esophagitis, cardiovascular disorders, disorders associated with wound healing, respiratory disorders, chronic obstructive pulmonary disease, emphysema, acute inflammatory conditions, atopic inflammatory disorders, bacterial, viral, fungal or protozoan infections, pulmonary diseases, systemic inflammatory response syndrome (SIRS), hemophagocytic lymphohistiocytosis (HLH), juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis, lupus nephritis, lupus-associated arthritis, ankylosing spondylitis, autoimmune diseases and related diseases or conditions.

[0159] Compositions within the scope of the present invention should contain the active agent (e.g. peptide, peptide derivative or peptidomimetic) in an amount effective to achieve the desired therapeutic effect while minimizing adverse side effects.
Pharmaceutically acceptable preparations and salts of the active agent are within the scope of the present invention and are well known in the art. For the administration of polypeptide antagonists and the like, the amount administered should be chosen so as to minimize adverse side effects. The amount of the therapeutic or pharmaceutical composition which is effective in the treatment of a particular disease, disorder or condition will depend on the nature and severity of the disease, the target site of action, the patient's weight, special diets being followed by the patient, concurrent medications being used, the administration route and other factors that will be recognized by those skilled in the art. The dosage will be adapted by the clinician in accordance with conventional factors such as the extent of the disease and different parameters from the patient. Typically, 0.001 to 100 mg/kg/day will be administered to the subject. Effective doses may be extrapolated from dose response curves derived from in vitro or animal model test systems. For example, in order to obtain an effective mg/kg dose for humans based on data generated from rat studies, the effective mg/kg dosage in rat is divided by six.

[0160] Various delivery systems are known and can be used to administer peptides, peptide derivatives or peptidomimetics or a pharmaceutical composition of the present invention. The pharmaceutical composition of the present invention can be administered by any suitable route including, intravenous or intramuscular injection, intraventricular or intrathecal injection (for central nervous system administration), orally, topically, subcutaneously, subconjunctivally, or via intranasal, intradermal, sublingual, vaginal, rectal or epidural routes.

[0161] Other delivery system well known in the art can be used for delivery of the pharmaceutical compositions of the present invention, for example via aqueous solutions, encapsulation in microparticles, or microcapsules.

[0162] In yet another embodiment, the pharmaceutical compositions of the present invention can be delivered in a controlled release system. In some embodiments, polymeric materials are used (see Smolen and Ball, Controlled Drug Bioavailability, Drug product design and performance, 1984, John Wiley & Sons; Ranade and Hollinger, Drug Delivery Systems, pharmacology and toxicology series, 2003, 2"d edition, CRRC Press);
in other embodiments, a pump is used (Saudek et al., 1989, N. Engl. J. Med. 321: 574).

[0163] Compounds of the present invention may also be delivered by the use of monoclonal antibodies as individual carriers to which the compound molecules are coupled.
The compounds of the present invention may also be coupled to a class of biodegradable polymers useful in achieving controlled release of the drug, non-limiting examples, include:
polylactic acid, polyorthoesters, cross-linked amphipathic block copolymers and hydrogels, polyhydroxy butyric acid and polydihydropyrans.

[0164] As mentioned above, pharmaceutical compositions of the present invention comprise a peptide, peptide derivative or peptidomimetic combined with a pharmaceutically acceptable carrier. The term carrier refers to diluents, adjuvants, excipients such as a filler or a binder, a disintegrating agent, a lubricant a silica flow conditioner a stabilizing agent or vehicles with which the peptide, peptide derivative or peptidomimetic is administered. Such pharmaceutical carriers include sterile liquids such as water and oils including mineral oil, vegetable oil (e.g., peanut oil, soybean oil, sesame oil, canola oil), animal oil or oil of synthetic origin. Aqueous glycerol and dextrose solutions as well as saline solutions may also be employed as liquid carriers of the pharmaceutical compositions of the present invention.
Of course, the choice of the carrier depends on the nature of the peptide, peptide derivative or peptidomimetic, its solubility and other physiological properties as well as the target site of delivery and application. For example, carriers that can penetrate the blood brain barrier are used for treatment, prophylaxis or amelioration of symptoms of diseases or conditions (e.g.

inflammation) in the central nervous system. Examples of suitable pharmaceutical carriers are described in Remington: The Science and Practice of Pharmacy by Alfonso R.
Gennaro, 2003, 21th edition, Mack Publishing Company.

[0165] Further pharmaceutically suitable materials that may be incorporated in pharmaceutical preparations of the present invention include absorption enhancers, pH
regulators and buffers, osmolarity adjusters, preservatives, stabilizers, antioxidants, surfactants, thickeners, emollient, dispersing agents, flavoring agents, coloring agents and wetting agents.

[0166] Examples of suitable pharmaceutical excipients include water, glucose, sucrose, lactose, glycol, ethanol, glycerol monostearate, gelatin, rice, starch flour, chalk, sodium stearate, malt, sodium chloride and the like. The pharmaceutical compositions of the present invention can take the form of solutions, capsules, tablets, creams, gels, powders sustained release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides (see Remington: The Science and Practice of Pharmacy by Alfonso R. Gennaro, 2003, 21th edition, Mack Publishing Company). Such compositions contain a therapeutically effective amount of the therapeutic composition, together with a suitable amount of carrier so as to provide the form for proper administration to the subject. The formulations are designed so as to suit the mode of administration and the target site of action (e.g., a particular organ or cell type).

[0167] The pharmaceutical compositions of the present invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those that form with free amino groups and those that react with free carboxyl groups. Non-toxic alkali metal, alkaline earth metal and ammonium salts commonly used in the pharmaceutical industry include sodium, potassium, lithium, calcium, magnesium, barium, ammonium, and protamine zinc salts, which are prepared by methods well known in the art. The term also includes non-toxic acid addition salts, which are generally prepared by reacting the compounds of the present invention with suitable organic or inorganic acid. Representative salts include the hydrobromide, hydrochloride, valerate, oxalate, oleate, laureate, borate, benzoate, sulfate, bisulfate, acetate, phosphate, tysolate, citrate, maleate, fumarate, tartrate, succinate, napsylate salts and the like.

[0168] Examples of fillers or binders that may be used in accordance with the present invention include acacia, alginic acid, calcium phosphate (dibasic), carboxymethylcellulose, carboxymethylcellulose sodium, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, dextrin, dextrates, sucrose, tylose, pregelatinized starch, calcium sulfate, amylose, glycine, bentonite, maltose, sorbitol, ethylcellulose, disodium hydrogen phosphate, disodium phosphate, disodium pyrosulfite, polyvinyl alcohol, gelatin, glucose, guar gum, liquid glucose, compressible sugar, magnesium aluminum silicate, maltodextrin, polyethylene oxide, polymethacrylates, povidone, sodium alginate, tragacanth micro crystalline cellulose, starch, and zein. In certain embodiments, a filler or binder is microcrystalline cellulose.

[0169] Examples of disintegrating agents that may be used include alginic acid, carboxymethylcellulose, carboxymethylcellulose sodium, hydroxypropylcellulose (low substituted), microcrystalline cellulose, powdered cellulose, colloidal silicon dioxide, sodium croscarmellose, crospovidone, methylcellulose, polacrilin potassium, povidone, sodium alginate, sodium starch glycolate, starch, disodium disulfite, disodium edathamil, disodium edetate, disodiumethylenediaminetetraacetate (EDTA) crosslinked polyvinylpyrollidines, pregelatinized starch, carboxymethyl starch, sodium carboxymethyl starch, microcrystalline cellulose.

[0170] Examples of lubricants include calcium stearate, canola oil, glyceryl palmitostearate, hydrogenated vegetable oil (type I), magnesium oxide, magnesium stearate, mineral oil, poloxamer, polyethylene glycol, sodium lauryl sulfate, sodium stearate fumarate, stearic acid, talc and, zinc stearate, glyceryl behapate, magnesium lauryl sulfate, boric acid, sodium benzoate, sodium acetate, sodium benzoate/sodium acetate (in combination), DL-leucine.

[0171] Examples of silica flow conditioners include colloidal silicon dioxide, magnesium aluminum silicate and guar gum. Another most preferred silica flow conditioner consists of silicon dioxide.

[0172] Examples of stabilizing agents include acacia, albumin, polyvinyl alcohol, alginic acid, bentonite, dicalcium phosphate, carboxymethylcellulose, hydroxypropylcellulose, colloidal silicon dioxide, cyclodextrins, glyceryl monostearate, hydroxypropyl methylcellulose, magnesium trisilicate, magnesium aluminum silicate, propylene glycol, propylene glycol alginate, sodium alginate, carnauba wax, xanthan gum, starch, stearate(s), stearic acid, stearic monoglyceride and stearyl alcohol.

[0173] The present invention also provides for modifications of peptides or peptide derivatives such that they are more stable once administered to a subject (i.e., once administered it has a longer half-life or longer period of effectiveness as compared to the unmodified form). Such modifications are well known to those skilled in the art to which this invention pertain (e.g., polyethylene glycol derivatization a.k.a. PEGylation, microencapsulation, etc).

[0174] The IL-1R/IL-lRacP antagonists or agonists of the present invention may be administered alone or in combination with other active agents useful for the treatment, prophylaxis or amelioration of symptoms of a IL-1, IL-1R/IL-1RacP associated disease or condition. Thus, the compositions and methods of the present invention can be used in combination with other agents exhibiting the ability to modulate IL-1 activity (e.g., synthesis, release and/or binding to IL-1R/IL-lRacP) or to reduce the symptoms of an IL-1 associated disease (e.g., rheumatoid arthritis and inflammatory bowel disease). Example of such agents include but are not limited to antirheumatic drugs such as chloroquine, auranofin (RidauraTM), dexamethasone, sodium aurothiomalate, methotrexate (see Lee et at., 1988, Proc. Int. Acad. Sci, 85:1204), probucol (see Ku et at., 1988, Am. J. Cardiol.
62:778), pentoxyfylline (e.g., Sullivan et at., 1988, Infect. Immun. 56 :1722), disulfiram (see Marx 1988, Science, 239:257), antioxidants such as nordihydroguaiaretic acid (lee et at., 1988, Int J. Immunopharm., 10:385), IL-1 Trap (see e.g., 2003, Curr. Opin. Inv. Drugs, 4(5): 593-597), Anakinra (KineretTM, PCT Application W000236152), leflunomide, corticosteroids (MedrolTM, DeltasoneTM, OrasoneTM) as well as other agents such as those described in Bender and Lee (1989) Annual Reports in Medicinal Chemistry, chapter 20:
Pharmacological Modulation of IL-1: 185-193). Other drugs may also be used in combination with the compounds of the present invention like anti-inflammatory drugs such as Non Steroidal Antiinflammatory Drugs (NSAIDS, e.g., Rofecoxib (VIOXXTM), Celecoxib (CelebrexTM), Valdecoxib (BextraTM), AspirinTM, advilTM), anti TNF-a drugs (Infliximab, etanercept, adalimumab), collagenase inhibitors and others. Of course a combination of two or more peptides, derivatives and peptide mimetics and their combination with one or more drug can also be used, in all combinations (e.g. one or more peptide with one or more mimetic, one or more mimetic with one or more derivative, one ore more peptide with one or more drug etc.).

[0175] The present invention is illustrated in further details by the following non-limiting examples. The examples are provided for illustration only and should not be construed as limiting the scope of the invention.

EXEMPLIFICATION

[0176] As depicted in the Examples below, in certain exemplary embodiments, compounds are prepared according to the following general procedures. It will be appreciated that, although the general methods depict the synthesis of certain compounds of the present invention, the following general methods, and other methods known to one of ordinary skill in the art, can be applied to all compounds and subclasses and species of each of these compounds, as described herein.

Synthesis of IL-1R antagonistic peptide 101.10 D-Agl analogs (a) (R)-Agl-D-Tyr-D-Thr-D-Val-D-Glu-D-Leu-D-Ala-NHz (54) (SEQ ID NO:61) O H O H O
H2N, -OH
N N,.= N
) ,r N N N NH2 O O H O H O
OH

[0177] (R)-Agl-D-Tyr-D-Thr-D-Val-D-Glu-D-Leu-D-Ala-NH2 (54) (SEQ ID NO:61) was prepared as described above, using microwave assisted annulation conditions B over 8 h, to give the desired lactam peptide TFA salt 59 (57 mg, 55% crude purity as analyzed by analytical RP-HPLC (UV 214), 2-40% MeCN in H20, 0.1 % FA, 15 min gradient).
Purification was then carried out by preparatory RP-HPLC (2-20% MeCN in H20, 0.1 % FA, 20 min gradient) to give the desired formic acid salt 54 (16 mg, 20%) as a white foam. The purified product was analyzed by analytical RP-HPLC (UV 214) using both MeCN
tR 10.98 (0-60% MeCN in H20, 0.1% FA, 30min gradient) and MeOH tR 15.30 (0-60% MeOH in H20, 0.1 % FA, 30min gradient) and revealed >99% purity. HRMS Calcd. for C36H57011Ns [M+H]+ 777.4141, found 777.4138.

(b) D-Arg-(R)-Agl-D-Thr-D-Val-D-Glu-D-Leu-D-Ala-NHz (55) (SEQ ID NO:62) rOHO1O
NH
HN=<

[0178] D-Arg-(R)-Agl-D-Thr-D-Val-D-Glu-D-Leu-D-Ala-NH2 (55) (SEQ ID NO:62) was prepared as described above, using microwave assisted annulation conditions B over 10 h, to give the desired lactam peptide TFA salt 55 (99 mg, 41 % crude purity as analyzed by analytical RP-HPLC (UV 214), 0-20% MeCN in H20, 0.1 % FA, 15 min gradient).
Purification was then carried out by preparatory RP-HPLC (0-20% MeCN in H20, 0.1 % FA, 30 min gradient) to give the desired formic acid salt 55 (22 mg, 15%) as a white foam. The purified product was analyzed by analytical RP-HPLC (UV 214) using both MeCN
tR 8.26 (0-60% MeCN in H20, 0.1% FA, 30 min gradient) and MeOH tR 9.36 (0-60% MeOH in H20, 0.1% FA, 30min gradient) and revealed >99% purity. HRMS Calcd. for C33H600i0Nii [M+H]+ 770.4519, found 770.4514.

(c) D-Arg-D-Tyr-(R)-Agl-D-Val-D-Glu-D-Leu-D-Ala-NHz (56) (SEQ ID NO:63) NH
_j-NH
HO O
H2NNH O ~ -O H O

O H O O
OH

[0179] D-Arg-D-Tyr-(R)-Agl-D-Val-D-Glu-D-Leu-D-Ala-NH2 (56) (SEQ ID NO:63) was prepared as described above, using microwave assisted annulation conditions B over 6 h, to give the desired lactam peptide TFA salt 56 (89 mg, 58% crude purity as analyzed by analytical RP-HPLC (UV 214), 0-40% MeCN in H20, 0.1 % FA, 20 min gradient).
Purification was then carried out by preparatory RP-HPLC (0-40% MeCN in H2O, 0.1 % FA, 30min gradient) to give the desired formic acid salt 56 (13 mg, 7%) as a white foam. The purified product was analyzed by analytical RP-HPLC (UV 214) using both MeCN
tR 9.84 (0-60% MeCN in H2O, 0.1% FA, 3 0min gradient) and MeOH tR 12.65 (0-60% MeOH in H20, 0.1% FA, 30min gradient) and revealed >99% purity. HRMS Calcd. for C3sH620i0Nii [M+H]+ 832.4676, found 832.4676.

(d) D-Arg-D-Tyr-D-Thr-(R)-Agl-D-Glu-D-Leu-D-Ala-NHz (57) (SEQ ID NO:64) OH H{NNH2 O H O N NH O

H O ,H ~ A
O O
HO
HNNH

[0180] D-Arg-D-Tyr-D-Thr-(R)-Agl-D-Glu-D-Leu-D-Ala-NH2 (57) (SEQ ID NO:64) was prepared as described above, using microwave assisted annulation conditions B over 4 h, to give the desired lactam peptide TFA salt 57 (79 mg, 50% crude purity as analyzed by analytical RP-HPLC (UV 214), 2-40% MeCN in H2O, 0.1 % FA, 15 min gradient).
Purification was then carried out by preparatory RP-HPLC (2-20% MeCN in H2O, 0.1 % FA, 20 min gradient) to give the desired formic acid salt 57 (21 mg, 11 %) as a white foam. The purified product was analyzed by analytical RP-HPLC (UV 214) using both MeCN
tR 9.19 (0-60% MeCN in H2O, 0.1% FA, 30 min gradient) and MeOH tR 11.15 (0-60% MeOH in H2O, 0.1 % FA, 30 min gradient) and revealed >99% purity. HRMS Calcd. for [M+H]+ 833.4468, found 833.4459.

(e) D-Arg-D-Tyr-D-Thr-D-Val- (R)-AgI-D-Leu-D-Ala-NHz (58) (SEQ ID NO:65) NH

H O H O O NH O
N
H2NN N N"
O O H O
OH

[0181] D-Arg-D-Tyr-D-Thr-D-Val-(R)-Agl-D-Leu-D-Ala-NH2 (58) (SEQ ID NO:65) was prepared as described above, using microwave assisted annulation conditions B over 4 h, to give the desired lactam peptide TFA salt 58 (157 mg, 36% crude purity as analyzed by analytical RP-HPLC (UV 214), 0-40% MeCN in H2O, 0.1% FA, 30 min gradient).
Purification was then carried out by preparatory RP-HPLC (0-40% MeCN in H2O, 0.1 % FA, 30 min gradient) to give the desired formic acid salt 58 (37 mg, 21%) as a white foam. The purified product was analyzed by analytical RP-HPLC (UV 214) using both MeCN
tR 9.93 (0-60% MeCN in H20, 0.1% FA, 30 min gradient) and MeOH tR 12.57 (0-60% MeOH in H20, 0.1% FA, 30 min gradient) and revealed >99% purity. HRMS Calcd. for C37H6209Ni i [M+H]+ 803.4727, found 803.4725.

69 D-Arg-D-Tyr-D-Thr-D-Val-D-Glu-(R)-Agl-D-Ala-NH2 (59) (SEQ ID NO:66) OH

H
O Fi O O
H2N HN HN H`,. N ~NH2 O "OH O O O

HN1~1 NH2 [0182] D-Arg-D-Tyr-D-Thr-D-Val-D-Glu-(R)-Agl-D-Ala-NH2 (59) (SEQ ID NO:66) was prepared as described above, using microwave assisted annulation conditions B over 3 h, to give the desired lactam peptide TFA salt 59 (70 mg, 81 % crude purity as analyzed by analytical RP-HPLC (UV 214), 0-40% MeCN in H2O, 0.1 % FA, 4 min gradient).
Purification was then carried out by preparatory RP-HPLC (0-40% MeCN in H2O, 0.1 % FA, 20 min gradient) to give the desired formic acid salt 59 (2.6 mg, 7%) as a white foam. The purified product was analyzed by analytical RP-HPLC (UV 214) using both MeCN tR 8.52 (0-60%
MeCN in H2O, 0.1 % FA, 30 min gradient) and MeOH tR 9.91 (0-60% MeOH in H2O, 0.1 %
FA, 30 min gradient) and revealed >99% purity. HRMS Calcd. for C36H58011Nii [M+H]+
820.4312, found 820.4307.

Optimized protocol for (R)-Bgl lactam peptide synthesis [0183] (S)- and (R)-Bgl 101.10 analogs were synthesized using optimized conditions.
Peptide synthesis was carried out in syringe tubes as described earlier. When the residue was reached for lactam synthesis, Fmoc deprotection was performed as usual and the resin was transferred into a 5 mL glass microwave vessel. A solution of cyclic sulfamidate (R)-17 (5 eq.) and DIEA (1 eq.) in THE (2 mL) was added, the reactor was sealed and the mixture was irradiated under microwave at 60 C for 1 h. The resin was then transferred into a 6 mL
syringe tube, washed with DMF (3 times), MeOH (3 times), DCM (3 times), and with three cycles comprising washing with 2% AcOH in DMSO for 30 min and washing with MeCN
for 5 minutes. The resin was transferred back to a 5 mL glass microwave vessel, a DMSO:H20:AcOH 75:23:2 (2 mL) was added, the reactor sealed and the mixture was irradiated under microwave at 100 C for 3 h. The resin was transferred back into a syringe tube and underwent an acetyl-capping step by treatment with a solution of acetic anhydride (5 eq.) in DMF (4 mL) in the presence of DIEA (5 eq.) for 1.5 h. The peptide synthesis was then continued as previously described.

(R)-Bab 101.10 analogs (R)-Bab-D-Tyr-D-Thr-D-Val-D-Glu-D-Leu-D-Ala-NH2 (71) (SEQ ID NO:67) H O OHH O H O
H2N~,N N". N N N N NH2 OHO O H O H O
OH

[0184] (R)-Bab-D-Tyr-D-Thr-D-Val-D-Glu-D-Leu-D-Ala-NH2 (71) (SEQ ID NO:67) was isolated while purifying compound 54, prepared as described above (28% of 54 crude mixture as analyzed by analytical RP-HPLC (column a, UV: 1= 214 nm, 2-40% MeCN
in H2O, 0.1 % FA, 15 min gradient). Purification was carried out by preparatory RP-HPLC
(column A, 2-20% MeCN in H2O, 0.1 % FA, 20 min gradient) to give the desired formic acid salt 71 (10 mg, 10%) as a white fluffy solid. The purified product was analyzed by analytical RP-HPLC (column a, UV: 1= 214 nm) using both MeCN tR 14.11 (5-40% MeCN in H2O, 0.1 % FA, 15 min gradient) and MeOH tR 12.69 (20-80% MeOH in H2O, 0.1 % FA, 15 min) and revealed >99% purity. HRMS Calcd. for C43H64012Ns [M+H]+ 885.4714, found 885.4716.

D-Arg-(R)-Bab-D-Thr-D-Val-D-Glu-D-Leu-D-Ala-NH2 (72) (SEQ ID NO:68) HNNH
H H O = H O = H O
H2N~NN N~N NTN(NH2 O OHO ,,OHH O H 0 [0185] D-Arg-(R)-Bab-D-Thr-D-Val-D-Glu-D-Leu-D-Ala-NH2 (72) (SEQ ID NO:68) was isolated while purifying compound 55, prepared as described above (45% of 55 crude mixture as analyzed by analytical RP-HPLC (column a, UV: 1= 214 nm, 0-20% MeCN
in H20, 0.1 % FA, 15 min gradient). Purification was carried out by preparatory RP-HPLC
(column A, 0-20% MeCN in H20, 0.1% FA, 30 min gradient) to give the desired formic acid salt 72 (27 mg, 15%) as a white fluffy solid. The purified product was analyzed by analytical RP-HPLC (column a, UV: 1= 214 nm) using both MeCN tR 9.04 (0-60% MeCN in H20, 0.1% FA, 30 min gradient) and MeOH tR 11.00 (0-60% MeOH in H20, 0.1% FA, 30 min) and revealed >99% purity. HRMS Calcd. for C40H68011Nii [M+H]+ 878.5092, found 878.5094.

D-Arg-D-Tyr-(R)-Bab-D-Val-D-Glu-D-Leu-D-Ala-NH2 (73) (SEQ ID NO:69) / Yi O = H H O = H O =

O oho O O

NH
H2N"ill NH

[0186] D-Arg-D-Tyr-D-Thr-(R)-Bab-D-Glu-D-Leu-D-Ala-NH2 (73) (SEQ ID NO:69) was isolated while purifying compound 56, prepared as described above (27% of 56 crude mixture as analyzed by analytical RP-HPLC (column a, UV: 1= 214 nm, 0-40% MeCN
in H20, 0.1 % FA, 15 min gradient). Purification was carried out by preparatory RP-HPLC

(column A, 0-40% MeCN in H20, 0.1% FA, 30 min gradient) to give the desired formic acid salt 73 (7 mg, 3%) as a white fluffy solid. The purified product was analyzed by analytical RP-HPLC (column a, UV: 1= 214 nm) using both MeCN tR 9.12 (0-60% MeCN in H20, 0.1 % FA, 15 min gradient) and MeOH tR 9.45 (0-60% MeOH in H20, 0.1 % FA, 15 min) and revealed >99% purity. HRMS Calcd. for C45H7001IN1I [M+H]+ 940.5240, found 940.5251.

D-Arg-D-Tyr-D-Thr-(R)-Bab-D-Glu-D-Leu-D-Ala-NH2 (74) (SEQ ID NO:70) NH HN N N N^,~,N NH2 H = H

HO N~Z HO 0 [0187] D-Arg-D-Tyr-D-Thr-(R)-Bab-D-Glu-D-Leu-D-Ala-NH2 (74) (SEQ ID NO:70) was isolated while purifying compound 57, prepared as described above (6% of 57 crude mixture as analyzed by analytical RP-HPLC (column a, UV: 1= 214 nm, 2-40% MeCN
in H20, 0.1 % FA, 15 min gradient). Purification was carried out by preparatory RP-HPLC
(column A, 2-20% MeCN in H20, 0.1 % FA, 20 min gradient) to give the desired formic acid salt 74 (8.5 mg, 4%) as a white fluffy solid. The purified product was analyzed by analytical RP-HPLC (column a, UV: 1= 214 nm) using both MeCN tR 12.27 (5-40% MeCN in H20, 0.1 % FA, 15 min gradient) and MeOH tR 17.09 (5-40% MeOH in H20, 0.1% FA, 15 min) and revealed >99% purity. HRMS Calcd. for C44H68012Nii [M+H]+ 942.5027, found 942.5043.

(R)-Bgl 101.10 scan compounds (R)-Bgl-D-Tyr-D-Thr-D-Val-D-Glu-D-Leu-D-Ala-NH2 (75) (SEQ ID NO:71) 0 O OHH O ) O =
HZN ,. =
N N,= N NN NNHZ ':)~ O H H O H O

OH

[0188] (R)-Bgl-D-Tyr-D-Thr-D-Val-D-Glu-D-Leu-D-Ala-NH2 (75) (SEQ ID NO:71) was prepared on a 0.072 mmol, in a syringe tube following the optimized protocol as described above, using microwave assisted annulation over 240 min, to give the desired lactam peptide TFA salt 75 (34 mg) in 71 % crude purity as determined by analytical RP-HPLC (column b, UV: 1= 214 nm, 0-80% MeCN in H20, 0.1% FA, 30 min gradient).
Purification was carried out by preparative RP-HPLC (column B, 10-30% MeCN in H2O, 0.1 % FA, 30 min gradient) to give the desired FA salt product 75 (13 mg, 22%) as a white fluffy solid. The purified product was analyzed by analytical RP-HPLC (column b, UV: 1=
214 nm) using both MeCN tR 11,8 (5-60% MeCN in H2O, 0.1% FA, 25 min gradient) and MeOH tR 16.41 (0-80% MeOH in H2O, 0.1 % FA, 25 min gradient) and revealed >99%
purity. HRMS Calcd. m/z for C36H57011Ns [M+H]+ 777.4141, found 777.4145.

D-Arg-(R)-Bgl-D-Thr-D-Val-D-Glu-D-Leu-D-Ala-NH2 (76) (SEQ ID NO:72) OHH O O
O - _ N N HEN H--Y
~` O O O
HNNH HN
O

[0189] D-Arg-(R)-Bgl-D-Thr-D-Val-D-Glu-D-Leu-D-Ala-NH2 (76) (SEQ ID NO:72) was prepared on a 0.078 mmol scale, in a syringe tube following the optimized protocol as described above, using microwave assisted annulation over 360 min, to give the desired lactam peptide TFA salt 76 (35 mg) in 87% crude purity as determined by analytical RP-HPLC (column a, UV: 1= 214 nm, 0-20% MeCN in H20, 0.1% FA, 30 min gradient).
Purification was carried out by preparative RP-HPLC (column A, 2-20% MeCN in H20, 0.1 % FA, 30 min gradient) to give the desired FA salt product 76 (16 mg, 24%) as a white fluffy solid. The purified product was analyzed by analytical RP-HPLC (column a, UV: 1=
214 nm) using both MeCN tR 7.79 (5-60% MeCN in H20, 0.1% FA, 25 min gradient) and MeOH tR 10.28 (0-80% MeOH in H20, 0.1 % FA, 25 min gradient) and revealed >99%
purity. HRMS Calcd. m/z for C33H600i0Nii [M+H]+ 770.4519, found 770.4521.

D-Arg-D-Tyr-(R)-Bgl-D-Val-D-Glu-D-Leu-D-Ala-NH2 (77) (SEQ ID NO:73) HZN
NH
NH
HO O
HZN--~- NH O
O O
O HN N H N H NHZ
O O
OH

[0190] D-Arg-D-Tyr-(R)-Bgl-D-Val-D-Glu-D-Leu-D-Ala-NH2 (77) (SEQ ID NO:73) was prepared on a 0.082 mmol scale, in a syringe tube following the optimized protocol as described above, using microwave assisted annulation over 240 min, to give the desired lactam peptide TFA salt 77 (34 mg) in 86% crude purity as determined by analytical RP-HPLC (column b, UV: 1= 214 nm, 0-20% MeCN in H20, 0.1% FA, 30 min gradient).
Purification was carried out by preparative RP-HPLC (column B, 0-20% MeCN in H2O, 0.1 % FA, 30 min gradient) to give the desired FA salt product 77 (17 mg, 23%) as a white fluffy solid. The purified product was analyzed by analytical RP-HPLC (column b, UV: 1=
214 nm) using both MeCN tR 9.55 (5-60% MeCN in H2O, 0.1% FA, 25 min gradient) and MeOH tR 12.99 (0-80% MeOH in H2O, 0.1 % FA, 25 min gradient) and revealed >99%
purity. HRMS Calcd. m/z for C3sH62010N11 [M+H]+ 832.4676, found 832.4677.

D-Arg-D-Tyr-D-Thr-(R)-Bgl-D-Glu-D-Leu-D-Ala-NH2 (78) (SEQ ID NO:74) N
NHOHH H ON, N O
HZNANH p O p-~NH
NH O
\1 O H
HN
HN

[0191] D-Arg-D-Tyr-D-Thr-(R)-Bgl-D-Glu-D-Leu-D-Ala-NH2 (78) (SEQ ID NO:74) was prepared on a 0.087 mmol scale, in a syringe tube following the optimized protocol as described above, using microwave assisted annulation over 420 min, to give the desired lactam peptide TFA salt 78 (62 mg) in 79% crude purity as determined by analytical RP-HPLC (column b, UV: 1= 214 nm, 0-30% MeCN in H20, 0.1% FA, 30 min gradient).
Purification was carried out by preparative RP-HPLC (column B, 18% MeCN in H20, 0.1 %
FA, isocratic) to give the desired FA salt product 78 (16 mg, 17%) as a white fluffy solid.
The purified product was analyzed by analytical RP-HPLC (column b, UV: 1= 214 nm) using both MeCN tR 8.00 (5-60% MeCN in H20, 0.1% FA, 25 min gradient) and MeOH tR
9.93 (0-80% MeOH in H20, 0.1 % FA, 25 min gradient) and revealed >99% purity. HRMS
Calcd.
m/z for C37H60011Nii [M+H]+ 834.4468, found 834.4470.

D-Arg-D-Tyr-D-Thr-D-Val-(R)-Bgl-D-Leu-D-Ala-NH2 (79) (SEQ ID NO:75) H O OHH O O p NH p HZNN H,. N H

OH

[0192] D-Arg-D-Tyr-D-Thr-D-Val-(R)-Bgl-D-Leu-D-Ala-NH2 (79) (SEQ ID NO:75) was prepared on a 0.098 mmol scale, in a syringe tube following the optimized protocol as described above, using microwave assisted annulation over 240 min, to give the desired lactam peptide TFA salt 79 (35 mg) in 87% crude purity as determined by analytical RP-HPLC (column a, UV: 1= 214 nm, 0-80% MeCN in H20, 0.1% FA, 30 min gradient).
Purification was carried out by preparative RP-HPLC (column A, 0-30% MeCN in H20, 0.1 % FA, 30 min gradient) to give the desired FA salt product 79 (6 mg, 7%) as a white fluffy solid. The purified product was analyzed by analytical RP-HPLC (column a, UV: 1=
214 nm) using both MeCN tR 9.67 (5-60% MeCN in H20, 0.1% FA, 25 min gradient) and MeOH tR 13.18 (0-80% MeOH in H20, 0.1 % FA, 25 min gradient) and revealed >99%
purity. HRMS Calcd. m/z for C37H6109N11 [M+Na]+ 826.4546, found 826.4544.

D-Arg-D-Tyr-D-Thr-D-Val-D-Glu-(R)-Bgl-D-Ala-NH2 (80) (SEQ ID NO:76) HZN'r NH

OH
H O H O = H
HzN qH1 N~N O
O O H O N
OH O

[0193] D-Arg-D-Tyr-D-Thr-D-Val-D-Glu-(R)-Bgl-D-Ala-NH2 (80) (SEQ ID NO:76) was prepared on a 0.105 mmol scale, in a syringe tube following the optimized protocol as described above, using microwave assisted annulation over 240 min, to give the desired lactam peptide TFA salt 80 (35 mg) in 50% crude purity as determined by analytical RP-HPLC (column b, UV: 1= 214 nm, 0-60% MeCN in H20, 0.1% FA, 25 min gradient).
Purification was carried out by preparative RP-HPLC (column B, 0-20% MeCN in H20, 0.1 % FA, 30 min gradient) to give the desired FA salt product 80 (13 mg, 14%) as a white fluffy solid. The purified product was analyzed by analytical RP-HPLC (column b, UV: 1=
214 nm) using both MeCN tR 7.27 (5-60% MeCN in H20, 0.1% FA, 25 min gradient) and MeOH tR 9.04 (0-80% MeOH in H20, 0.1 % FA, 25 min gradient) and revealed >99%
purity.
HRMS Calcd. m/z for C36H58011N11 [M+H]+ 820.4312, found 820.4313.

(S)-Bgl 101.10 scan compounds (S)-Bgl-D-Tyr-D-Thr-D-Val-D-Glu-D-Leu-D-Ala-NH2 (81) (SEQ ID NO:77) N N~,= N :)~ NN N NH2 O H O H O
OH

[0194] (S)-Bgl-D-Tyr-D-Thr-D-Val-D-Glu-D-Leu-D-Ala-NH2 (81) (SEQ ID NO:77) was prepared on a 0.102 mmol scale, in a syringe tube following the optimized protocol as described above, using microwave assisted annulation over 600 min, to give the desired lactam peptide TFA salt 81 (34 mg) in 70% crude purity as determined by analytical RP-HPLC (column a, UV: 1= 214 nm, 0-80% MeCN in H20, 0.1% FA, 30 min gradient).
Purification was carried out by preparative RP-HPLC (column A, 10-30% MeCN in H2O, 0.1% FA, 30 min gradient) to give the desired FA salt product 81 (11 mg, 13%) as a white fluffy solid. The purified product was analyzed by analytical RP-HPLC (column a, UV: 1=
214 nm) using both MeCN tR 12.00 (5-60% MeCN in H2O, 0.1% FA, 25 min gradient) and MeOH tR 19.49 (0-60% MeOH in H2O, 0.1 % FA, 25 min gradient) and revealed >99%
purity. HRMS Calcd. m/z for C36H57011Ns [M+H]+ 777.4141, found 777.4138.

D-Arg-(S)-Bgl-D-Thr-D-Val-D-Glu-D-Leu-D-Ala-NH2 (82) (SEQ ID NO:78) OHH O H O
H
N N N HNHZ
O O O O
HN HN
NH O
H2N -t [0195] D-Arg-(S)-Bgl-D-Thr-D-Val-D-Glu-D-Leu-D-Ala-NH2 (82) (SEQ ID NO:78) was prepared on a 0.105 mmol scale, in a syringe tube following the optimized protocol as described above, using microwave assisted annulation over 600 min, to give the desired lactam peptide TFA salt 82 (33 mg) in 82% crude purity as determined by analytical RP-HPLC (column a, UV: 1= 214 nm, 0-20% MeCN in H20, 0.1% FA, 30 min gradient).
Purification was carried out by preparative RP-HPLC (column A, 0-20% MeCN in H20, 0.1 % FA, 30 min gradient) to give the desired FA salt product 82 (13 mg, 16%) as a white fluffy solid. The purified product was analyzed by analytical RP-HPLC (column a, UV: 1=
214 nm) using both MeCN tR 7.14 (5-60% MeCN in H20, 0.1% FA, 25 min gradient) and MeOH tR 8.62 (0-80% MeOH in H20, 0.1 % FA, 25 min gradient) and revealed >99%
purity.
HRMS Calcd. m/z for C33H600ioNii [M+H]+ 770.4519, found 770.4518.

D-Arg-D-Tyr-(S)-Bgl-D-Val-D-Glu-D-Leu-D-Ala-NH2 (83) (SEQ ID NO:79) HZN
NH
NH
HO O
HZN--~- NH O
O O
O HN -CfN' H N NH2 O H O
OH

[0196] D-Arg-D-Tyr-(S)-Bgl-D-Val-D-Glu-D-Leu-D-Ala-NH2 (83) (SEQ ID NO:79) was prepared on a 0.105 mmol scale, in a syringe tube following the optimized protocol as described above, using microwave assisted annulation over 480 min, to give the desired lactam peptide TFA salt 83 (52 mg) in 60% crude purity as determined by analytical RP-HPLC (column a, UV: 1= 214 nm, 0-80% MeCN in H20, 0.1% FA, 30 min gradient).
Purification was carried out by preparative RP-HPLC (column A, 5-15% MeCN in H20, 0.1 % FA, 50 min gradient) to give the desired FA salt product 83 (15 mg, 17%) as a white fluffy solid. The purified product was analyzed by analytical RP-HPLC (column a, UV: 1=
214 nm) using both MeCN tR 9.43 (5-60% MeCN in H20, 0.1% FA, 25 min gradient) and MeOH tR 14.71 (0-60% MeOH in H20, 0.1 % FA, 25 min gradient) and revealed >99%
purity. HRMS Calcd. m/z for C3sH620i0Nii [M+H]+ 832.4676, found 832.4674.

D-Arg-D-Tyr-D-Thr-(S)-Bgl-D-Glu-D-Leu-D-Ala-NH2 (84) (SEQ ID NO:80) NH OH HN, HNA
2 O NH O O ~-NH O
OH
HN
HN

[0197] D-Arg-D-Tyr-D-Thr-(S)-Bgl-D-Glu-D-Leu-D-Ala-NH2 (84) (SEQ ID NO:80) was prepared on a 0.105 mmol scale, in a syringe tube following the optimized protocol as described above, using microwave assisted annulation over 420 min, to give the desired lactam peptide TFA salt 84 (28 mg) in 87% crude purity as determined by analytical RP-HPLC (column a, UV: 1= 214 nm, 0-80% MeCN in H20, 0.1% FA, 30 min gradient).
Purification was carried out by preparative RP-HPLC (column A, 0-20% MeCN in H20, 0.1 % FA, 20 min gradient) to give the desired FA salt product 84 (12 mg, 14%) as a white fluffy solid. The purified product was analyzed by analytical RP-HPLC (column a, UV: 1=
214 nm) using both MeCN tR 7.56 (5-60% MeCN in H20, 0.1% FA, 25 min gradient) and MeOH tR 8.91 (0-80% MeOH in H20, 0.1 % FA, 25 min gradient) and revealed >99%
purity.
HRMS Calcd. m/z for C37H6001IN11 [M+H]+ 834.4468, found 834.4469.

D-Arg-D-Tyr-D-Thr-D-Val-(S)-Bgl-D-Leu-D-Ala-NH2 (85) (SEQ ID NO:81) H O OHH O O O NH p H2N~N H` N H

OH

[0198] D-Arg-D-Tyr-D-Thr-D-Val-(S)-Bgl-D-Leu-D-Ala-NH2 (85) (SEQ ID NO:81) was prepared on a 0.105 mmol scale, in a syringe tube following the optimized protocol as described above, using microwave assisted annulation over 240 min, to give the desired lactam peptide TFA salt 85 (40 mg) in 90% crude purity as determined by analytical RP-HPLC (column a, UV: 1= 214 nm, 0-80% MeCN in H20, 0.1% FA, 30 min gradient).
Purification was carried out by preparative RP-HPLC (column A, 0-20% MeCN in H20, 0.1% FA, 30 min gradient) to give the desired FA salt product 85 (11 mg, 13%) as a white fluffy solid. The purified product was analyzed by analytical RP-HPLC (column a, UV: 1=
214 nm) using both MeCN tR 9.48 (5-60% MeCN in H20, 0.1% FA, 25 min gradient) and MeOH tR 10.47 (0-80% MeOH in H20, 0.1 % FA, 25 min gradient) and revealed >99%
purity. HRMS Calcd. m/z for C37H6109N11 [M+H]+ 804.4726, found 804.4724.

D-Arg-D-Tyr-D-Thr-D-Val-D-Glu-(S)-Bgl-D-Ala-NH2 (86) (SEQ ID NO:82) HZN'r NH

OH
H O H O = H
HzNN qH1 N N~NO
O O H O N
OH O

[0199] D-Arg-D-Tyr-D-Thr-D-Val-D-Glu-(S)-Bgl-D-Ala-NH2 (86) (SEQ ID NO:82) was prepared on a 0.105 mmol scale, in a syringe tube following the optimized protocol as described above, using microwave assisted annulation over 240 min, to give the desired lactam peptide TFA salt 86 (29 mg) in 96% crude purity as determined by analytical RP-HPLC (column a, UV: 1= 214 nm, 0-20% MeCN in H20, 0.1% FA, 30 min gradient).
Purification was carried out by preparative RP-HPLC (column A, 0-20% MeCN in H20, 0.1 % FA, 30 min gradient) to give the desired FA salt product 86 (10 mg, 11 %) as a white fluffy solid. The purified product was analyzed by analytical RP-HPLC (column a, UV: 1=
214 nm) using both MeCN tR 7.6 (5-60% MeCN in H20, 0.1% FA, 25 min gradient) and MeOH tR 8.9 (0-80% MeOH in H20, 0.1 % FA, 25 min gradient) and revealed >99%
purity.
HRMS Calcd. m/z for C36H58011N11 [M+H]+ 820.4131, found 820.4136.

(R)-Bgl insertion 101.10 compounds D-Arg-(S)-Bgl-D-Tyr-D-Thr-D-Val-D-Glu-D-Leu-D-Ala-NH2 (87) (SEQ ID NO:83) NH
HZN-~ NH HO O

HN N N ~~[H ---r HZN O O H V H

OH

[0200] D-Arg-(S)-Bgl-D-Tyr-D-Thr-D-Val-D-Glu-D-Leu-D-Ala-NH2 (87) (SEQ ID
NO:83) was prepared on a 0.048 mmol scale, in a syringe tube following the optimized protocol as described above, using microwave assisted annulation over 600 min, to give the desired lactam peptide TFA salt 87 (27 mg) in 56% crude purity as determined by analytical RP-HPLC (column a, UV: 1= 214 nm, 0-40% MeCN in H20, 0.1% FA, 30 min gradient).
Purification was carried out by preparative RP-HPLC (column A, 2-20% MeCN in H20, 0.1 % FA, 25 min gradient) to give the desired FA salt product 87 (9 mg, 18%) as a white fluffy solid. The purified product was analyzed by analytical RP-HPLC (column a, UV: 1=
214 nm) using both MeCN tR 10.23 (5-60% MeCN in H20, 0.1% FA, 25 min gradient) and MeOH tR 15.99 (0-60% MeOH in H20, 0.1 % FA, 25 min gradient) and revealed >99%
purity. HRMS Calcd. m/z for C42H69012N12 [M+H]+ 933.5152, found 933.5152.

D-Arg-D-Tyr-D-(S)-Bgl-Thr-D-Val-D-Glu-D-Leu-D-Ala-NH2 (88) (SEQ ID NO: 84) OHH O H O
NH
H2N4 N'- N N N N NH2 NH H H
NH
HN OH

[0201] D-Arg-D-Tyr-D-(S)-Bgl-Thr-D-Val-D-Glu-D-Leu-D-Ala-NH2 (88) (SEQ ID
NO:84) was prepared on a 0.052 mmol scale, in a syringe tube following the optimized protocol as described above, using microwave assisted annulation over 600 min, to give the desired lactam peptide TFA salt 88 (6 mg) in 55% crude purity as determined by analytical RP-HPLC (column b, UV: 1= 214 nm, 0-60% MeCN in H20, 0.1 % FA, 25 min gradient).
Purification was carried out by preparative RP-HPLC (column B, 2-15% MeCN in H20, 0.1 % FA, 30 min gradient) to give the desired FA salt product 88 (3 mg, 6%) as a white fluffy solid. The purified product was analyzed by analytical RP-HPLC (column b, UV: 1=
214 nm) using both MeCN tR 9.22 (0-60% MeCN in H20, 0.1% FA, 25 min gradient) and MeOH tR 14.01 (0-60% MeOH in H20, 0.1 % FA, 25 min gradient) and revealed >99%
purity. HRMS Calcd. m/z for C42H69012N12 [M+H]+ 933.5152, found 933.5137.

D-Arg-D-Tyr-D-Thr-(S)-Bgl-D-Val-D-Glu-D-Leu-D-Ala-NH2 (89) (SEQ ID NO:85) HO O

NH OH O O O
--~H
N H NH, O N

HN OH

[0202] D-Arg-D-Tyr-D-Thr-(S)-Bgl-D-Val-D-Glu-D-Leu-D-Ala-NH2 (89) (SEQ ID
NO:85) was prepared on a 0.055 mmol scale, in a syringe tube following the optimized protocol as described above, using microwave assisted annulation over 480 min, to give the desired lactam peptide TFA salt 89 (16 mg) in 71 % crude purity as determined by analytical RP-HPLC (column a, UV: 1= 214 nm, 0-60% MeCN in H2O, 0.1% FA, 25 min gradient).
Purification was carried out by preparative RP-HPLC (column A, 2-20% MeCN in H2O, 0.1 % FA, 20 min gradient) to give the desired FA salt product 89 (6 mg, 10%) as a white fluffy solid. The purified product was analyzed by analytical RP-HPLC (column a, UV: 1=
214 nm) using both MeCN tR 10.38 (5-60% MeCN in H2O, 0.1% FA, 25 min gradient) and MeOH tR 16.25 (0-60% MeOH in H2O, 0.1 % FA, 25 min gradient) and revealed >99%
purity. HRMS Calcd. m/z for C42H69012N12 [M+H]+ 933.5152, found 933.5152.

D-Arg-D-Tyr-D-Thr-D-Val-(S)-Bgl-D-Glu-D-Leu-D-Ala-NH2 (90) (SEQ ID NO:86) NH HO
O
= H O O Hr' O .~
HZN~-N H"

':~ H 0 HOH O

[0203] D-Arg-D-Tyr-D-Thr-D-Val-(S)-Bgl-D-Glu-D-Leu-D-Ala-NH2 (90) (SEQ ID
NO:86) was prepared on a 0.058mmol scale, in a syringe tube following the optimized protocol as described above, using microwave assisted annulation over 420 min, to give the desired lactam peptide TFA salt 90 (7 mg) in 68% crude purity as determined by analytical RP-HPLC (column b, UV: 1= 214 nm, 5-30% MeCN in H20, 0.1% FA, 30 min gradient).
Purification was carried out by preparative RP-HPLC (column B, 5-15% MeCN in H20, 0.1 % FA, 30 min gradient) to give the desired FA salt product 90 (3 mg, 6%) as a white fluffy solid. The purified product was analyzed by analytical RP-HPLC (column b, UV: 1=
214 nm) using both MeCN tR 9.90 (5-60% MeCN in H20, 0.1% FA, 25 min gradient) and MeOH tR 15.20 (0-60% MeOH in H20, 0.1 % FA, 25 min gradient) and revealed >99%
purity. HRMS Calcd. m/z for C42H69012N12 [M+H]+ 933.5152, found 933.5147.

D-Arg-D-Tyr-D-Thr-D-Val-D-Glu-(S)-Bgl-D-Leu-D-Ala-NH2 (91) (SEQ ID NO:87) OH
H O H O = H
H2NN qHI N N~NO
O O H O N O

OH HN
~-NH2 O

[0204] D-Arg-D-Tyr-D-Thr-D-Val-D-Glu-(S)-Bgl-D-Leu-D-Ala-NH2 (91) (SEQ ID
NO:87) was prepared on a 0.065 mmol scale, in a syringe tube following the optimized protocol as described above, using microwave assisted annulation over 240 min, to give the desired lactam peptide TFA salt 91 (20 mg) in 37% crude purity as determined by analytical RP-HPLC (column b, UV: 1= 214 nm, 2-30% MeCN in H20, 0.1% FA, 25 min gradient).
Purification was carried out by preparative RP-HPLC (column B, 5-15% MeCN in H20, 0.1 % FA, 25 min gradient) to give the desired FA salt product 91 (2 mg, 3%) as a white fluffy solid. The purified product was analyzed by analytical RP-HPLC (column b, UV: 1=
214 nm) using both MeCN tR 9.96 (5-60% MeCN in H20, 0.1% FA, 25 min gradient) and MeOH tR 15.43 (0-60% MeOH in H20, 0.1 % FA, 25 min gradient) and revealed >99%
purity. HRMS Calcd. m/z for C42H69012N12 [M+H]+ 933.5152, found 933.5154.

D-Arg-D-Tyr-D-Thr-D-Val-D-Glu-D-Leu-(S)-Bgl-D-Ala-NH2 (92) (SEQ ID NO:88) NH HO O

H O O H O = H O
H2N^ H H N HNH2 o O O I

OH

[0205] D-Arg-D-Tyr-D-Thr-D-Val-D-Glu-D-Leu-(S)-Bgl-D-Ala-NH2 (92) (SEQ ID
NO:88) was prepared on a 0.070 mmol scale, in a syringe tube following the optimized protocol as described above, using microwave assisted annulation over 240 min, to give the desired lactam peptide TFA salt 92 (27 mg) in 97% crude purity as determined by analytical RP-HPLC (column a, UV: 1= 214 nm, 5-30% MeCN in H2O, 0.1% FA, 30 min gradient).
Purification was carried out by preparative RP-HPLC (column A, 2-30% MeCN in H2O, 0.1 % FA, 30 min gradient) to give the desired FA salt product 92 (13 mg, 18%) as a white fluffy solid. The purified product was analyzed by analytical RP-HPLC (column a, UV: 1=
214 nm) using both MeCN tR 10.04 (5-60% MeCN in H2O, 0.1% FA, 25 min gradient) and MeOH tR 15.69 (0-60% MeOH in H2O, 0.1 % FA, 25 min gradient) and revealed >99%
purity. HRMS Calcd. m/z for C42H69012N12 [M+H]+ 933.5152, found 933.5150.

[3H]thymidine incorporation for TF-1 cell proliferation measurements [0206] Human TF-1 cells (5x104 cells/well) were cultured in complete RPMI
medium (GIBCO RPMI Medium 1640, Invitrogen) supplemented with GM-CSF (Granulocyte Macrophage Colony Stimulating Factor, 2 ng/ml; BD Biosource). Cells were deprived of growth factors for 18 h before preincubation with lactam peptide (1 M) followed by treatment with IL-1(3 (10 or 25 ng/mL). After 24 h incubation at 37 C, [3H]thymidine (1 gCi/mL; Amersham) was added and the cells were incubated for another 24 h.
Cells were harvested, washed two times with PBS (10 mM Na2HPO4, 2 mM KH2PO4, 137 mM NaCl, 2.7 mM KC1; pH 7.4) and lysed with a 0.1 N NaOH/ 0.1% Triton X-100 solution.
Scintillation cocktail (Fisher Scientific, 8 mL/sample) was added to the lysate, and after 3 h, radioactivity was measured (Beckman Multi-Purpose Scintillation Coulter Counter LS6500).
Results were analyzed by one- or two-way ANOVA factoring for concentration or treatments. Postanova comparisons among means were performed using the Tukey-Framer method. Statistical significance was set at p < 0.05. Data are presented as mean SEM.
Inhibition of thymocytes TF-1 proliferation by 101.10 lactam analogs 54-59 [0207] The efficacy of Agl peptides 54-59 was ascertained by measuring their influence on IL-1 induced human thymocyte TF-1 proliferation as assessed by incorporation of [3H]thymidine as previously described.

[0208] Among the analogs tested, five maintained some inhibitory effect on TF-proliferation (Figure 1). Peptide 56 failed to block proliferation of TF-1 cells treated with IL-1. Relative to 101.10, lactams 59, 58, 57, and 55, all exhibited similar efficacy, suggesting that the balance between side chain removal and conformational constraint at positions 2 and 4-6 does not perturb activity. Replacement of the N-terminal D-Arg residue by (R)-Agl in compound 54 led to 2.2 fold increase in efficacy compared to 101.10, suggesting that the Argi side chain may not be necessary for activity.

[0209] The efficacy of Agl peptides 75-78 as allosteric negative modulators of the IL-1 receptor I was also ascertained by measuring their influence on IL-1 induced human thymocyte TF-1 proliferation as assessed by incorporation of [3H]thymidine (Figure 2).

[0210] Results show that all (R)-Bab analogs tested exhibited an improvement in efficacy compared to 101.10, from a 2.4 fold (compound 78) to a 1.7 fold (compound 76) increase. Inhibition of TF-1 proliferation obtained with compound 75 confirms that the Arg' side chain does not seem to be necessary for activity. Reasons for the increase of efficacy observed with compounds 76-78 remains unclear and requires further investigation regarding the influence of features brought by a Bab residue, which, while not wishing to be limited by theory, may include the character of the aromatic ester and the increased conformational flexibility from insertion of an amino alkyl chain into the peptide backbone.

Synthesis of D-Arg-D-Tyr-(S)-Agl-D-Val-D-Glu-D-Leu-D-Ala-NH2 (93) (SEQ ID
NO:89): lantern-resin comparison and synthesis of multiple lactam residues on SynPhase Lanterns (a) Rink Amide-MBHA resin Swelling and Fmoc-deprotection [0211] A 12 mL plastic filtration tube with polyethylene frit was charged with Rink Amide-MBHA resin (200 mg, 0.134 mmol, 0.67 mmol/g) and DCM (7 mL). The tube was sealed and shaken for 0.5 h. The resin was then filtered and taken up in freshly prepared 20%
piperidine in DMF solution (7 mL), shaken for 30 min, filtered, retreated with 20%
piperidine/DMF solution (7 mL) and shaken for 30 min. A positive Kaiser colour test indicated qualitatively the presence of free amine.

Washing [0212] Washing steps after coupling or deprotection steps were performed by successive agitations for 1 min and filtration from DMF (3 x 7 mL), MeOH (3 x 7 mL) and DCM (3 x 7 mL).

Amino Acid couplings [0213] A solution of N-(Fmoc)amino acid (3 equiv.), HBTU (3 equiv.) and DIEA
(6 equiv.) in DMF (7 mL) was prepared in a small sample vial, stirred for 3 min and then added to the resin. The reaction mixture was shaken for 1 h with Fmoc-D-Ala, for 3 h with Fmoc-D-Val, Fmoc-D-Thr(tBu), Fmoc-D-Tyr(tBu) and Fmoc-D-Arg(Pbf) and for 4 h with Fmoc-D-Leu, Fmoc-D-Glu(tBu), at room temperature. The completeness of each coupling was verified by the Kaiser test on few resin beads. (Kaiser, E.; Colescott, R. L.;
Bossinger C. D.;
Cook P. I. Anal. Biochem. 1970, 34, 595.) Silylation and Alkylation [0214] After Fmoc protecting group removal, resin was dried in vacuo for at least 3 h.
The anhydrous resin, in a 12 mL plastic filtration tube with polyethylene frit, was flushed with argon, swollen in THE (7 mL), treated with BSA (5 equiv.), shaken for 6 h, filtered under argon and treated with a solution of sulfamidate (5 equiv.) in THE (7 mL). After shaking for 18 h at room temperature, the resin was filtered and washed under argon with THE (3 x 7 mL).

Microwave Assisted Annulation [0215] A 2 mL glass microwave vial was charged with resin and a freshly prepared I% AcOH/DMSO solution (2 mL). The vial was sealed, heated in the microwave at (pressure 1 bar) for 6 h. The resin was then washed from the microwave vessel into a 12 mL
plastic filtration tube with polyethylene frit.

Cleavage test [0216] Monitoring of reaction progress was performed by LC-MS analyses of material cleaved from resin. Typically, a small resin sample (3-5 mg) was treated with a mixture of TFA/H20/TES (1 mL, 95/2.5/2.5, v/v/v) for lh and filtered. The filtrate was evaporated, dissolved in water and acetonitrile and examined by LC-MS.

Final Cleavage [0217] The peptide was cleaved from the resin by shaking in TFA/H20/TES (7 mL, 95/2.5/2.5, v/v/v) for 3 h. The resin was filtered and washed with TFA. The combined filtrate and washings were concentrated in vacuo. The resulting residue was dissolved in a minimum volume of TFA (-1 mL), transferred to a centrifuge tube and precipitated by the addition of ice-cold diethyl ether (40 mL). The peptide was separated by centrifugation and the diethyl ether was carefully decanted from the tube. The precipitated peptide was washed twice with cold diethyl ether. The resulting white solid was dissolved in water and freeze-dried to give a white powder that was purified by preparative RP-HPLC, using the specified conditions.

(b) Polystyrene Rink amide lanterns [0218] D-Sized polystyrene Rink amide lanterns with a 35-gmol loading were used for the synthesis of 93 and 94 and A-sized polystyrene Rink amide lanterns with a 75-gmol loading were used for the synthesis of 95-97. A 20-mL sample vials with a cover in which seven 0,3 mm diameter holes were drilled was used and solutions were removed simply by reversing the flask.

Swelling and Fmoc-Deprotection [0219] Swelling and Fmoc-deprotection steps were respectively performed by immersing lanterns for 30 min in DCM and in DMF/Pip (80/20, v/v) solution. A
positive Kaiser colour test on a sliver of lantern indicated qualitatively the presence of free amine.
Washing [0220] Washing steps after coupling and deprotection steps were performed by dipping the lanterns in DMF (3 x 3 min), MeOH (1 x 3 min) and DCM (3 x 3 min), successively.

Amino acid coupling [0221] A DMF solution containing the Fmoc-protected amino acid (3 equiv.), HBTU
(3 equiv.), and DIEA (6 equiv.) were freshly prepared in a 20-mL flask and lanterns were immersed in the coupling solution for 3 h with Fmoc-D-Ala, Fmoc-D-Leu, Fmoc-D-Val, Fmoc-D-Tyr(tBu), Fmoc-D-Pro and Fmoc-D-Arg(Pbf) and for 4 h with Fmoc-D-Glu(tBu) and Fmoc-D-Thr(tBu), at room temperature. The completeness of each coupling was verified by a Kaiser test on a sliver of lantern.

Silylation and Alkylation [0222] After Fmoc protecting group deprotection, lanterns were then dried in vacuo for at least 3 h. The anhydrous lanterns, in 2- or 5- mL glass microwave vials, were flushed with argon, suspended in THF, treated with a solution of sulfamidate (4 equiv.) and DIEA (0-1.1 equiv) in THF and heated in the microwave at 60-70 C for 1-2h. The lanterns were washed under argon with THE Alternately, the alkylation with sulfamidate was preceded by a treatment with BSA (5 equiv) in THF for 1-6 h.

Microwave Assisted Annulation [0223] A 2- or 5-mL glass microwave vial was charged with lanterns and a freshly prepared solution of DMSO/AcOH (99:1, v/v) or DMSO/H20/AcOH (75:23:2, v/v/v).
The vial was sealed, heated in the microwave at 80 C for 5-10 h. The lanterns were then washed as previously described.

Capping [0224] After lactam annulation, a capping of the deletion sequence from incomplete sulfamidate alkylation was performed by immersing the lantern in a DMF
solution of acetic anhydride (10 equiv.) and DIEA (10 equiv) for 1 h.

Cleavage test [0225] Monitoring of reaction progress was performed by LC-MS analyses of material cleaved from lantern. Typically, a slice of lantern was treated with a mixture of TFA/H20/TES (1 mL, 95/2.5/2.5, v/v/v) for lh and filtered. The filtrate was evaporated, dissolved in water and acetonitrile and examined by LC-MS.

Peptide cleavage [0226] The peptide was cleaved by immersing the lantern in TFA/H20/TES (3 mL, 95/2.5/2.5, v/v/v) for 3 h. The cleavage cocktail was removed directly from the tubes, peptides were precipitated with ice-cold diethyl ether, centrifuged, and decanted.
Precipitation, centrifugation, and decantation operations were repeated twice.
The resulting white solid was dissolved in water (10 mL) and freeze-dried to give a white powder that was analysed for purity and purified by preparative RP-HPLC, using the specified conditions.
Discussion for Example 26 A comparative study was first made between Rink amide MBHA resin and Rink-amide SynPhase lantern as supports in the synthesis of lactam peptide 93, in which the D-Thr3 residue was replaced by (S)-Agl (Scheme 10). Assembly of the C-terminal peptide fragment (vela) was done by standard SPPS protocols. The amino terminal of the peptide was then alkylated with (4S)-(Fmoc)oxathiazinane ester (S)-8, which along with its enantiomer (R)-8, were synthesized in solution from L- and D-Met as described above. Peptide alkylation was preceded by N-silylation of the peptide amine with N,O-bis(trimethylsilyl)acetamide (BSA) to minimize bis-alkylation. Lactam annulation was performed by microwave irradiation, the Fmoc group was removed and the sequence was elongated to provide the supported, protected Agl peptide 93. Lactam peptide 93 was obtained by side-chain deprotection and cleavage from the resin and lantern upon treatment with TFA. The crude peptides were analyzed by RP-HPLC and comparable crude purity was obtained using resin and lantern (33% and 32%, respectively) indicating that Agl peptide synthesis was unaffected by the support. After cleavage from the resin, the main impurities in crude material 93 were the deletion sequence from incomplete sulfamidate alkylation (30%) and uncyclized product (17%). After cleavage from lantern, the main impurity was the acetylated (S)-Agl-vela peptide (39%) probably due to an undesired partial Agl Fmoc deprotection prior to treatment with the acetic anhydride. The residual amine capping was performed on lantern after lactam annulation to avoid complications from a deletion sequence due to incomplete sulfamidate alkylation.

Scheme 10 o OtBu sPPS 0 _ 0 Fmoc-HN SS = H
H2N H^~N HNH SS
SS = Rink amide resin or lantern O OtBu 1) BSA, THF, RT O O
BnO2C HN N
2) p THE HN ~ H/~NH SS
I
f0 N" CO2Bn NHFmoc 0 Fmoc (8) O OtBu MW, 1%AcOH/DMSO
O O
H
FmocHN N HeN HNH SS
O IOI
O

HN
O OH
1) SPPS O
O O
2) TFA/TES/H20 = H = H =
(95:2.5:2.5, v/v/v) H2N N H N H N H

O O
O
O
OH
D-Arg-D-Tyr-(S)-Agl-D-Val-D-Glu-D-Leu-D-Ala-NH2 SEQ ID NO: 89 [0227] The rytvela analog 93 was synthesized in parallel on color-tagged lanterns with the analog 94, in which the D-Va14 residue was replaced by (S)-Agl. The C-and N-terminal peptide fragments were assembled by standard SPPS protocols. The alkylation with sulfamidate (S)-8 was performed after treatment of the peptide lanterns with BSA for 1 h, and comparable results were obtained by overnight alkylation at room temperature and heating at 60 C with microwave-irradiation for 1 h. Lactam annulation was accomplished in I% AcOH

in DMSO with heating at 110 C under microwave irradiation for 6 h, or in an oil bath for longer times. As previously reported on resin conversion to lactam was cleaner using the microwave; however, irradiation at 110 C caused the lanterns to change morphology and degrade. Although lactam product could be recovered after cleavage from the altered lantern, it was observed that at 80 C, annulation could be effected without melting lantern and good conversion was achieved after 10 h.

[0228] In light of the improved activity of the (R)-Agli and maintained potency of (R)-Ag12 and (R)-Ag14 analogs of rytvela, the combinations of (R)-Agli-(R)-Ag12 and (R)-Agli-(R)-Ag14 in rytvela analogs 96 and 97 were explored. For comparison, the (R)-Agl'-D-Pro4 analog 95 was also synthesized. The three analogs were constructed using a split-and-pool approach on SynPhase-lanterns equipped with a Rink amide linker. The lanterns were equipped with coloured spindles as a visual tagging system. (R)-Cyclic sulfamidate (R)-8 was used to alkylate peptides bound to the lanterns suspended in THE at 60 C for lh of microwave irradiation. Monitoring the reaction progress by LC-MS analyses after TFA-mediated cleavage of a lantern slice, it was found that, in contrast to synthesis on resin, the silylation with BSA had little effect on the amount of bis-alkylation product formed on lantern and comparable percentages of alkylated peptides 100 and 101 (27-30%
and 50-52%, respectively) were achieved. This prompted the investigation of peptide alkylation without a prior BSA treatment. Longer heating in the microwave (2h) at higher temperature (70 C) and the addition of 1.1 equiv of DIEA to the sulfamidate solution increased the amount of 100 and 101 (>40% and >90%, respectively); however bis-alkylation of 98 was significant (50%).

[0229] Subsequently, the condition for lactam annulation on both 100 and 101 were optimized, and it was found that the addition of water to the DMSO (I% AcOH) solution gave, in both cases, quantitative lactam cyclization at 80 C under microwave irradiation.

[0230] Investigating the feasibility of introducing a second lactam motif into the same peptide, alkylation and lactam annulation were performed using conditions previously effective for installing Agl residues: alkylation with (R)-8 (4 equiv) in THE
(0.1 M) with microwave irradiation for lh at 70 C; lactam formation in a solution of DMSO/AcOH/H20 (75 : 2 : 23, v/v/v) with microwave irradiation for 10 h at 80 C. These conditions gave respectively 50, 8 and 26% conversions to 107, 108, 109. The addition of a second (R)-Agl residue presented a more difficult challenge than the first (R)-Agl residue.
Different conditions were pursued to improve conversion in the second alkylation step including solvent, temperature and microwave irradiation time, without much success. The addition of 0.5 equiv of DIEA to the sulfamidate solution favoured alkylation. This amount of DIEA was chosen to avoid bis-alkylation, which was promoted with more base (1.1 equiv).
Lactam formation by microwave irradiation at 80 C, Fmoc-deprotection and cleavage gave the crude peptides, which were analyzed by RP-HPLC to have 55, 30 and 45% crude purity for 95, 96 and 97, respectively (Table 9 and Scheme 11).

[0231] The efficacy of Agl peptides 93 and 94 was ascertained by measuring their influence on IL-1 induced human thymocyte TF-1 proliferation as assessed by incorporation of [3H]thymidine as in Example 25. The percentages of proliferation of TF-1 cells (150% and 132%, respectively) pre-treated with peptides 93 and 94 demonstrated that both these analogs lost inhibitory activity on TF-1 proliferation exhibited by rytvela (69%).

Synthesis of (R)-Agl-D-Tyr-D-Thr-D-Val-(R)-Agl-D-Leu-D-Ala-NH2 (98) (SEQ ID
NO:94) OH O
YO
N H =
H2Nl" N\\\**' N Ni"'" N ,-YNH2 H H
O O O O
OH

[0232] (R)-Agl-D-Tyr-D-Thr-D-Val-(R)-Agl-D-Leu-D-Ala-NH2 (98) (SEQ ID
NO:94) was prepared on an A-sized polystyrene Rink amide lantern as outlined in Example 26. The crude peptide purity (30%) was assessed by RP-HPLC-MS (UV 214), 5-90%
MeOH
in H20, 0.1% FA, 20 min gradient, tR 13.76 min., MS calcd. for C35H54Ng09 [M+H]+ 731.4, found 731.2).

alpha-Amino-beta-hydroxy-gamma-lactams, constrained Serine and Threonine dipeptide mimics Synthesis (a) Synthesis of N-(Fmoc) Oxiranylglycine reagent 106 CI Me -q-O
~S,,,,'-,S,,,,'i CI ^yCOZMe m-CPBA O~COZMe 191 C, 2h 1,2-DCE NFmoc I
H NFmoc HFmoc 40 C, 17h H

[0233] From known compound 107, an adapted literature procedure was followed:
N-(Fmoc)Vinylglycine methyl ester [0234] A 2,4-dichlorotoluene (30 mL) solution of N-(Fmoc)Met(S=O)-OMe 107 (4.01 g, 10.0 mmol) was heated to 191 C for 2h under Argon. The unconcentrated crude was loaded directly onto a 6.5 cm diameter X 13 cm high pad of silica, which was eluted using a step gradient of 3--->6--->9--->12--->20--->35--->60% ethyl acetate: hexanes, 400mL per step) yielding 2.81 g (84%) of N(Fmoc)Vinylglycine methyl ester.

N-(Fmoc)Oxiranylglycine methyl ester (106) [0235] N-(Fmoc)Vinylglycine methyl ester (1.69g, 5.00 mmol) and m-CPBA
(commercial <77%, 5.6g, 25 mmol) were heated in 1,2-dichloroethane at 40 C
for 17h. The crude was filtered over a frit, concentrated and purified by flash chromatography (7.5 cm wide X 8.5 cm high silica pad, eluded by step gradient of 3->6->9->15->20->25%
ethyl acetate: toluene, 400 mL per step) yielding 833 mg (47%) of N-(Fmoc)oxiranylglycine methyl ester.

Representative procedure for the synthesis ofl3-hydroxy-a-amino-y-lactams [0236] D-Phe-OMe=HCl (77.6 mg, 360 mmol) in dilute Na2CO3 was extrated with CHC13 (3 X 1 mL), dried over MgSO4, concentrated, and dried under high vacuum for a few hours to yield 40.8 mg (78%) of D-Phe-OMe. The free base (26.1 mg, 0.180 mmol) was immediately used in a reaction with N-(Fmoc)oxiranylglycine methyl ester 106 (18.4 mg, 0.052 mmol) at 76 C in 2,2,2-trifluoroethanol (0.3 mL) for 15h. The crude was concentrated in vacuo, and purified by flash chromatography (2.2 cm wide X 2 cm high silica pad, step gradient of 28-*31-*34-*37-*50-*70% ethyl acetate: hexanes, 20 mL per step) yielding 21.9 mg (91%) product (Rfproduct = 0.2 with 60% ethyl acetate: hexanes eluent, Rf oxiranylglycine = 0.55).

Spectroscopic Data (a) (2S)-[(9H-Fluoren-9-ylmethoxycarbonylamino)]-oxiranyl-acetic acid methyl ester (106) Oj~ ~CO2Me ~NFmoc H

[0237] 1H NMR (400 MHz, CDC13) 6 7.77 (d, J= 7.4 Hz, 2H), 7.60 (t, J= 6.7 Hz, 2H), 7.41 (t, J = 7.4 Hz, 2H), 7.32 (td, J = 7.2, 2.5 Hz, 2H), 5.35 (d, J =
9.0 Hz, 1 H), 4.74 (d, J= 8.9 Hz, 1H), 4.43 (d, J= 7.1 Hz, 2H), 4.22 (t, J= 6.8 Hz, 1H), 3.83 (s, 3H), 3.51-3.47 (m, 1H), 2.79 (t, J= 4.3 Hz, 1H), 2.63 (dd, J= 4.6, 2.6 Hz, 1H). 13C NMR (75 MHz, CDC13) 170.3, 156.3, 143.9, 143.7, 141.51, 141.47, 127.9, 127.27, 127.26, 125.2, 120.2, 76.8, 67.4, 53.2, 51.3, 47.3, 44Ø HRMS(ESI+) for MH+ = C2oH2oNO5+; calculated: 354.1336, found:
354.1331 (dif m/z = 1.3 ppm).

(b) 2-[3-(9H-Fluoren-9ylmethoxycarbonylamino)-4-hydroxy-2-oxo pyrrolidin-l-ylJ propionic acid benzyl ester (108) HO
FmocNH N-_/C02Bn O Me [0238] 1H NMR (300 MHz, CDC13) 6 7.78 (d, J= 7.3 Hz, 2H), 7.59 (d, J= 7.3 Hz, 2H), 7.46-7.29 (m, 9H), 5.70 (br s, 1 H), 5.16 (dd, J = 15.3, 12.2, 2H), 5.00-4.82 (m, 2H), 4.58-4.32 (m, 3H), 4.23 (t, J= 7.0 Hz, 1H), 4.13 (pseudo t, J= 7.2 Hz, 1 H), 3.69 (t, J= 3.69 Hz, 1H), 3.24 (t, J= 8.7 Hz, 1H), 1.47 (d, J= 7.2 Hz, 3H). 13C NMR (75 MHz, CDC13): 6 (ppm) 170.6, 169.3, 158.2, 143.7, 141.5, 135.3, 128.9, 128.8, 128.4, 128.1, 127.3, 125.2, 120.3, 73.7, 67.9, 67.5, 60.7, 49.7, 47.6, 47.1, 15.1. HRMS (ESI+) for MH+ =
C29H29N2O6+;
calculated: 501.2020, found: 501.2032 (error m/z = 2.4 ppm).

2-[3-(9H-Fluoren-9 ylmethoxycarbonylamino)-4-hydroxy-2-oxo pyrrolidin-1 ylJ-4-methyl-pentanoic acid methyl ester (109) HO
FmocNH N C02Me O

[0239] iH NMR (400 MHz, CDC13): 6 (ppm) 7.77 (d, J= 7.5 Hz, 2H), 7.59 (d, J=
7.5 Hz, 2H), 7.41 (t, J= 7.5 Hz, 2H), 7.32 (t, J= 7.4 Hz), 5.77 (br s, 1H), 4.88 (pseudo t, J= 8.2 Hz, 1 H), 4.44 (d, J = 7.1 Hz, 2H), 4.29 (q, J = 8.2 Hz, 1 H), 4.22 (t, J =
7.0 Hz, 1 H), 4.13 (d, J
= 8.0 Hz, 1H), 3.72 (s, 1H), 3.56 (t, J= 8.8 Hz, 1H), 3.38 (t. J= 9.0 Hz, 1H), 1.83-1.35 (m, 3H), 1.01-0.87 (m, 6H). ESI+ for MH+ = C26H3,N206+ calculated and found:
467.2; for MNa+
= C26H30N206Na+ calculated and found: 489.2.

(d) [3-(9H-Fluoren-9 ylmethoxycarbonylamino)-4-hydroxy-2-oxo pyrrolidin-1-ylJ-acetic acid benzyl ester (110) HO
FmocNH N-_/C02Bn [0240] iH NMR (300 MHz, CDC13): 6 (ppm) 7.78 (d, J= 7.5 Hz, 2H), 7.59 (d, J=
7.4 Hz, 2H), 7.45-7.30 (m, 9H), 5.73 (s, 1H), 5.18 (s, 2H), 4.97 (very br), 4.48-4.36 (m, 3H), 4.28-4.07 (m, 4H), 3.63 (dd, J= 9.4, 8.3 Hz, 1H), 3.44 (dd, J= 9.3, 8.1 Hz, 1H). ESI+ for MH+= C2gH27N206+ calculated and found: 487.2. For MNa+ = C28H26N2O6Na+
calculated and found: 509.2.

(e) 2-[3-(9H-Fluoren-9 ylmethoxycarbonylamino)-4-hydroxy-2-oxo pyrrolidin-l -ylJ-3phenylpropionic acid methyl ester (111) HO
FmocNH N-_/C02Me 0 ~-Ph [0241] iH NMR (300 MHz, CDC13): 6 (ppm) 7.74 (d, J= 7.5 Hz, 2H), 7.55 (d, J=
7.2 Hz, 2H),7.39 (t, J= 7.3 Hz, 2H), 7.33-7.13 (m, 7H), 5.68 (s, 1H), 5.06 (dd, J=
11.4, 4.9 Hz, 1H), 4.57-4.41 (m, 1H), 4.37 (d, J= 7.1 Hz, 2H), 4.30 (q, J= 8.1 Hz, 1H), 4.18 (t, J= 6.9 Hz, 1 H), 3.82 (dd, J = 8.0, 1.8 Hz, 1 H), 3.75 (s, 3 H), 3.69 (dd, J = 9.0, 8.0 Hz, 1 H), 3.3 9 (dd, J =

14.7, 5.0 Hz, I H), 3.14 (t, J = 3.4 Hz, I H), 2.96 (dd, J = 14.7, 11.5, I H).
13C NMR (75 MHz, CDC13): 6 (ppm) 170.3, 169.6, 158.2, 143.64, 143.56, 141.5, 141.4, 135.9, 129.0, 128.6, 128.0, 127.4, 127.25, 127.26, 125.14, 125.12, 120.2, 73.5, 67.8, 60.3, 55.1, 52.8, 48.0, 47.1, 35.4.
ESI+ for C29H29N2O6+ calculated and found: 501.2. For C29H28N2O6Na+ calculated and found 523.2. HRMS (ESI+) for MH+ = C29H29N2O6+ calculated: 501.2020, found: 501.2027 (diff.
m/z = 1.4 ppm).

(f) 2-[3-(9H-Fluoren-9 ylmethoxycarbonylamino)-4-hydroxy-2-oxo pyrrolidin-1-ylJ-3-methyl-butyric acid methyl ester (112) HO
FmocNH N-_/CO2Me O

[0242] 1H NMR (700 MHz, CDC13): 6 (ppm) 7.77 (d, J= 7.7 Hz, 2H), 7.59 (dd, J=
7.5, 4.0 Hz, 2H), 7.41 (t, J= 7.5 Hz, 2H), 7.33 (tt, J= 7.5, 1.1 Hz, 2H), 5.78 (s, 1H), 5.02 (br s, 1 H), 4.52 (d, J = 9.5 Hz, 1 H), 4.44 (dd, J = 10.7, 7.1, 1 H), 4.42 (dd, J
= 10.7, 7.1, 1 H), 4.36 (q, J = 8.0 Hz, 1 H), 4.23 (t, J = 7.1 Hz, 1 H), 4.14 (ddd, J = 8.1, 1.9, 1.0 Hz, 1 H), 4.03 (dd, J =
9.5, 8.0 Hz, 1H), 3.73 (s, 3H), 3.25 (dd, J= 9.6, 8.2 Hz, 1H), 2.23 (doublet of septuplets, J=
9.5, 6.7 Hz, 1H), 0.99 (d, J= 6.7 Hz, 3H), 0.96 (d, J= 6.7 Hz, 3H). 13C NMR
(100 MHz, CDC13): 6 (ppm) 170.6, 169.8, 158.2, 143.7, 143.6, 141.5, 128.0, 127.3, 125.1, 120.2, 73.6, 67.9, 60.5, 59.9, 52.3, 48.0, 47.1, 28.1, 19.4, 19.3 (diasteriotopic Me). HRMS
(ESI+) for MH+
= C25H29N2O6+; calculated 453.2020, found: 453.2028 (dif m/z = 1.8 ppm).

(g) 2-[3-(9H-Fluoren-9 ylmethoxycarbonylamino)-4-hydroxy-2-oxo pyrrolidin-l -ylJ-3-(4-hydroxyphenyl) propionic acid methyl ester (113) HO
FmocNH N-,/CO2Me O
OH

[0243] 1H NMR (300 MHz, CDC13): 6 (ppm) 7.77 (d, J= 7.6 Hz, 2H), 7.57 (d, J=
7.6 Hz, 2H), 7.41 (t, J= 7.6, 2H), 7.36 (br, 1H), 7.32 (t, J= 6.8, 2H), 7.02 (d, J= 8.3 Hz, 2H), 6.70 (d, J = 8.4 Hz, 2H), 6.20 (very br s, 1 H), 5.63 (br s, 1 H), 5.09 (dd, J
= 11.7, 4.8 Hz, 1 H), 4.45-2.48 (m, 3H), 4.20 (t, J= 6.9 Hz, 1H), 3.92 (d, J= 8.3 Hz, 1H), 3.77 (s, 3H), 3.83-3.68 (m, 1 H), 3.31 (dd, J = 11.5, 4.7 Hz, 1 H), 3.15 (t, J = 8.6 Hz, 1 H), 2.8 8 (t, J = 11.8 Hz, 1 H).

ESI+ for MH+ = C29H29N207+ calculated and found: 517.2. For MNa+ =
C29H28N2O7Na+
calculated: 539.2, found: 539.3.

(h) 2-[3-(9H-Fluoren-9 ylmethoxycarbonylamino)-4-hydroxy-2-oxo pyrrolidin-l-ylJ-3-(JH-indol-3yl)propionic acid methyl ester (114) HO
FmocNH N-_/CO2Me O

N
H

[0244] 1H NMR (300 MHz, CDC13): 6 (ppm) 8.17 (br s, 1H), 7.77 (d, J= 7.7 Hz, 2H), 7.62-7.53 (m, 3H), 7.45-7.10 (m, 7H), 7.00 Hz (br s, 1H), 5.65 (br s, 1H), 5.18 (dd, J= 11.2, 4.7 Hz, 1 H), 4.82 (very br s, 1 H), 4.3 8-4.26 (m, 3H), 4.18 (t, J = 6.9 Hz, 1 H), 3.94 (dd, J =
8.2, 2.4 Hz, 1 H), 3.79 (s, 3H), 3.72 (t, J = 8.6 Hz, 1 H), 3.49 (dd, J =
15.5, 4.7 Hz, 1 H), 3.27-3.14 (m, 2H). 13C NMR (75.5 MHz, CDC13): 6 (ppm) 170.7, 169.7, 158.1, 143.7, 143.6, 141.46,141.44,136.3, 128.0,127.3, 127.1, 125.1, 122.6,122.0,120.2,119.9,118.4,111.5, 110.5, 73.5, 67.8, 60.4, 54.4, 52.8, 47.8, 47.1, 25.5. HRMS (ESI) for MH+ =
C3,H3oN3O6+;
calculated: 540.2129, found: 540.2141 (diff. m/z = 2.2 ppm).

(i) 3-[3-(9H-Fluoren-9 ylmethoxycarbonylamino)-4-hydroxy-2-oxo pyrrolidin-l -ylJ propionic acid benzyl ester (115) HO
FmocNH N~~CO2Bn [0245] 1H NMR (300 MHz, CDC13): 6 (ppm) 7.78 (d, J= 7.5 Hz, 2H), 7.59 (d, J=
7.3 Hz, 2H), 7.45-7.29 (m, 9H), 5.68 (br s, I H), 5.13 (s, 2H), 4.87 (br s, I H), 4.44 (d, J= 6.8 Hz, I H), 4.31-4.19 (m, 2H), 4.00 (d, J= 8.0 Hz, I H), 3.79-3.67 (m, I H), 3.62-3.46 (m, 2H), 3.28 (t, J= 8.8 Hz, 1H), 2.63 (t, J= 6.6 Hz, 2H). 13C NMR (300 MHz, CDC13): 6 (ppm) 171.1, 168.9, 158.2, 143.7, 143.6, 141.48, 141.47, 135.6, 128.8, 128.6, 128.0, 127.27, 127.26, 125.14, 125.11, 120.24, 120.22, 73.5, 67.8, 67.0, 60.8, 50.9, 47.1, 39.0, 32.5. HRMS
(ESI) for MH+ _ C29H29N2O6 ; calculated: 501.2020, found: 501.2032 (dif m/z = 2.3).

(j) 3-[3-(9H-Fluoren-9 ylmethoxycarbonylamino)-4-hydroxy-2-oxo pyrrolidin-1-ylJ-benzoic acid methyl ester (116) HO
CO2Me FmocNH N
O I

[0246] 1H NMR (300 MHz, CDC13): 6 (ppm) 8.15 (pseudo t, J= 1.9 Hz, 1H), 7.94 (ddd, J = 8.1, 2.4, 1.0 Hz, 1 H), 7.89 (dt, J = 7.9, 1.3 Hz, 1 H), 7.78 (d, J
= 7.6 Hz, 2H), 7.61 (d, J = 7.5 Hz, 2H), 7.48 (t, J = 8.0 Hz, 1 H), 7,43 (t, J = 7.3 Hz, 2H), 7.34 (td, J = 7.5, 1.2 Hz, 2H), 5.84 (br s, 1H), 5.09 (br s, 1H), 4.54-4.42 (m, 3H), 4.31-4.21 (m, 2H), 4.08 (dd, J= 9.8, 8.1 Hz, 1H), 3.94 (s, 3H), 3.79 (dd, J= 9.6, 8.3 Hz, 1H). ESI+ for MH+=
C27H25N2O6+;
calculated and found: 473.2. For MNa+ = C27H24N2O6Na+; calculated and found:
495.2.
Discussion [0247] Enantiomerically pure (2S,3R)-N-(Cbz)-oxiranylglycine was prepared from L-Met according to literature procedures and examined in reactions with Ala-013n. In acetonitrile at 90 C, the desired sequential alkylation / lactam formation occurred producing target (3-hydroxy-a-amino-y-lactam, albeit in 30% yield. The utility of Fmoc protection in peptide synthesis compelled further examination using N-(Fmoc)oxiranylglycine 106, which was prepared in an analogous manner from L-Met. Epoxide 106 reacted with Ala-013n to produce lactam 108 in 10% yield. Little improvement was obtained in attempts to yield lactam 108 using Lewis and Bronsted acid catalysts. In the reaction between N-(Fmoc)Oxiranylglycine 106 and different amino acid analogs, 2,2,2-trifluoroethanol (TFE) as solvent proved optimum.

[0248] For example, substrates with sterically demanding side chains such as the methyl esters of Phe, Val and Trp, reacted well with 106. In the case of Gly-OBn, lower reaction temperatures mitigated losses from Fmoc deprotection. The nucleophilic phenol of unprotected Tyr-OMe was tolerated, however the ester-protected glutamate side competed for lactamization producing N-alkyl pyroglutamate. In addition to proteinogenic examples, the methyl ester of aminobenzoic acid as well as the benzyl ester of beta-Ala gave respectively 58 and 67% yield.

[0249] The stereogenic carbons in these dipeptide mimics are derived from the chiral pool with diasteroselective induction to set the 3-hydroxy center by way of selective epoxidation of vinylglycine. The oxiranylglycine diasteriomers 106 were separable, allowing all possible sterioisomers to be obtained by choice of chirality in the starting material.

Preparation of (3-hydroxy-a-amino-y-lactam peptides on polystyrene Rink amide lantern Synthesis of rytvela /3-hydroxy-a-amino-y-lactam analogue D-Arg-D-Tyr-Agl(4-OH)-D-Val-D-Glu-D-Leu-D-Ala-NH2 (99) (SEQ ID NO:95) NH
NH

H2N b-NH O OH
O O = H O
HN N Nib N NH2 O H O O
OH

[0250] D-Arg-D-Tyr-Agl(4-OH)-D-Val-D-Glu-D-Leu-D-Ala-NH2 (99) (SEQ ID
NO:95) was prepared on an A-sized polystyrene Rink amide lantern as outlined in Example 25 except the sulfamidate alkylation step was substituted by the following:

[0251] After Fmoc protecting group deprotection, the lanterns were treated with an oxiranylglycine 106 (3 equiv.) in 2,2,2-trifluoroethanol (0.06 M) and heated to 80 C under microwave irradiation for 12h. Lantern washing and subsequent elongation was done as described, providing the desired lactam peptide as the TFA salt 99 (50% crude purity by analytical RP-HPLC-MS (UV 214), 5-80% MeOH in H2O, 0.1% FA, 20 min gradient, tR
8.45 min., MS calcd. for C3sH62011Nii [M+H]+ 848.4, found 848.6.).

[0252] Alternatively, the hydroxylactam dipeptidyl esters may be hydrolyzed and used as dipeptide building blocks in standard peptide synthesis.

[0253] These scaffolds may find application in medicinal chemistry, especially because the hydroxy and protected amino groups are ideally suited for orthogonally elaborating these structures further. In the context of solid supported peptide synthesis, elaboration of the hydroxy group would allow for mimicry of other constrained amino acid residues, by the attachment of carbohydrates, phosphonate, sulfate and other ester and ether types.

Solid-Phase Synthesis of Indolizidinone Analogs of API-101.10 Rink Amide Resin swelling and deprotection [0254] A 12 mL plastic filtration tube with polyethylene frit was charged with Rink resin (300 mg, 0.09 mmol, 0.3 mmol/g) and DMF (7 mL). The tube was sealed and shaken for 0.5 h. The resin was then filtered and taken up in freshly prepared 20%
piperidine in DMF solution (7 mL), shaken for 30 min, filtered, retreated with 20%
piperidine/DMF
solution (7 mL) and shaken for 30 min. The resin was washed by successive agitations for 1 min and filtered from DMF (3 x 7 mL), MeOH (3 x 7 mL) and DCM (3 X 7 mL). A
positive Kaiser colour test indicated qualitatively the presence of free amine.

Amino Acid couplings H
FmocHN\\ , N
O O^OH
Fmoc-I2aa-OH

[0255] The resin was first swollen in DMF (7 mL) for 15 min. Meanwhile, a solution of N-(Fmoc)amino acid (Fmoc-Xaa-OH, 3 equiv.), HBTU (3 equiv.) and DIEA (6 equiv.) in DMF (7 mL) was prepared in a small sample vial, stirred for 10 min and then added to the resin. The reaction mixture was shaken for 8 h with Fmoc-D-Leu-OH, 12 h with Fmoc-D-Glu(tBu)-OH and Fmoc-D-Gln(Trt)-OH, 18 h with Fmoc-I2aa-OH, 10 h with Fmoc-D-Tyr(tBu)-OH, 11 h with Fmoc-D-Om(Boc)-OH, HOOC(CH2)5NH(Boc) and HOOC(CH2)4NH(Fmoc), at room temperature. The resin was then filtered and respectively washed by shaking for 1 min with DMF (3 x 7 mL), MeOH (3 x 7 mL) and DCM (3 x mL). A negative Kaiser test response indicated completion of the reaction. The resin was then dried in vacuo.

Silylation and Alkylation (for peptides WC144 and WC145) I
O ~/ i N "/CO2Bn Fmoc 6-membered cyclic D-sulfamidate (N-(Fmoc)oxathiazinane) O

C02Bn O0// 'N

Fmoc 6-membered cyclic L-sulfamidate (N-(Fmoc)oxathiazinane) [0256] After swelling the resin, the Fmoc protecting group was removed as described above. The resin was then dried in vacuo for at least 3 h. The anhydrous resin, in a 12 mL
plastic filtration tube with polyethylene frit, was then flushed with argon, swollen in THE (7 mL), treated with BSA (5 equiv.), shaken for 16 h, filtered under argon and treated with a solution of sulfamidate (5 equiv.) in THE (7 mL). After shaking for 24 h, the resin was filtered and washed under argon with THE (3 x 7 mL), MeOH (3 x 7 mL) and DCM
(3 X 7 mL) and dried in vacuo.

Microwave Assisted Annulation (for peptides WC144 and WC145) [0257] A 2 mL glass microwave vial was charged with resin and either DMF (2 mL) or a freshly prepared 1% acetic acid/DMSO solution (2 mL). The vial was sealed, heated in the microwave at 100 C (pressure 1 bar) for 3 h and then cooled using a jet of air. The resin was then washed from the microwave vessel into a 12 mL plastic filtration tube with polyethylene frit and washed by shaking for 1 min with DMF (3 x 7 mL), MeOH (3 x 7 mL) and DCM (3 x 7 mL) and then dried in vacuo.

Alkylation (for peptides WC146 and WC147) CO2Me FmocN
O1i-O
O
5-membered cyclic D-sulfamidate (N-(Fmoc)oxathiazolidine) CO2Me FmocN

O~:II-O
O
5-membered cyclic L-sulfamidate (N-(Fmoc)oxathiazolidine) [0258] The resin was swollen in DMF (4 mL) for 20 min and filtered. A 20%
piperidine solution in DMF (4 mL) was added and the suspension was shaken for 20 min and filtered. This operation was repeated and the resin was washed with DMF (3 x 3 mL), MeOH
(3 x 3 mL) and DCM (3 x 3 mL). At this point the Kaiser test gave a positive response.

[0259] The peptidyl resin was then transferred into a dry 2 mL microwave vessel. A
solution of freshly prepared sulfamidate in THE (1.5 mL) was added under argon, followed by DIEA. The mixture was then heated under microwave irradiation at 60 C for 2.5 hours.
The resin was washed with DCM (3 x 6 mL), MeOH (3 x 6 mL) and DCM (3 x mL).

Microwave Assisted Annulation (for peptides WC146 and WC147) [0260] A 2 mL glass microwave vial was charged with resin and a mixture of DMSO/H20/AcOH (75:23:2, 2 mL). The vial was sealed, heated in the microwave at (pressure 1 bar) for 4h and then cooled using a jet of air. The resin was then washed from the microwave vessel into a 12 mL plastic filtration tube with polyethylene frit and washed by shaking for 1 min with DMF (3 x 7 mL), MeOH (3 x 7 mL) and DCM (3 x 7 mL) and then dried in vacuo.

Resin Capping [0261] The resin was swollen in a solution of di-tent-butyl dicarbonate (5 equiv.) in DMF (7 mL), treated with DIEA (10 equiv.), shaken for 1 h, filtered and washed by shaking for 1 min with DMF (3 x 7 mL), MeOH (3 x 7 mL) and DCM (3 X 7 mL) and then dried in vacuo.

Peptide Cleavage [0262] The resin was first swollen in a plastic filtration tube with polyethylene frit, as described for Fmoc removal above, treated with a freshly prepared 20%
piperidine / DMF
solution (7 mL), shaken for 15 min, filtered, treated with a second portion of 20%

piperidine/DMF solution (7 mL) and shaken for 15 min. The resin was then filtered and washed by shaking for 1 min with DMF (3 x 7 mL), MeOH (3 x 7 mL) and DCM (3 x mL). A positive Kaiser colour test indicated qualitatively the presence of free amine. The peptide was then cleaved from the resin by shaking in TFA/H20/TES (7 mL, 95/2.5/2.5, v/v/v) for 2 h. The resin was filtered, washed with TFA (7 mL) and the combined filtrate and washings were concentrated in vacuo. The resulting residue was dissolved in a minimum volume of TFA (-1 mL), transferred to a centrifuge tube and precipitated by the addition of ice-cold diethyl ether (40 mL). The peptide was then centrifuged and the diethyl ether was carefully decanted from the tube. The treatment of the precipitated peptide with cold diethyl ether wash was repeated twice. The resulting white solid was dissolved in water (10 mL) and freeze-dried to give a white foam that was purified by preparatory RP-HPLC, using the specified conditions.

D-Orn-D-Tyr-haa-D-Glu-D-Leu-NHz (WC115) (SEQ ID NO:96) NHZ
H

H
H NN N\`~N = H O
Z O H N
O Ce-N NHZ
O
OH

[0263] D-Orn-D-Tyr-12 aa-D-Glu-D-Leu-NHz (WC115) (SEQ ID NO:96) was prepared as described above to give the desired peptide TFA salt (38 mg, 71%
crude purity as analyzed by analytical RP-HPLC (UV 214), 0-40 MeCN, 8 min gradient).
Purification was then carried out by preparatory RP-HPLC (5-40 MeCN, 15 min gradient) to give the desired formic acid salt WC115 (11.5 mg, 21%) as a white foam. The purified product was analyzed by analytical RP-HPLC (UV 214) using both MeCN TR 13.57 (2-40 MeCN, 20 min gradient) and MeOH TR 14.99 (10-60 MeOH, 20 min gradient) and revealed >99% purity. HRMS
Calcd. for C34H5209Ns [M+H]+ 717.3930, found 717.3936.

H2N(CH2)4C0-D-Tyr-haa-D-Glu-D-Leu-NH2 (WC116) (SEQ ID NO:97) H

O
(;N ' H O
H2N N N\`., p H O p^HN NH2 O
OH

[0264] H2N(CH2)4CO-D-Tyr-haa-D-Glu-D-Leu-NH2 (WC116) (SEQ ID NO: 97) was prepared as described above to give the desired peptide TFA salt (15 mg, 47%
crude purity as analyzed by analytical RP-HPLC (UV 214), 0-40 MeCN, 8 min gradient).
Purification was then carried out by preparatory RP-HPLC (5-40 MeCN, 25 min gradient) to give the desired formic acid salt WC116 (5.3 mg, 10%) as a white foam. The purified product was analyzed by analytical RP-HPLC (UV 214) using both MeCN TR 16.43 (2-40 MeCN, 20 min gradient) and MeOH TR 19.39 (10-60 MeOH, 20 min gradient) and revealed >99% purity. HRMS
Calcd. for C34H52O9N7 [M+H]+ 702.3821, found 702.3827.

H2N(CH2)5CO -D-Tyr-haa-D-Glu-D-Leu-NHz (WC117) (SEQ ID NO:98) H
_J02H
H O
H N N N\`~, N H O
2 O H O p^N^ N NH2 \ H CIO

/ OH

[0265] H2N(CH2)5CO-D-Tyr-haa-D-Glu-D-Leu-NH2 (WC117) (SEQ ID NO:98) was prepared as described above to give the desired peptide TFA salt (23.4 mg, 44%
crude purity as analyzed by analytical RP-HPLC (UV 214), 0-40 MeCN, 8 min gradient).
Purification was then carried out by preparatory RP-HPLC (2-30 MeCN, 20 min gradient) to give the desired formic acid salt WC117 (9.8 mg, 18%) as a white foam. The purified product was analyzed by analytical RP-HPLC (UV 214) using both MeCN TR 16.80 (2-40 MeCN, 20 min gradient) and MeOH TR 17.95 (10-60 MeOH, 20 min gradient) and revealed >99% purity. HRMS
Calcd. for C35H54O9N7 [M+H]+ 716.3978, found 716.3980.

H2N(CH2)4C0-D-Tyr-haa-D-Gln-D-Leu-NH2 (WC125) (SEQ ID NO:99) H
COZNHZ
O
(;N ' H O
HZN N N\`., p H O H N NHZ
O
OH

[0266] H2N(CH2)4CO-D-Tyr-haa-D-Gln-D-Leu-NH2 (WC125) (SEQ ID NO:99) was prepared as described above to give the desired peptide TFA salt (33.0 mg, 62%
crude purity as analyzed by analytical RP-HPLC (UV 214), 0-40 MeCN, 8 min gradient).
Purification was then carried out by preparatory RP-HPLC (5-40 MeCN, 20 min gradient) to give the desired formic acid salt WC125 (10.7 mg, 20%) as a white foam. The purified product was analyzed by analytical RP-HPLC (UV 214) using both MeCN TR 16.21 (0-40 MeCN, 25min gradient) and MeOH TR 17.95 (0-80 MeOH, 25 min gradient) and revealed >97% purity. HRMS
Calcd. for C34H53OsNs [M+H]+ 701.3981, found 701.3979.

H2N(CH2)5CO -D-Tyr-haa-D-Gln-D-Leu-NHz (WC126) (SEQ ID NO: 100) H

O
H QN O
H N N N\\\, H

O
OH

[0267] H2N(CH2)5CO-D-Tyr-haa-D-Gln-D-Leu-NH2 (WC 126) (SEQ ID NO: 100) was prepared as described above to give the desired peptide TFA salt (38.0 mg, 71% crude purity as analyzed by analytical RP-HPLC (UV 214), 0-40 MeCN, 8 min gradient).
Purification was then carried out by preparatory RP-HPLC (5-40 MeCN, 15 min gradient) to give the desired formic acid salt WC126 (11.0 mg, 20%) as a white foam. The purified product was analyzed by analytical RP-HPLC (UV 214) using both MeCN TR 15.37 (0-40 MeCN, 25min gradient) and MeOH TR 18.38 (0-60 MeOH, 25 min gradient) and revealed >99% purity. HRMS Calcd. for C35H5508Ns [M+H]+ 715.4137, found 715.4156.

D-Orn-D-Tyr-haa-D-Gln-D-Leu-NHz (WC127) (SEQ ID NO: 101) f 0 H CO2NH2 = H
N N\\\, H O
H N

,*~'Y , O
OH

[0268] D-Orn-D-Tyr-12 aa-D-Gln-D-Leu-NHz (WC127) (SEQ ID NO:101) was prepared as described above to give the desired peptide TFA salt (45.0 mg, 84%
crude purity as analyzed by analytical RP-HPLC (UV 214), 0-40 MeCN, 8 min gradient).
Purification was then carried out by preparatory RP-HPLC (5-40 MeCN, 15 min gradient) to give the desired formic acid salt WC127 (8.7 mg, 17%) as a white foam. The purified product was analyzed by analytical RP-HPLC (UV 214) using both MeCN TR 14.71 (0-60 MeCN, 20 min gradient) and MeOH TR 11.12 (0-60 MeOH, 25 min gradient) and revealed >99% purity. HRMS
Calcd. for C34H54O8N9 [M+H]+ 716.4090, found 716.4089.
D-Agl-D-Tyr-haa-D-Glu-D-Leu-NHz (WC144) (SEQ ID NO: 102) H

H2NW N NN = H O
p H O 0~- N NH2 O
OH

[0269] D-Agl-D-Tyr-I 2aa-D-Glu-D-Leu-NHz (WC144) (SEQ ID NO:102) was prepared as described above to give the desired peptide TFA salt (34.0 mg, 44%
crude purity as analyzed by analytical RP-HPLC (UV 214), 0-40 MeCN, 8 min gradient).
Purification was then carried out by preparatory RP-HPLC (2-40 MeCN, 25 min gradient) to give the desired formic acid salt WC144 (4.4 mg, 6%) as a white foam. The purified product was analyzed by analytical RP-HPLC (UV 214) using both MeCN TR 16.47 (2-40 MeCN, 25 min gradient) and MeOH TR 19.14 (0-60 MeOH, 25 min gradient) and revealed >96% purity. HRMS
Calcd. for C33H48O9N7 [M+H]+ 686.3508, found 686.3512.

Agl-D-Tyr-haa-D-Glu-D-Leu-NHz (WC145) (SEQ ID NO: 103) H

H2N N = H O
~~~' Q N
O H O J~N NH2 O
OH

[0270] Agl-D-Tyr-haa-D-Glu-D-Leu-NHz (WC145) (SEQ ID NO: 103) was prepared as described above to give the desired peptide TFA salt (30.0 mg, 39% crude purity as analyzed by analytical RP-HPLC (UV 214), 0-40 MeCN, 8 min gradient).
Purification was then carried out by preparatory RP-HPLC (2-40 MeCN, 25 min gradient) to give the desired formic acid salt WC145 (5.0 mg, 6%) as a white foam. The purified product was analyzed by analytical RP-HPLC (UV 214) using both MeCN TR 16.18 (2-40 MeCN, 25 min gradient) and MeOH TR 18.73 (0-60 MeOH, 25 min gradient) and revealed >99% purity. HRMS
Calcd. for C33H48O9N7 [M+H]+ 686.3508, found 686.3514.

D-Bgl-D-Tyr-12 aa-D-Glu-D-Leu-NHz (WC146) (SEQ ID NO: 104) O H

H NV C N N\\\, N' = H O
2 H O O-)'-HN NH2 O
OH

[0271] D-Bgl-D-Tyr-I aa-D-Glu-D-Leu-NHz (WC146) (SEQ ID NO: 104) was prepared as described above to give the desired peptide TFA salt (37.0 mg, 48%
crude purity as analyzed by analytical RP-HPLC (UV 214), 0-40 MeCN, 8 min gradient).
Purification was then carried out by preparatory RP-HPLC (2-40 MeCN, 25 min gradient) to give the desired formic acid salt WC146 (10.5 mg, 13%) as a white foam. The purified product was analyzed by analytical RP-HPLC (UV 214) using both MeCN TR 15.85 (2-40 MeCN, 25 min gradient) and MeOH TR 18.80 (0-60 MeOH, 25 min gradient) and revealed >98% purity. HRMS
Calcd. for C33H48O9N7 [M+H]+ 686.3508, found 686.3514.

Bgl-D-Tyr-haa-D-Glu-D-Leu-NHz (WC147) (SEQ ID NO: 105) H N N N\`", N H O

O

OH

[0272] Bgl-D-Tyr-I 2aa-D-Glu-D-Leu-NHz (WC147) (SEQ ID NO:105) was prepared as described above to give the desired peptide TFA salt (37.0 mg, 48% crude purity as analyzed by analytical RP-HPLC (UV 214), 0-40 MeCN, 8 min gradient).
Purification was then carried out by preparatory RP-HPLC (2-40 MeCN, 25 min gradient) to give the desired formic acid salt WC147 (9.8 mg, 13%) as a white foam. The purified product was analyzed by analytical RP-HPLC (UV 214) using both MeCN TR 16.04 (2-40 MeCN, 25 min gradient) and MeOH TR 18.83 (0-60 MeOH, 25 min gradient) and revealed >99% purity. HRMS
Calcd. for C33H48O9N7 [M+H]+ 686.3508, found 686.3508.

[0273] While we have described a number of embodiments of this invention, it is apparent that our basic examples may be altered to provide other embodiments that utilize the compounds and methods of this invention. Therefore, it will be appreciated that the scope of this invention is to be defined by the appended claims rather than by the specific embodiments that have been represented by way of example. The present application also refers to a number of publications or other documents, the contents of all of which are incorporated by reference herein in their entireties.

[0274] We claim:

Claims (43)

1. A peptidomimetic comprising one or more lactams and/or Bab residues and at least four out of seven contiguous amino acids that appear in an extracellular region of the IL-1 receptor (SEQ ID NO: 1) or IL-1 receptor accessory protein (IL-1RacP) (SEQ ID
NO:2), wherein the at least four amino acids maintain their relative positions as they appear in the extracellular region of the IL-1 receptor or IL-1 receptor accessory protein (IL-1RacP).

2. A peptidomimetic comprising one or more lactams and/or Bab residues and at least four out of seven contiguous amino acids that appear in an extracellular region of the IL-1 receptor (SEQ ID NO: 1) or IL-1 receptor accessory protein (IL-1RacP) (SEQ ID
NO:2), wherein the at least four amino acids maintain their relative positions, but in the inverse configuration, as they appear in the extracellular region of the IL-1 receptor or IL-1 receptor accessory protein (IL-1RacP).

3. The peptidomimetic of claim 1 or 2, wherein the one or more lactams and/or Bab residues replace one or more amino acids of the seven contiguous amino acids.

4. The peptidomimetic of claim 1 or 2, wherein the one or more lactams and/or Bab residues are inserted at the N-terminus, C-terminus or internally.

5. A peptidomimetic comprising one or more lactams and/or Bab residues and at least four amino acids from any peptide selected from Table 1(SEQ ID NOs:3-40), wherein the at least four amino acids maintain their relevant positions as they appear in said peptide.

6. A peptidomimetic comprising one or more lactams and/or Bab residues and at least four amino acids from RYTVELA (SEQ ID NO: 12), wherein the at least four amino acids maintain their relevant positions as they appear in RYTVELA (SEQ ID NO: 12).

7. The peptidomimetic of claim 6, comprising one or more lactams.

8. The peptidomimetic of claim 7, wherein the one or more lactams replace one or more amino acids of RYTVELA (SEQ ID NO:12).

9. The peptidomimetic of claim 7, wherein the one or more lactams are inserted at the N-terminus, C-terminus or internally.

10. The peptidomimetic of any one of the preceding claims, wherein at least one of the one or more lactams is of formula (I):

wherein:

each R X is independently -R, halogen, -OR, -SR, -N(R)2, -CN, -NO2, -C(O)R, -CO2R, -C(O)N(R)2, -C(O)C(O)R, -C(O)CH2C(O)R, -S(O)R, -SO2R, -SO2N(R)2, -NRC(O)R, -NRC(O)N(R)2, -NRSO2R, -NRSO2N(R)2, -N(R)N(R)2, -C=NN(R)2, -C=NOR, -OC(O)R, or -OC(O)N(R)2;

each R is independently hydrogen or an optionally substituted group selected from C1-6 aliphatic; phenyl; a 3- to 7-membered saturated or partially unsaturated carbocyclic ring;
a 5- to 6-membered monocyclic heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; or a 4- to 7-membered saturated or partially unsaturated heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; or two R groups attached to the same nitrogen atom may be taken together with their intervening atoms to form a 4- to 7- membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independent selected from nitrogen, oxygen, and sulfur;

m is 0, 1, or 2; and n is 0, 1, 2, or 3.

11. The peptidomimetic of claim 10, wherein m is 0.

12. The peptidomimetic of claim 10, wherein n is 1.

13. The peptidomimetic of claim 10, wherein the at least one of the one or more lactams is alpha-amino-gamma-lactam (Agl), beta-amino-gamma-lactam (Bgl), and/or beta-hydroxy-alpha-amino-gamma-lactam (Agl(4-OH)).

14. The peptidomimetic of any one of the preceding claims, wherein at least one of the one or more lactams is of formula (II):

wherein:

each R y is independently -R, halogen, -OR, -SR, -N(R)2, -CN, -NO2, -C(O)R, -CO2R, -C(O)N(R)2, -C(O)C(O)R, -C(O)CH2C(O)R, -S(O)R, -SO2R, -SO2N(R)2, -NRC(O)R, -NRC(O)N(R)2, -NRSO2R, -NRSO2N(R)2, -N(R)N(R)2, -C=NN(R)2, -C=NOR, -OC(O)R, or -OC(O)N(R)2;

each R is independently hydrogen or an optionally substituted group selected from C1-6 aliphatic; phenyl; a 3- to 7-membered saturated or partially unsaturated carbocyclic ring;
a 5- to 6-membered monocyclic heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; or a 4- to 7-membered saturated or partially unsaturated heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; or two R groups attached to the same nitrogen atom may be taken together with their intervening atoms to form a 4- to 7- membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independent selected from nitrogen, oxygen, and sulfur;

p is 0, 1, or 2; and each q is independently 0, 1, 2, or 3.

15. The peptidomimetic of claim 14, wherein p is 0.

16. The peptidomimetic of claim 14, wherein each q is 1 or 2.

17. The peptidomimetic of claim 14, wherein the at least one of the one or more lactams is indolizidin-2-one amino acid (I2haa), indolizidin-9-one amino acid (I9aa), and/or quinolizidinone amino acid(Qaa).

18. The peptidomimetic of claim 14, wherein the at least one of the one or more lactams is indolizidin-2-one amino acid (haa).

19. The peptidomimetic of claim 6, comprising one or more Bab residues.

20. The peptidomimetic of claim 19, wherein the one or more Bab residues replace one or more amino acids of RYTVELA (SEQ ID NO: 12).

21. The peptidomimetic of any one of the preceding claims, comprising at least one D-amino acid.

22. The peptidomimetic of claim 21, wherein all amino acids in the peptidomimetic are D-amino acids.

23. A peptidomimetic modified from any one of SEQ ID NO:3-40, wherein the peptidomimetic comprises at least four amino acids from the corresponding peptide sequence and a lactam replacement or insertion.

24. The peptidomimetic of claim 23, wherein the at least four amino acids maintain their relative positions as they appear in the corresponding sequence.

25. The peptidomimetic of claim 23 or 24, wherein the lactam is selected from the group consisting of alpha-amino-gamma-lactam (Agl), beta-amino-gamma-lactam (Bgl), beta-hydroxy-alpha-amino-gamma-lactam (Agl(4-OH)), indolizidin-2-one amino acid (I2aa), and combination thereof.

26. A peptidomimetic modified from any one of SEQ ID NO:3-40, wherein the peptidomimetic comprises at least four amino acids from the corresponding peptide sequence and a Bab replacement or insertion.

27. The peptidomimetic of claim 26, wherein the at least four amino acids maintain their relative positions as they appear in the corresponding sequence.

28. A peptidomimetic comprising any one of SEQ ID NO:61-105.

29. The peptidomimetic of any one of the preceding claims, wherein the peptidomimetic further comprises one or more modifications to increase protease resistance, serum stability and/or bioavailability.

30. The peptidomimetic of claim 29, wherein the one or more modifications are selected from N- and/or C-terminal acetylation, glycosylation, biotinylation, amidation, substitution with D-amino acid, or un-natural amino acid, and/or cyclization of the peptide.

31. A method of inhibiting the activity of an IL-1 receptor in a cell, the method comprising contacting the cell with a peptidomimetic of any one of the preceding claims.

32. A method of treating an IL-1 related disease, disorder or condition, the method comprising administering to a subject in need of treatment a peptidomimetic of any one of claims 1 through 30.

33. The method of claim 32, wherein the IL-1 related disease, disorder or condition is an inflammatory disease, disorder or condition.

34. The method of claim 33, wherein the inflammatory disease, disorder or condition is selected from rheumatoid arthritis, inflammatory bowel disease, septic shock, osteoarthritis, psoriasis, encephalitis, glomerulonephritis, respiratory distress syndrome, Reiter's syndrome, systemic lupus erythematosus, scleroderma, Crohn's disease, ulcerative colitis, inflammatory joint disease, cachexia in certain leukemias, Alzheimer's disease, numerous types of cancers, diabetes mellitus (type I), pulmonary hypertension, stroke, periventricular leucopenia, meningitis, CNS demyelinating diseases, multiple sclerosis, acute disseminated encephalomyelitis (ADEM), idiopathic inflammatory demyelinating disease, transverse myelitis, Devic's disease, progressive multifocal leukoencephaly, Guillain-Barre syndrome, chronic inflammatory demyelinating polyneuropathy, anti-MAG neuropathy, inflammatory bowel disease, sepsis, septic shock, adult respiratory distress syndrome, pancreatitis, trauma-induced shock, asthma, bronchial asthma, allergic rhinitis, cystic fibrosis, stroke, acute bronchitis, chronic bronchitis, acute bronchiolitis, chronic bronchiolitis, gout, spondylarthropathris, ankylosing spondylitis, Reiter's syndrome, psoriatic arthropathy, enterapathric spondylitis, juvenile arthropathy or juvenile ankylosing spondylitis, reactive arthropathy, infectious or post-infectious arthritis, gonoccocal arthritis, tuberculous arthritis, viral arthritis, fungal arthritis, syphilitic arthritis, Lyme disease, arthritis associated with vasculitic syndromes, polyarteritis nodosa, hypersensitivity vasculitis, Luegenec's granulomatosis, polymyalgin rheumatica, joint cell arteritis, calcium crystal deposition arthropathris, pseudo gout, non-articular rheumatism, bursitis, tenosynomitis, epicondylitis (tennis elbow), carpal tunnel syndrome, repetitive use injury, miscellaneous forms of arthritis, neuropathic joint disease, hemarthrosis, Henoch-Schonlein purpura, hypertrophic osteoarthropathy, multicentric reticulohistiocytosis, arthritis associated with certain diseases, surcoilosis, hemochromatosis, sickle cell disease and other hemoglobinopathries, hyperlipoproteineimia, hypogammaglobulinemia, hyperparathyroidism, acromegaly, familial Mediterranean fever, Behat's Disease, systemic lupus erythrematosis, and relapsing polychondritis, inflammatory conditions resulting from harmful stimuli, such as pathogens, damaged cells, or irritants, sarcoidosis, disseminated intravascular coagulation, atherosclerosis, Kawasaki's disease, macrophage activation syndrome (MAS), HIV, graft-versus-host disease, Sjogren's syndrome, vasculitis, autoimmune thyroiditis, dermatitis, atopic dermatitis, myasthenia gravis, inflammatory conditions of the skin, cardiovascular system, nervous system, liver, kidney and pancreas, cirrhosis, eosinophilic esophagitis, cardiovascular disorders, disorders associated with wound healing, respiratory disorders, chronic obstructive pulmonary disease, emphysema, acute inflammatory conditions, atopic inflammatory disorders, bacterial, viral, fungal or protozoan infections, pulmonary diseases, systemic inflammatory response syndrome (SIRS), hemophagocytic lymphohistiocytosis (HLH), juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis, lupus nephritis, lupus-associated arthritis, ankylosing spondylitis, and/or autoimmune diseases.

35. A pharmaceutical composition comprising a peptidomimetic of any one of claims 1 through 30 and a pharmaceutically acceptable carrier.

36. A method of modifying an IL-1 R modulatory peptide, the method comprising steps of:

(a) providing a peptide that modulates the IL-1R activity;

(b) modifying the peptide by introducing one or more lactams and/or Bab residues into the peptide; and (c) testing the IL-1R modulatory activity of the modified peptide.

37. The method of claim 36, wherein the peptide modulates the IL-1R activity non-competitively.

38. The method of claim 37, wherein the peptide is a Negative Allosteric Modulator (NAM) of the IL-1R activity.

39. The method of claim 37, wherein the peptide is a Positive Allosteric Modulator (PAM) of the IL-1R activity.

40. The method of claim 37, wherein the peptide is both a NAM and a PAM of the activity.

41. The method of any one of claims 36-40 further comprising a step of identifying a modified peptide having improved ability to modulate the IL-1R activity as compared to a control.

42. The method of claim 41, wherein the control is the corresponding unmodified parent peptide.

43. A modified IL-1R modulator peptide according to the method of any one of claims 36 through 42.