HK1161742A - Peptidomimetic marcrocycles - Google Patents

Description

Peptidomimetic macrocycles

Cross referencing

This application claims priority to U.S. provisional application 61/099,167 filed on 22/9/2008, which is incorporated herein by reference.

Background

Nuclear transcription factor NF-kB has a key role in immune function in the pathogenesis of inflammatory and autoimmune diseases. NF-kB plays a key role in the autoimmune, inflammatory and destructive mechanisms that contribute to the development of disease (e.g., rheumatoid arthritis). This includes adjustments to: (i) cell recruitment by NF-kB-dependent expression of cell adhesion molecules (such as intercellular adhesion molecule (ICAM) -1 and Vascular Cell Adhesion Molecule (VCAM) -1) and chemoattractants (such as Monocyte Chemotactic Protein (MCP) -1 and Interleukin (IL) -8); (ii) NF-kB dependent production of proinflammatory cytokines such as Tumor Necrosis Factor (TNF) -a, IL-1b, IL-6, IL-17 and IL-23, and resulting NF-kB activation on cells where these cytokines function; (iii) production of IL-2, which promotes T cell proliferation; (iv) modulation of immunoglobulin secretion, cytokine expression, and class switching (class switching) in B cells; (v) the production of receptor activators of NF-kB ligand (RANK-L), which, together with other cytokines, promotes osteoclast differentiation; (vi) expression of Matrix Metalloproteinases (MMPs) that degrade cartilage and bone; and (vii) inhibition of apoptosis of inflammatory cells (Strnad j. and Burke JR, Trends in pharmaceutical Science, vol 28, 2007). Because of the key role of NF-kB in these mechanisms, inhibitors that inhibit the activation of NF-kB should be effective in treating diseases including, but not limited to, cancer, autoimmune diseases, and inflammatory diseases (such as rheumatoid arthritis, inflammatory bowel disease, lupus, and multiple sclerosis).

Activation of NF-kB requires the activity of an inhibitor of the kB (IkB) kinase (IKK) complex, which comprises two kinases (IKKA and IKKb) and the regulatory protein NEMO (NF-kB key regulator). In unstimulated cells, NF-kB is sequestered in the cytoplasm as an inactive complex with an IkB inhibitory protein (Whiteside, s.t. and Israel, a. (1997) se. cancer biol.8, 75-82). In the case of IkBa, stimulation of receptors such as TNF receptors, Toll-like receptors (TLRs) or T cell receptors activates the so-called "canonical" pathway of NF-kB activation, in which a multi-subunit IkB kinase (IKK) complex catalyzes phosphorylation of IkBa at Ser32 and Ser 36. This phosphorylation is critical for subsequent signaling of ubiquitination and proteolysis of IkBa, thereby allowing NF-kB to translocate freely to the nucleus (Ghosh, S. and Karin, M. (2002) Cell 109, S81-S96).

As dysregulation of NFkB has been linked to a variety of diseases (including inflammation, autoimmune diseases and cancer), the NF-kB activation pathway has become a major target for the development of new therapies for inflammatory diseases and cancer. Drugs that selectively target inflammation-induced NF-kB activity, but at the same time do not affect the protective function of the underlying NF-kB activity, would be of greater therapeutic value and likely show fewer undesirable side effects. NEMO binds to C-terminal fragments of both IKK β and IKK α, known as NEMO Binding Domains (NBDs). Cell permeable peptides (cell permeable peptides) spanning NBD disrupt binding of NEMO to two IKKs and block TNF-alpha induced activation of NFkB in various cell types, effectively improving responses in animal models of inflammation (May MJ et al Science 289, 2000). Thus, drugs targeting NBD of the IkB complex have several advantages over drugs targeting the IKK kinase domain. First, NBD-targeting drugs do not affect basal NFkB activity. Second, they have well-defined molecular sites of action. Third, they are specific for the classical NFkB activation pathway and do not affect the alternative NFkB activation pathway. Fourth, NBD-targeting drugs do not target the active domain of kinases and are therefore less likely to affect other key kinases. Finally, such drugs have a wide range of disease indications, including but not limited to: atherosclerosis, asthma, acquired immunodeficiency syndrome, cancer, diabetes, auditory disorders, muscular dystrophy, pigmentary disorders, rheumatoid arthritis, alzheimer's disease, inflammatory bowel disease, multiple sclerosis and inflammatory bone resorption (inflammatory bone resorption).

Disclosure of Invention

In one aspect, the invention provides peptidomimetic macrocycles comprising an amino acid sequence that is at least about 60%, 80%, 90%, or 95% identical to an amino acid sequence selected from the group consisting of those in Table 1. Alternatively, the amino acid sequence of the peptidomimetic macrocycle is selected from the amino acid sequences in Table 1. In certain embodiments, the peptidomimetic macrocycle comprises a helix, such as an alpha-helix. In other embodiments, the peptidomimetic macrocycle comprises an α, α -disubstituted amino acid. The peptidomimetic macrocycles of the invention may comprise a cross-linker connecting the alpha-positions of at least two amino acids. At least one of the two amino acids may be an α, α -disubstituted amino acid.

In some embodiments, the peptidomimetic macrocycle has the formula:

wherein:

A. c, D and E are each independently a natural or unnatural amino acid;

b is natural or non-natural amino acid, amino acid analogue,[-NH-L₃-CO-]、[-NH-L₃-SO₂-]Or [ -NH-L ]₃-]；

R₁And R₂independently-H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-;

R₃is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl (cycloaryl) or heterocycloaryl (heterocycloaryl), optionally substituted with R₅Substitution;

l is of the formula-L₁-L₂-a macrocycle-forming linker;

L₁and L₂Independently is alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene, heterocycloalkylene or [ -R ]₄-K-R₄-]_nEach optionally substituted by R₅Substitution;

each R is₄Is alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;

each K is O, S, SO₂、CO、CO₂Or CONR₃；

Each R is₅Independently halogen, alkyl, -OR₆、-N(R₆)₂、-SR₆、-SOR₆、-SO₂R₆、-CO₂R₆A fluorescent moiety, a radioisotope or a therapeutic agent;

each R is₆independently-H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope, or a therapeutic agent;

R₇is-H, alkyl, alkenyl, alkynylArylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl or heterocycloaryl, optionally substituted with R₅Substituted or part of a cyclic structure with a D residue;

R₈is-H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R₅Substituted or part of a cyclic structure with an E residue;

v and w are independently integers from 1 to 1000;

u, x, y and z are independently integers from 0 to 10; and

n is an integer of 1 to 5.

In other embodiments, the peptidomimetic macrocycle may comprise a cross-linker connecting a backbone amino group of a first amino acid to a second amino acid in the peptidomimetic macrocycle. For example, the invention provides peptidomimetic macrocycles of formula (IV) or (IVa):

wherein:

A. c, D and E are each independently a natural or unnatural amino acid;

b is natural or non-natural amino acid, amino acid analogue,[-NH-L₃-CO-]、[-NH-L₃-SO₂-]Or [ -NH-L ]₃-]；

R₁And R₂independently-H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo, or part of a cyclic structure with an E residue;

R₃is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl or heterocycloaryl, optionally substituted with R₅Substitution;

L₁and L₂Independently is alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene, heterocycloarylene, or [ -R ]₄-K-R₄-]_nEach optionally substituted by R₅Substitution;

each R is₄Is alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;

each K is O, S, SO₂、CO、CO₂Or CONR₃；

Each R is₅Independently halogen, alkyl, -OR₆、-N(R₆)₂、-SR₆、-SOR₆、-SO₂R₆、-CO₂R₆A fluorescent moiety, a radioisotope or a therapeutic agent;

each R is₆independently-H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope, or a therapeutic agent;

R₇is-H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R₅Substitution;

v and w are independently integers from 1 to 1000;

u, x, y and z are independently integers from 0 to 10; and

n is an integer of 1 to 5.

In addition, the present invention provides a method of treating an inflammatory disease in a subject comprising administering to the subject a peptidomimetic macrocycle of the invention. Also provided are methods of modulating HEMO activity in a subject comprising administering to the subject a peptidomimetic macrocycle of the invention; or antagonizing the interaction between IKK β and NEMO protein in a subject, comprising administering to the subject such a peptidomimetic macrocycle.

Is incorporated by reference

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Brief Description of Drawings

The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

figure 1 shows a possible binding pattern of the NBD α helix to NEMO of the IKK β peptidomimetic macrocycle precursors of the invention. Residues 701 to 730 of the NBD alpha helix of IKK beta are PAKKSEELVAEAHNLCTLLENAIQDTVREAnd Q. The solvent-exposed side chains available for crosslinking are underlined.

Figure 2 shows a possible binding pattern of the NBD α helix to NEMO of the IKK β peptidomimetic macrocycle precursors of the invention. Residues 701 to 730 of the NBD alpha helix of IKK beta are PAKKSEELVAEAHNLCTLLENAIQDTVREAnd Q. Solvent for crosslinkingThe exposed side chain is underlined.

FIG. 3 shows several peptidomimetic macrocycles of the invention.

Detailed Description

As used herein, the term "macrocyclic compound" refers to a molecule having a chemical structure comprising a ring or toroid formed from at least 9 covalently bonded atoms.

As used herein, the term "peptidomimetic macrocycle" or "crosslinked polypeptide" refers to a compound comprising a plurality of amino acid residues joined by a plurality of peptide bonds and at least one macrocycle-forming linker that forms a macrocycle between a first naturally-occurring or non-naturally-occurring amino acid residue (or analog) and a second naturally-occurring or non-naturally-occurring amino acid residue (or analog) in the same molecule. Peptidomimetic macrocycles include embodiments in which a macrocycle-forming linker connects the alpha carbon of a first amino acid residue (or analog) and the alpha carbon of a second amino acid residue (or analog). The peptidomimetic macrocycles optionally comprise one or more non-peptide bonds between one or more amino acid residues and/or amino acid analog residues, and optionally one or more non-naturally occurring amino acid residues or amino acid analog residues in addition to any residues that form the macrocycle. When referring to a peptidomimetic macrocycle, "corresponding non-crosslinked polypeptide" is understood to relate to a polypeptide having the same length as the macrocycle and comprising equivalent natural amino acids corresponding to the wild-type sequence of the macrocycle.

The term "stability" as used herein refers to the maintenance of a particular secondary structure of the peptidomimetic macrocycle of the invention in solution, or resistance to proteolytic degradation in vitro or in vivo as measured by circular dichroism, NMR or another biophysical measurement. Non-limiting examples of secondary structures contemplated by the present invention are alpha-helices, beta-turns, and beta-pleated sheets (pleated sheets).

As used herein, the term "helix stability" refers to the maintenance of the α -helical structure of the peptidomimetic macrocycle of the invention as measured by circular dichroism or NMR. For example, in certain embodiments, the peptidomimetic macrocycles of the invention exhibit at least a 1.25, 1.5, 1.75, or 2-fold increase in alpha helicity as determined by circular dichroism spectroscopy compared to the corresponding non-crosslinked macrocycles.

The term "alpha-amino acid" or simply "amino acid" refers to a molecule that contains both an amino group and a carboxyl group bound to a carbon called the alpha-carbon. Suitable amino acids include, but are not limited to, the D-and L-isomers of naturally occurring amino acids, as well as non-naturally occurring amino acids prepared by organic synthesis or other metabolic pathways. The term amino acid as used herein is intended to include amino acid analogs unless the context specifically indicates otherwise.

The term "naturally occurring amino acid" refers to any of the 20 amino acids commonly found in naturally synthesized peptides, abbreviated in one letter as A, R, N, C, D, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y and V.

The term "amino acid analog" or "unnatural amino acid" refers to a molecule that is structurally similar to an amino acid and can be substituted for an amino acid in forming a peptidomimetic macrocycle. Amino acid analogs include, but are not limited to, compounds that are structurally identical to the amino acids defined herein except that they include one or more additional methylene groups between the amino and carboxyl groups (e.g., α -amino β -carboxylic acid) or substitution of the amino or carboxyl groups with similar reactive groups (e.g., substitution of a primary amine with a secondary or tertiary amine, or substitution of the carboxyl group with an ester).

A "non-essential" amino acid residue is one that can be altered from the wild-type sequence of a polypeptide (e.g., the BH3 domain or the p53 MDM2 binding domain) without abolishing or substantially altering its essential biological or biochemical activity (e.g., receptor binding or activation). An "essential" amino acid residue is a residue that, when altered from the wild-type sequence of a polypeptide, results in the elimination or substantial elimination of the major biological or biochemical activity of the polypeptide.

A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. The prior art has defined families of amino acid residues with similar side chains. These families include amino acids with basic side chains (e.g., K, R, H), acidic side chains (e.g., D, E), uncharged polar side chains (e.g., G, N, Q, S, T, Y, C), nonpolar side chains (e.g., A, V, L, I, P, F, M, W), beta-branched side chains (e.g., T, V, I), and aromatic side chains (e.g., Y, F, W, H). Thus, for example, a predicted nonessential amino acid residue in a polypeptide is preferably replaced with another amino acid residue from the same side chain family. Other examples of acceptable substitutions are those based on isosteric considerations (e.g., norleucine for methionine) or other properties (e.g., 2-thienylalanine for phenylalanine).

The term "element" as used herein in relation to a macrocycle or a macrocycle-forming linker refers to an atom that forms or can form a macrocycle, with the exception of substituents or side chain atoms. By analogy, cyclodecane, 1, 2-difluoro-decane and 1, 3-dimethyl-cyclodecane are all considered ten-membered macrocyclic compounds, since hydrogen or fluoro substituents or methyl side chains do not participate in the formation of the macrocycle. When used as part of a molecular structure, the symbolsRefers to a single bond or a trans or cis double bond.

The term "amino acid side chain" refers to the moiety attached to the alpha-carbon in an amino acid. For example, the amino acid side chain of alanine is methyl, the amino acid side chain of phenylalanine is benzyl, the amino acid side chain of cysteine is thiomethyl, the amino acid side chain of aspartic acid is carboxymethyl, the amino acid side chain of tyrosine is 4-hydroxybenzyl, and the like. Other non-naturally occurring amino acid side chains are also included, for example, those occurring in nature (e.g., amino acid metabolites) or synthetically prepared amino acid side chains (e.g., alpha disubstituted amino acids).

The term "α, α disubstituted amino acid" refers to a molecule or moiety comprising an amino group and a carboxyl group bound to the carbon (α -carbon) connecting the side chains of two natural or unnatural amino acids.

The term "polypeptide" includes two or more naturally or non-naturally occurring amino acids joined by covalent bonds (e.g., amide bonds). The polypeptides described herein include full-length proteins (e.g., fully processed proteins) as well as shorter amino acid sequences (e.g., fragments of naturally occurring proteins or synthetic polypeptide fragments).

As used herein, the term "macrocyclization reagent" or "macrocycle-forming reagent" refers to any reagent that can be used to prepare the peptidomimetic macrocycles of the invention by mediating a reaction between two reactive groups. The reactive group may be, for example, azides and alkynes, in which case macrocyclization reagents include, but are not limited to: cu reagents, such as reagents that provide reactive Cu (i) species, such as CuBr, CuI, or CuOTf; and Cu (II) salts which can be converted in situ to active Cu (I) agents by the addition of reducing agents such as ascorbic acid or sodium ascorbate, e.g. Cu (CO)₂CH₃)₂、CuSO₄And CuCl₂. Macrocyclization reagents may additionally include, for example, Ru reagents known in the art, such as Cp RuCl (PPh)₃)₂、[Cp*RuCl]₄Or other Ru reagents that can provide reactive Ru (ii) species. In other cases, the reactive group is a terminal alkene. In such embodiments, the macrocyclization reagent or macrocycle-forming reagent is a metathesis catalyst, including but not limited to a stable late transition metal carbene complex catalyst, such as a group VIII transition metal carbene catalyst. For example, such catalysts are Ru and Os metal centers having an oxidation state of +2, an electron count of 16, and penta-coordination. In Grubbs et al, "Ring cloning methods and Related Processes in Organic Synthesis" Acc.chem.Res.1995, 2Additional catalysts are disclosed in 8,446-. In still other cases, the reactive group is a thiol group. In such embodiments, the macrocyclization reagent is, for example, a linker functionalized with two thiol-reactive groups (e.g., halogen groups).

The term "halo" or "halogen" refers to fluorine, chlorine, bromine or iodine or a group thereof.

The term "alkyl" refers to a straight or branched hydrocarbon chain containing the specified number of carbon atoms. E.g. C₁-C₁₀Representing 1 to 10 (inclusive) carbon atoms in the group. In the absence of any numerical designation, "alkyl" is a chain (straight or branched) having from 1 to 20 (inclusive) carbon atoms therein.

The term "alkylene" refers to a divalent alkyl group (i.e., -R-).

The term "alkenyl" refers to a hydrocarbon chain that is straight or branched having one or more carbon-carbon double bonds. The alkenyl moiety contains the indicated number of carbon atoms. E.g. C₂-C₁₀Representing 2 to 10 (inclusive) carbon atoms in the group. The term "lower alkenyl" means C₂-C₆An alkenyl chain. In the absence of any numerical designation, "alkenyl" is a chain (straight or branched) having 2 to 20 (inclusive) carbon atoms therein.

The term "alkynyl" refers to a hydrocarbon chain that is straight or branched having one or more carbon-carbon triple bonds. Alkynyl moieties contain the indicated number of carbon atoms. E.g. C₂-C₁₀Representing 2 to 10 (inclusive) carbon atoms in the group. The term "lower alkynyl" refers to C₂-C₆An alkynyl chain. In the absence of any numerical designation, "alkynyl" is a chain (straight or branched) having 2 to 20 (inclusive) carbon atoms therein.

The term "aryl" refers to a 6-carbon monocyclic or 10-carbon bicyclic aromatic ring system wherein 0, 1, 2,3 or 4 atoms of each ring are substituted with a substituent. Examples of aryl groups include phenyl, naphthyl, and the like. The term "arylalkyl" or the term "aralkyl" refers to an alkyl group substituted with an aryl group. The term "arylalkoxy" refers to an alkoxy group substituted with an aryl group.

"alkylaryl" refers to a radical C wherein one of the aryl's hydrogen atoms is as defined above₁-C₅Alkyl-substituted aryl as defined above. Typical examples of alkylaryl groups include, but are not limited to: 2-methylphenyl, 3-methylphenyl, 4-methylphenyl, 2-ethylphenyl, 3-ethylphenyl, 4-ethylphenyl, 2-propylphenyl, 3-propylphenyl, 4-propylphenyl, 2-butylphenyl, 3-butylphenyl, 4-butylphenyl, 2-pentylphenyl, 3-pentylphenyl, 4-pentylphenyl, 2-isopropylphenyl, 3-isopropylphenyl, 4-isopropylphenyl, 2-isobutylphenyl, 3-isobutylphenyl, 4-isobutylphenyl, 2-sec-butylphenyl, 3-sec-butylphenyl, 4-sec-butylphenyl, 2-tert-butylphenyl, 3-tert-butylphenyl and 4-tert-butylphenyl.

"Amidoaryl" refers to an aryl in which one of the aryl's hydrogen atoms is replaced by one or more-C (O) NH groups₂A group-substituted aryl group as defined above. Typical examples of amidoaryl groups include: 2-C (O) NH₂Phenyl, 3-C (O) NH₂Phenyl, 4-C (O) NH₂Phenyl, 2-C (O) NH₂Pyridyl, 3-C (O) NH₂Pyridyl and 4-C (O) NH₂-a pyridyl group.

"Heterocyclylalkyl" refers to a compound wherein C is₁-C₅C as defined above with one of the hydrogen atoms of the alkyl radical being substituted by a heterocyclic ring₁-C₅An alkyl group. Typical examples of heterocyclylalkyl groups include, but are not limited to: -CH₂CH₂-morpholine, -CH₂CH₂-piperidine, -CH₂CH₂CH₂-morpholine and-CH₂CH₂CH₂-imidazole.

"Amidoalkyl" refers to a compound wherein C is₁-C₅One of the hydrogen atoms of the alkyl group being substituted by-C (O) NH₂C as defined above substituted by radicals₁-C₅An alkyl group. Typical examples of amidoalkyl groups include, but are not limited to: -CH₂-C(O)NH₂、-CH₂CH₂-C(O)NH₂、-CH₂CH₂CH₂C(O)NH₂、-CH₂CH₂CH₂CH₂C(O)NH₂、-CH₂CH₂CH₂CH₂CH₂C(O)NH₂、-CH₂CH(C(O)NH₂)CH₃、-CH₂CH(C(O)NH₂)CH₂CH₃、-CH(C(O)NH₂)CH₂CH₃、-C(CH₃)₂CH₂C(O)NH₂、-CH₂-CH₂-NH-C(O)-CH₃、-CH₂-CH₂-NH-C(O)-CH₃-CH₃and-CH₂-CH₂-NH-C(O)-CH＝CH₂。

"hydroxyalkyl (alkanol)" refers to a compound wherein C is₁-C₅C as defined above with one of the hydrogen atoms of the alkyl radical being substituted by a hydroxyl group₁-C₅An alkyl group. Typical examples of hydroxyalkyl groups include, but are not limited to: -CH₂OH、-CH₂CH₂OH、-CH₂CH₂CH₂OH、-CH₂CH₂CH₂CH₂OH、-CH₂CH₂CH₂CH₂CH₂OH、-CH₂CH(OH)CH₃、-CH₂CH(OH)CH₂CH₃、-CH(OH)CH₃and-C (CH)₃)₂CH₂OH。

"carboxyalkyl" refers to where C is₁-C₅C, as defined above, having one of the hydrogen atoms of the alkyl group replaced by a-COOH group₁-C₅An alkyl group. Typical examples of carboxyalkyl groups include, but are not limited to: -CH₂COOH、-CH₂CH₂COOH、-CH₂CH₂CH₂COOH、-CH₂CH₂CH₂CH₂COOH、-CH₂CH(COOH)CH₃、-CH₂CH₂CH₂CH₂CH₂COOH、-CH₂CH(COOH)CH₂CH₃、-CH(COOH)CH₂CH₃and-C (CH)₃)₂CH₂COOH。

The term "cycloalkyl" as used herein includes saturated and partially unsaturated cyclic hydrocarbon groups having 3 to 12 carbons, preferably 3 to 8 carbons, more preferably 3 to 6 carbons, wherein the cycloalkyl group is additionally optionally substituted. Some cycloalkyl groups include, but are not limited to: cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl.

The term "heteroaryl" refers to an aromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic; if bicyclic, having 1-6 heteroatoms; or 1-9 heteroatoms selected from O, N or S (e.g., carbon atoms and 1-3, 1-6, or 1-9O, N or S heteroatoms, respectively, if monocyclic, bicyclic, or tricyclic) if tricyclic, wherein 0, 1, 2,3, or 4 atoms of each ring are substituted with a substituent. Examples of heteroaryl groups include: pyridyl, furyl (furyl or furanyl), imidazolyl, benzimidazolyl, pyrimidinyl, thiophenyl or thienyl, quinolyl, indolyl, thiazolyl, and the like.

The term "heteroarylalkyl" or the term "heteroaralkyl" refers to an alkyl group substituted with a heteroaryl group. The term "heteroarylalkoxy" refers to an alkoxy group substituted with a heteroaryl group.

The term "heteroarylalkyl" or the term "heteroaralkyl" refers to an alkyl group substituted with a heteroaryl group. The term "heteroarylalkoxy" refers to an alkoxy group substituted with a heteroaryl group.

The term "heterocyclyl" refers to a non-aromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic; if bicyclic, having 1-6 heteroatoms; or 1-9 heteroatoms selected from O, N or S (e.g., carbon atoms and 1-3, 1-6, or 1-9O, N or S heteroatoms, respectively, if monocyclic, bicyclic, or tricyclic) if tricyclic, wherein 0, 1, 2, or 3 atoms of each ring are substituted with a substituent. Examples of heterocyclyl groups include piperazinyl (piperazinyl), pyrrolidinyl, dioxanyl, morpholinyl (morpholinonyl), tetrahydrofuranyl and the like.

The term "substituent" refers to a group that replaces another atom or group (e.g., a hydrogen atom) on any molecule, compound, or moiety. Suitable substituents include, but are not limited to: halogen, hydroxy, mercapto, oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, thioalkoxy, aryloxy, amino, alkoxycarbonyl, amido, carboxy, alkanesulfonyl, alkylcarbonyl, and cyano.

In certain embodiments, the compounds of the present invention contain one or more asymmetric centers and thus exist as racemates or racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. Unless expressly indicated otherwise, the present invention includes all such isomeric forms of these compounds. In certain embodiments, the compounds of the present invention are also represented in multiple tautomeric forms, in such cases, the present invention includes all tautomeric forms of the compounds described herein (e.g., if alkylation of a ring system results in alkylation at multiple positions, the present invention includes all such reaction products). Unless expressly indicated otherwise, the present invention includes all such isomeric forms of these compounds. Unless expressly indicated otherwise, the present invention includes all crystalline forms of the compounds described herein.

As used herein, the term "increase" or "decrease" means resulting in a statistically significant (i.e., p < 0.1) increase or decrease, respectively, of at least 5%.

As used herein, reference to a numerical range for a variable is meant to indicate that the invention can be practiced with the variable equaling any value within the range. Thus, for a variable that is inherently discontinuous, the variable is equal to any integer value within the numerical range, including the endpoints of the range. Similarly, for a variable that is itself continuous, the variable is equal to any real value within the numerical range, including the endpoints of the range. By way of example, but not limitation, if the variable itself is discontinuous, the variable described as having a value between 0-2 takes a value of 0, 1, or 2; and if the variable itself is continuous, then take the value 0.0, 0.1, 0.01, 0.001 or any other real value ≧ 0 and ≦ 2.

As used herein, the word "or" is used in the inclusive sense of "and/or" and not the exclusive sense of "any/or," unless specifically indicated otherwise.

The term "average" means the average obtained by performing at least 3 independent repetitions for each data point.

The term "biologically active" includes the structural and functional properties of the macrocyclic compounds of the present invention. The biological activity is, for example, structural stability, alpha-helicity, affinity for a target, resistance to proteolytic degradation, cell permeability, intracellular stability, in vivo stability, or any combination thereof.

The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

In some embodiments, the peptidomimetic macrocycles of the invention are associated with Nuclear Factor (NF) -kB. Nuclear Factor (NF) -kB is a key transcription factor that is widely implicated in disorders associated with a variety of diseases, including inflammation and cancer. The NF-kB activation pathway has become a major target for the development of new therapies for inflammatory diseases and cancer. The indispensable role played by NF-kB in many biological processes has led to the following concerns: complete shutdown of this pathway would have a significant detrimental effect on normal cell function. Drugs that selectively target inflammation-induced NF-kB activity, but at the same time do not affect the protective function of the underlying NF-kB activity, would be of greater therapeutic value and likely exhibit fewer undesirable side effects.

Activation of NF-kB may require the activity of an inhibitor of the kB (ikb) -kinase (IKK) complex. The IKK complex comprises two catalytic subunits, i.e., two kinases (IKKa and IKKb), and a regulatory subunit called NEMO (NF-kB key regulator), which converts upstream signals into activation of the catalytic subunits (Schomer-Miller b. et al 2006). In vitro, IKK α and IKK β have similar substrate specificities, targeting two specific serines in the N-terminal regulatory domain of the IkB protein. However, IKK β is a more potent IkB kinase than IKK α. The N-terminal alpha-helical region of NEMO is thought to bind to C-terminal fragments of IKK alpha and IKK beta, known as NEMO binding domains (NEBs). The minimum requirement for binding of NEMO and IKK kinases has been identified. Mutational analysis of IKK β NBD demonstrated that residues W739 and W741 are critical for NEMO binding, while mutations of the other four amino acids did not affect binding (Strickland I and Ghosh S, Ann Rheum Dis, 2006). Thus, the small size of NBD may allow the design of peptides that can disrupt the interaction of NEMO with IKK.

In view of the attractiveness of NFkB as a therapeutic target for many diseases, including but not limited to inflammatory diseases, autoimmune diseases and cancer, small molecule IKK inhibitors have been developed, such as TRCA1, BMS345541, ML120B, Bayer's compound A, PS1145 and IMD 0354. Each of these inhibitors appears to have superior selectivity for IKK β over IKK α and does not inhibit the other kinases tested. These IKK inhibitors block a variety of biological responses in vitro, all of which are based on the role of the classical IKK β -NFkB pathway in the regulation of these responses. Such reactions include: (i) stimulation of inflammatory cytokines production; (ii) TNF α and IL1 β induced production of pro-inflammatory chemokines; (iii) expression of growth factors and cytokines (e.g., IL-2, IL-4, IL5, and IFN γ) in concanavalin-A-stimulated T cells; (iv) TNF α -induced expression of adhesion molecules; (v) IL-1 β -induced production of GM-CSF and IL-8 in airway smooth muscle cells, and (vi) LPS-stimulated proliferation of B-cells and Con-A-stimulated T-cells (Strnad J and Burke JR, Trends in Pharm Sciences, vol 28, 2007). These drugs, which target the kinase domain of IKK, can abrogate important basal NFkB activity and inhibit both the classical and alternative NFkB activation pathways. These drugs may be associated with toxicity due to potential cross-reactivity with other kinases.

Selective inhibition of NFkB using peptides targeted to the NEMO Binding Domain (NBD) represents a preferred approach to inhibit NFkB-induced biological responses. NBD peptides can block NFkB-induced osteoclastogenesis in vitro and in vivo, and reduce the production of pro-inflammatory cytokines and inflammatory bone loss in collagen-induced arthritis (Jimi E, et al, nat. med, vol 10, 2004).

The predicted domain structure of NEMO includes two coiled coil regions (CC1 and CC2), a Leucine Zipper (LZ), and a C-terminal Zinc Finger (ZF). The N-terminal CC1 region interacts with the C-terminal tail of IKK kinase (Ghosh and Karin, 2002; Leonardi et al, 2000). Although the CC2 and LZ regions of NEMO are considered potential sites for trimerization (Agou et al, 2002) or tetramerization (Tegethoff et al, 2003), the N-terminal domain comprising CC1 has been reported to exist as a dimer (Marienfeld et al, 2006). The IKK binding region of NEMO is defined at residues 47-120(Marienfeld et al, 2006). Replacement of the phosphate receptor Ser68 in this region with a phosphoserine mimetic (glutamate) reduces dimerization of NEMO and reduces IKK β binding in vitro, while replacement with cysteine or alanine increases dimerization (Palkowitsch et al, 2008). Mutation analysis has determined that D738, W739 and W741 in NBD are key binding determinants (May et al, 2002). In addition, phosphorylation of S740 within NBD decreases binding to NEMO (May et al, 2002; Schomer-Miller et al, 2006). The lowest complex of NEMO and IKK was determined using a reasonably truncated NEMO protein and IKK-derived fragments. Residues 44-111 of NEMO constitute the lowest IKK interaction domain (Rushe m. et al, Structure 16, 2008). The NEMO44-111-IKK peptide complex and the larger N-terminal fragment NEMO1-196 observed in the crystal structure were dimeric. The molecular details of the binding between NEMO and IKK kinases explain how phosphorylation of specific serine residues in this region can affect complex formation and suggest an approach for developing targeted small molecule therapeutics.

The present invention provides peptidomimetic macrocycles that block NEMO binding to the IKK complex and inhibit acute and chronic inflammation in a patient. For example, the peptidomimetic macrocycles of the invention may affect only the classical/typical NF-kB activation pathway, but not the alternative/non-classical pathway, and thus inhibit inflammation-induced NF-kB activation, but not the underlying NF-kB activity.

A non-limiting, exemplary list of suitable IKK β/NEMO (IKK γ) peptides for use in the present invention is given below:

TABLE 1

Peptidomimetic macrocycles of the invention

In some embodiments, the peptidomimetic macrocycle of the invention has the formula (I):

wherein:

A. c, D and E are each independently a natural or unnatural amino acid;

b is natural or non-natural amino acid, amino acid analogue,[-NH-L₃-CO-]、[-NH-L₃-SO₂-]Or [ -NH-L ]₃-]；

R₁And R₂independently-H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-;

R₃is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl or heterocycloaryl, optionally substituted with R₅Substitution;

l is of the formula-L₁-L₂-a macrocycle-forming linker;

L₁and L₂Independently is alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene, heterocycloarylene, or [ -R ]₄-K-R₄-]_nEach optionally substituted by R₅Substitution;

each R is₄Is alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;

each K is O, S, SO₂、CO、CO₂Or CONR₃；

Each R is₅Independently halogen, alkyl, -OR₆、-N(R₆)₂、-SR₆、-SOR₆、-SO₂R₆、-CO₂R₆A fluorescent moiety, a radioisotope or a therapeutic agent;

each R is₆independently-H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope, or a therapeutic agent;

R₇is-H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R₅Substituted or part of a cyclic structure with a D residue;

R₈is-H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R₅Substituted or part of a cyclic structure with an E residue;

v and w are independently integers from 1 to 1000;

u, x, y and z are independently integers from 0 to 10; and

n is an integer of 1 to 5.

In one example, R₁And R₂Is an alkyl group which is unsubstituted or substituted by halogen. In another example, R₁And R₂Both are independently alkyl, unsubstituted or substituted with halogen. In certain embodiments, R₁And R₂At least one of which is methyl. In other embodiments, R₁And R₂Is methyl.

In certain embodiments of the invention, x + y + z is at least 3. In other embodiments of the invention, x + y + z is 1, 2,3, 4, 5, 6,7, 8, 9 or 10. Each specific value of A, B, C, D or E in the macrocycle or macrocycle precursor of the invention is independently selected. For example, when x is 3, formula [ A]_xSequences represented include embodiments in which the amino acids are not identical, e.g., Gln-Asp-Ala; and embodiments in which the amino acids are the same, e.g., Gln-Gln-Gln. This applies to any value of x, y or z within the specified range. Similarly, when u is greater than 1, each compound of the invention can comprise the same or different peptidomimetic macrocycles. For example, the compounds of the invention may include compounds comprising different linker lengths or groupsA peptidomimetic macrocycle of chemical composition.

In certain embodiments, the peptidomimetic macrocycles of the invention comprise a secondary structure that is an alpha-helix, and R is₈is-H, allowing hydrogen bonding within the helix. In certain embodiments, at least one of A, B, C, D or E is an α, α -disubstituted amino acid. In one example, B is an α, α -disubstituted amino acid. For example, at least one of A, B, C, D or E is 2-aminoisobutyric acid. In other embodiments, at least one of A, B, C, D or E is

In other embodiments, the length of the macrocycle-forming linker L, as measured from the first ca to the second ca, is selected to stabilize a desired secondary peptide structure, such as an a-helix formed by residues of the peptidomimetic macrocycle (including but not necessarily limited to residues located between the first ca and the second ca).

In one embodiment, the peptidomimetic macrocycle of formula (I) is:

wherein R is₁And R₂Each independently is-H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-.

In a related embodiment, the peptidomimetic macrocycle of formula (I) is:

in other embodiments, the peptidomimetic macrocycle of formula (I) is a compound of any one of the formulae shown below:

wherein "AA" represents any natural or unnatural amino acid side chain, andis [ D ] as defined above]_v、[E]_wAnd n is an integer between 0 and 20, 50, 100, 200, 300, 400 or 500. In certain embodiments, n is 0. In other embodiments, n is less than 50.

Exemplary embodiments of the macrocycle-forming linker L are shown below.

In certain embodiments, the peptidomimetic macrocycle of the invention has the formula (II):

wherein:

A. c, D and E are each independently a natural or unnatural amino acid;

b is natural or non-natural amino acid, amino acid analogue,[-NH-L₃-CO-]、[-NH-L₃-SO₂-]Or [ -NH-L ]₃-]；

R₁And R₂independently-H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-;

R₃is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl or heterocycloaryl, optionally substituted with R₅Substitution;

l is formulaA macrocycle-forming linker of (a);

L₁、L₂and L₃Independently is alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene, heterocycloarylene, or [ -R ]₄-K-R₄-]_nEach optionally substituted by R₅Substitution;

each R is₄Is alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;

each K is O, S, SO₂、CO、CO₂Or CONR₃；

Each R is₅Independently halogen, alkyl, -OR₆、-N(R₆)₂、-SR₆、-SOR₆、-SO₂R₆、-CO₂R₆A fluorescent moiety, a radioisotope or a therapeutic agent;

each R is₆independently-H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope, or a therapeutic agent;

R₇is-H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R₅Substituted or part of a cyclic structure with a D residue;

R₈is-H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R₅Substituted or part of a cyclic structure with an E residue;

v and w are independently integers from 1 to 1000;

u, x, y and z are independently integers from 0 to 10; and

n is an integer of 1 to 5.

In one example, R₁And R₂Is an alkyl group which is unsubstituted or substituted by halogen. In another example, R₁And R₂Independently an alkyl group which is unsubstituted or substituted by halogen. In certain embodiments, R₁And R₂At least one of which is methyl. In other embodiments, R₁And R₂Is methyl.

In certain embodiments of the invention, x + y + z is at least 3. In other embodiments of the invention, x + y + z is 1, 2,3, 4, 5, 6,7, 8, 9 or 10. The specific values of A, B, C, D or E in the macrocycle or macrocycle precursor of the invention are independently selected. For example, when x is 3, formula[A]_xSequences represented include embodiments in which the amino acids are not identical, e.g., Gln-Asp-Ala; and embodiments in which the amino acids are the same, e.g., Gln-Gln-Gln. This applies to any value of x, y or z within the specified range.

In certain embodiments, the peptidomimetic macrocycles of the invention comprise a secondary structure that is an alpha-helix, and R is₈is-H, allowing hydrogen bonding within the helix. In certain embodiments, at least one of A, B, C, D or E is an α, α -disubstituted amino acid. In one example, B is an α, α -disubstituted amino acid. For example, at least one of A, B, C, D or E is 2-aminoisobutyric acid. In other embodiments, at least one of A, B, C, D or E is

In other embodiments, the length of the macrocycle-forming linker L, as measured from the first ca to the second ca, is selected to stabilize a desired secondary peptide structure, such as an a-helix formed by residues of a peptidomimetic macrocycle (including but not necessarily limited to residues between the first ca and the second ca).

Exemplary embodiments of the macrocycle-forming linker L are shown below.

In other embodiments, the invention provides peptidomimetic macrocycles of formula (III):

formula (III)

Wherein:

A. c, D and E are each independently a natural or unnatural amino acid;

b is natural or non-natural amino acid, amino acid analogue,[-NH-L₄-CO-]、[-NH-L₄-SO₂-]Or [ -NH-L ]₄-]；

R₁And R₂independently-H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-;

R₃is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl or heterocycloaryl, which are unsubstituted or substituted by R₅Substituted;

L₁、L₂、L₃and L₄Independently is alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene, heterocycloarylene, or [ -R ]₄-K-R₄-]_nEach being unsubstituted or substituted by R₅Substituted;

k is O, S, SO₂、CO、CO₂Or CONR₃；

Each R is₄Is alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;

each R is₅Independently halogen, alkyl, -OR₆、-N(R₆)₂、-SR₆、-SOR₆、-SO₂R₆、-CO₂R₆A fluorescent moiety, a radioisotope or a therapeutic agent;

each R is₆independently-H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope, or a therapeutic agent;

R₇is-H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, unsubstituted or substituted with R₅Substituted or part of a cyclic structure with a D residue;

R₈is-H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, unsubstituted or substituted with R₅Substituted or part of a cyclic structure with an E residue;

v and w are independently integers from 1 to 1000;

u, x, y and z are independently integers from 0 to 10; and

n is an integer of 1 to 5.

In one example, R₁And R₂Is an alkyl group which is unsubstituted or substituted by halogen. In another example, R₁And R₂Independently an alkyl group which is unsubstituted or substituted by halogen. In certain embodiments, R₁And R₂At least one of which is methyl. In other embodiments, R₁And R₂Is methyl.

In certain embodiments of the invention, x + y + z is at least 3. In other embodiments of the invention, x + y + z is 3, 4, 5, 6,7, 8, 9 or 10. Each specific value of A, B, C, D or E in the macrocycle or macrocycle precursor of the invention is independently selected. For example, when x is 3, formula [ A]_xSequences represented include embodiments in which the amino acids are not identical, e.g., Gln-Asp-Ala; and whereinAmino acids are the same, e.g., Gln-Gln-Gln. This applies to any value of x, y or z within the specified range.

In certain embodiments, the peptidomimetic macrocycles of the invention comprise a secondary structure that is an alpha-helix, and R is₈is-H, allowing hydrogen bonding within the helix. In certain embodiments, at least one of A, B, C, D or E is an α, α -disubstituted amino acid. In one example, B is an α, α -disubstituted amino acid. For example, at least one of A, B, C, D or E is 2-aminoisobutyric acid. In other embodiments, at least one of A, B, C, D or E is

In other embodiments, a macrocycle-forming linker [ -L ] is selected as measured from a first ca to a second ca₁-S-L₂-S-L₃-]To stabilize a desired secondary peptide structure, such as an alpha-helix formed by residues of a peptidomimetic macrocycle (including but not necessarily limited to residues between a first C.alpha.and a second C.alpha.).

For example, the macrocycle or macrocycle precursor is synthesized by solution phase or solid phase methods and may comprise naturally occurring and non-naturally occurring amino acids. See, for example, "The Non-Protein Amino Acids" in Hunt, Chemistry and Biochemistry of The Amino Acids, eds by G.C. Barrett, Chapman and Hall, 1985. In certain embodiments, the sulfhydryl moiety is the side chain of the amino acid residue L-cysteine, D-cysteine, α -methyl-L-cysteine, α -methyl-D-cysteine, L-homocysteine, D-homocysteine, α -methyl-L-homocysteine or α -methyl-D-homocysteine. The bisalkylating reagent has X-L₂-Y is of the formula, wherein L₂Is a linker moiety and X and Y are replaced by a-SH moiety to react with L₂A bond-forming leaving group. In certain embodiments, X and Y are halogen such as I, Br or Cl.

In other embodiments, D and/or E in the compound of formula I, II or III is further modified in order to facilitate cellular uptake. In certain embodiments, lipidation (1) or PEGylation (PEGylation) of the peptidomimetic macrocycle facilitates cellular uptake, increases bioavailability, increases blood circulation, alters pharmacokinetics, decreases immunogenicity, and/or decreases the frequency of administration required.

In other embodiments, at least one of [ D ] and [ E ] in the compound of formula I, II or III represents a moiety comprising an additional macrocycle-forming linker, such that the peptidomimetic macrocycle comprises at least two macrocycle-forming linkers. In a specific embodiment, the peptidomimetic macrocycle comprises two macrocycle-forming linkers.

In the peptidomimetic macrocycles of the invention, any of the macrocycle-forming linkers described herein can be used in any combination with any of the sequences shown in tables 1-4, and also in any combination with any of the R-substituents described herein.

In certain embodiments, the peptidomimetic macrocycle comprises at least one alpha-helix motif. For example, A, B and/or C in the compound of formula I, II or III comprises one or more a-helices. In general, the α 0-helix comprises 3-4 amino acid residues/turn. In certain embodiments, the α 1-helix of the peptidomimetic macrocycle comprises 1 to 5 turns and thus comprises 3 to 20 amino acid residues. In particular embodiments, the alpha-helix comprises 1 turn, 2 turns, 3 turns, 4 turns, or 5 turns. In certain embodiments, the macrocycle-forming linker stabilizes the alpha-helical motif included within the peptidomimetic macrocycle. Thus, in certain embodiments, the length of the macrocycle-forming linker L from the first ca to the second ca is selected to increase the stability of the a-helix. In certain embodiments, the macrocycle-forming linker spans from 1 to 5 turns of the alpha-helix. In certain embodiments, the macrocycle-forming linker spans about 1, 2,3, 4, or 5 turns of the a-helix. In certain embodiments, the length of the macrocycle-forming linker is about every turn of the alpha-helixOr about a-helix per turnThe length is equal to about 5-13 carbon-carbon bonds, about 7-11 carbon-carbon bonds, or about 9 carbon-carbon bonds when the macrocycle-forming linker spans about 1 turn of the alpha-helix. When the macrocycle-forming linker spans about 2 turns of the alpha-helix, the length is equal to about 8-16 carbon-carbon bonds, about 10-14 carbon-carbon bonds, or about 12 carbon-carbon bonds. When the macrocycle-forming linker spans about 3 turns of the α 0-helix, the length is equal to about 14-22 carbon-carbon bonds, about 16-20 carbon-carbon bonds, or about 18 carbon-carbon bonds. When the macrocycle-forming linker spans about 4 turns of the α 1-helix, the length is equal to about 20-28 carbon-carbon bonds, about 22-26 carbon-carbon bonds, or about 24 carbon-carbon bonds. When the macrocycle-forming linker spans about 5 turns of the alpha-helix, the length is equal to about 26-34 carbon-carbon bonds, about 28-32 carbon-carbon bonds, or about 30 carbon-carbon bonds. When the macrocycle-forming linker spans about 1 turn of the alpha-helix, the linker comprises about 4-12 atoms, about 6-10 atoms, or about 8 atoms. When the macrocycle-forming linker spans about 2 turns of the alpha-helix, the linker comprises about 7-15 atoms, about 9-13 atoms, or about 11 atoms. When the macrocycle-forming linker spans about 3 turns of the alpha-helix, the linker comprises about 13-21 atoms, about 15-19 atoms, or about 17 atoms. When the macrocycle-forming linker spans about 4 turns of the alpha-helix, the linker comprises about 19-27 atoms, about 21-25 atoms, or about 23 atoms. When the macrocycle-forming linker spans about 5 turns of the alpha-helix, the linker comprises about 25-33 atoms, about 27-31 atoms, or about 29 atoms. When the macrocycle-forming linker spans about 1 turn of the alpha-helix, the macrocycle that is produced forms a cycle comprising about 17-25 members, about 19-23 members, or about 21 members. When the macrocycle-forming linker spans about 2 turns of the alpha-helix, the macrocycle formation produced comprises about 29-37, about 31-35, or about 3 membersA 3-membered ring. When the macrocycle-forming linker spans about 3 turns of the alpha-helix, the macrocycle that is produced forms a cycle comprising about 44-52 members, about 46-50 members, or about 48 members. When the macrocycle-forming linker spans about 4 turns of the alpha-helix, the macrocycle that is produced forms a cycle comprising about 59-67 members, about 61-65 members, or about 63 members. When the macrocycle-forming linker spans about 5 turns of the alpha-helix, the macrocycle that is produced forms a loop comprising about 74-82 members, about 76-80 members, or about 78 members.

In other embodiments, the invention provides peptidomimetic macrocycles of formula (IV) or (IVa):

wherein:

A. c, D and E are each independently a natural or unnatural amino acid;

b is natural or non-natural amino acid, amino acid analogue,[-NH-L₃-CO-]、[-NH-L₃-SO₂-]Or [ -NH-L ]₃-]；

R₁And R₂independently-H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo, or part of a cyclic structure with an E residue;

R₃is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl or heterocycloaryl, optionally substituted with R₅Substitution;

l is of the formula-L₁-L₂-a macrocycle-forming linker;

L₁and L₂Independently is alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene, heterocycloarylene, or [ -R ]₄-K-R₄-]_nEach optionally substituted by R₅Substitution;

each R is₄Is alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;

each K is O, S, SO₂、CO、CO₂Or CONR₃；

Each R is₅Independently halogen, alkyl, -OR₆、-N(R₆)₂、-SR₆、-SOR₆、-SO₂R₆、-CO₂R₆A fluorescent moiety, a radioisotope or a therapeutic agent;

each R is₆independently-H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope, or a therapeutic agent;

R₇is-H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R₅Substitution;

v and w are independently integers from 1 to 1000;

u, x, y and z are independently integers from 0 to 10; and

n is an integer of 1 to 5.

In one example, R₁And R₂Is an alkyl group which is unsubstituted or substituted by halogen. In another example, R₁And R₂Independently an alkyl group which is unsubstituted or substituted by halogen. In certain embodiments, R₁And R₂At least one of which is methyl. In other embodiments, R₁And R₂Is methyl.

In certain embodiments of the invention, x + y + z is at least 1. In other embodiments of the invention, x + y + z is at least 2. In other embodiments of the invention, x + y + z is 3, 4, 5, 6,7, 8, 9 or 10. Each specific value of A, B, C, D or E in the macrocycle or macrocycle precursor of the invention is independently selected. For example, when x is 3, formula [ A]_xSequences represented include embodiments in which the amino acids are not identical, e.g., Gln-Asp-Ala; and embodiments in which the amino acids are the same, e.g., Gln-Gln-Gln. This applies to any value of x, y or z within the specified range.

In certain embodiments, the peptidomimetic macrocycles of the invention comprise a secondary structure that is an alpha-helix, and R is₈is-H, allowing hydrogen bonding within the helix. In certain embodiments, at least one of A, B, C, D or E is an α, α -disubstituted amino acid. In one example, B is an α, α -disubstituted amino acid. For example, at least one of A, B, C, D or E is 2-aminoisobutyric acid. In other embodiments, at least one of A, B, C, D or E is

In other embodiments, the length of the macrocycle-forming linker L, as measured from the first ca to the second ca, is selected to stabilize a desired secondary peptide structure, such as an a-helix formed by residues of a peptidomimetic macrocycle (including but not necessarily limited to residues between the first ca and the second ca).

Macrocycle-forming linker-L₁-L₂Exemplary embodiments of (a) are as follows.

Peptidomimetic macrocyclesPreparation of

The peptidomimetic macrocycles of the invention can be prepared by any of a variety of methods known in the art. For example, any residue represented by an "X" in tables 1, 2,3 or 4 may be replaced by a residue capable of forming a cross-linker with a second residue in the same molecule or a precursor of such a residue.

Various methods for achieving the preparation of peptidomimetic macrocycles are known in the art. For example, schaffeister et al, j.am.chem.soc.122: 5891 while 5892 (2000); schafmeister & Verdine, j.am.chem.soc.122: 5891 (2005); walensky et al, Science 305: 1466 and 1470 (2004); the preparation of peptidomimetic macrocycles of formula I is described in U.S. Pat. No. 7,192,713 and PCT application WO 2008/121767. The α, α -disubstituted amino acids and amino acid precursors disclosed in the cited references can be used in the synthesis of peptidomimetic macrocycle precursor polypeptides. For example, "S5-ene amino acid" is (S) -alpha- (2 '-pentenyl) alanine, and "S8-ene amino acid" is (S) -alpha- (2' -octenyl) alanine. After incorporation of such amino acids into the precursor polypeptide, the terminal alkene is reacted with a metathesis catalyst, resulting in the formation of a peptidomimetic macrocycle.

In other embodiments, the peptidomimetic macrocycle of the invention has formula IV or IVa. For example, U.S. patent 7,202,332 describes a method for preparing such macrocyclic compounds.

In certain embodiments, the synthesis of these peptidomimetic macrocycles involves a multi-step process characterized by: synthesizing a peptidomimetic precursor comprising an azide moiety and an alkyne moiety; the peptidomimetic precursor is then contacted with a macrocyclization reagent to produce a triazole-linked peptidomimetic macrocycle. Such methods are described, for example, in U.S. application 12/037,041 filed on 25/2/2008. For example, the macrocycle or macrocycle precursor is synthesized by solution phase or solid phase methods and may comprise naturally occurring and non-naturally occurring amino acids. See, for example, Hunt,Chemistry and Biochemistry of the Amino AcidstheNon-Protein Amino Acids ", by G.C. Barrett, Chapman and Hall, 1985.

In certain embodiments, the azide is attached to the α -carbon of a residue and the alkyne is attached to the α -carbon of another residue. In certain embodiments, the azide moiety is an azido analog of the amino acids L-lysine, D-lysine, α -methyl-L-lysine, α -methyl-D-lysine, L-ornithine, D-ornithine, α -methyl-L-ornithine, or α -methyl-D-ornithine. In another embodiment, the alkyne moiety is L-propargylglycine. In yet other embodiments, the alkyne moiety is an amino acid selected from the group consisting of: l-propargylglycine, D-propargylglycine, (S) -2-amino-2-methyl-4-pentynoic acid, (R) -2-amino-2-methyl-4-pentynoic acid, (S) -2-amino-2-methyl-5-hexynoic acid, (R) -2-amino-2-methyl-5-hexynoic acid, (S) -2-amino-2-methyl-6-heptynoic acid, (R) -2-amino-2-methyl-6-heptynoic acid, (S) -2-amino-2-methyl-7-octynoic acid, (R) -2-amino-2-methyl-7-octynoic acid, and, (S) -2-amino-2-methyl-8-nonynoic acid and (R) -2-amino-2-methyl-8-nonynoic acid.

In certain embodiments, the present invention provides a method of synthesizing a peptidomimetic macrocycle, the method comprising the step of contacting a peptidomimetic precursor of formula V or formula VI with a macrocyclization reagent:

wherein v, w, x, y, z, A, B, C, D, E, R₁、R₂、R₇、R₈、L₁And L₂As defined above for formula (II); when the macrocyclization reagent is a Cu reagent, R₁₂is-H, and when the macrocyclization reagent is a Ru reagent, R₁₂is-H or alkyl; and go intoA step wherein the contacting step results in the formation of a covalent linkage between the alkyne moiety and the azide moiety in formula III or formula IV. For example, when the macrocyclization reagent is a Ru reagent, R₁₂May be a methyl group.

In the peptidomimetic macrocycles of the invention, R₁And R₂Is alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-. In certain embodiments, R₁And R₂Independently is alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-. In certain embodiments, at least one of A, B, C, D or E is an α, α -disubstituted amino acid. In one example, B is an α, α -disubstituted amino acid. For example, at least one of A, B, C, D or E is 2-aminoisobutyric acid.

For example, R₁And R₂Is an alkyl group which is unsubstituted or substituted by halogen. In another example, R₁And R₂Independently an alkyl group which is unsubstituted or substituted by halogen. In certain embodiments, R₁And R₂At least one of which is methyl. In other embodiments, R₁And R₂Is methyl. The macrocyclization reagent may be a Cu reagent or a Ru reagent.

In certain embodiments, the peptidomimetic precursor is purified prior to the contacting step. In other embodiments, the peptidomimetic macrocycle is purified after the contacting step. In still other embodiments, the peptidomimetic macrocycle is refolded after the contacting step. The process may be carried out in solution or, alternatively, the process may be carried out on a solid support.

It is also envisioned herein that the methods of the invention are carried out in the presence of a target macromolecule that binds to a peptidomimetic precursor or peptidomimetic macrocycle under conditions that favor such binding. In certain embodiments, the methods of the invention are performed in the presence of a target macromolecule that preferentially binds to a peptidomimetic precursor or peptidomimetic macrocycle under conditions that favor such binding. The method is also applicable to the synthesis of libraries of peptidomimetic macrocycles.

In certain embodiments, the alkyne moiety of the peptidomimetic precursor of formula V or formula VI is a side chain of an amino acid selected from the group consisting of: l-propargylglycine, D-propargylglycine, (S) -2-amino-2-methyl-4-pentynoic acid, (R) -2-amino-2-methyl-4-pentynoic acid, (S) -2-amino-2-methyl-5-hexynoic acid, (R) -2-amino-2-methyl-5-hexynoic acid, (S) -2-amino-2-methyl-6-heptynoic acid, (R) -2-amino-2-methyl-6-heptynoic acid, (S) -2-amino-2-methyl-7-octynoic acid, (R) -2-amino-2-methyl-7-octynoic acid, and, (S) -2-amino-2-methyl-8-nonynoic acid and (R) -2-amino-2-methyl-8-nonynoic acid. In other embodiments, the azide moiety of the peptidomimetic precursor of formula V or formula VI is a side chain of an amino acid selected from the group consisting of: epsilon-azido-L-lysine, epsilon-azido-D-lysine, epsilon-azido-alpha-methyl-L-lysine, epsilon-azido-alpha-methyl-D-lysine, delta-azido-alpha-methyl-L-ornithine and delta-azido-alpha-methyl-D-ornithine.

In certain embodiments, x + y + z is 3, and A, B and C are independently natural or unnatural amino acids. In other embodiments, x + y + z is 6, and A, B and C are independently natural or unnatural amino acids.

In certain embodiments, the contacting step is carried out in a solvent selected from the group consisting of protic solvents, aqueous solvents, organic solvents, and mixtures thereof. For example, the solvent may be selected from H₂O、THF、THF/H₂O、tBuOH/H₂O、DMF、DIPEA、CH₃CN or CH₂Cl₂、ClCH₂CH₂Cl or a mixture thereof. The solvent may be one that favours helix formation.

Alternative but equivalent protecting groups, leaving groups or reagents, and performing specific synthetic steps in an alternative sequence or order to produce the desired compound. Synthetic chemical transformations and protecting group methods for synthesizing the compounds described hereinProtection and deprotection) include, for example, those described in, for example, Larock,Comprehensive Organic TransformationsVCH Publishers (1989); greene and Wuts, respectively, in the form of tablets,Protective Groups in Organic Synthesis2 nd edition, John Wiley and Sons (1991); the Fieser and the Fieser,Fieser and Fieser′s Reagents for Organic Synthesisjohn Wiley and Sons (1994); and the one by Paquette, and,Encyclopedia of Reagents for Organic Synthesismethods described in John Wiley and Sons (1995) and its subsequent versions.

For example, by chemical synthesis methods, such as Fields et al,Synthetic Peptides：A User′s Guidechapter 3 of (1), Grant, w.h. authoring, Freeman&The peptidomimetic macrocycles of the invention are prepared by the methods described in Co., New York, N.Y., 1992, page 77. Thus, for example, peptides are synthesized using side chain protected amino acids on, for example, an automated peptide synthesizer (e.g., Applied Biosystems (Foster City, CA), model 430A, 431 or 433) using automated Merrifield solid phase synthesis techniques with amines protected by tBoc or Fmoc chemistry.

One way described herein to generate the peptidomimetic precursors and peptidomimetic macrocycles described herein uses Solid Phase Peptide Synthesis (SPPS). The C-terminal amino acid is attached to the cross-linked polystyrene resin via an acid-labile bond to a linker molecule. Such resins are insoluble in the solvents used for synthesis, making washing away of excess reagents and by-products relatively simple and rapid. The N-terminus is protected with an Fmoc group that is stable in acid but removable with base. If necessary, the side chain functional groups are protected with base-stable, acid-labile groups.

For example, longer peptidomimetic precursors are generated by combining individual synthetic peptides using natural chemical linkages. Alternatively, longer synthetic peptides are biosynthesized by well-known recombinant DNA and protein expression techniques. Detailed protocols for these techniques are provided in well-known standard manuals. To construct a gene encoding a peptidomimetic precursor of the invention, the amino acid sequence is reverse translated to obtain a nucleic acid sequence encoding the amino acid sequence, preferably using codons optimized for the organism in which the gene is to be expressed. Next, the synthetic gene is typically prepared by synthesizing the oligonucleotide encoding the peptide and any regulatory elements, if necessary. The synthesized gene is inserted into an appropriate cloning vector and transfected into a host cell. The peptide is then expressed under suitable conditions for the chosen expression system and host. The peptide was purified and characterized by standard methods.

For example, peptidomimetic precursors are prepared in a high-throughput combinatorial manner using, for example, a high-throughput multichannel combinatorial synthesizer (e.g., the Thuramed TETRAS multichannel peptide synthesizer from CreoSalus, Louisville, KY or the Apex 396 multichannel peptide synthesizer from AAPPTEC, inc.

The following synthetic schemes are provided only to illustrate the present invention and are not intended to limit the scope of the invention described herein. To simplify the figure, the exemplary scheme shows the azido amino acid analogs epsilon-azido-alpha-methyl-L-lysine and epsilon-azido-alpha-methyl-D-lysine, and the alkyne amino acid analogs L-propargylglycine, (S) -2-amino-2-methyl-4-pentynoic acid and (S) -2-amino-2-methyl-6-heptynoic acid. Thus, in the following synthetic schemes, each R is₁、R₂、R₇And R₈is-H; each L₁Is- (CH)₂)₄-; and each L₂Is- (CH)₂) -. However, as noted throughout the detailed description above, many other amino acid analogs can be employed, wherein R is₁、R₂、R₇、R₈、L₁And L₂May be independently selected from the various structures disclosed herein.

Synthesis scheme 1:

synthesis scheme 1 describes the preparation of several compounds of the present invention. Such as Belokon et al (1998), Tetrahedron Asymm.9: 4249-4252 Ni (II) complexes of Schiff bases derived from the chiral auxiliary (S) -2- [ N- (N' -benzylprolyl) amino ] benzophenone (BPB) and amino acids such as glycine or alanine. The resulting complex is then reacted with an alkylating agent comprising an azide moiety or an alkyne moiety to produce an enantiomerically enriched (enantiomerically enriched) compound of the invention. The resulting compounds can be protected for peptide synthesis, if desired.

Synthesis scheme 2:

in the general procedure for the synthesis of peptidomimetic macrocycles as illustrated in FIG. 2, the peptidomimetic precursors comprise an azide moiety and an alkyne moiety and are synthesized by solution phase or Solid Phase Peptide Synthesis (SPPS) using the commercially available N- α -Fmoc-L-propargyl glycine and N- α -Fmoc protected forms of the amino acids (S) -2-amino-2-methyl-4-pentynoic acid, (S) -2-amino-6-heptynoic acid, (S) -2-amino-2-methyl-6-heptynoic acid, N-methyl- ε -azido-L-lysine and N-methyl- ε -azido-D-lysine. The peptidomimetic precursor is then deprotected and cleaved from the solid phase resin by standard conditions (e.g., strong acids such as 95% TFA). The peptidomimetic precursors are reacted as crude mixtures or purified in organic or aqueous solution prior to reaction with a macrocyclization reagent such as Cu (I) (Rostovtsev et al (2002), Angew. chem. Int. Ed.41: 2596-. In one embodiment, the triazole formation reaction is conducted under conditions that favor the formation of an alpha-helix. In one embodiment, in the group selected from H₂O、THF、CH₃CN, DMF, DIPEA, tBuOH or a mixture thereof. In another embodiment, in DMFA macrocyclization step is performed. In certain embodiments, the macrocyclization step is performed in a buffered aqueous solvent or a partially aqueous solvent.

Synthesis scheme 3:

in the general procedure for the synthesis of peptidomimetic macrocycles as illustrated in FIG. 3, the peptidomimetic precursors comprise an azide moiety and an alkyne moiety and are synthesized by Solid Phase Peptide Synthesis (SPPS) using the commercially available N- α -Fmoc-L-propargylglycine and N- α -Fmoc-protected forms of the amino acids N- α -Fmoc-L-propargylglycine and (S) -2-amino-2-methyl-4-pentynoic acid, (S) -2-amino-6-heptynoic acid, (S) -2-amino-2-methyl-6-heptynoic acid, N-methyl- ε -azido-L-lysine and N-methyl- ε -azido-D-lysine. The peptidomimetic precursors are reacted as a crude mixture with a macrocyclization reagent on a resin, such as a Cu (I) reagent (Rostovtsev et al (2002), Angew. chem. Int. Ed.41: 2596-. The synthesized triazole-containing peptidomimetic macrocycle is then deprotected and cleaved from the solid phase resin by standard conditions (e.g., strong acids such as 95% TFA). In certain embodiments, in a group selected from CH₂Cl₂、ClCH₂CH₂Cl, DMF, THF, NMP, DIPEA, 2, 6-lutidine, pyridine, DMSO, H₂O or a mixture thereof in a solvent. In certain embodiments, the macrocyclization step is performed in a buffered aqueous solvent or a partially aqueous solvent.

Synthesis scheme 4:

in the general procedure for the synthesis of peptidomimetic macrocycles as illustrated in FIG. 4, the peptidomimetic precursors comprise an azide moiety and an alkyne moiety and are synthesized by solution phase or Solid Phase Peptide Synthesis (SPPS) using the commercially available N- α -Fmoc-L-propargyl glycine and N- α -Fmoc protected forms of the amino acids (S) -2-amino-2-methyl-4-pentynoic acid, (S) -2-amino-6-heptynoic acid, (S) -2-amino-2-methyl-6-heptynoic acid, N-methyl- ε -azido-L-lysine and N-methyl- ε -azido-D-lysine. The peptidomimetic precursor is then deprotected and cleaved from the solid phase resin by standard conditions (e.g., strong acids such as 95% TFA). The peptidomimetic precursors are reacted as a crude mixture or with a macrocyclization reagent such as Cu (II) reagent (e.g., Cp RuCl (PPh)₃)₂Or [ Cp RuCl ]]₄) Purification was performed prior to the reaction (Rasmussen et al (2007), org.lett.9: 5337-5339; zhang et al (2005), j.am.chem.soc.127: 15998-15999). In certain embodiments, the compound is selected from DMF, CH₃The macrocyclization step was performed in a solvent of CN and THF.

Synthesis scheme 5:

in the general procedure for the synthesis of peptidomimetic macrocycles as illustrated in FIG. 5, the peptidomimetic precursors comprise an azide moiety and an alkyne moiety and are synthesized by Solid Phase Peptide Synthesis (SPPS) using the commercially available N- α -Fmoc-L-propargylglycine and N- α -Fmoc-protected forms of the amino acids N- α -Fmoc-L-propargylglycine and (S) -2-amino-2-methyl-4-pentynoic acid, (S) -2-amino-6-heptynoic acid, (S) -2-amino-2-methyl-6-heptynoic acid, N-methyl- ε -azido-L-lysine and N-methyl- ε -azido-D-lysine. The peptidomimetic precursor is reacted as a crude mixture with a macrocyclization reagent (e.g., a Ru (II) reagent) on the resin. For example, the reagent may be Cp RuCl (PPh)₃)₂Or [ Cp RuCl ]]₄(Rasmussen et al (2007), org.Lett.9: 5337-5339; Zhang et al (2005), J.Am.Che.m.Soc.127: 15998-15999). In certain embodiments, in a group selected from CH₂Cl₂、ClCH₂CH₂Cl、CH₃The macrocyclization step was performed in a solvent of CN, DMF and THF.

The present invention encompasses the synthesis of the peptidomimetic macrocycles described herein using non-naturally occurring amino acids and amino acid analogs. Any amino acid or amino acid analog suitable for the synthetic procedures used to synthesize the stable triazole-containing peptidomimetic macrocycles can be used in the present invention. For example, L-propargylglycine is expected to be a useful amino acid in the present invention. However, other alkyne-containing amino acids having different amino acid side chains may also be used in the present invention. For example, L-propargylglycine contains a methylene unit between the α -carbon of the amino acid and the alkyne in the amino acid side chain. The invention also includes the use of amino acids having multiple methylene units between the alpha-carbon and the alkyne. Similarly, the amino acids L-lysine, D-lysine, alpha-methyl-L-lysine and alpha-methyl-D-lysine azide analogues are also considered to be useful amino acids in the present invention. However, other terminal azide amino acids containing different amino acid side chains may also be used in the present invention. For example, an azide analog of L-lysine contains 4 methylene units between the α -carbon of the amino acid and the terminal azide group of the amino acid side chain. The invention also includes the use of amino acids having less than or more than 4 methylene units between the alpha-carbon and the terminal azide group. Table 2 shows some amino acids that may be used to prepare the peptidomimetic macrocycles of the invention.

TABLE 2

Table 2 shows exemplary amino acids used to prepare the peptidomimetic macrocycles of the invention.

In certain embodiments, the amino acids and amino acid analogs are in the D-configuration. In other embodiments, they are in the L-configuration. In certain embodiments, certain amino acids and amino acid analogs contained in the peptidomimetic are in the D-configuration and certain amino acids and amino acid analogs are in the L-configuration. In certain embodiments, the amino acid analogs are α, α -disubstituted, such as α -methyl-L-propargylglycine, α -methyl-D-propargylglycine, epsilon-azido- α -methyl-L-lysine, and epsilon-azido- α -methyl-D-lysine. In certain embodiments, the amino acid analogs are N-alkylated, e.g., N-methyl-L-propargylglycine, N-methyl-D-propargylglycine, N-methyl-e-azido-L-lysine, and N-methyl-e-azido-D-lysine.

In certain embodiments, the-NH moiety of an amino acid is protected using a protecting group (including, but not limited to, -Fmoc and-Boc). In other embodiments, the amino acids are not protected prior to synthesis of the peptidomimetic macrocycle.

In other embodiments, the peptidomimetic macrocycle of formula III is synthesized. Such methods are described, for example, in U.S. application 11/957,325 filed on 17.12.2007. The following synthetic schemes describe the preparation of such compounds. To simplify the diagram, the exemplary scheme depicts amino acid analogs derived from L-or D-cysteine, wherein L₁And L₃Are all- (CH)₂) -. However, as noted throughout the detailed description above, many other amino acid analogs can be employed, wherein L is₁And L₃May be independently selected from the various structures disclosed herein. Symbol "[ AA ]]_m”、“[AA]_n”、“[AA]_o"represents the sequence of an amide linking moiety (e.g., a natural or unnatural amino acid). As previously mentioned, each instance of "AA" is independent of any other instance of "AA" and is as "[ AA ]]_mThe formula (a) includes, for example, sequences of non-identical amino acids as well as sequences of identical amino acids.

Synthesis scheme 6:

in FIG. 6, the peptidomimetic precursor contains 2-SH moieties and is synthesized by Solid Phase Peptide Synthesis (SPPS) using commercially available N- α -Fmoc amino acids such as N- α -Fmoc-S-trityl-L-cysteine or N- α -Fmoc-S-trityl-D-cysteine. The alpha-methylated form of D-cysteine or L-cysteine is produced by known methods (Seebach et al (1996), Angew. chem. int. Ed. Engl. 35: 2708-Bioorganic Chemistry：Peptides and Proteins", Oxford University Press, New York: 1998, incorporated herein by reference in its entirety) into appropriately protected N- α -Fmoc-S-trityl monomers. The precursor peptidomimetic is then deprotected by standard conditions (e.g., strong acid such as 95% TFA) and cleaved from the solid phase resin. The precursor peptoid is reacted as a crude mixture, or in organic or aqueous solution with X-L₂Purification was carried out before the-Y reaction. In certain embodiments, the alkylation reaction is conducted under dilute conditions (i.e., 0.15mmol/L) to facilitate macrocyclization and avoid polymerization. In certain embodiments, in an organic solution such as liquid NH₃(Mosberg et al (1985), J.Am.chem.Soc.107: 2986-2987; Szewczuk et al (1992), int.J.peptide Protein Res.40: 233-242), NH2₃In MeOH or NH₃Alkylation reactions were carried out in DMF (Or et al (1991), J.Org.chem.56: 3146-3149). In other embodiments, the alkylation reaction is carried out in an aqueous solution such as 6M guanidine hydrochloride at pH 8 (Brunel et al (2005), chem. Commun. (20): 2552-. In other embodiments, the solvent used for the alkylation reaction is DMF or dichloroethane.

Synthesis scheme 7:

in FIG. 7, the precursor peptidomimetic contains2 or more-SH moieties, 2 of which are specifically protected to allow selective deprotection and subsequent alkylation thereof to form a macrocycle. The precursor peptide mimetics were synthesized by Solid Phase Peptide Synthesis (SPPS) using commercially available N- α -Fmoc amino acids such as N- α -Fmoc-S-p-methoxytrityl-L-cysteine or N- α -Fmoc-S-p-methoxytrityl-D-cysteine. The alpha-methylated form of D-cysteine or L-cysteine is produced by known methods (Seebach et al (1996), Angew. chem. int. Ed. Engl. 35: 2708-Bioorganic Chemistry：Peptides and ProteinsOxford University Press, New York: 1998, incorporated herein by reference in its entirety) into appropriately protected N- α -Fmoc-S-p-methoxytrityl monomers. The Mmt protecting group of the peptidomimetic precursor is then selectively cleaved off by standard conditions (e.g., a weak acid such as 1% TFA in DCM). The precursor peptide mimic is then reacted with X-L in organic solution on a resin₂-Y reaction. For example, the reaction takes place in the presence of a hindered base (hinded base) such as diisopropylethylamine. In certain embodiments, in an organic solution such as liquid NH₃(Mosberg et al (1985), J.Am.chem.Soc.107: 2986-2987; Szewczuk et al (1992), int.J.peptide Protein Res.40: 233-242), NH2₃In MeOH or NH₃The alkylation reaction was carried out in DMF (Or et al (1991), J.Org.chem.56: 3146-3149). In other embodiments, the alkylation reaction is carried out in DMF or dichloroethane. The peptidomimetic macrocycle is then deprotected and cleaved from the solid phase resin by standard conditions (e.g., strong acids such as 95% TFA).

Synthesis scheme 8:

in FIG. 8, the peptidomimetic precursor contains 2 or more-SH moieties, 2 of which are specifically protected to allow selective deprotection and subsequent alkylation to form the macrocycle. Make itThe peptidomimetic precursors are synthesized by Solid Phase Peptide Synthesis (SPPS) using commercially available N- α -Fmoc amino acids such as N- α -Fmoc-S-p-methoxytrityl-L-cysteine, N- α -Fmoc-S-p-methoxytrityl-D-cysteine, N- α -Fmoc-S-t-butyl-L-cysteine, and N- α -Fmoc-S-S-t-butyl-D-cysteine. The alpha-methylated form of D-cysteine or L-cysteine is produced by known methods (Seebach et al (1996), Angew. chem. int. Ed. Engl. 35: 2708-Bioorganic Chemistry：Peptides and ProteinsOxford University Press, New York: 1998, incorporated herein by reference in its entirety) into an appropriately protected N- α -Fmoc-S-p-methoxytrityl or N- α -Fmoc-S-t-butyl monomer. The S-S-tert-butyl protecting group of the peptidomimetic precursor is then selectively cleaved off by known conditions (e.g., 20% 2-mercaptoethanol in DMF, ref: Galande et al (2005), J.Comb.chem.7: 174-177). The precursor peptidomimetic is then contacted with a molar excess of X-L in an organic solution on a resin₂-Y reaction. For example, the reaction takes place in the presence of a sterically hindered base such as diisopropylethylamine. The Mmt protecting group of the peptidomimetic precursor is then selectively cleaved off by standard conditions (e.g., a weak acid such as 1% TFA in DCM). The peptidomimetic precursor is then cyclized on the resin by treatment with a hindered base in an organic solution. In certain embodiments, in e.g. NH₃/MeOH or NH₃The alkylation reaction was carried out in organic solution with/DMF (Or et al (1991), J.Org.chem.56: 3146-3149). The peptidomimetic macrocycle is then deprotected by standard conditions (e.g., strong acids such as 95% TFA) and cleaved from the solid phase resin.

Synthetic scheme 9:

in FIG. 9, the peptidomimetic precursor contains 2L-cysteine moieties. By known biological expression systems in living cells or by knownIn vitro cell-free expression methods synthesize peptidomimetic precursors. The precursor peptoid is reacted as a crude mixture, or in organic or aqueous solution with X-L₂Purification was carried out before the-Y reaction. In certain embodiments, the alkylation reaction is conducted under dilute conditions (i.e., 0.15mmol/L) to facilitate macrocyclization and avoid polymerization. In certain embodiments, in an organic solution such as liquid NH₃(Mosberg et al (1985), J.Am.chem.Soc.107: 2986-2987; Szewczuk et al (1992), int.J.peptide Protein Res.40: 233-242), NH2₃In MeOH or NH₃Alkylation reactions were carried out in DMF (Or et al (1991), J.Org.chem.56: 3146-3149). In other embodiments, the alkylation reaction is carried out in an aqueous solution such as 6M guanidine hydrochloride at pH 8 (Brunel et al (2005), chem. Commun. (20): 2552-. In other embodiments, the alkylation reaction is carried out in DMF or dichloroethane. In another embodiment, the alkylation is carried out in a non-denaturing aqueous solution; in yet another embodiment, the alkylation is conducted under conditions that favor the formation of an alpha-helical structure. In yet another embodiment, the alkylation is carried out under conditions that favor binding of the precursor peptidomimetic to another protein, thereby inducing formation of a bound alpha-helix conformation during the alkylation process.

The present invention contemplates various embodiments of X and Y suitable for reaction with a thiol group. Typically, X or Y are each independently selected from the general classes shown in table 5. For example, X and Y are halogen such as-Cl, -Br or-I. Any of the macrocycle-forming linkers described herein may be used in any combination with any of the sequences shown in tables 1-4, and also in any combination with any of the R-substituents indicated herein.

Table 3: examples of reactive groups capable of reacting with thiol groups and resulting linkages

X or Y	The resulting covalent linkage
		Acrylamide	Thioethers
Halides (e.g. alkyl or aryl halides)	Thioethers
		Sulfonic acid (sulfonate)	Thioethers
Aziridine (aziridine)	Thioethers
		Epoxide compound	Thioethers
Haloacetamide	Thioethers
		Maleimide	Thioethers
Sulfonate (sulfonate ester)	Thioethers

The invention encompasses the use of naturally occurring and non-naturally occurring amino acids and amino acid analogs in the synthesis of peptidomimetic macrocycles of formula (III). Any amino acid or amino acid analog suitable for use in the synthetic procedures used to synthesize the stable dimercapto-containing peptidomimetic macrocycles may be used in the present invention. For example, cysteine is expected to be a useful amino acid in the present invention. However, in addition to cysteine, other sulfur-containing amino acids containing different amino acid side chains are also useful. For example, cysteine contains a methylene unit between the α -carbon of the amino acid and the terminal-SH of the amino acid side chain. The present invention also includes the use of amino acids having multiple methylene units between the alpha-carbon and the terminal-SH. Non-limiting examples include alpha-methyl-L-homocysteine and alpha-methyl-D-homocysteine. In certain embodiments, the amino acids and amino acid analogs are in the D-configuration. In other embodiments, they are in the L-configuration. In certain embodiments, certain amino acids and amino acid analogs contained in the peptidomimetic are in the D-configuration and certain amino acids and amino acid analogs are in the L-configuration. In certain embodiments, the amino acid analogs are α, α -disubstituted, such as α -methyl-L-cysteine and α -methyl-D-cysteine.

The invention includes macrocycles in which a macrocycle-forming linker is used to link two or more-SH moieties in a peptidomimetic precursor to form a peptidomimetic macrocycle of the invention. As described above, the macrocycle-forming linker confers conformational rigidity, increased metabolic stability, and/or increased cell permeability. In addition, in certain embodiments, the macrocycle-forming linkage stabilizes the α -helical secondary structure of the peptidomimetic macrocycle. The macrocycle-forming linker has the formula X-L₂-Y, wherein X and Y are the same or different moieties as defined above. Both X and Y have a linker-L allowing one to form a macrocycle₂Chemical characterization of the double alkylation of the peptoid precursors containing a double thiol group. As defined above, linker-L₂Including alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, cycloalkylene or heterocycloarylene or-R₄-K-R₄-, all of which may optionally be R as defined above₅And (4) substituting the group. In addition, a macrocyclic linker-L is formed in addition to the carbon linking the-SH group of the sulfhydryl-containing amino acid₂-wherein 1-3 carbon atoms are optionally replaced by a heteroatom such as N, S or O.

Macrocycle-forming linker X-L₂of-YL₂The moieties may vary in length depending, inter alia, on the distance between the positions of the two amino acid analogs used to form the peptidomimetic macrocycle. In addition, L following the macrocycle-forming linker₁And/or L₃The length of the part varies, L₂Can also be varied to create linkers of suitable overall length to form stable peptidomimetic macrocycles. For example, if the direction of the channel is L₁And L₃Each additional methylene unit being added to alter the amino acid analog used, then L₂A reduction in length equivalent to about two methylene units to offset L₁And L₃Is increased.

In certain embodiments, L₂Is of the formula- (CH)₂)_n-wherein n is an integer between about 1 and about 15. For example, n is 1, 2,3, 4, 5, 6,7, 8, 9, or 10. In other embodiments, L₂Is an alkenylene group. In still other embodiments, L₂Is an aryl group.

Table 4 shows X-L₂-further embodiments of the Y group.

TABLE 4 exemplary X-L of the invention₂-Y group

For example, X and Y in the table are each independently Cl-, Br-or I-.

Additional methods of forming peptidomimetic macrocycles contemplated for use in carrying out the present invention include those disclosed in the following references: mustapa, M.Firouz Mohd et al, J.org.chem (2003), 68, pages 8193-8198; yang, Bin et al Bioorg Med.chem.Lett. (2004), 14, p. 1403-1406; us patent 5,364,851; us patent 5,446,128; us patent 5,824,483; us patent 6,713,280 and us patent 7,202,332. In such embodiments, amino acid precursors containing additional R-substituents at the α -position are used. Such amino acids are incorporated into the macrocycle precursor at desired positions, which may be at positions where the cross-linker is substituted, or, alternatively, at other positions in the macrocycle precursor sequence. Cyclization of the precursor is then effected according to the specified method.

Analysis of

For example, the properties of the peptidomimetic macrocycles of the invention are analyzed using the methods described below. In some embodiments, the peptidomimetic macrocycles of the invention have improved biological properties relative to corresponding polypeptides lacking the substituents described herein.

Assay for determining alpha helicity

In solution, the secondary structure of a polypeptide having an alpha-helical domain reaches a dynamic equilibrium between a random coil structure and an alpha-helical structure, which is commonly referred to as "percent helicity". Thus, for example, the unmodified α -helical domain may be predominantly random coil in solution, with α -helix content typically below 25%. In another aspect, the peptidomimetic macrocycle with the optimized linker has, for example, at least 2-fold higher alpha helicity than the corresponding non-crosslinked polypeptide. In certain embodiments, the macrocycles of the invention have an alpha helicity of greater than 50%. To analyze the helicity of the peptidomimetic macrocycles of the invention, the compounds are dissolved in an aqueous solution (e.g., 50mM potassium phosphate solution at pH 7 or distilled water to a concentration of 25-50. mu.M). Circular Dichroism (CD) spectra were obtained on a spectropolarimeter (e.g., Jasco J-710) using standard measurement parameters (e.g., temperature, 20 ℃; wavelength, 190-. The alpha-helix content of each peptide was calculated by dividing the mean residue ellipticity (e.g., [ phi ]222obs) by the reported value for the model helical decapeptide (Yang et al (1986), Methods enzymol.130: 208).

Analysis for determination of melting temperature (Tm)

The peptidomimetic macrocycles of the invention containing a secondary structure (e.g., an alpha-helix) exhibit, for example, a higher melting temperature than the corresponding non-crosslinked polypeptides. In general, the peptidomimetic macrocycles of the invention exhibit a Tm of > 60 ℃, indicating a highly stable structure in aqueous solution. To analyze the effect of macrocycle formation on the melting temperature, the Tm is determined by dissolving the peptidomimetic macrocycle or unmodified peptide in distilled water (e.g., to a final concentration of 50 μ M) and measuring the change in ellipticity over a range of temperatures (e.g., 4-95 ℃) on a spectroscopic polarimeter (e.g., Jasco J-710) using standard parameters (e.g., wavelength, 222 nm; step resolution, 0.5 nm; speed, 20 nm/second; accumulation, 10; response, 1 second; bandwidth, 1 nm; temperature ramp rate, 1 ℃/minute; path length, 0.1 cm).

Protease resistance assay

The amide bond of the peptide backbone is susceptible to hydrolysis by proteases, thus rendering the peptidal compound susceptible to rapid degradation in vivo. However, formation of the peptide helix typically embeds the amide backbone, which can be protected from proteolytic cleavage. The peptidomimetic macrocycles of the invention can be subjected to proteolysis by trypsin in vitro to assess any change in the rate of degradation compared to the corresponding non-crosslinked polypeptides. For example, the peptidomimetic macrocycle and the corresponding non-crosslinked polypeptide are incubated with trypsin agarose and the reaction is terminated by centrifugation at various time points and subsequently HPLC injection is performed to quantify the residual substrate based on UV absorption at 280 nm. Briefly, the peptidomimetic macrocycle and peptidomimetic precursor (5mcg) were incubated with trypsin agarose (Pierce) (S/E-125) for 0, 10, 20, 90 and 180 minutes. Terminating the reaction by high-speed centrifugation through a desk centrifuge; the residual substrate in the separated supernatant was quantified by HPLC-based peak detection at 280 nm. The proteolytic reaction shows first order kinetics and the rate constant k is determined from the plot of ln S versus time (k ═ 1X slope).

In vitro stability assay

The peptidomimetic macrocycles with optimized linkers have, for example, an in vitro half-life that is at least 2-fold greater than a corresponding non-crosslinked polypeptide, and have an in vitro half-life of 12 hours or more. For in vitro serum stability studies, a variety of analytical methods can be used. For example, the peptidomimetic macrocycle and the corresponding non-crosslinked polypeptide (2mcg) are incubated with fresh mouse, rat, and/or human serum (2mL) at 37 ℃ for 0, 1, 2, 4,8, and 24 hours. To determine the content of intact compound, the following procedure can be used: samples were extracted by transferring 100. mu.l of serum to a 2ml centrifuge tube, followed by addition of 10. mu.l of 50% formic acid and 500. mu.l acetonitrile and centrifugation at 14,000RPM for 10 minutes at 4. + -. 2 ℃. The supernatant was then transferred to a new 2ml tube and incubated under N₂Evaporate at < 10psi, 37 ℃ on Turbovap. Samples were reconstituted in 100. mu.L of 50: 50 acetonitrile: water and analyzed by LC-MS/MS.

In vitro binding assays

To assess the binding and affinity of peptidomimetic macrocycles and peptidomimetic precursors to receptor proteins (receptor proteins), for example, Fluorescence Polarization Analysis (FPA) is used. The FPA technique measures molecular orientation and molecular mobility using polarized light and fluorescent tracers. When excited with polarized light, fluorescent tracers (e.g., FITC) attached to molecules with high apparent molecular weights (e.g., FITC-labeled peptides bound to large proteins) emit higher levels of polarized fluorescence due to their slower rotational velocity compared to fluorescent tracers attached to smaller molecules (e.g., FITC-labeled peptides free in solution).

For example, a fluorescently labeled (fluorosylated) peptidomimetic macrocycle (25nM) is incubated with the receptor protein (25-1000nM) in binding buffer (140mM NaCl, 50mM Tris-HCL, pH 7.4) for 30 minutes at room temperature. Binding activity is measured, for example, by fluorescence polarization using a luminescence spectrophotometer (e.g., Perkin-Elmer LS 50B). Kd values can be determined by nonlinear regression analysis using, for example, Graphpad Prism Software (Graphpad Software, inc., San Diego, CA). In certain instances, the peptidomimetic macrocycles of the invention exhibit Kd's that are similar to or lower than the Kd's of the corresponding non-crosslinked polypeptide.

In vitro displacement assay to identify antagonists of peptide-protein interactions

To evaluate the binding and affinity of compounds that antagonize the interaction between the peptide and the receptor protein, for example, Fluorescence Polarization Assays (FPAs) using fluorescently labeled peptidomimetic macrocycles derived from a peptidomimetic precursor sequence are used. The FPA technique measures molecular orientation and molecular mobility using polarized light and fluorescent tracers. When excited with polarized light, fluorescent tracers (e.g., FITC) attached to molecules with high apparent molecular weights (e.g., FITC-labeled peptides bound to large proteins) emit higher levels of polarized fluorescence due to their lower rotational velocity compared to fluorescent tracers attached to smaller molecules (e.g., FITC-labeled peptides free in solution). Compounds that antagonize the interaction between a fluorescently labeled peptidomimetic macrocycle and a receptor protein are detected in a competitive binding FPA assay.

For example, putative antagonist compounds (1nM to 1mM) and fluorescently labeled peptidomimetic macrocycles (25nM) were incubated with receptor proteins (50nM) in binding buffer (140mM NaCl, 50mM Tris-HCL, pH 7.4) for 30 minutes at room temperature. For example, the binding activity of the antagonist is measured by fluorescence polarization using a luminescence spectrophotometer (e.g., Perkin-Elmer LS 50B). Kd values are determined by nonlinear regression analysis using, for example, Graphpad Prism Software (Graphpad Software, inc., San Diego, CA).

Any type of molecule (e.g., small organic molecule, peptide, oligonucleotide, or protein) can be tested as a putative antagonist in this assay.

Binding assays in intact cells

It is possible to measure the binding of a peptide or peptidomimetic macrocycle to its native receptor in intact cells by immunoprecipitation experiments. For example, intact cells are incubated with fluorescently labeled (FITC-labeled) compounds for 4 hours in the absence of serum, followed by serum replacement and further incubation for 4-18 hours. The cells were then pelleted and incubated in lysis buffer (50mM Tris [ pH 7.6], 150mM NaCl, 1% CHAPS and protease inhibitor cocktail) for 10 minutes at 4 ℃. The extract was centrifuged at 14,000rpm for 15 minutes, the supernatant was collected and incubated with 10. mu.l goat anti-FITC antibody for 2 hours, spun at 4 ℃ and further incubated with protein A/G Sepharose (50. mu.l of 50% microsphere slurry) for 2 hours at 4 ℃. After rapid centrifugation, the pellet is washed in lysis buffer containing increasing salt concentrations (e.g., 150, 300, 500 mM). The microspheres were then re-equilibrated with 150mM NaCl before adding SDS-containing sample buffer and boiling. After centrifugation, the supernatant is optionally electrophoresed using a 4% -12% gradient of Bis-Tris gel, followed by transfer to Immobilon-P membrane. Following blocking, the blot is optionally incubated with an antibody that detects FITC, and also with one or more antibodies that detect proteins bound to the peptidomimetic macrocycle.

Cell permeability analysis

The peptidomimetic macrocycles, for example, have better cell permeability than the corresponding non-crosslinked macrocycles. The peptidomimetic macrocycles with optimized linkers have, for example, a cell permeability at least 2-fold higher than the corresponding non-crosslinked macrocycles, and typically 20% or more of the applied peptidomimetic macrocycles have been observed to penetrate into the cells after 4 hours. To measure the cell permeability of the peptidomimetic macrocycle and the corresponding non-crosslinked macrocycle, intact cells were incubated with the fluorescently labeled peptidomimetic macrocycle or the corresponding non-crosslinked macrocycle (10 μ M) in serum-free medium for 4 hours at 37 ℃, washed 2 times with medium, and incubated with trypsin (0.25%) for 10 minutes at 37 ℃.The cells were washed again and resuspended in PBS. For example, by using a FACSCalibur flow cytometer or Cellomics' KineticScanThe HCS reader analyzes cell fluorescence.

Cell potency assay

In cell-based assays, the efficacy of certain peptidomimetic macrocycles is determined using a variety of human and mouse cell lines and primary cells derived from human or mouse cell populations. Several assays can be used to assess anti-inflammatory effects, and an osteoclastogenesis assay is listed as an example. Inhibition of osteoblast differentiation stimulated by basal (RANKL) and M-CSF was monitored as a decrease in TRAP-positive osteoblasts due to incubation with peptidomimetic macrocycles (0.5-50 μ M) to determine those peptidomimetic macrocycles with EC50 < 10 μ M. Briefly, mouse bone marrow cells seeded in 48-well cell culture plates were incubated with RANKL (100ng/ml) with or without human macrophage colony stimulating factor (M-CSF; 20ng/ml) for 4 days in the presence or absence of various concentrations of peptidomimetic macrocycles (0.5-50 μ M). The cells were then fixed and stained for the osteoclast phenotype marker tartrate-resistant acid phosphatase (TRAP), TRAP-positive multinucleated cells containing more than 3 nuclei were scored as osteoclasts (oesteoclast). Several standard assays for measuring TNF-or LPS-stimulated cytokine release are commercially available and are optionally used to assess the efficacy of the peptidomimetic macrocycles. In addition, assays for measuring the translocation of NF-. kappa.Bp 65 from the cytoplasm to the nucleus and the NF-. kappa.B-dependent transcriptional activity of luciferase reporter assays can be used to assess whether the peptidomimetic macrocycles affect cells by targeting the NF-. kappa.B mechanism.

In vivo stability assay

To study the in vivo stability of peptidomimetic macrocycles, for example, the compounds are administered to mice and/or rats by the IV, IP, PO or inhalation routes at concentrations of 0.1-50mg/kg and blood samples are drawn at 0 ', 5', 15 ', 30', 1 hour, 4 hours, 8 hours and 24 hours post-injection. The content of intact compounds in 25 μ L of fresh serum was then measured by LC-MS/MS as above.

In vivo efficacy in animal models

To determine the anti-inflammatory activity of the peptidomimetic macrocycles of the invention in vivo, for example, the compounds are administered alone (IP, IV, PO, by inhalation or nasal routes) or in combination with sub-optimal doses of the relevant anti-inflammatory drugs (e.g., dexamethasone). In one embodiment, an animal model of rheumatoid arthritis (i.e., collagen-induced arthritis (CIA)) is used. Briefly, male DBA/1J mice were sensitized with 200 μ g of collagen type II in Freund's complete adjuvant by s.c injection at the tail root. 21 days after the initial immunization, mice were fortified with type II collagen (200 μ g) in Freund's complete adjuvant (s.c). Treatment of the peptidomimetic macrocycle alone or in combination with suboptimal doses of the relevant anti-inflammatory drug is initiated with a second immunization and the peptidomimetic macrocycle is administered daily via tail vein IV or daily via IP route at a dose ranging from 0.1mg/kg to 50mg/kg until 40 days after the first immunization. Mice were analyzed every other day by two independent blindness (blinder) testers and monitored for signs of arthritis using the following clinical parameters: paw swelling and clinical score. For histological analysis, mice were sacrificed by cervical dislocation at day 45 post immunization, and the hindpaws from 5-9 mice were randomly collected by two independent experimenters and stained with TRAP or H & E. Commercially available kits were used to detect cytokine levels (TNF-. alpha., IL-1. beta. and IL-10) in serum and joint samples prepared 35 days after the initial immunization. Anti-type II collagen IgG was measured on the same serum samples using ELISA. These in vivo tests optionally yield preliminary pharmacokinetic, pharmacodynamic and toxicology data.

Clinical trial

To determine the suitability of the peptidomimetic macrocycles of the invention for human therapy, clinical trials have been conducted. For example, patients diagnosed with rheumatoid arthritis, inflammatory bone resorption, inflammatory bowel disease, asthma or multiple sclerosis in need of treatment are selected and divided into a treatment group to which a peptidomimetic macrocycle of the invention is administered and one or more control groups that receive a placebo or a known anti-inflammatory drug. Thus, the safety and efficacy of treatment with the peptidomimetic macrocycles of the invention can be evaluated by comparing patient groups for factors such as clinical scores for inflammation. In this example, the group of patients treated with the peptidomimetic macrocycle shows a reduced inflammatory parameter/clinical score compared to a placebo-treated control group of patients.

Pharmaceutical compositions and routes of administration

The peptidomimetic macrocycles of the invention also include pharmaceutically acceptable derivatives or prodrugs thereof. "pharmaceutically acceptable derivative" refers to any pharmaceutically acceptable salt, ester, salt of an ester, prodrug, or other derivative of a compound of the invention that, upon administration to a recipient, is capable of providing (directly or indirectly) a compound of the invention. Particularly advantageous pharmaceutically acceptable derivatives may enhance the bioavailability of the compounds of the invention when administered to a mammal (e.g., by enhancing absorption of an orally administered compound into the blood), or increase delivery of the active compound to a biological compartment (e.g., the brain or lymphatic system) relative to the parent species. Some pharmaceutically acceptable derivatives contain chemical groups that enhance water solubility or active transport across the gastrointestinal mucosa.

In certain embodiments, the peptidomimetic macrocycles of the invention are modified by suitable functional groups attached, covalently or non-covalently, to enhance selective biological properties. Such modifications include those that increase biological permeability into specific biological compartments (e.g., blood, lymphatic system, central nervous system), increase oral availability, increase solubility to allow administration by injection, alter metabolism, and alter rate of excretion.

The inventionPharmaceutically acceptable salts of the compounds of (a) include those derived from pharmaceutically acceptable inorganic and organic acids and bases. Examples of suitable acid salts include acetate, adipate, benzoate, benzenesulfonate, butyrate, citrate, digluconate, dodecylsulfate, formate, fumarate, glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, lactate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, palmitate (palmoate), phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate, tartrate, tosylate, and undecanoate (uncanoate). Salts derived from suitable bases include alkali metal salts (e.g., sodium salts), alkaline earth metal salts (e.g., magnesium salts), ammonium salts, and N- (alkyl)₄ ⁺And (3) salt.

For preparing pharmaceutical compositions from the compounds of the invention, pharmaceutically acceptable carriers include solid or liquid carriers. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. A solid carrier can be one or more substances which may also act as diluents, flavoring agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material. Details of formulation and administration techniques are described in detail in the scientific and patent literature, see, e.g., the latest versions of Remington's Pharmaceutical Sciences, Maack Publishing Co, Easton PA.

In powders, the carrier is a finely divided solid which is in admixture with the finely divided active ingredient. In tablets, the active ingredient is mixed with the carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired.

Suitable solid excipients are carbohydrate or protein fillers including, but not limited to: sugars, including lactose, sucrose, mannitol, or sorbitol; starches from corn, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxypropyl methylcellulose, or sodium carboxymethyl cellulose; and gums including gum arabic and tragacanth; and proteins such as gelatin and collagen. If desired, disintegrating or solubilizing agents are added, such as cross-linked polyvinylpyrrolidone, agar, alginic acid or a salt thereof (e.g., sodium alginate).

Liquid form preparations include solutions, suspensions and emulsions, for example, water or water/propylene glycol solutions. For parenteral injection, liquid preparations may be formulated as solutions in aqueous polyethylene glycol solutions.

The pharmaceutical preparation is preferably in unit dosage form. In such forms, the preparation is subdivided into unit doses containing appropriate quantities of the active ingredient. The unit dosage form may be a packaged preparation, the package containing discrete quantities of the preparation, such as packeted tablets, capsules, and powders in vials or ampoules. In addition, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or any of the same in the form of an appropriate number of packages.

When the compositions of the present invention comprise a peptidomimetic macrocycle in combination with one or more additional therapeutic or prophylactic agents, both the compound and the additional agent should be present at dosage levels of about 1-100%, more preferably about 5-95%, of the dosages typically administered in a monotherapy regimen. In certain embodiments, the additional agent is administered separately from the compound of the invention as part of a multiple dose regimen. Alternatively, these agents are part of a single dosage form, mixed together with the compounds of the present invention in a single composition.

Application method

In one aspect, the invention provides novel peptidomimetic macrocycles that can be used in competitive binding assays to identify substances that bind to the natural ligands of proteins or peptides that the peptidomimetic macrocycles mimic. For example, in the IKK β/NEMO system, IKK β -based labeled peptidomimetic macrocycles are used in NEMO binding assays with small molecules that compete for binding to NEMO. Competitive binding studies allow rapid evaluation and determination of drug candidates specific for the IKK β/NEMO system in vitro. Such binding studies can be performed using any of the peptidomimetic macrocycles disclosed herein and their binding partners.

The invention further provides for the generation of antibodies against the peptidomimetic macrocycles. In certain embodiments, these antibodies specifically bind to peptidomimetic macrocycles and peptidomimetic macrocycle-related precursor peptides such as IKK β. For example, such antibodies disrupt natural protein-protein interactions, such as the binding between IKK β and NEMO.

In other aspects, the invention provides prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disease associated with aberrant (e.g., insufficient or excessive) expression or activity of a molecule, including NFkB.

In another embodiment, the disease is caused, at least in part, by an abnormal level (e.g., over-or under-expression) of NFkB, or by the presence of NFkB exhibiting abnormal activity. Thus, a decrease in the level and/or activity of NFkB or an increase in the level and/or activity of NFkB resulting from IKK β derived peptidomimetic macrocycles for binding NEMO is used, for example, to alleviate or mitigate the negative symptoms of a disease.

In another aspect, the invention provides methods of treating or preventing diseases, including hyperproliferative diseases and inflammatory conditions, by interfering with the interaction or binding between binding partners (e.g., between IKK β and NEMO). These methods comprise administering to a warm-blooded animal, including man, an effective amount of a compound of the present invention. In certain embodiments, administration of a compound of the invention induces cell growth arrest or apoptosis.

The term "treating" as used herein is defined as applying or administering a therapeutic agent to a patient, or to a tissue or cell line isolated from a patient, who has a disease, disease symptoms, or a predisposition toward a disease, with the goal of curing, restoring, alleviating, relieving, altering, correcting, alleviating, ameliorating, or affecting the disease, disease symptoms, or predisposition toward a disease.

In certain embodiments, the peptidomimetic macrocycles of the invention are used to treat, prevent and/or diagnose cancer and neoplastic disorders. As used herein, the terms "cancer," "hyperproliferative," and "neoplastic" refer to cells that have the ability to grow spontaneously, i.e., an abnormal state or disease characterized by rapidly proliferating cell growth. Hyperproliferative and neoplastic disease states can be classified as pathological, i.e., representing or constituting a disease state; or may be classified as non-pathological, i.e., deviation from normal but not associated with a disease state. The term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues or organs, regardless of histopathological type or stage of invasion. Metastatic tumors can arise from a number of primary tumor types, including but not limited to: tumor types of breast, lung, liver, colon and ovarian origin. "pathologically hyperproliferative" cells occur in disease states characterized by malignant tumor growth. Examples of non-pathological hyperproliferative cells include cell proliferation associated with wound repair. Examples of cell proliferative and/or differentiative disorders include cancer, e.g., carcinoma, sarcoma, or metastatic disease. In certain embodiments, the peptidomimetic macrocycles are novel therapeutic agents for controlling breast cancer, ovarian cancer, colon cancer, lung cancer, metastases of these cancers, and the like.

Examples of cancer or neoplastic disorders include, but are not limited to: fibrosarcoma, myosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, gastric cancer, esophageal cancer, rectal cancer, pancreatic cancer, ovarian cancer, prostate cancer, uterine cancer, head and neck cancer, skin cancer, brain cancer, squamous cell cancer, sebaceous gland cancer, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary cancer, bronchial cancer, renal cell cancer, hepatoma, bile duct cancer, choriocarcinoma, seminoma, embryonal cancer, Wilms' tumor, cervical cancer, testicular cancer, small cell lung cancer, non-small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, neuroblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, leukemia, lymphoma or kaposi's sarcoma.

Examples of proliferative diseases include neoplastic diseases of the hematopoietic system. As used herein, the term "hematopoietic neoplastic disease" includes diseases involving hyperplastic/neoplastic cells of hematopoietic origin (e.g., derived from myeloid, lymphoid or erythroid lineages) or their precursor cells. Preferably, the disease results from poorly differentiated acute leukemias, e.g., erythroblastic leukemia and acute megakaryoblastic leukemia. Additional exemplary bone marrow diseases include, but are not limited to: acute promyelocytic leukemia (APML), Acute Myelogenous Leukemia (AML) and Chronic Myelogenous Leukemia (CML) (reviewed in Vaickus (1991), Crit Rev. Oncol./Hemotol. 11: 267-97); lymphoid malignancies include, but are not limited to, Acute Lymphocytic Leukemia (ALL), including B-lineage ALL and T-lineage ALL, Chronic Lymphocytic Leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HLL), and Waldenstrom's Macroglobulinemia (WM). Other forms of malignant lymphoma include, but are not limited to: non-Hodgkin's lymphoma and variants thereof, peripheral T-cell lymphoma, adult T-cell leukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), large granular lymphocytic Leukemia (LGF), Hodgkin's disease, and Reed-Stenberg disease.

Examples of cell proliferation and/or differentiation disorders of the breast include, but are not limited to: proliferative breast diseases including, for example, epithelial cell hyperplasia, sclerosing adenopathy, and tubulocytoma; tumors, for example, stromal tumors such as fibroadenoma, phyllodes, and sarcomas, and epithelial tumors such as large ductal papillomas; carcinomas of the breast, including carcinoma in situ (non-invasive) including ductal carcinoma in situ (including paget's disease) and lobular carcinoma in situ) and invasive (invasive) carcinomas (including, but not limited to, invasive ductal carcinoma, invasive lobular carcinoma, medullary carcinoma, glue-like (mucus) carcinoma, tubular carcinoma, and invasive papillary carcinoma); and mixed malignancies. Gynecomastia includes, but is not limited to, gynecomastia and cancer.

Examples of cellular proliferative and/or differentiative disorders of the lung include, but are not limited to: bronchial cancer, including paraneoplastic syndrome, bronchioloalveolar carcinoma, neuroendocrine tumors, e.g., bronchial carcinoid tumors, miscellaneous tumors, and metastatic tumors; pathologies of the pleura include inflammatory pleural effusions, noninflammatory pleural effusions, pneumothorax, and pleural tumors, including solitary fibrous tumors (pleural fibroma) and malignant mesothelioma.

Examples of cellular proliferative and/or differentiative disorders of the colon include, but are not limited to: non-neoplastic polyps, adenomas, familial syndromes, colorectal carcinogenesis, colorectal cancer and carcinoid tumors.

Examples of cellular proliferative and/or differentiative disorders of the liver include, but are not limited to: nodular hyperplasia, adenomas and malignancies, including primary and metastatic tumors of the liver.

Examples of cellular proliferative and/or differentiative disorders of the ovary include, but are not limited to: ovarian tumors, e.g., coelomic epithelial tumors, serous tumors, myxoma, endometrioid tumors, clear cell adenocarcinoma, cystic fibroma, brenner's tumor, superficial epithelial tumors; germ cell tumors, e.g., mature (benign) teratomas, single germ layer teratomas, immature malignant teratomas, asexual cell tumors, endoblastoma, choriocarcinoma; cord-matrix tumors (sex cord-stromal tumors), such as, for example, granulosa-theca cell tumors, theca cell fibroids (thecomafilomas), andropatomas (androbastomas), hilt cell tumors (hill cell tumors), and gonadal cell tumors; and metastatic tumors such as Klukenberg tumor.

In other or further embodiments, the peptidomimetic macrocycles described herein are used for the treatment, prevention, or diagnosis of conditions characterized by overactive cell death or cell death due to physiological damage, and the like. Some examples of conditions characterized by premature or undesirable cell death or unwanted or excessive cell proliferation include, but are not limited to, cytoreductive/cytoreductive, acellular/aplastic or hypercellular/hyperplastic conditions. Some examples include hematological disorders including, but not limited to, fanconi anemia, aplastic anemia, thalassemia, congenital neutropenia, and myelodysplasia.

In other or further embodiments, the peptidomimetic macrocycles of the invention that function to reduce apoptosis are useful for treating conditions associated with undesirable levels of cell death. Thus, in certain embodiments, the anti-apoptotic peptidomimetic macrocycles of the invention are used to treat conditions that lead to cell death, such as those associated with viral infections (e.g., infections associated with Human Immunodeficiency Virus (HIV) infections). Many neurological diseases are characterized by the gradual loss of a particular set of neurons. One example is Alzheimer's Disease (AD). Alzheimer's disease is characterized by neuronal and synaptic loss in the cerebral cortex and certain subcortical regions. This loss results in total atrophy of the affected area. Amyloid plaques and neurofibrillary tangles are visible in the brain of patients with AD. Alzheimer's disease has been identified as a disease of protein misfolding due to the accumulation of aberrantly folded A-beta and tau proteins in the brain. Plaques are composed of beta-amyloid. Beta-amyloid is a fragment from a larger protein called Amyloid Precursor Protein (APP). APP is critical for nerve growth, survival and post-injury repair. In AD, an unknown process results in proteolytic cleavage of APP into smaller fragments by enzymes. One of these fragments is the fibrils of beta-amyloid, which form masses (called senile plaques) that deposit by formation of an outer matrix outside the neuron. Plaque continues to grow into insoluble twisted fibers within the nerve cells, commonly referred to as tangles. Thus, disruption of the interaction between β -amyloid and its original receptor is important in the treatment of AD. In some embodiments, the anti-apoptotic peptidomimetic macrocycles of the invention are used in the treatment of AD and other neurological diseases associated with apoptosis. These neurological disorders include alzheimer's disease, parkinson's disease, Amyotrophic Lateral Sclerosis (ALS), retinitis pigmentosa, spinal muscular atrophy, and various forms of cerebellar degeneration. Cell loss in these diseases does not trigger an inflammatory response, and apoptosis appears as a mechanism of cell death.

In addition, many hematological diseases are associated with a decrease in blood cell production. These disorders include anemia associated with chronic disease, aplastic anemia, chronic neutropenia, and myelodysplastic syndromes. Disorders of blood cell production (such as myelodysplastic syndrome and certain forms of aplastic anemia) are associated with increased apoptotic cell death in the bone marrow. These disorders may result from gene activation that promotes apoptosis, acquired defects in stromal cells or hematopoietic survival factors, or direct action of toxins and mediators of immune response. Two common conditions associated with cell death are myocardial infarction and stroke. In both conditions, cells in the central region of ischemia (generated in the event of acute loss of blood flow) appear to die rapidly due to necrosis. However, outside the ischemic central region, cells die over a longer period of time and morphologically appear to die due to apoptosis. In other or further embodiments, the anti-apoptotic peptidomimetic macrocycles of the invention are useful for treating all conditions associated with unwanted cell death.

Some examples of neurological disorders treated with the peptidomimetic macrocycles described herein include, but are not limited to, Alzheimer's disease, Down's Syndrome, amyloidosis with hereditary cerebral hemorrhage of the Dutch type, reactive amyloidosis, familial amyloid nephropathy with urticaria and deafness, Mukle-Wells Syndrome (Muckle-Wells Syndrome), idiopathic myeloma; macroglobulinemia-associated myeloma, Familial Amyloid Polyneuropathy, Familial Amyloid cardiomyopathy, Isolated Cardiac Amyloid (Isolated Cardiac Amyloid), systemic senile amyloidosis, adult onset diabetes mellitus, insulinoma, Isolated anterior chamber Amyloid (Isolated Amyloid), medullary carcinoma of the thyroid gland, Familial amyloidosis, hereditary cerebral hemorrhage with amyloidosis, Familial amyloidosis Polyneuropathy (Familial Amyloid Polyneuropathy), scrapie, Creutzfeldt-Jacob Disease (Creutzfeldt-Jacob Disease), Getmrsan-Straussler-Scheinker syndrome, bovine spongiform encephalopathy, prion-mediated diseases, and Huntington's Disease.

In another embodiment, the peptidomimetic macrocycles described herein are used for the treatment, prevention or diagnosis of an inflammatory disorder. There are various types of inflammatory disorders. Certain inflammatory diseases are associated with the immune system, such as autoimmune diseases. Autoimmune diseases result from an overactive immune response of the body to substances and tissues (i.e., self-antigens) that are normally present in the body. In other words, the immune system attacks its own cells. Autoimmune diseases are the major cause of immune-mediated diseases. Rheumatoid arthritis is an autoimmune disease in which the immune system attacks the joints, in which case it causes inflammation (e.g., arthritis) and destruction. It can also damage some organs such as the lungs and skin. Rheumatoid arthritis can result in a significant loss of function and mobility. Rheumatoid arthritis is diagnosed by blood tests, especially rheumatoid factor tests. Some examples of autoimmune diseases treated with the peptidomimetic macrocycles described herein include, but are not limited to: acute Disseminated Encephalomyelitis (ADEM), Addison's disease, ankylosing spondylitis, antiphospholipid antibody syndrome (APS), autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, Behcet's disease, bullous pemphigoid, chylemia, Chagas's disease, Churg-Strauss syndrome, Chronic Obstructive Pulmonary Disease (COPD), Crohn's disease, dermatomyositis, type 1 diabetes, endometriosis, Goodpasture's syndrome, Graves ' disease, Guillain Barre Syndrome (GBS), Hashimoto's disease, hidradenitis suppurativa, idiopathic thrombocytopenic purpura, Inflammatory Bowel Disease (IBD), interstitial cystitis, lupus erythematosus, hard spots, multiple sclerosis, myasthenia gravis, lethargy, neuromyotonia, pemphigus vulgaris, pernicious anemia, polymyositis, polymyalgia rheumatica, primary biliary cirrhosis, Psoriasis, rheumatoid arthritis, schizophrenia, scleroderma, sjogren's syndrome, temporal arteritis (also known as "giant cell arteritis"), takayasu's arteritis, vasculitis, vitiligo and wegener's granulomatosis.

Some examples of other types of inflammatory conditions that are treated with the peptidomimetic macrocycles described herein include, but are not limited to: allergies, including allergic rhinitis/sinusitis, skin allergies (urticaria/hives, angioedema, allergic dermatitis), food allergies, drug allergies, insect allergies; and rare allergic conditions such as mastocytosis, asthma, arthritis including osteoarthritis, rheumatoid arthritis and spondyloarthropathies, primary vasculitis of the central nervous system, sarcoidosis, organ transplant rejection, fibromyalgia, fibrosis, pancreatitis and pelvic inflammatory disease.

Some examples of cardiovascular disorders (e.g., inflammatory disorders) treated or prevented with the peptidomimetic macrocycles described herein include, but are not limited to, aortic valve stenosis, atherosclerosis, myocardial infarction, stroke, thrombosis, aneurysm, heart failure, ischemic heart disease, angina, sudden cardiac death, hypertensive heart disease; non-coronary vascular diseases such as arteriosclerotic disease, small vessel disease, renal disease, hypertriglyceridemia, hypercholesterolemia, hyperlipidemia, xanthoma, asthma, hypertension, emphysema and chronic lung disease; or cardiovascular disorders associated with interventional procedures ("procedural vascular trauma"), such as restenosis following angioplasty and placement of shunts, stents, synthetic or natural resected grafts, indwelling catheters, valves or other implantable devices. Preferred cardiovascular diseases include atherosclerosis, myocardial infarction, aneurysm and stroke.

Example 1 design of the Peptidomimetic macrocycle of formula (I)

Figures 1 and 2 show a possible binding pattern of PAKKSEELVAEAHNLCTLLENAIQDTVREQ (which represents residues 701 to 730 of the NBD α helix of IKK β) to NEMO of the wild type sequence fragment peptide. The peptidomimetic macrocycles of the invention are prepared starting from the corresponding uncrosslinked polypeptide sequence PAKKSEELVAEAHNLCTLLENAIQDTVREQ and substituting the 7 th and 11 th amino acids with α, α -disubstituted amino acids (e.g., S5 alkene amino acids). An alkene metathesis reaction is performed, thereby producing a peptidomimetic macrocycle comprising i to i +4 crosslinks.

Example 2 Synthesis of a Peptidomimetic macrocycle of formula (I)

Alpha-helix cross-linked polypeptides were synthesized, purified and analyzed as previously described (Schaffeister et al (2000), J.Am.chem.Soc.122: 5891-5892; Walensky et al (2004) Science 305: 1466-70; Walensky et al (2006) Mol Cell 24: 199-210) and as shown below. The following macrocyclic compounds derived from the human IKK β peptide sequence were used in this study:

in the above sequence, Nle represents norleucine, Aib represents 2-aminoisobutyric acid, Abu represents (S) -2-aminobutanoic acid, Ac represents an N-terminal acetyl group, and NH2 represents a C-terminal amino group. The amino acid represented by $ is (S) -alpha- (2 '-pentenyl) alanine ("S5-enamino acid") and the amino acid represented by $ R8 is (R) -alpha- (2' -octenyl) alanine ("R8 enamino acid"). After incorporation of such amino acids into the precursor polypeptide, the terminal alkene moiety reacts with the metathesis catalyst, resulting in the formation of the peptidomimetic macrocycle. A macrocycle connecting two amino acids has an all-carbon cross-linker comprising 8 carbon atoms between the alpha carbon atoms of each amino acid and 1 double bond between the fourth and fifth carbon atoms, and wherein each alpha-carbon atom to which the cross-linker connects is additionally substituted with a methyl group. The macrocycle connecting one $ r8 amino acid and one $ amino acid has an all-carbon cross-linker comprising 11 carbon atoms between the alpha carbon atoms of each amino acid and 1 double bond between the seventh and eighth carbon atoms, and wherein each alpha-carbon atom to which the cross-linker is attached is additionally substituted with a methyl group. If no metathesis reaction is performed, the olefinic amino acids in the resulting polypeptide are labeled $/and $ R8/, indicating unmodified polypeptides comprising unmodified (S) - α - (2 '-pentenyl) alanine ("S5-olefinic amino acid") or unmodified (R) - α - (2' -octenyl) alanine, respectively. Predicted and measured m/z spectra are provided.

The α, α -disubstituted amino acids and amino acid precursors disclosed in the cited references can be used in the synthesis of peptidomimetic macrocycle precursor polypeptides. According to Williams et al (1991) J.am.chem.Soc.113: 9276; and Schaffeister et al (2000) J.am.chem Soc.122: 5891 the method synthesizes an alpha, alpha-disubstituted unnatural amino acid containing olefinic side chains. The cross-linked polypeptides were designed by substituting the corresponding synthetic amino acids for the 2 natural amino acids (see above). Permutations are made at i and i +4 bits and at i and i +7 bits.

Unnatural amino acids (R and S enantiomers of 5-carbon olefinic amino acids and S enantiomer of 8-carbon olefinic amino acids) were identified by Nuclear Magnetic Resonance (NMR) spectroscopy (Varian Mercury 400) and mass spectrometry (Micromass LCT). Peptide synthesis was performed manually or on an automated peptide synthesizer (Applied Biosystems, model 433A) using solid phase conditions, rink amide AM resin (Novabiochem) and Fmoc backbone protecting group chemistry. For the coupling of the native Fmoc-protected amino acid (Novabiochem), 10 equivalents of amino acid and 1: 2 molar ratio of coupling reagents HBTU/HOBt (Novabiochem)/DIEA were used. Unnatural amino acids (4 equiv.) were coupled using a 1: 2 molar ratio of HATU (applied biosystems)/HOBt/DIEA. In the solid phase, olefin metathesis was carried out using 10mM Grubbs catalyst (Blackewell et al 1994, supra) (Materia) in degassed dichloromethane and reacted at room temperature for 2 hours. Isolation of the metathesized compound was achieved by trifluoroacetic acid mediated deprotection and cleavage, yielding the crude product by ether precipitation, and high performance liquid phase (HPLC) on reverse phase C18 column (Varian prosar) to yield the pure compound. The chemical composition of the pure product was confirmed by LC/MS mass spectrometry (Micromass LCT, coupled to Agilent 1100 HPLC system) and amino acid analysis (Applied Biosystems, model 420A).

While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims (15)

1. A peptidomimetic macrocycle comprising an amino acid sequence which is at least about 60% identical to an amino acid sequence selected from the group consisting of the amino acid sequences in Table 1.

2. The peptidomimetic macrocycle of claim 1, wherein the amino acid sequence of said peptidomimetic macrocycle is at least about 80% identical to an amino acid sequence chosen from the group consisting of the amino acid sequences in Table 1.

3. The peptidomimetic macrocycle of claim 1, wherein the amino acid sequence of said peptidomimetic macrocycle is at least about 90% identical to an amino acid sequence chosen from the group consisting of the amino acid sequences in Table 1.

4. The peptidomimetic macrocycle of claim 1, wherein the amino acid sequence of said peptidomimetic macrocycle is selected from the group consisting of the amino acid sequences in Table 1.

5. The peptidomimetic macrocycle of claim 1, wherein said peptidomimetic macrocycle comprises a helix.

6. The peptidomimetic macrocycle of claim 1, wherein said peptidomimetic macrocycle comprises an alpha-helix.

7. The peptidomimetic macrocycle of claim 1, wherein said peptidomimetic macrocycle comprises an α, α -disubstituted amino acid.

8. The peptidomimetic macrocycle of claim 1, wherein said peptidomimetic macrocycle comprises a crosslinker linking the alpha-positions of at least two amino acids.

9. The peptidomimetic macrocycle of claim 8, wherein at least one of said two amino acids is an α, α -disubstituted amino acid.

10. The peptidomimetic macrocycle of claim 8, wherein the peptidomimetic macrocycle has the formula:

wherein:

A. c, D and E are each independently a natural or unnatural amino acid;

b is natural or non-natural amino acid, amino acid analogue,[-NH-L₃-CO-]、[-NH-L₃-SO₂-]Or [ -NH-L ]₃-]；

R₁And R₂independently-H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-;

R₃is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl or heterocycloaryl, optionally substituted with R₅Substitution;

l is of the formula-L₁-L₂-a macrocycle-forming linker;

L₁and L₂Independently is alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene, heterocycloarylene, or [ -R ]₄-K-R₄-]_nEach optionally substituted by R₅Substitution;

each R is₄Is alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;

each K is O, S, SO₂、CO、CO₂Or CONR₃；

Each R is₅Independently halogen, alkyl, -OR₆、-N(R₆)₂、-SR₆、-SOR₆、-SO₂R₆、-CO₂R₆A fluorescent moiety, a radioisotope or a therapeutic agent;

each R is₆independently-H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope, or a therapeutic agent;

R₇is-H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycleAlkyl, cycloaryl or heterocycloaryl, optionally substituted by R₅Substituted or part of a cyclic structure with a D residue;

R₈is-H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R₅Substituted or part of a cyclic structure with an E residue;

v and w are independently integers from 1 to 1000;

u, x, y and z are independently integers from 0 to 10; and

n is an integer of 1 to 5.

11. The peptidomimetic macrocycle of claim 1, wherein said peptidomimetic macrocycle comprises a linker connecting a backbone amino group of a first amino acid to a second amino acid in the peptidomimetic macrocycle.

12. The peptidomimetic macrocycle of claim 11, wherein said peptidomimetic macrocycle has formula (IV) or (IVa):

wherein:

A. c, D and E are each independently a natural or unnatural amino acid;

b is natural or non-natural amino acid, amino acid analogue,[-NH-L₃-CO-]、[-NH-L₃-SO₂-]Or [ -NH-L ]₃-]；

R₁And R₂independently-H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halogen, or a member of a cyclic structure with the E residueA moiety;

R₃is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl or heterocycloaryl, optionally substituted with R₅Substitution;

L₁and L₂Independently is alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene, heterocycloarylene, or [ -R ]₄-K-R₄-]_nEach optionally substituted by R₅Substitution;

each R is₄Is alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;

each K is O, S, SO₂、CO、CO₂Or CONR₃；

Each R is₅Independently halogen, alkyl, -OR₆、-N(R₆)₂、-SR₆、-SOR₆、-SO₂R₆、-CO₂R₆A fluorescent moiety, a radioisotope or a therapeutic agent;

each R is₆independently-H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope, or a therapeutic agent;

R₇is-H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R₅Substitution;

v and w are independently integers from 1 to 1000;

u, x, y and z are independently integers from 0 to 10; and

n is an integer of 1 to 5.

13. A method of treating an inflammatory disease in a subject comprising administering to the subject a peptidomimetic macrocycle of claim 1.

14. A method of modulating NEMO activity in a subject comprising administering to the subject a peptidomimetic macrocycle of claim 1.

15. A method of antagonizing the interaction between IKK β and NEMO in a subject comprising administering to the subject a peptidomimetic macrocycle of claim 1.