# Patent

## Title
Oligooxopiperazines and methods of making and using them

## Abstract

The present invention relates to oligooxopiperazines and their use. Methods for preparing oligooxopiperazines are also disclosed.

## Background
This application is a continuation of U.S. patent application Ser. No. 12/917,176, filed Nov. 1, 2010, which claims the priority benefit of U.S. Provisional Patent Application Ser. No. 61/373,108, filed Aug. 12, 2010, which are hereby incorporated by reference in their entirety.


This invention was made with government support under National Science Foundation grant number CHE-0848410. The government has certain rights in this invention.


FIELD OF THE INVENTION
This invention is directed generally to oligooxopiperazines and methods for preparing oligooxopiperazines from amino acids.
BACKGROUND OF THE INVENTION
A fundamental limitation of current drug development centers on the inability of traditional pharmaceuticals to target spatially extended protein interfaces. The majority of modern pharmaceuticals are small molecules that target enzymes or protein receptors with defined pockets. However, in general they cannot target protein-protein interactions involving large contact areas with the required specificity. Examination of complexes of proteins with other biomolecules reveals that proteins tend to interact with partners via folded sub-domains, in which the backbone possesses secondary structure. These protein sub-domains rarely remain structured once excised from the protein; much of their ability to specifically bind their intended targets is lost because they assume a manifold of shapes rather than the biologically relevant one. The α-helix is the most prevalent protein secondary structure.
α-Helices play fundamental roles in mediating protein-protein interactions. Several approaches for stabilizing peptides in helical conformations or mimicking this conformation with nonnatural oligomers have been described (Henchey et al., Curr. Opin. Chem. Biol. 12: 692-697 (2008); Home et al., Acc. Chem. Res. 41: 1399-1408 (2008); Seebach et al., J. Acc. Chem. Res. 41: 1366-1375 (2008); Patgiri et al., Acc. Chem. Res. 41: 1289-1300 (2008); Garner et al., Org. Biomol. Chem. 5: 3577-3585 (2007); Goodman et al., Nat. Chem. Biol. 3: 252-262 (2007); Chin et al., Am. Chem. Soc. 123: 2929-2930 (2001)). Examination of complexes of proteins with other biomolecules reveals that often one face of the helix featuring the i, i+4 and i+7 residues is involved in binding. Synthetic scaffolds that display protein-like functionality and reproduce the arrangement of key side chains on an α-helix would be invaluable as inhibitors of selective protein interactions.
The present invention is directed to overcoming these and other deficiencies in the art.
SUMMARY OF THE INVENTION
One aspect of the present invention relates to an oligooxopiperazine of Formula I:




wherein:

    each of R1, R2, R3, and R4 is independently an amino acid side chain, H, N(R)2, OR, halogen, an alkyl, or an aryl; wherein each R is independently H, an alkyl, or an aryl;
    each R6 is independently H, N(R)2, OR, halogen, an alkyl, or an aryl; wherein each R is independently H, an alkyl, or an aryl;
    A is X1 or C, wherein:
    
        X1 is H, COR′, CO2R′, CONR′, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a protecting group for protection of an amine, a targeting moiety, or a tag; wherein R′ is H, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety, or a tag; and
        C is a moiety of the formula
    
    






    
    
         wherein:
        
            each X′ is independently H, COR′, CO2R′, CONR′, N(R′′)2, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety, or a tag; wherein:
            
                R′ is H, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety, or a tag; and
                each R′′ is independently H, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety, or a tag;
            
            
            R0 is an amino acid side chain, H, N(R)2, OR, halogen, an alkyl, or an aryl; wherein each R is independently H, an alkyl, or an aryl; and
            R6 is H, N(R)2, OR, halogen, an alkyl, or an aryl; wherein each R is independently H, an alkyl, or an aryl; and
        
        
    
    
    B is Y or D, wherein:
    
        Y is OR′, COR′, N(R′′′)2, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a protecting group for protection of a carboxylic acid, a targeting moiety, or a tag; wherein:
        
            R′ is H, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety, or a tag; and
            each R′′′ is independently H, CO2R′, CONR′, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety, or a tag; and
        
        
        D is a moiety of the formula
    
    






    
    
         wherein:
        
            R5 is an amino acid side chain, H, N(R)2, OR, halogen, an alkyl, or an aryl; wherein each R is independently H, an alkyl, or an aryl;
            R6 is H, N(R)2, OR, halogen, an alkyl, or an aryl; wherein each R is independently H, an alkyl, or an aryl; and
            E is X2 or F, wherein:
            
                X2 is H, COR′, CO2R′, CONR′, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a protecting group for protection of an amine, a targeting moiety, or a tag; wherein R′ is H, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety, or a tag; and
                F is a moiety of the formula
            
            
        
        
    
    






    
    
        
        
            
            
                 wherein:
                
                    R6 is H, N(R)2, OR, halogen, an alkyl, or an aryl; wherein each R is independently H, an alkyl, or an aryl;
                    R7 is an amino acid side chain; and
                    Y is OR′, COR′, N(R′′)2, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a protecting group for protection of a carboxylic acid, a targeting moiety, or a tag; wherein:
                     R′ is H, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety, or a tag; and
                     each R′′′ is independently H, CO2R′, CONR′, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety, or a tag;

with the proviso that A and B are not both, respectively, C and D.

                
                
            
            
        
        
    
    


The present invention is further directed to pharmaceutical formulations containing the oligooxopiperazine of Formula I and methods of inhibiting protein activity or protein-protein interactions using the oligooxopiperazine of Formula I.
Another aspect of the present invention relates to a method of inhibiting a protein-protein interaction. This method involves contacting at least one of the proteins involved in the protein-protein interaction with an oligooxopiperazine under conditions effective to inhibit the protein-protein interaction. In one embodiment of this aspect of the present invention, the protein-protein interaction is mediated by a first hot spot amino acid residue and a second hot spot amino acid residue, and the oligooxopiperazine comprises an oligooxopiperazine of Formula II:




wherein:

    R1 and R2 are independently an amino acid side chain, H, N(R)2, OR, halogen, an alkyl, or an aryl; wherein each R is independently H, an alkyl, or an aryl;
    each R6 is independently H, N(R)2, OR, halogen, an alkyl, or an aryl; wherein each R is independently H, an alkyl, or an aryl;
    A is X1 or C, wherein:
    
        X1 is H, COR′, CO2R′, CONR′, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a protecting group for protection of an amine, a targeting moiety, or a tag; wherein R′ is H, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety, or a tag; and
        C is a moiety of the formula
    
    






    
    
         wherein:
        
            each X′ is independently H, COR′, CO2R′, CONR′, N(R′′)2, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety, or a tag; wherein:
            R′ is H, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety, or a tag; and
            each R′′ is independently H, CO2R′, CONR′, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety, or a tag;
            R0 is an amino acid side chain, H, N(R)2, OR, halogen, an alkyl, or an aryl; wherein each R is independently H, an alkyl, or an aryl; and
            R6 is H, N(R)2, OR, halogen, an alkyl, or an aryl; wherein each R is independently H, an alkyl, or an aryl; and
        
        
    
    
    Y is OR′, COR′, N(R′′′)2, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a protecting group for protection of a carboxylic acid, a targeting moiety, or a tag;
    
        wherein:
        R′ is H, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety, or a tag; and
        each R′′′ is independently H, CO2R′, CONR′, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety, or a tag.
    
    


The present invention is further directed to methods of solid phase and solution phase synthesis of the oligooxopiperazines of the present invention.
A fundamental limitation of current drug development centers on the inability of traditional pharmaceuticals to target spatially extended protein surfaces. The intrinsic conformational and chemical instabilities of peptides limit their potential as reagents in molecular biology and drug discovery. Accordingly, there is a need to develop nonpeptidic oligomers that display protein-like side chains as alternatives to peptides and have superior pharmacological properties. The present invention describes the design and synthesis of nonpeptidic oxopiperazine oligomers that are non-aromatic helix mimetics that are easily synthesized from α-amino acids. These scaffolds present chiral backbones, as compared to the aromatic templates, that are more effective in discriminating between chiral protein pockets Importantly, because the oligooxopiperazines of the present invention are obtained by linking neighboring amide nitrogen atoms in peptides with ethylene bridges, the amide bond, that may be the chief culprit leading to the poor cellular uptake of peptides, is removed. Molecular modeling studies, 2D NMR, and circular dichroism provide strong support for the design features of the oligooxopiperazines described herein.



BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A-1H depict suitable methods of coupling and cyclizing amino acid residues using various alkylating agents. FIGS. 1A-1B show the steps of coupling and cyclizing amino acid residues using X—CH2—CH═CH (designated as alkylating agent A) in the solid phase (designated Sd) or solution phase (designated Sn) synthesis of the oligooxopiperazines of the present invention. FIGS. 1C-1D show the steps of coupling and cyclizing amino acid residues using X—CH2—CH(OR11)2 (designated as alkylating agent B) in the solid phase (designated Sd) or solution phase (designated Sn) synthesis schemes of the present invention. FIGS. 1E-1F show the steps of coupling and cyclizing amino acid residues using X—(CH2)2—X (designated as alkylating agent C) in the solid phase (designated Sd) or solution phase (designated Sn) synthesis schemes of the present invention. FIGS. 1G-1H shows the steps of coupling and cyclizing amino acid residues using X—(CH2)2—OH (designated as alkylating agent D) in the solid phase (designated Sd) or solution phase (designated Sn) synthesis schemes of the present invention.
FIGS. 2A-2C illustrate the design and predicted structure of amino acid-derived oligooxopiperazines. The oligooxopiperazines are obtained by linking neighboring amide nitrogen atoms in peptides with ethylene bridges as depicted in FIG. 2A. FIG. 2B shows an 8mer canonical α-helix with side chain residues depicted as dark grey spheres (left). The predicted structure of an oligooxopiperazine dimer with side chain residues depicted as light grey spheres (FIG. 2B, right) and an overlay of the piperazine dimer and the α-helix (FIG. 2B, center) is also shown. FIG. 2C (left) and FIG. 2C (right) show a top-down view of the structures shown in FIG. 2B (left) and FIG. 2B (center), respectively.
FIGS. 3A-3C depict the rotatable bonds and favored geometries of an oligooxopiperazine dimer. The rotatable bonds (i.e., φ, ψ, and ω) of an oligooxopiperazine dimer are show in FIG. 3A. The favored chair and amide bond geometries are shown in FIGS. 3B and 3C, respectively. The values were calculated with Macromodel MMFF force field in chloroform.
FIGS. 4A-4B show three oligooxopiperazine helix mimetics of the present invention (FIG. 4A; oxopiperazine 1a, 1b, and 1c) and their synthesis via reductive amination (FIG. 4B). Synthesis of dimers 1a-c: (a) O3, (b) Me2S, (c) TFA and triethylsilane. Combined yield for steps a-c: 3a, 81%; 3b, 80%; 3c, 85%; (d) Boc2O: 4a, 98%; 4b, 94%; 4c, 97%; (e) LiOH3, DCC, HOBt: 1a, 73%; 1b, 70%; 1c, 71%. a: R1═CH2CH(CH3)2, R2═CH3. b: R1═CH2Ph, R2═(CH2)4NHCbz. c: R1═CH2CH(CH3)2, R2═CH2CH(CH3)2.
FIGS. 5A-5C show the solution conformation and thermal stabilities of oligooxopiperazines 1a, 1b, and 1c shown in FIG. 4A. The circular dichroism (CD) spectra of oxopiperazines 1a-1c in acetonitrile and methanol is depicted in FIGS. 5A and 5C, respectively. The effect of temperature on the stability of compounds 1a-1c is shown in FIG. 5B.
FIGS. 6A-6B show a cross-section of the NOESY spectra of oligooxopiperazine 1a in CDCl3 (FIG. 6A) and an overlay of key NOEs on the predicted oligooxopiperazine conformation (FIG. 6B) (Side chain groups not shown for clarity.)
FIGS. 7A-7B are graphs showing the low energy φ (FIG. 7A) and ψ (FIG. 7B) angles for oligooxopiperazine dimer 30 calculated using the macromodel “dihedral drive” function.
FIGS. 8A-8D depict a 10mer alpha helix and the predicted structure of an oligooxopiperazine trimer. The 10mer alpha helix of FIG. 8A displays i and i+1, and i and i+4 distances. FIG. 8B shows the predicted structure of an oligooxopiperazine trimer. An overlay of the trimer and the α-helix (gray stick model) is shown in FIG. 8C. The spheres represent amino acid side chains. FIG. 8D illustrates the numbering of side chain residues on the oligooxopiperazine trimer.
FIGS. 9A-9D show the design and structure of model oligooxopiperazine dimers A-C (FIGS. 9B-9D) and a model oligooxopiperazine trimer (FIG. 9A) of the present invention. An overlay of the predicted structure of each model oligooxopiperazine and its target α-helix is also shown.
FIGS. 10A-10C show oligooxopiperazine 38 of the present invention designed to target the p53 transactivation domain, which adopts a helical conformation to target Mdm2. Three key hydrophobic residues of p53 (F19, W23, and L26) bind in the Mdm2 pocket as depicted in FIG. 10A. FIG. 10B shows an overlay of oligooxopiperazine 38 and the p53 helix. FIG. 10C shows the structures of oligooxopiperazine 38 (FIG. 10C; left), and the negative control oligooxopiperazine 39 (FIG. 10C; right), which lacks the key tryptophan residue.
FIGS. 11A1-11AA2 contain a table of α-helices involved in modulating protein-protein interactions that are suitable targets for oligooxopiperazines design. The table sets forth the α-helices by, inter alia, their RSC Protein Data Bank (an online database that includes proteins involved in protein-protein interactions; “PDB”) code (column A), title (column D), function (column E), the chains in the protein-protein complex featuring a helix at the interface (column B), and the chain containing the candidate helix to be mimicked (column C). Also shown in the table are the number of hot spot residues in the helix (column J), the relative position of the hot spot residues within the chain (column K) and within the helix (column L), the length of the candidate helix to be mimicked (column N), the first (column O) and last (column P) residue of the helix to be mimicked, and the amino acid sequence of the helix to be mimicked (column Q).



DETAILED DESCRIPTION OF THE INVENTION
A first aspect of the present invention is directed to an oligooxopiperazine of Formula I:




wherein:

    each of R1, R2, R3, and R4 is independently an amino acid side chain, H, N(R)2, OR, halogen, an alkyl, or an aryl; wherein each R is independently H, an alkyl, or an aryl;
    each R6 is independently H, N(R)2, OR, halogen, an alkyl, or an aryl; wherein each R is independently H, an alkyl, or an aryl;
    A is X1 or C, wherein:
    
        X1 is H, COR′, CO2R′, CONR, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a protecting group for protection of an amine, a targeting moiety, or a tag; wherein R′ is H, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety, or a tag; and
        C is a moiety of the formula
    
    






    
    
         wherein:
        
            each X′ is independently H, COR′, CO2R′, CONR′, N(R′′)2, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety, or a tag; wherein:
            
                R′ is H, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety, or a tag; and
                each R′′ is independently H, CO2R′, CONR′, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety, or a tag;
            
            
            R0 is an amino acid side chain, H, N(R)2, OR, halogen, an alkyl, or an aryl; wherein each R is independently H, an alkyl, or an aryl; and
            R6 is H, N(R)2, OR, halogen, an alkyl, or an aryl; wherein each R is independently H, an alkyl, or an aryl; and
        
        
    
    
    B is Y or D, wherein:
    
        Y is OR′, COR′, N(R′′′)2, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a protecting group for protection of a carboxylic acid, a targeting moiety, or a tag; wherein:
        
            R′ is H, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety, or a tag; and
            each R′′′ is independently H, CO2R′, CONR′, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety, or a tag; and
        
        
        D is a moiety of the formula
    
    






    
    
         wherein:
        
            R5 is an amino acid side chain, H, N(R)2, OR, halogen, an alkyl, or an aryl; wherein each R is independently H, an alkyl, or an aryl;
            R6 is H, N(R)2, OR, halogen, an alkyl, or an aryl; wherein each R is independently H, an alkyl, or an aryl; and
            E is X2 or F, wherein:
            
                X2 is H, COR′, CO2R′, CONR′, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a protecting group for protection of an amine, a targeting moiety, or a tag; wherein R′ is H, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety, or a tag; and
                F is a moiety of the formula
            
            
        
        
    
    






    
    
        
        
            
            
                 wherein:
                
                    R6 is H, N(R)2, OR, halogen, an alkyl, or an aryl; wherein each R is independently H, an alkyl, or an aryl;
                    R7 is an amino acid side chain; and
                    Y is OR′, COR′, N(R′′′)2, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a protecting group for protection of a carboxylic acid, a targeting moiety, or a tag; wherein:
                     R′ is H, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety, or a tag; and
                     each R′′′ is independently H, CO2R′, CONR′, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety, or a tag;

with the proviso that A and B are not both, respectively, C and D.

                
                
            
            
        
        
    
    


Amino acid side chains according to this and all aspects of the present invention can be any amino acid side chain—from natural or nonnatural amino acids—including alpha amino acids, beta amino acids, gamma amino acids, L-amino acids, and D-amino acids.
As used herein, the term “alkyl” means an aliphatic hydrocarbon group which may be straight or branched having about 1 to about 6 carbon atoms in the chain. Branched means that one or more lower alkyl groups such as methyl, ethyl or propyl are attached to a linear alkyl chain. Exemplary alkyl groups include methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, n-pentyl, and 3-pentyl.
As used herein, “cycloalkyl” refers to a non-aromatic saturated or unsaturated mono- or polycyclic ring system which may contain 3 to 6 carbon atoms, and which may include at least one double bond. Exemplary cycloalkyl groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, anti-bicyclopropane, or syn-bicyclopropane.
As used herein, the term “aryl” refers to an aromatic monocyclic or polycyclic ring system containing from 6 to 19 carbon atoms, where the ring system may be optionally substituted. Aryl groups of the present invention include, but are not limited to, groups such as phenyl, naphthyl, azulenyl, phenanthrenyl, anthracenyl, fluorenyl, pyrenyl, triphenylenyl, chrysenyl, and naphthacenyl.
The term “arylalkyl” refers to a radical of the formula —RaRb where Ra is an alkyl radical as defined above and Rb is an aryl radical as defined above. The alkyl radical and the cycloalkyl radical may be optionally substituted as defined above.
As used herein, “heteroaryl” refers to an aromatic ring radical which consists of carbon atoms and from one to five heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur. Examples of heteroaryl groups include, without limitation, pyrrolyl, pyrazolyl, imidazolyl, triazolyl, furyl, thiophenyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, oxadiazolyl, thiadiazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazinyl, thienopyrrolyl, furopyrrolyl, indolyl, azaindolyl, isoindolyl, indolinyl, indolizinyl, indazolyl, benzimidazolyl, imidazopyridinyl, benzotriazolyl, benzoxazolyl, benzoxadiazolyl, benzothiazolyl, pyrazolopyridinyl, triazolopyridinyl, thienopyridinyl, benzothiadiazolyl, benzofuyl, benzothiophenyl, quinolinyl, isoquinolinyl, tetrahydroquinolyl, tetrahydroisoquinolyl, cinnolinyl, quinazolinyl, quinolizilinyl, phthalazinyl, benzotriazinyl, chromenyl, naphthyridinyl, acrydinyl, phenanzinyl, phenothiazinyl, phenoxazinyl, pteridinyl, and purinyl. Additional heteroaryls are described in COMPREHENSIVE HETEROCYCLIC CHEMISTRY: THE STRUCTURE, REACTIONS, SYNTHESIS AND USE OF HETEROCYCLIC COMPOUNDS (Katritzky et al. eds., 1984), which is hereby incorporated by reference in its entirety.
The oligooxopiperazines of Formula I may comprise a protecting group that is suitable for the protection of an amine or a carboxylic acid. Such protecting groups function primarily to protect or mask the reactivity of functional groups. Protecting groups that are suitable for the protection of an amine group are well known in the art, including without limitation, carbamates, amides, N-alkyl and N-aryl amines, imine derivatives, enamine derivatives, and N-hetero atom derivatives as described by THEODORA W. GREENE & PETER G. M. WUTS, PROTECTIVE GROUPS IN ORGANIC SYNTHESIS 494-615 (1999), which is hereby incorporated by reference in its entirety. Protecting groups that are suitable for the protection of a carboxylic acid are also well known in the art. Suitable carboxylic acid protecting groups include, without limitation, esters (e.g., substituted methyl esters, 2-substituted ethyl esters, 2,6-dialkylphenyl esters, substituted benzyl esters, silyl esters, and stannyl esters), amides, and hydrazides as described by THEODORA W. GREENE & PETER G. M. WUTS, PROTECTIVE GROUPS IN ORGANIC SYNTHESIS 372-450 (1999), which is hereby incorporated by reference in its entirety. Methods of protecting and deprotecting amine and carboxylic acids vary depending on the chosen protecting group; however, these methods are well known in the art and described in THEODORA W. GREENE & PETER G. M. WUTS, PROTECTIVE GROUPS IN ORGANIC SYNTHESIS 372-450 and 494-615 (1999), which is hereby incorporated by reference in its entirety.
A “tag” as used herein includes any labeling moiety that facilitates the detection, quantitation, separation, and/or purification of the oligooxopiperazine of the present invention. Suitable tags include purification tags, radioactive or fluorescent labels, and enzymatic tags.
Purification tags, such as poly-histidine (His6-), a glutathione-S-transferase (GST-), or maltose-binding protein (MBP-), can assist in oligomer purification or separation but can later be removed, i.e., cleaved from the oligooxopiperazine following recovery. Protease-specific cleavage sites can be used to facilitate the removal of the purification tag. The desired oligooxopiperazine product can be purified further to remove the cleaved purification tags.
Other suitable tags include radioactive labels, such as, 125I, 131I, 111In, or 99TC. Methods of radiolabeling compounds, are known in the art and described in U.S. Pat. No. 5,830,431 to Srinivasan et al., which is hereby incorporated by reference in its entirety. Radioactivity is detected and quantified using a scintillation counter or autoradiography. Alternatively, the oligooxopiperazine can be conjugated to a fluorescent tag. Suitable fluorescent tags include, without limitation, chelates (europium chelates), fluorescein and its derivatives, rhodamine and its derivatives, dansyl, Lissamine, phycoerythrin and Texas Red. The fluorescent labels can be conjugated to the oligooxopiperazine using techniques disclosed in CURRENT PROTOCOLS IN IMMUNOLOGY (Coligen et al. eds., 1991), which is hereby incorporated by reference in its entirety. Fluorescence can be detected and quantified using a fluorometer.
Enzymatic tags generally catalyze a chemical alteration of a chromogenic substrate which can be measured using various techniques. For example, the enzyme may catalyze a color change in a substrate, which can be measured spectrophotometrically. Alternatively, the enzyme may alter the fluorescence or chemiluminescence of the substrate. Examples of suitable enzymatic tags include luciferases (e.g., firefly luciferase and bacterial luciferase; see e.g., U.S. Pat. No. 4,737,456 to Weng et al., which is hereby incorporated by reference in its entirety), luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidases (e.g., horseradish peroxidase), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (e.g., uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like. Techniques for conjugating enzymes to proteins and peptides are described in O'Sullivan et al., Methods for the Preparation of Enzyme—Antibody Conjugates for Use in Enzyme Immunoassay, in METHODS IN ENZYMOLOGY 147-66 (Langone et al. eds., 1981), which is hereby incorporated by reference in its entirety. Such tags may be particularly useful for detecting inhibition of protein-protein interactions using the oligooxopiperazine of the present invention, as described more fully, infra.
A targeting moiety according to the present invention functions to (i) promote the cellular uptake of the oligooxopiperazine, (ii) target the oligooxopiperazine to a particular cell or tissue type (e.g., signaling peptide sequence), or (iii) target the oligooxopiperazine to a specific sub-cellular localization after cellular uptake (e.g., transport peptide sequence).
To promote the cellular uptake of an oligooxopiperazine of the present invention, the targeting moiety may be a cell penetrating peptide (CPP). CPPs translocate across the plasma membrane of eukaryotic cells by a seemingly energy-independent pathway and have been used successfully for intracellular delivery of macromolecules, including antibodies, peptides, proteins, and nucleic acids, with molecular weights several times greater than their own. Several commonly used CPPs, including polyarginines, transportant, protamine, maurocalcine, and M918, are suitable targeting moieties for use in the present invention and are well known in the art (see Stewart et al., “Cell-Penetrating Peptides as Delivery Vehicles for Biology and Medicine,” Organic Biomolecular Chem 6:2242-2255 (2008), which is hereby incorporated by reference in its entirety). Additionally, methods of making CPP are described in U.S. Patent Application Publication No. 20080234183 to Hallbrink et al., which is hereby incorporated by reference in its entirety.
Another suitable targeting moiety useful for enhancing the cellular uptake of the oligooxopiperazine is an “importation competent” signal peptide as disclosed by U.S. Pat. No. 6,043,339 to Lin et al., which is hereby incorporated by reference in its entirety. An importation competent signal peptides is generally about 10 to about 50 amino acid residues in length, typically hydrophobic residues, that render the oligooxopiperazine capable of penetrating through the cell membrane from outside the cell to the interior of the cell. An exemplary importation competent signal peptide includes the signal peptide from Kaposi fibroblast growth factor (see U.S. Pat. No. 6,043,339 to Lin et al., which is hereby incorporated by reference in its entirety). Other suitable peptide sequences can be selected from the SIGPEP database (see von Heijne G., “SIGPEP: A Sequence Database for Secretory Signal Peptides,” Protein Seq. Data Anal. 1(1):41-42 (1987), which is hereby incorporated by reference in its entirety).
Another suitable targeting moiety is a signal peptide sequence capable of targeting the oligooxopiperazine to a particular tissue or cell type. The signaling peptide can include at least a portion of a ligand binding protein. Suitable ligand binding proteins include high-affinity antibody fragments (e.g., Fab, Fab′ and F(ab)2), single-chain Fv antibody fragments), nanobodies or nanobody fragments, fluorobodies, or aptamers. Other ligand binding proteins include biotin-binding proteins, lipid-binding proteins, periplasmic binding proteins, lectins, serum albumins, enzymes, phosphate and sulfate binding proteins, immunophilins, metallothionein, or various other receptor proteins. For cell specific targeting, the signaling peptide is preferably a ligand binding domain of a cell specific membrane receptor. Thus, when the modified oligooxopiperazine is delivered intravenously or otherwise introduced into blood or lymph, the oligooxopiperazine will adsorb to the targeted cell, and the targeted cell will internalize the oligooxopiperazine. For example, if the target cell is a cancer cell, the oligooxopiperazine may be conjugated to an anti-C3B(I) antibody as disclosed by U.S. Pat. No. 6,572,856 to Taylor et al., which is hereby incorporated by reference in its entirety. Alternatively, the oligooxopiperazine may be conjugated to an alphafeto protein receptor as disclosed by U.S. Pat. No. 6,514,685 to Moro, or to a monoclonal GAH antibody as disclosed by U.S. Pat. No. 5,837,845 to Hosokawa, which are hereby incorporated by reference in their entirety. For targeting an oligooxopiperazine to a cardiac cell, the oligooxopiperazine may be conjugated to an antibody recognizing elastin microfibril interfacer (EMILIN2) (Van Hoof et al., “Identification of Cell Surface for Antibody-Based Selection of Human Embryonic Stem Cell-Derived Cardiomyocytes,” J Proteom Res 9:1610-18 (2010), which is hereby incorporated by reference in its entirety), cardiac troponin I, connexin-43, or any cardiac cell-surface membrane receptor that is known in the art. For targeting an oligooxopiperazine to a hepatic cell, the signaling peptide may include a ligand domain specific to the hepatocyte-specific asialoglycoprotein receptor. Methods of preparing such chimeric proteins and peptides are described in U.S. Pat. No. 5,817,789 to Heartlein et al., which is hereby incorporated by reference in its entirety.
Another suitable targeting moiety is a transport peptide that directs intracellular compartmentalization of the oligooxopiperazine once it is internalized by a target cell or tissue. For example, if the protein activity or protein-protein interaction that is sought to be inhibited occurs in the endoplasmic reticulum (ER), the oligooxopiperazine can be conjugated to an ER transport peptide sequence. A number of such signal peptides are known in the art, including the signal peptide MMSFVSLLLVGILFYATEAEQLTKCEVFQ (SEQ ID NO:182). Other suitable ER signal peptides include the N-terminus endoplasmic reticulum targeting sequence of the enzyme 17β-hydroxysteroid dehydrogenase type 11 (Horiguchi et al., “Identification and Characterization of the ER/Lipid Droplet-Targeting Sequence in 17β-hydroxysteroid Dehydrogenase Type 11,” Arch. Biochem. Biophys. 479(2):121-30 (2008), which is hereby incorporated by reference in its entirety), or any of the ER signaling peptides (including the nucleic acid sequences encoding the ER signal peptides) disclosed in U.S. Patent Publication No. 20080250515 to Reed et al., which is hereby incorporated by reference in its entirety. Additionally, the oligooxopiperazine of the present invention can contain an ER retention signal, such as the retention signal KEDL (SEQ ID NO:183). Methods of modifying the oligooxopiperazines of the present invention to incorporate transport peptides for localization of the oligomers to the ER can be carried out as described in U.S. Patent Publication No. 20080250515 to Reed et al., which is hereby incorporated by reference in its entirety.
If the protein activity or protein-protein interaction that is sought to be inhibited occurs in the nucleus, the oligooxopiperazine can include a nuclear localization transport signal. Suitable nuclear transport peptide sequences are known in the art, including the nuclear transport peptide PPKKKRKV (SEQ ID NO:184). Other nuclear localization transport signals include, for example, the nuclear localization sequence of acidic fibroblast growth factor and the nuclear localization sequence of the transcription factor NF-KB p50 as disclosed by U.S. Pat. No. 6,043,339 to Lin et al., which is hereby incorporated by reference in its entirety. Other nuclear localization peptide sequences known in the art are also suitable for use in the accordance with this aspect of the invention.
Suitable transport peptide sequences for targeting to the mitochondria include MLSLRQSIRFFKPATRTLCSSRYLL (SEQ ID NO:185). Other suitable transport peptide sequences suitable for selectively targeting the oligooxopiperazine of the present invention to the mitochondria are disclosed in U.S. Published Patent Application No. 20070161544 to Wipf, which is hereby incorporated by reference in its entirety.
In one embodiment of the present invention, the oligooxopiperazine of Formula I has a formula of Formula IA:




Exemplary oligooxopiperazine compounds of Formula IA include, without limitation,




where X is H, COCH3, or any amino acid, and Y is OH, NH2, OMe, or any amino acid.

In another embodiment of the present invention, the oligooxopiperazine of Formula I has a formula of Formula IB:




Exemplary oligooxopiperazine compounds of Formula IB include, without limitation,




where X is H, COCH3, or any amino acid, and Y is OH, NH2, OMe, or any amino acid.

In another embodiment of the present invention, the oligooxopiperazine of Formula I has a formula of Formula IC:




Exemplary oligooxopiperazine compounds of Formula IC include, without limitation,




where X is H, COCH3, or any amino acid, and Y is OH, NH2, OMe, or any amino acid.

In a preferred embodiment of the present invention, the oligooxopiperazine of Formula I, including oligooxopiperazines of Formulas IA, IB, and IC, are designed to mimic an α-helix that is involved in a protein-protein interaction. α-Helices involved in modulating protein-protein interactions that are suitable for mimicking are shown in the table of FIGS. 11A1-11AA2. This table sets forth predicted targets by, inter alia, their RSC Protein Data Bank (an online database that includes proteins involved in protein-protein interactions; “PDB”) code (column A), title (column D), function (column E), the chains in the protein-protein complex featuring a helix at the interface (column B), and the chain containing the candidate helix to be mimicked (column C). Also shown in the table of FIGS. 11A1-11AA2 are the number of hot spot residues in the helix (column J), the relative position of the hot spot residues within the chain (column K) and within the helix (column L), the length of the candidate helix to be mimicked (column N), the first (column O) and last (column P) residue of the helix to be mimicked, and the amino acid sequence of the helix to be mimicked (column Q). Additional α-helices suitable for mimicking are disclosed in Jochim et al., “Assessment of Helical Interfaces in Protein-Protein Interactions,” Mol. Biosyst. 5(9):924-26 (2009), which is hereby incorporated by reference in its entirety, which describes the identification and classification of over 2,500 helical interface protein-protein interactions and the hot spot residues involved in these interactions.
Oligooxopiperazines of the present invention that are designed to mimic an α-helix of a protein, e.g., an α-helix involved in a protein-protein interaction, can be designed to mimic every side chain of the α-helix. Alternatively, if the hot spot residues of the α-helix are known, the oligooxopiperazine can be designed to mimic only the hot spot residues, in which case the remaining oligooxopiperazine side groups can be any side group that does not interfere with the oligooxopiperazine's function.
In accordance with this embodiment of the present invention, R1, R2, R4, and R5 of the oligooxopiperazine of Formula IA can mimic the amino acid side chain of, respectively, residues i, i+4, i+6, and i+7, of the α-helix. Suitable oligooxopiperazines of Formula IA that mimic an α-helix involved in a protein-protein interaction include, without limitation,




where X is H, COCH3, or any amino acid, and Y is OH, NH2, OMe, or any amino acid.

The oligooxopiperazine of Formula IB can also be designed to mimic an α-helix involved in a protein-protein interaction. In one embodiment, R1, R2, and R4 of the oligooxopiperazine of Formula IB can mimic the amino acid side chain of, respectively, residues i, i+4, and i+7, of the α-helix. Such suitable oligooxopiperazines of Formula IB include, without limitation,




where X is H, COCH3, or any amino acid, and Y is OH, NH2, OMe, or any amino acid. Alternatively, R1, R2, and R4 can mimic the amino acid side chain of, respectively, residues i, i+4, and i+6 of the α-helix. Such suitable oligooxopiperazines of Formula IB include, without limitation,





where X is H, COCH3, or any amino acid, and Y is OH, NH2, OMe, or any amino acid.

The oligooxopiperazine of Formula IC can also be designed to mimic an α-helix involved in protein-protein interactions. For example, R0, R1, R2, R3, and R4 of Formula IC can mimic the amino acid side chain of, respectively, residues i, i+2, i+3, i+4, and i+7 of the α-helix. Suitable oligooxopiperazines of Formula IC that mimic an α-helix involved in a protein-protein interaction include, without limitation,




where X is H, COCH3, or any amino acid, and Y is OH, NH2, OMe, or any amino acid.

Another aspect of the present invention relates to pharmaceutical formulations comprising any of the above described oligooxopiperazines of Formula I, including the oligooxopiperazines of Formulas IA, IB, and IC of the present invention and a pharmaceutically acceptable carrier. Acceptable pharmaceutical carriers include solutions, suspensions, emulsions, excipients, powders, or stabilizers. The carrier should be suitable for the desired mode of delivery.
In addition, the pharmaceutical formulations of the present invention may further comprise one or more pharmaceutically acceptable diluents, adjuvants, excipients, or vehicles, such as preserving agents, fillers, disintegrating agents, wetting agents, emulsifying agents, suspending agents, sweetening agents, flavoring agents, perfuming agents, antibacterial agents, antifungal agents, lubricating agents and dispensing agents, depending on the nature of the mode of administration and dosage forms. Examples of suspending agents include ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, for example sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monosterate and gelatin. Examples of suitable carriers, diluents, solvents, or vehicles include water, ethanol, polyols, suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters such as ethyl oleate. Examples of excipients include lactose, milk sugar, sodium citrate, calcium carbonate, and dicalcium phosphate. Examples of disintegrating agents include starch, alginic acids, and certain complex silicates. Examples of lubricants include magnesium stearate, sodium lauryl sulphate, talc, as well as high molecular weight polyethylene glycols.
Another aspect of the present invention relates to a method of inhibiting activity of a protein that involves contacting the protein with an oligooxopiperazine of the present invention under conditions effective to inhibit activity of the protein. The oligooxopiperazine according to this aspect of the present invention is an oligooxopiperazine of Formula I (e.g, an oligooxopiperazine of Formula IA, IB, or IC), preferably designed to mimic an α-helix involved in a protein-protein interaction as described supra.
Another aspect of the present invention relates to a method of inhibiting a protein-protein interaction that involves contacting at least one of the proteins involved in the protein-protein interaction with an oligooxopiperazine under conditions effective to inhibit the protein-protein interaction. The oligooxopiperazine according to this aspect of the present invention is an oligooxopiperazine of Formula I (e.g, an oligooxopiperazine of Formula IA, IB, or IC), or, if the protein-protein interaction is mediated by a first hot spot residue and a second hot spot residue, an oligooxopiperazine Formula II:




wherein:

    
    
        R1 and R2 are independently an amino acid side chain, H, N(R)2, OR, halogen, an alkyl, or an aryl; wherein each R is independently H, an alkyl, or an aryl;
        each R6 is independently H, N(R)2, OR, halogen, an alkyl, or an aryl; wherein each R is independently H, an alkyl, or an aryl;
        A is X1 or C, wherein:
        
            X1 is H, COR′, CO2R′, CONR′, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a protecting group for protection of an amine, a targeting moiety, or a tag; wherein R′ is H, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety, or a tag; and
            C is a moiety of the formula
        
        
    
    






    
    
        
        
            wherein:
            
                each X′ is independently H, COR′, CO2R′, CONR′, N(R′′)2, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety, or a tag; wherein:
                
                    R′ is H, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety, or a tag; and
                    each R′′ is independently H, CO2R′, CONR′, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety, or a tag;
                
                
                R0 is an amino acid side chain, H, N(R)2, OR, halogen, an alkyl, or an aryl; wherein each R is independently H, an alkyl, or an aryl; and
                R6 is H, N(R)2, OR, halogen, an alkyl, or an aryl; wherein each R is independently H, an alkyl, or an aryl; and
            
            
        
        
        Y is OR′, COR′, N(R′′′)2, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a protecting group for protection of a carboxylic acid, a targeting moiety, or a tag; wherein:
        
            R′ is H, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety, or a tag; and
            each R′′′ is independently H, CO2R′, CONR′, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety, or a tag.
        
        
    
    


Preferably, the oligooxopiperazine is designed to mimic an α-helix involved in the protein-protein interaction. Oligooxopiperazines of Formula I can be used to mimic α-helices containing 3-5 hot spot residues, such as the α-helices identified in FIGS. 11A1-11AA2. Oligooxopiperazines of Formula II can be used to mimic α-helices containing only 2 hot spot residues. For example, the first and second hot spot residues can be, respectively, residues i and i+4 of an alpha helix, and R1 and R2 of Formula II can mimic the amino acid side chain of, respectively, residues i and i+4 of the α-helix.
Another aspect of the present invention is directed to a method of treating a disorder in a subject, where the disorder is mediated by p53. This method involves administering to the subject a pharmaceutical composition containing an oligooxopiperazine that mimics the α-helix of p53 under conditions effective to treat the disorder. In accordance with this aspect of the invention, the oligooxopiperazine is preferably an oligooxopiperazine of Formula IA, where R1, R2, R4, and R5 mimic the amino acid side chain of, respectively, residues i, i+4, i+6, and i+7 of a p53 α-helix.
In accordance with this aspect of the invention, an oligooxopiperazine of Formula IA that is suitable for treating a disorder mediated by p53 in a subject is oligooxopiperazine 38 as described in the Examples herein and shown in FIG. 10C. Oligooxopiperazines mimicking an α-helix of p53 that disrupt p53 complex formation with, for example, Mdm2, would be suitable for treating cancer. Therapeutic inhibition of p53 is also suitable for the treatment of ischemia induced apoptosis, myocardial infarction, cholestasis, and a variety of neurodegenerative diseases including AID-associated neurodegeneration, stroke, Parkinson's disease, Alzheimer's disease, and Huntington's disease (see Amaral J., “The Role of p53 in Apoptosis,” Discov. Med. 9(45):145-53 (2010), which is hereby incorporated by reference in its entirety).
Another aspect of the present invention is directed to methods of making oligooxopiperazines, including the oligooxopiperazines of Formulas IA, IB, and IC. The oligooxopiperazines can be synthesized via solution phase synthesis, or alternatively, via solid phase synthesis.
Accordingly, one aspect of the present invention is directed to a method of solid phase synthesis of the oligooxopiperazine of Formula IA. This method of synthesis involves providing a compound of Formula III:




where PG is a protecting group for the protection of an amine; R8 is an amino acid side chain, H, N(R)2, OR, halogen, an alkyl, or an aryl, where each R is independently H, an alkyl, or an aryl; and R9 is —O-Res or —NH-Res, where Res is a solid phase peptide synthesis resin. This method further involves providing a compound of Formula IV1:





PG is a protecting group for the protection of an amine and where R10 is —OH or a halide. The compound of Formula III is reacted with a first alkylating agent and the compound of Formula IV1 under conditions effective to produce a compound of Formula V:





If necessary, —CR6R8—CO—R9 in the compound of Formula V can be converted to E of Formula IA using standard methods known in the art. In addition, if necessary the N-terminal hydrogen in the compound of Formula V can be converted to X1 of Formula IA. As will be appreciated by one of skill in the art, according to this and all aspects of the present invention that call for converting a first moiety to a second moiety, said converting can be carried out, for example, by chemically transforming the first moiety to the second moiety or by entirely replacing the first moiety with the second moiety.

This and other synthesis methods described herein include the use of individual amino acid residues. Typically, individual amino acid residues are obtained protected at the N-terminal and unprotected at the C-terminal. The C-terminal can then be protected using standard methods known in the art (see e.g., THEODORA W. GREENE & PETER G. M. WUTS, PROTECTIVE GROUPS IN ORGANIC SYNTHESIS 372-450 and 494-615 (1999), which is hereby incorporated by reference in its entirety). If desired, the N-terminal protecting group in the amino acid residue can be replaced with a different amino protecting group for use in these methods.
In accordance with this and subsequent solid phase synthesis embodiments of the invention, solid phase peptide synthesis resins suitable for use include, without limitation, polystyrene resins, polyamide resins, PEG hybrid polystyrene resins, and PEG-based resins as described in Fluka Chemie GmbH, “Resins for Solid-Phase Peptide Synthesis,” ChemFiles 3(4):5-6 (2003), which is hereby incorporated by reference in its entirety.
In this and all synthesis methods described herein, suitable protecting groups for the protection of an amine include any of those described supra. Exemplary protecting groups include Boc, Cbz, Ns, and Fmoc. Likewise, in all synthesis methods described herein, suitable protecting groups for the protection of a carboxylic acid include any of those described supra.
In accordance with this and all aspects of the present invention, suitable halides include Br, Cl, and F. Preferably, the halide is Br.
In all the synthesis methods described herein involving the use of an alkylating agent, suitable alkylating agents include those selected from the group consisting of X—CH2—CH═CH, X—CH2—CH(OR11)2, X—(CH2)2—X and X—(CH2)2—OH wherein each X is independently a leaving group and each R11 is independently an alkyl (e.g., halogens, OMs, or OTs). Suitable methods of using the above alkylating agents are depicted in FIGS. 1A-1H and are described infra. Where more than one alkylating step is called for, the alkylating agent for each step can be the same or different.
In accordance with the above method of making the oligooxopiperazine of Formula IA, the compound of Formula III can be provided by providing a compound of Formula VI:




and a compound of Formula IV2:





The compound of Formula VI is reacted with the compound of Formula IV2 under conditions effective to produce a compound of Formula III using methods that will be apparent to one of ordinary skill in the art.

The compound of Formula VI above can be provided by providing a compound of Formula VII:




and a compound of Formula IV3:





The compound of Formula VII is reacted with a second alkylating agent and the compound of Formula IV3 under conditions effective to produce a compound of Formula VI using methods that will be apparent to one of ordinary skill in the art.

The compound of Formula VII above can be provided by providing and reacting a compound of Formula VIII:




and a compound of Formula IV4:





under conditions effective to produce a compound of Formula VII using methods that will be apparent to one of ordinary skill in the art.

The compound of Formula VIII above can be provided by providing a compound of Formula IX:




and a compound of Formula IV5:





The compound of Formula IX is reacted with a third alkylating agent and the compound of Formula IV5 under conditions effective to produce a compound of Formula VIII using methods that will be apparent to one of ordinary skill in the art.

Another aspect of the present invention is directed to the solid phase synthesis of the oligooxopiperazines of Formula IB and IC. This method of synthesis involves providing a compound of Formula VII′:




where PG is a protecting group for the protection of an amine, and R9 is —O-Res or —NH-Res. The method further involves providing a compound of Formula IV1:





where PG is a protecting group for the protection of an amine and R10 is —OH or a halide. The compound of Formula VII′ is reacted with a first alkylating agent and the compound of Formula IV1 under conditions effective to produce a compound of Formula VI′:





using methods that will be apparent to one of ordinary skill in the art.

If necessary, —R9 of Formula VI′ can be converted to Y using standard methods known in the art. Further, when synthesizing an oligooxopiperazine of Formula 1B, if necessary, the N-terminal hydrogen in the compound of Formula VI′ can be converted to X1 using standard methods known in the art; when synthesizing an oligooxopiperazine of Formula IC, the N-terminal hydrogen in the compound of Formula VI′ can be converted to a moiety of formula




using standard methods.

In accordance with the above method of making the oligooxopiperazines of Formulas IB and IC, the compound of Formula VII′ can be provided by providing a compound of Formula VIII′:




and providing a compound of Formula IV2:





The compound of Formula VIII′ is reacted with the compound of Formula IV2 under conditions effective to produce a compound of Formula VII′ using methods that will be apparent to one of ordinary skill in the art.

A compound of Formula VIII′ can be provided by providing a compound of Formula IX′:




and providing a compound of Formula IV3:





The compound of Formula IX′ is reacted with a second alkylating agent and the compound of Formula IV3 under conditions effective to produce a compound of Formula VIII′ using methods that will be apparent to one of ordinary skill in the art.

Another aspect of the present invention is directed to a method of solution phase synthesis of the oligooxopiperazines of Formula IA. This method of synthesis involves providing a compound of Formula X:




where PG1 is a protecting group for the protection of an amine and R10 is —OH or a halide, and providing a compound of Formula XI5/8:





where PG2 is a protecting group for the protection of a carboxylic acid; and R8 is an amino acid side chain, H, N(R)2, OR, halogen, an alkyl, or an aryl, where each R is independently H, an alkyl, or an aryl. The compound of Formula X is reacted with the compound of Formula XI5/8 under conditions effective to produce a compound of Formula XII:





If necessary, —CR6R8—CO—PG2 in the compound of Formula XII can be converted to E of Formula IA using standard methods known in the art. Additionally, if necessary PG1 in the compound of Formula XII can be converted to X1 of Formula IA using standard methods known in the art.

The compound of Formula X above can be provided by providing a compound of Formula X′:




and converting PG1 of Formula X′ to hydrogen and converting PG2 of Formula X′ to R10. Methods for removing protecting groups are well known in the art (see e.g., THEODORA W. GREENE & PETER G. M. WUTS, PROTECTIVE GROUPS IN ORGANIC SYNTHESIS 372-450 and 494-615 (1999), which is hereby incorporated by reference in its entirety).

The compound of Formula X′ above can be provided by providing a compound of Formula XIII:




and providing a compound of Formula XI3/4:





The compound of Formula XIII is reacted with the compound of Formula XI3/4 under conditions effective to produce a compound of Formula X′ using methods that will be apparent to one of ordinary skill in the art.

The compound of Formula XIII above can be provided by providing a compound of Formula XI1/2:




and reacting it with a protecting group under conditions effective to produce a compound of Formula XIII using methods that will be apparent to one of ordinary skill in the art. Suitable methods for adding protecting groups are well known in the art (see e.g., THEODORA W. GREENE & PETER G. M. WUTS, PROTECTIVE GROUPS IN ORGANIC SYNTHESIS 372-450 and 494-615 (1999), which is hereby incorporated by reference in its entirety).

The compound of Formula XI1/2 can be provided by providing a compound of Formula XIV:




and providing a compound of Formula XV:





The compound of Formula XIV is reacted with an alkylating agent and the compound of Formula XV under conditions effective to produce a compound of Formula XI1/2.

Another aspect of the present invention is directed to the solution phase synthesis of the oligooxopiperazines of Formula IB and IC. This method of synthesis involves providing a compound of Formula XIII:




where PG1 is a protecting group for the protection of an amine and R10 is —OH or a halide. Suitable methods of making the compound of Formula XIII are described supra. A compound of Formula XI3/4:





where PG2 is a protecting group for the protection of a carboxylic acid, is also provided. The compound of Formula XIII is reacted with the compound of Formula XI3/4 under conditions effective to produce a compound of Formula X′:





If necessary, PG2 in the compound of Formula X′ can be converted to Y using standard methods known in the art. Further, when synthesizing the oligooxopiperazine of Formula IB, if necessary, PG1 in the compound of Formula X′ can be converted to X1 using standard methods; when synthesizing the oligooxopiperazine of Formula IC, PG1 in the compound of Formula X′ can be converted to a moiety of formula




using standard methods.

The above described solid phase and solution phase methods of oligooxopiperazine synthesis sometimes call for reacting compounds with an alkylating agent. The alkylating agent is used to facilitate coupling and cyclization of the oligooxopiperazine. Suitable methods of coupling and cyclization using the exemplary alkylating agents disclosed herein include the methods shown in FIGS. 1A-1H.
In particular, FIGS. 1A-1B depict coupling and cyclization using X—CH2—CH═CH as the alkylating agent (alkylating agent A) in solid phase (Sd; left) and solution phase (Sn; right) methods of synthesis. Step A involves the alkylation of the amino acid residue (1ASd or 1ASn). PG3, which is a protecting group for the protection of an amine, allows the alkylating agent to react with the hydrogen on the amine. Ns is a preferred protecting group for this purpose. PG3 can then be replaced with hydrogen to facilitate coupling with another residue. Typically, a mild base is used during alkylation to facilitate hydrogen removal. Suitable bases include triethylamine, N,N-diisopropylethylamine, 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU), 2,4,6-trimethylpyridine, potassium carbonate, and cesium carbonate.
Step B involves the coupling of a second amino acid residue (2ASd or 2ASn) to the alkylated amino acid residue (1′ASd or 1′ASn). In Step C, the coupled amino acid residues (3ASd or 3ASn) are cyclized upon the simultaneous or sequential addition of an oxidizing agent, an acid, and a hydride donor. The oxidizing agent, preferably ozone, converts the allyl to an aldehyde. The acid is one that removes the protecting group to provide for cyclization with the aldehyde. Suitable acids include TFA, HCl, HBr, HCOOH, and CH3COOH. The hydride donor ensures that the cyclization reaction takes place in excess hydrogen so the resulting ring is saturated. Suitable hydride donors include triethylsilane and NaBH3CN.
FIGS. 1C-1D depict coupling and cyclization using X—CH2—CH(OR11)2 as the alkylating agent (alkylating agent B) in solid phase (Sd; left) and solution phase (Sn; right) methods of synthesis. These steps are similar to the steps of coupling and cyclization using alkylating agent A described above. Step A involves the alkylation of the amino acid residue (1BSd or 1BSn). As in FIGS. 1A-1B, PG3, which is a protecting group for the protection of an amine, allows the alkylating agent to react with the hydrogen on the amine. Ns is a preferred protecting group for this purpose. PG3 can then be replaced with hydrogen to facilitate coupling with another residue. Typically, a mild base is used during alkylation to facilitate hydrogen removal. Suitable bases include those described supra.
Step B in FIG. 1C involves the coupling of a second amino acid residue (2Bsd or 2BSn) to the alkylated amino acid residue (1′BSd or 1′BSn). In step C, the coupled amino acid residues (3BSd or 3BSn) are cyclized upon the simultaneous or sequential addition of an acid and a hydride donor. The acid is one that removes the protecting group to provide for cyclization with the aldehyde. Suitable acids include TFA, HCl, HBr, HCOOH, and CH3COOH. The hydride donor ensures that the cyclization reaction takes place in excess hydrogen so the resulting ring is saturated. Suitable hydride donors include triethylsilane and NaBH3CN.
FIGS. 1E-1F and 1G-1H depict coupling and cyclization using X—(CH2)2—X) (alkylating agent C) or X—(CH2)2—OH (alkylating agent D), respectively, in solid phase (Sd; left) and solution phase (Sn; right) methods of synthesis. Using either agent C or D, step A involves the coupling of two amino acid residues (1CSd/CSn+2CSd/CSn→3CSd/CSn; 1DSd/DSn+2DSd/DSn→3DSd/DS). Similar to the previous methods, PG3 allows the alkylating agent to react with the hydrogen on the amine during alkylation. Ns is preferred. PG3 can be present as PG in compound 2CSd or PG1 in compound 2CSn, or can be added after coupling.
Step B involves the alkylation of one of the coupled amino acid residues (3CSd/CSn or 3DSd/DSn). Typically, a mild base is used during alkylation to facilitate hydrogen removal. Suitable bases include triethylamine, N,N-diisopropylethylamine, 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU), 2,4,6-trimethylpyridine, potassium carbonate, and cesium carbonate. In Step C, the alkylated coupled amino acid residues (3′CSd/CSn or 3′DSd/DSn) are cyclized upon the addition of a base. Suitable bases include triethylamine, N,N-diisopropylethylamine, 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU), 2,4,6-trimethylpyridine, potassium carbonate, and cesium carbonate.
The present invention may be further illustrated by reference to the following examples.
EXAMPLES
Example 1
Materials and Reagents
Commercial-grade reagents and solvents were used without further purification except as indicated. All reactions were stirred magnetically; moisture-sensitive reactions were performed under nitrogen in flame-dried glassware. Unless indicated, all reactions were performed at 25° C. Thin-layer chromatography (TLC), using ethyl acetate: hexane, diethyl ether: ethyl acetate, diethyl ether: hexane, DCM: methanol as solvent systems, was used to monitor reactions. Visualization was accomplished by either ultraviolet light or immersing the plate in 1% aqueous solution of potassium permanganate followed by heating. Flash chromatography with silica gel was performed following the conditions described by Still et al., J. Org. Chem. 43, 2923-2925 (1978), which is hereby incorporated by reference in its entirety. Solvents were removed by rotary evaporation under reduced pressure. Where appropriate, the residue was further dried using vacuum. One-dimensional Proton (400 MHz) and carbon (100 MHz) NMR spectra were obtained on a Bruker AV-400 spectrometer. Two-dimensional 1H NMR spectra were obtained on a Bruker AV-600 (600 MHz) spectrometer. Proton chemical shifts are reported as values relative to tetramethylsilane (0.00 ppm) or the particular solvent used in the experiment. Carbon chemical shifts are reported as values relative to the solvent used in the experiment (CDCl3; 77.0 ppm). Data is reported as follows: chemical shift, multiplicity (s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet, dd=doublet of doublet, ddt=doublet of doublet of triplet, and br=broad), coupling constant, and integration. The following abbreviations are used in the examples described infra: DCM=dichloromethane, THF=tetrahydrofuran, DIPEA=N,Ndiisopropylethylamine, TEA=triethylamine, TFA=trifluoroacetic acid, HOBt=hydroxybenzotriazole, DCC=N,N′-dicyclohexylcarbodiimide.
Example 2
Synthesis of Oligooxopiperazine 1a
A schematic of oligooxopiperazine 1a synthesis via the reductive amination route (Tong et al., J. Org. Chem. 65:2484-2493 (2000), which is hereby incorporated by reference in its entirety), is shown in Scheme 1 below.







The synthesis of (S)—N-Allyl-Leu-OMe (5a) was carried out as follows. Allyl bromide (137.0 mmol, 11.6 mL) was added to a solution of H-Leu-OMe (55.0 mmol, 10.0 g), DMF (120 mL) and TEA (192.0 mmol, 26.5 mL) at 0° C. The resulting mixture was warmed to 25° C. and stirred for 48 h. The reaction mixture was diluted with water (250 mL) and extracted with diethyl ether (3×, 250 mL). The combined organic layers were washed with saturated aqueous sodium bicarbonate and saturated brine, dried with MgSO4 and concentrated under vacuum. The residue was purified by column chromatography (15% ethyl acetate in hexane) to afford compound 5a as a light yellow oil (7.4 g, 73%). 1H NMR (400 MHz, CDCl3) 5.88 (ddt, J=17.1, 10.2, 6.1 Hz, 1H), 5.19 (dd, J=17.1, 1.5 Hz, 1H), 5.09 (dd, J=10.2, 1.5 Hz, 1H), 3.65 (s, 3H), 3.25 (t, J=7.4 Hz, 1H), 3.15 (ddt, J=6.2, 1.4 Hz, 1H), 2.96 (ddt, J=6.2, 1.4 Hz, 1H), 1.72 (m, 1H), 1.42 (t, J=6.7 Hz, 2H), 0.88 (d, J=6.6 Hz, 3H), 0.78 (d, J=6.7 Hz, 3H); 13C NMR (100 MHz, CDCl3) 176.4, 136.3, 116.4, 59.0, 51.5, 50.7, 42.8, 24.9, 22.6, 22.3; HRMS m/z for C10H19NO2[M+H]+, calcd 186.1494. found 186.1486.
The synthesis of Boc-Ala-N(allyl)-Leu-OMe (2a) was carried out as follows. A solution of Boc-Ala-OH (80.0 mmol, 15.1 g), HOBt (80.0 mmol, 10.8 g) and DCC (80.0 mmol, 16.5 g) in DMF (200 mL) was stirred at 25° C. After 15 min, a solution of 5a (40.0 mmol, 7.4 g) in DMF (5 mL) was added, and the resulting mixture heated at reflux overnight. The reaction mixture was cooled to 25° C., diluted with 400 mL of water and extracted with diethyl ether (250 mL, 3×). The combined organic layers were sequentially washed with 1M NaOH (250 mL, 3×), water (250 mL, 3×), 1M HCl (250 mL, 3×), and saturated brine (250 mL), dried with anhydrous MgSO4 and concentrated under vacuum. The residue was purified by column chromatography using 20% ethyl acetate in hexane. The purified product yielded compound 2a as a yellow oil (6.2 g, 44%). 1H NMR (400 MHz, CDCl3) 5.90-5.80 (m, 1H), 5.25-5.15 (m, 2H), 5.04 (q, J=5.2 Hz, 1H), 4.55 (t, J=7.3 Hz, 1H), 3.94 (d, J=5.3 Hz, 2H), 3.62 (s, 3H), 1.73-1.60 (m, 2H), 1.60-1.50 (m, 1H), 1.45 (s, 9H), 1.21 (d, J=6.8 Hz, 3H), 0.84 (d, J=7.9 Hz, 3H), 0.78 (d, J=6.7 Hz, 3H); 13C NMR (100 MHz, CDCl3) 174.4, 172.2, 155.1, 134.2, 117.7, 79.6, 55.5, 52.1, 48.4, 46.7, 37.7, 28.3, 24.7, 22.9, 21.8, 19.0; HRMS m/z for C18H32N2O5 [M+Na]+, calcd 379.2209. found 379.2210.
The synthesis of (AlaLeu)Oxopiperazine methyl ester (3a) was carried out as follows. Ozone was bubbled into a solution of 2a (17.4 mmol, 6.21 g) in anhydrous methanol (200 mL) at −78° C. and ambient pressure. The reaction mixture turned light blue after 2 h. After an additional 30 min, nitrogen was bubbled into the solution until the blue color disappeared. Dimethyl sulfide (61.0 mmol, 4.5 g) was added and the mixture was stirred at room temperature. After 16 h, the mixture was concentrated under vacuum, and the residue (6.2 g) was dissolved in DCM (125 mL), and triethylsilane (34.6 mmol, 5.5 mL) and TFA (260.0 mmol, 19.3 mL) was added. The reaction mixture was stirred for 24 h at 25° C. and then concentrated under vacuum. The residue was redissolved in DCM (60 mL) and TEA (60 mL) at 0° C. and stirred at 25° C. After one hour, the solvent was concentrated under vacuum. The residue was dissolved in DCM and the organic layer was washed with saturated aqueous sodium bicarbonate. The aqueous layer was washed 3× with DCM. The residue was purified by column chromatography (95% diethyl ether 5% methanol and 0.1% TEA) to obtain compound 3a as a colorless oil (2.6 g, 81%). 1H NMR (400 MHz, CDCl3) 5.20 (t, J=8.2 Hz, 1H), 3.68 (s, 3H), 3.53 (q, J=6.8 Hz, 1H), 3.28-3.20 (m, 2H), 3.10-3.02 (m, 2H), 1.63 (t, J=7.5 Hz, 2H), 1.52-1.46 (septet, J=7.8 Hz, 1H), 1.32 (d, J=6.9 Hz, 3H), 0.88 (d, J=6.7 Hz, 3H), 0.78 (d, J=6.5 Hz, 3H); 13C NMR (100 MHz, CDCl3) 172.3, 171.4, 55.4, 53.8, 52.2, 44.9, 42.2, 36.7, 24.9, 23.2, 21.3, 18.99; HRMS m/z for C12H22N2O3[M+Na]+, calcd 265.1528. found 265.1523.
The synthesis of Boc-(AlaLeu)oxopiperazine-methyl ester (4a) was carried out as follows. To a solution of 3a (6.6 mmol, 1.6 g) in DCM (22 mL) at 0° C. was added 4-methylmorpholine (10.0 mmol, 1.1 mL) and ditert-butyl dicarbonate (16.6 mmol, 3.6 g) in 50 mL of DCM. The mixture was allowed to warm to 25° C. and then heated at reflux. After 6 h, the mixture was concentrated and the residue purified by column chromatography (40% hexane in diethyl ether) to yield 2.2 g (98%) of compound 4a as a colorless oil. 1H NMR (400 MHz, CDCl3) 5.21 (q, J=5.72 Hz, 1H), 4.51 (br, 1H), 3.89 (br, 1H), 3.64 (s, 3H), 3.46-3.35 (m, 1H), 3.26 (br, 1H), 3.16-3.11 (m, 1H), 1.73-1.60 (m, 2H), 1.45 (br, 1H), 0.41 (s, 9H), 1.3 (d, J=6.9 Hz, 3H), 0.88 (d, J=6.7 Hz, 3H), 0.78 (d, J=6.8 Hz, 3H); 13C NMR (100 MHz, CDCl3) 172.2, 168.0, 151.3, 78.3, 52.1, 40.3, 34.2, 25.8, 22.5, 20.6, 18.8, 15.5; HRMS m/z for C17H30N2O5 [M+Na]+, calcd 365.2052. found 365.2049.
The synthesis of oxopiperazine dimer (1a) was carried out as follows. To solution of 4a (6.5 mmol, 2.2 g) in THF/MeOH/H2O (12:4:1, total volume of 120 mL) at 0° C. was added lithium hydroxide monohydrate (16.3 mmol, 0.7 g). The mixture was stirred for 2 h at 0° C. and then acidified to pH 3 with saturated aqueous sodium bisulfate. The mixture was concentrated and the residue was dissolved in ethyl acetate and washed with brine (2:1). The aqueous layer was extracted 3× with ethyl acetate, the combined organic layers were dried with anhydrous sodium sulfate, and concentrated under vacuum to yield 2.2 g of product. The residue was used in the next step without further purification.
A portion of the residue from above (1.3 mmol, 0.40 g), HOBt (2.6 mmol, 0.40 g) and DCC (1.3 mmol, 0.30 g) were dissolved in 50 mL of DMF. The reaction mixture was stirred for 15 min at room temperature followed by the addition of 3b (0.6 mmol, 0.3 g) in DMF (5 mL). The reaction mixture was heated at 55° C. for 48 h. Then, the reaction mixture was cooled to 25° C. and diluted with 100 mL of water and extracted with diethyl ether (100 mL, 3×). The combined organic layers were washed sequentially with 1M NaOH (50 mL, 3×), water (50 mL), 1M HCl (50 mL, 3×), and brine (50 mL). The solution was dried with anhydrous MgSO4 and concentrated under vacuum. The residue was purified by column chromatography with 20% ethyl acetate in hexane to yield compound 1a as a yellow oil (0.2 g, 73%). 1H NMR (400 MHz, CDCl3) 7.29-7.08 (m, 10H), 5.31 (t, J=7.2 Hz, 1H), 5.25-5.15 (m, 1H), 5.03 (s, 2H), 4.65 (t, J=7.0 Hz, 1H), 4.46 (br, 1H), 3.77-3.72 (m, 2H), 3.69 (s, 3H), 3.38-3.33 (m, 2H), 3.31-3.15 (m, 4.5H), 3.15-2.92 (m, 3.5H), 1.50 (t, J=7.1 Hz, 2H), 1.38 (s, 9H), 1.34 (d, J=7.0 Hz, 3H), 1.34-1.15 (m, 5H), 1.02-0.93 (m, 2H), 0.87 (d, J=6.6 Hz, 3H), 0.83 (d, J=6.6 Hz, 3H); 13C NMR (100 MHz, CDCl3) 168.0, 167.2, 166.1, 166.0, 153.9, 151.1, 134.2, 133.6, 126.3, 126.2, 126.0, 125.6, 124.6, 113.4, 76.4, 64.1, 54.9, 53.3, 50.8, 50.0, 47.2, 41.1, 39.0, 38.3, 38.2, 35.3, 34.9, 32.0, 29.2, 25.8, 22.3, 20.4, 20.2, 19.8, 15.6; HRMS m/z for C42H59N5O9 [M+Na]+, calcd 800.4210. found 800.4248.
Example 3
Synthesis of Oligooxopiperazine 1b
The synthesis of oligooxopiperazine dimer 1b of the present invention is illustrated in Scheme 1 above.
The synthesis of (S)—N-allyl-Phe-OMe (5b) was carried out as follows. Allyl bromide (140.0 mmol, 9.8 mL) was added to a solution of H-Phe-OMe (46.0 mmol, 10.0 g), DMF (130 mL) and TEA (164.0 mmol, 22.6 mL) at 0° C., and the reaction mixture was warmed to 25° C. After 48 h, the reaction mixture was diluted with 250 mL of water and extracted with diethyl ether (200 mL, 3×). The combined organic layers were washed with saturated aqueous sodium bicarbonate and saturated brine, dried with MgSO4 and concentrated under vacuum. The residue was purified with column chromatography (15% ethyl acetate in hexane) to afford compound 5b as a light yellow oil 6.7 g (66%). 1H NMR (400 MHz, CDCl3) 7.32-7.16 (m, 5H), 5.83 (ddt, J=17.1, 10.2, 6.1 Hz, 1H), 5.14 (dd, J=17.1, 1.5 Hz, 1H), 5.09 (dd, J=10.2, 1.5 Hz, 1H), 3.64 (s, 3H), 3.56 (t, J=6.8 Hz, 1H), 3.26 (ddt, J=6.8, 1.4 Hz, 1H), 3.15 (ddt, J=6.8, 1.4 Hz, 1H), 2.96 (d, J=6.9 Hz, 2H), 1.59 (br, 1H); 13C NMR (100 MHz, CDCl3) 175.0, 137.2, 136.0, 129.2, 128.4, 126.8, 116.5, 62.0, 51.6, 50.6, 39.7; HRMS m/z for C13H17NO2 [M+H]+, calcd 220.1338. found 220.1344.
The synthesis of Boc-Lys(Z)—N(allyl)-Phe-OMe (2b) was carried out as follows. A solution of Boc-Lys(Z)—OH (29.7 mmol, 11.3 g), HOBt (29.7 mmol, 4.0 g) and DCC (29.7 mmol, 6.1 g) in 200 mL of DMF was stirred at 25° C. After 15 min, a solution of 5b (22.8 mmol, 5.0 g) in DMF (5 mL) was added. The mixture was heated at 55° C. After 48 h, the mixture was cooled to 25° C., diluted with 400 mL of water and extracted with diethyl ether (300 mL, 3×). The combined organic layers were sequentially washed with 1M NaOH (400 mL, 3×), water (400 mL), 1M HCl (400 mL, 3×), and brine (400 mL). The organic layer was dried with MgSO4 and concentrated under vacuum. The residue was purified by column chromatography using 20% ethyl acetate in hexane. The purified product yielded compound 2b as a yellow oil (3.5 g, 26%). 1H NMR (400 MHz, CDCl3) 7.37-7.14 (m, 10H), 5.63-5.53 (m, 1H), 5.22-5.08 (m, 4H), 5.07 (s, 2H), 5.01 (br, 1H), 4.47-4.39 (m, 2H) 3.91-3.81 (br, 1H), 3.69 (s, 3H), 3.50-3.31 (m, 2H), 3.21-3.09 (m, 2H), 1.77 (br, 2H), 1.57-1.48 (m, 4H), 1.41 (s, 9H); 13C NMR (100 MHz, CDCl3) 170.8, 168.9, 154.5, 153.6, 135.7, 134.7, 131.2, 127.6, 127.4, 126.9, 126.6, 126.3, 126.2, 125.0, 116.8, 114.5, 77.8, 64.7, 58.7, 50.4, 49.4, 48.3, 38.9, 32.9, 31.3, 27.4, 26.4, 20.6. HRMS m/z for C32H43N3O7 [M+Na]+, calcd 604.2999. found 604.3005.
The synthesis of (LyszPhe)Oxopiperazine-methylester (3b) was carried out as follows. Ozone was bubbled through a solution of 2b (3.3 mmol, 1.9 g) in anhydrous methanol (12 mL) at −78° C. and ambient pressure. The reaction mixture turned light blue after 2 h. After an additional 30 min, nitrogen was bubbled through until the blue color disappeared. Dimethyl sulfide (11.6 mmol, 0.9 mL) was added to the mixture and the reaction was stirred for 12 h at 25° C. The reaction mixture was concentrated under vacuum, and the residue (1.9 g) was redissolved in 23.7 mL of DCM, and triethylsilane (6.7 mmol, 1.1 mL) and TFA (49.9 mmol, 3.7 mL) were added. The mixture was stirred for 24 h at 25° C. and then concentrated under vacuum. The residue was dissolved in DCM (12 mL) and TEA (12 mL) at 0° C. and stirred for 1 h at 25° C. The solution was re-concentrated under vacuum and the residue dissolved in DCM (200 mL). The DCM solution was washed with saturated aqueous sodium bicarbonate (150 mL). The aqueous layer was extracted with DCM (150 mL) The organic layers were combined, dried over anhydrous MgSO4, and concentrated. The residue was purified with column chromatography (95% diethyl ether, 5% methanol and 0.1% of TEA) to obtain compound 3b as a colorless oil (0.9 g, 80%). 1H NMR (400 MHz, CDCl3) 7.26-7.09 (m, 10H), 5.07-5.03 (m, 1H), 5.00 (s, 2H), 4.84 (br, 1H), 3.64 (s, 3H), 3.45-3.26 (m, 2H), 3.26-3.21 (m, 1H), 3.19-3.17 (m, 3H), 3.03-2.99 (m, 2H), 2.96-2.86 (m, 1H), 1.58-1.45 (m, 3H), 1.34-1.31 (m, 2H), 1.16-0.95 (br, 2H); 13C NMR (100 MHz, CDCl3) 170.0, 169.5, 155.4, 135.9, 135.7, 127.8, 127.7, 127.5, 127.4, 127.1, 127.0, 125.8, 65.5, 58.0, 57.2, 51.3, 45.6, 40.8, 39.6, 33.1, 30.7, 28.6, 21.1; HRMS m/z for C26H33N3O5 [M+H]+, calcd 468.2498. found 468.2500.
The synthesis of Boc-Oxopiperazine-methylester (4b) was carried out as follows. To a solution of 3b (3.2 mmol, 1.5 g) in 11.7 mL of DCM at 0° C. was added 4-methylmorpholine (4.8 mmol, 0.5 mL) and di-tert-butyl dicarbonate (8.0 mmol, 1.8 g) in 24 mL of DCM. The mixture was allowed to warm to 25° C. and then heated at reflux for 6 h. The mixture was concentrated and the residue was purified by column chromatography (40% hexane in diethyl ether) to yield 1.7 g (94%) of compound 5c as a colorless oil. 1H NMR (400 MHz, CDCl3) 7.41-7.08 (m, 10H), 5.30-5.26 (m, 1H), 5.02 (s, 2H), 4.74 (br, 1H), 4.32 (br, 1H), 3.68 (s, 3H), 3.35-3.29 (m, 1H), 3.16-3.08 (m, 1H), 3.07-2.93 (m, 5H), 1.45-1.29 (m, 5H), 1.34 (s, 9H), 1.13-0.93 (br, 2H); 13C NMR (100 MHz, CDCl3) 170.7, 168.9, 156.4, 153.9, 136.7, 136.4, 128.9, 128.6, 128.5, 128.1, 128.0, 127.0, 80.6, 66.6, 57.2, 52.5, 43.3, 40.8, 34.4, 32.2, 28.3, 22.8; HRMS m/z for C31H41N3O7 [M+Na]+, calcd 590.2842. found 590.2845.
The synthesis of oxopiperazine dimer (1b) was carried out as follows. Lithium hydroxide monohydrate (4.4 mmol, 0.2 g) was added to solution of 4b (1.8 mmol, 1 g) in 12:4:1 THF/MeOH/H2O (32 mL) at 0° C. The reaction mixture was stirred for 2 h at 0° C. and then acidified to pH 3 with saturated aqueous sodium bisulfate. The reaction mixture was concentrated and the residue was dissolved in ethyl acetate (15 mL) and washed with brine (10 mL). The aqueous layer was extracted with ethyl acetate (15 mL), and the combined organic layers were dried with anhydrous sodium sulfate and concentrated under vacuum to yield 1.1 g of residue. The residue was used in the next step without further purification.
A portion of the residue from above (0.6 mmol, 0.3 g), HOBt (1.2 mmol, 0.2 g) and DCC (0.6 mmol, 0.1 g) were dissolved in 100 mL of DMF. The reaction mixture was stirred for 15 min at 25° C. and 3a (0.3 mmol, 0.1 g) in DMF (5 mL) was added. The mixture was heated at 55° C. After 48 h, the solution was cooled to room temperature and diluted with 100 mL of water and extracted with diethyl ether (100 mL, 3×). The combined organic layers were washed sequentially with 1M NaOH (50 mL, 3×), water (50 mL), 1M HCl (50 mL, 3×) and brine (50 mL), dried with MgSO4 and concentrated under vacuum. The residue was purified by column chromatography with 20% ethyl acetate in hexane to obtain compound 1b as a yellow oil (0.1 g, 70%). 1H NMR (400 MHz, CDCl3) 7.29-7.11 (m, 10H), 5.72-5.63 (m, 1H), 5.22-5.16 (m, 1H), 5.02 (s, 2H), 4.88-4.80 (m, 1H), 4.34 (br, 1H), 3.91 (br, 1H), 3.64 (s, 3H), 3.59-3.54 (m, 2H), 3.46-3.41 (m, 1H), 3.38-3.21 (m, 2H), 3.15-2.90 (m, 6H), 1.93-1.57 (m, 5H), 1.36-1.22 (m, 15H), 1.19-1.06 (m, 2H), 0.87 (d, J=6.6 Hz, 3H), 0.81 (d, J=6.6 Hz, 3H); 13C NMR (100 MHz, CDCl3) 170.6, 168.5, 168.2, 167.5, 166.2, 135.2, 134.9, 128.4, 128.3, 128.2, 127.6, 127.5, 127.48, 127.46, 127.1, 126.0, 79.9, 65.6, 52.4, 51.4, 51.3, 51.2, 41.3, 39.8, 39.4, 35.9, 35.8, 34.1, 33.9, 28.9, 28.7, 27.3, 27.27, 24.0, 22.2, 22.1, 21.9, 20.2; HRMS m/z for C42H59N5O9 [M+Na]+, calcd 800.4210. found 800.4233.
Example 4
Synthesis of Oligooxopiperazine 1c
The synthesis of oligooxopiperazine dimer 1c of the present invention is illustrated in Scheme 1 above.
The synthesis of Boc-Leu-N(allyl)-Leu-OMe (2c) was carried out as follows. A solution of Boc-Leu-OH (78.6 mmol, 24.1 g), HOBt (78.6 mmol, 10.6 g) and DCC (78.6 mmol, 16.2 g) in 200 mL of DMF was stirred at 25° C. After 15 min, a solution of 5a (39.3 mmol, 7.3 g) was added in DMF (5 mL), and the resulting mixture was heated at 55° C. for 12 h. The mixture was cooled to 25° C., diluted with 400 mL of water and extracted with diethyl ether (300 mL, 3×). The combined organic layers were washed sequentially with 1M NaOH (500 mL, 3×), water (500 mL), 1M HCl (500 mL, 3×), and brine (500 mL). The organic layer was dried with anhydrous MgSO4 and concentrated under vacuum. The residue was purified by column chromatography (20% ethyl acetate in hexane) to afford 2c as a yellow oil (6.2 g, 40%). 1H NMR (400 MHz, CDCl3) 5.92-5.74 (m, 1H), 5.22-5.16 (m, 2H), 5.06-4.97 (m, 2H), 4.57-4.54 (m, 1H), 4.01-3.91 (br, 2H), 3.65 (s, 3H), 1.79-1.56 (m, 3H), 1.54-1.43 (m, 3H), 1.35 (s, 9H), 0.93-0.74 (m, 12H); 13C NMR (100 MHz, CDCl3) 174.4, 172.2, 155.5, 134.2, 117.7, 79.4, 55.4, 52.1, 49.3, 48.3, 42.4, 37.8, 28.3, 24.6, 23.5, 22.9, 21.7; HRMS m/z for C21H38N2O5 [M+H]+, calcd 398.2859. found 399.2862.
The synthesis of (LeuLeu)Oxopiperazine methyl ester (3c) was carried out as follows. Ozone was bubbled through a solution of 2c (11.0 mmol, 4.4 g) in anhydrous methanol (75 mL) at −78° C. and ambient pressure. The reaction mixture turned light blue after 2 h. After an additional 30 min, nitrogen was bubbled through until the blue color disappeared. Dimethyl sulfide (38.5 mmol, 2.8 mL) was added and the mixture stirred for 12 h at 25° C. The mixture was concentrated under vacuum, and the residue was redissolved in DCM (78 mL), triethylsilane (21.9 mmol, 3.5 mL) and TFA (165.0 mmol, 12.2 mL). The reaction mixture was stirred for 24 h at 25° C. and then concentrated under vacuum. The residue was dissolved in 39 mL of DCM and 39 mL of TEA at 0° C. and stirred for h at 25° C. The solvent was then concentrated under vacuum. The residue was redissolved in DCM (300 mL), and the solution washed with saturated aqueous sodium bicarbonate (300 mL). The aqueous layer was extracted with DCM (200 mL, ×3). The combined organic layers were concentrated and the residue was purified with column chromatography (95% diethyl ether, 5% methanol, and 0.1% of TEA) to obtain compound 3c as a colorless oil (2.2 g, 85%). 1H NMR (400 MHz, CDCl3) 5.26-5.22 (t, J=7.8 Hz, 1H), 3.65 (s, 3H), 3.48-3.38 (m, 1H), 3.27-3.17 (m, 2H), 3.14-3.09 (m, 1H), 3.02-2.95 (m, 1H), 1.83-1.76 (m, 1H), 1.74-1.61 (m, 4H), 1.52-1.41 (m, 2H), 0.89-0.85 (m, 12H); 13C NMR (100 MHz, CDCl3) 172.3, 171.5, 57.7, 53.8, 52.1, 44.7, 42.0, 41.6, 36.7, 24.9, 24.5, 23.5, 23.2, 21.3, 21.1; HRMS m/z for C15H28N2O3 [M+H]+, calcd 285.2178. found 285.2182.
The synthesis of Boc-(LeuLeu)oxopiperazine-methyl ester (4c) was carried out as follows. To a solution of 3c (7.7 mmol, 2.2 g) in 25 mL of DCM at 0° C. was added 4-methylmorpholine (11.5 mmol, 1.3 mL) and ditert-butyl dicarbonate (19.2 mmol, 4.2 g) in 50 mL of DCM. The mixture was allowed to warm to 25° C., and then heated at reflux. After 6 h, the mixture was concentrated and the residue was purified by column chromatography (40% hexane in diethyl ether) to yield 2.9 g (97%) of compound 4c as a colorless oil. 1H NMR (400 MHz, CDCl3) 5.21-5.17 (m, 1H), 4.53 (br, 1H), 3.95 (br, 1H), 3.66 (s, 3H), 3.44-3.34 (m, 1H), 3.23 (br, 1H), 3.15-3.10 (m, 1H), 1.70-1.62 (m, 3H), 1.60-1.52 (m, 3H), 1.45 (s, 9H), 0.91-0.81 (m, 12H); 13C NMR (100 MHz, CDCl3) 171.6, 169.7, 154.1, 80.7, 56.2, 53.6, 52.1, 41.9, 41.8, 37.7, 36.8, 28.3, 24.9, 24.6, 23.2, 22.8, 22.3, 21.2; HRMS m/z for C20H36N2O5 [M+Na]+, calcd 407.2522. found 407.2510.
The synthesis of oxopiperazine dimer (1c) was carried out as follows. To solution of 4c (5.4 mmol, 1.9 g) in 12:4:1 THF/MeOH/H2O (100 mL) at 0° C. was added lithium hydroxide monohydrate (16.4 mmol, 0.7 g). The reaction mixture was stirred for 2 h at 0° C. and then acidified to pH 3 with saturated aqueous sodium bisulfate. The reaction mixture was concentrated and the residue was dissolved in ethyl acetate (100 mL) and washed with brine (50 mL). The aqueous layer was extracted with ethyl acetate (100 mL), and the combined organic layers were dried with anhydrous sodium sulfate and concentrated to yield 2 g of product residue. The residue was used in the next step without further purification.
A portion of the residue from above (1.8 mmol, 0.70 g), HOBt (3.5 mmol, 0.50 g) and DCC (1.8 mmol, 0.40 g) were dissolved in 50 mL of DMF. The solution was stirred for 15 min at 25° C., and 3c (0.9 mmol, 0.3 g) was added in DMF (5 mL). The mixture was heated at 55° C. for 48 h and then cooled to 25° C. and diluted with water (100 mL) and extracted with diethyl ether (100 mL, 3×). The combined organic layers were washed sequentially with 1M NaOH (50 mL, 3×), water (50 mL), 1M HCl (50 mL, 3×) and brine (50 mL), dried with MgSO4, and concentrated under vacuum. The residue was purified by column chromatography with 20% ethyl acetate in hexane to obtain compound 1c as a yellow oil (0.2 g, 71%). 1H NMR (400 MHz, CDCl3) 5.60-5.58 (t, J=7.4 Hz, 1H), 5.28-5.25 (m, 1H), 5.12-5.08 (m, 1H), 4.58 (br, 1H), 4.23 (br, 1H), 4.12 (br, 1H), 3.66 (s, 3H), 3.48-3.42 (m, 1H), 3.39-3.24 (m, 4H), 3.20-3.11 (m, 1H), 1.73-1.49 (m, 10H), 1.45 (s, 9H), 1.44-1.36 (m, 2H), 0.97-0.88 (m, 24H); 13C NMR (100 MHz, CDCl3) 171.8, 171.7, 169.6, 169.2, 154.1, 54.9, 54.5, 53.5, 53.3, 52.3, 52.2, 42.5, 42.2, 41.7, 41.5, 41.4, 41.2, 40.6, 39.5, 37.6, 37.4, 37.0, 36.8, 28.3, 28.0, 25.1, 25.0, 24.9, 24.8, 24.7; HRMS m/z for C34H60N4O7 [M+Na]+, calcd 659.4360. found 659.4350.
Example 5
Two-Dimensional NMR Spectroscopy of Oligooxopiperazine 1a
COSY spectrum of 1a was recorded on a Bruker Avance 400 at 20° C. by collecting 2048 complex data points in the t2 domain by averaging 32 scans and 256 increments in the t1 domain with States-TPPI mode. The original free induction decays (FIDs) were zero-filled to give a final matrix of 1024 by 1024 real data points. A 0° sine-bell window function was applied in both dimensions. NOESY spectrum of 1a was recorded on a Bruker Avance 600 at 20° C. by collecting 4096 complex data points in the t2 domain by averaging 48 scans and 512 increments in the t1 domain with States-TPPI mode and the mixing time of 750 ms. The original free induction decays (FIDs) were zero-filled to give a final matrix of 2048 by 1024 real data points. A 90° sine-square window function was applied in both dimensions. All the data were processed and analyzed using Bruker TOPSPIN 1.3 program.
Example 6
Circular Dichroism (CD) Spectroscopy Studies
CD spectra were recorded on AVIV 202SF CD spectrometer equipped with a temperature controller using 1 mm length cells and a scan speed of 15 nm/min. The spectra were averaged over 10 scans with the baseline subtracted from analogous conditions as that for the samples. The samples were prepared in acetonitrile or methanol with the final peptide concentration of 100 M. The amount of oxopiperazines were determined by dry weight.
Example 7
Conformational Analysis of Oligooxopiperazines
The present invention relates to the design and synthesis of nonaromatic helix mimetics which feature a chiral backbone and are easily synthesized from α-amino acids. The piperazine skeleton was an attractive design choice because it is considered a privileged scaffold for peptidomimetic research and drug discovery (Patchett et al., Ann. Rep. Med. Chem. 35: 289-298 (2000), which is hereby incorporated by reference in its entirety). Specifically, the 2-oxopiperazine and the diketopiperazines have a rich history in medicinal chemistry and are considered to be “drug-like” scaffolds (Herrero et al., J. Org. Chem. 67:3866-3873 (2002); Kitamura et al., J. Med. Chem. 44:2438-2450 (2001); Gante, J., “Peptidomimetics—Tailored Enzyme-Inhibitors,” Angew. Chem. Int. Ed. Engl. 33:1699-1720 (1994); Giannis et al., Angew. Chem. Int. Ed. 32:1244-1267 (1993), which are hereby incorporated by reference in their entirety). Initial computational studies of the oligooxopiperazines predicted stable structures due to the conformational constraints inherent in the system. Molecular modeling studies indicate that an oxopiperazine dimer spans the length of an 8mer α-helix and superimposes amino acid functionality onto the i, i+4, and i+7 residues of the helix (FIG. 2B). Oligooxopiperazines do not contain hydrogen bond donors in the backbone; however, this omission is not expected to be detrimental for helix mimetics because helices typically do not utilize backbone hydrogen bonding functionality for interaction with other biomolecules.
The analysis of oligooxopiperazines was started by searching the Cambridge Structural Database for examples of oxopiperazine derivatives. This search resulted in five hits (CSD codes: KEMXUV, ZOZTUD, ZARZOH, FOBFEH, and KEMXUV) of single piperazine ring systems relevant to the system. Although this is a narrow set to base hypotheses upon, these hits provided invaluable insights regarding the φ and ψ dihedral angles favored in the amino acid residue linking two piperazine rings and corroborated the molecular modeling calculations (FIGS. 3A-3C).
The oxopiperazine rings may adopt the half-chair or the boat conformation, but the half-chair conformation is substantially lower in energy,  ̃2.9 Kcal/mol (FIG. 3B). A dimer of oxopiperazines contains three rotatable bonds φ, ψ, and ω. The tertiary amide bond may adopt a cis or trans amide conformation like proline as shown in FIG. 3C. Macromodel calculations suggest that the trans conformation is roughly 1 Kcal/mol more stable than the cis conformation in tetraalanine systems. The trans to cis ratio is expected to increase in dimers built from bulkier amino acid residues.
To examine the preferred φ and ψ dihedral angles in an oxopiperazine dimer, dimer 30 (see FIG. 7), the “dihedral drive” functionality in Macromodel was utilized (Mohamadi et al., J. Comp. Chem. 11:440-467 (1990), which is hereby incorporated by reference in its entirety). The results of these calculations intimate a limited number and a narrow range of φ and ψ values in the lowest energy conformations (Table 1 below and FIGS. 7A-7B). Importantly, the dihedral angles predicted by Macromodel were also found in the crystal structures of relevant compounds in CSD (Table 1). The calculations indicate that oligooxopiperazines will favor φ and ψ angles of −128°±25° and 76°±15°, respectively. The favored φ values show direct correlation with allyl 1,2 and 1,3 strains.







TABLE 1







Calculated Low Energy and Values for Oxopiperazine Dimer 30.


30


 








 











Dihedral
Relative energy
Cambridge structure



angle (°)
(Kcal/mol)
database code*













 
−150
0.95
—



−128
0
KEMXUV, ZOZTUD



−90
1.26
ZARZOH


ψ
60
0.64
—



76.76
0
ZARZOH



90
0.34
—



120
1.87
—





*the corresponding dihedral value was found in the indicated CSD structure.






The predicted low energy structure of the oxopiperazine dimer arrays functionality to match side chain patterns on a canonical α-helix (FIG. 2). Similarly, the predicted low energy structure of the oxopiperazine trimer arrays functionality to match side chain patterns on a canonical α-helix (FIG. 8). Positions 1, 2, 3, and 4 overlay well onto the i+1, i+2, i+3 and i+7 residues on a 10mer α-helix; while the i+1, i+4 and i+7 positions are best mimicked by positions 1, 2, and 5 of an oligooxopiperazine (FIG. 8). This level of structural versatility has not been observed with other nonpeptidic helix mimetics, which typically only mimic one face of the helix (Davis et al., Chem. Soc. Rev. 36:326-334 (2007); Yin et al., Angew. Chem. Int. Ed., 44: 4130-4163 (2005), which are hereby incorporated by reference in their entirety.
Oligooxopiperazines 1a-c were designed to test the impact of different side chain combinations on the stability of the oxopiperazine dimer conformation. Several synthetic routes to piperazines are known, which were anticipated to allow rapid synthesis and evaluation of the desired compounds (Franceschini et al., Org. Biomol. Chem. 3:787-793 (2005); Tong et al., J. Org. Chem. 65:2484-2493 (2000); Sugihara et al., J. Med. Chem. 41:489-502 (1998), which are hereby incorporated by reference in their entirety). While a number of these synthetic routes were evaluated, it was discovered that the reductive amination route described by Tong et al., J. Org. Chem., 65:2484-93 (2000), which is hereby incorporated by reference in its entirety, can afford short oligomers in respectable yields (FIG. 4B and Scheme 1).
The solution conformation of dimers 1a-c was investigated by CD spectroscopy in methanol and acetonitrile solutions. FIGS. 5A and 5C show CD spectra of oxopiperazine 1a, 1b, and 1c in acetonitrile and methanol, respectively. The CD spectra of 1a-c display double minima near 220 and 230 nm and maxima at 200 nm. Surprisingly, the overall shape is reminiscent of CD spectra of α-helices; although, the maxima and minima are red-shifted by 10 nm. Although CD spectra of artificial systems are often difficult to interpret (Driver et al., Org. Lett. 11:3092-3095 (2009), which is hereby incorporated by reference in its entirety), the spectra of 1a-c indicate a high degree of preorganization. The thermal stabilities of 1a-c were investigated by monitoring the temperature-dependent change in the intensity of the 220 nm bands in the CD spectra (FIG. 5B). A gradual increase in the signal intensity was observed at 220 nm with temperature, but the dimers retain over 70% of their room-temperature elipticity at 75° C. Similar non-cooperative denaturation behavior has been observed with other conformationally defined oligomers (Saludes et al., Am. Chem. Soc. 131:5495-5505 (2009); Wang et al., Org. Biomol. Chem. 4:4074-4081 (2006). Overall, the CD studies demonstrate that helix mimetics 1a-c adopt stable conformations confirming the molecular modeling analysis.
Two-dimensional NMR spectroscopy was also utilized to analyze the conformations adopted by 1a as a model oxopiperazine helix mimetic, specifically to determine the geometry adopted by the tertiary amide bond linking two piperazine rings. A combination of COSY and NOESY spectroscopy was used to assign 1H NMR resonances for 1a. The NOESY spectrum reveals several NOEs in the two-ring system, which would be expected from a trans-amide geometry in 1a but not from the cis-amide conformation (FIG. 6A). NOE crosspeaks were not observed between protons on neighboring piperazine rings (FIG. 6B). This absence of NOEs is expected based on the proposed low energy conformation in which these protons lie outside the 5 Å distance typically required to observe the nuclear Overhouser effect. Thus, the NOESY studies strongly corroborate the modeling analysis. Significantly, the NMR spectra did not display peaks indicative of a minor cis-amide isomer, suggesting that the trans conformation is substantially more stable than the cis analog.
Example 8
Representative Solid Phase Synthesis of Oligooxopiperazines
An alternative route of oligooxopiperazine synthesis was investigated. Scheme 2 below illustrates a representative solid phase synthesis scheme for the synthesis of oligooxopiperazine dimers (i.e., dimers A, B, and C) and trimers. FIGS. 9A-9D show the predicted structures of the oligooxopiperazine dimers A, B, and C, and trimer as they overlay with the target α-helix. Exemplary dimers and trimers produced via this synthesis approach are shown in Tables 2 and 3 below. The biological protein target of the oligooxopiperazine, the helical sequence of the target protein, and the oligooxopiperazine structure are provided in Tables 2 and 3.
Representative Solid Phase Synthesis of Oxopiperazine Dimer A-D and Trimer

















TABLE 2







Exemplary Oligooxopiperazines and their Helical Targets












Sequence of Helical



Target
Model Type
Partner
Oligooxopiperazine Structure





 


HDM2
Trimer
p5317_28   


 (SEQ ID NO: 186)






 


HDM2
Dimer A
p5317-28   


 (SEQ ID NO: 186)






 


p300-TAZ1
Dimer B
Hifl140-147   


 (SEQ ID NO: 187)






 


p300-KIX
Dimer C
cMyb91-103   


 (SEQ ID NO: 188)






 


p300-SID
Dimer C
p1605-16   


 (SEQ ID NO: 189)






 


p300-IBid
Dimer C
IRF3372-381   


 (SEQ ID NO: 190)









X = H, COCH3, amino acid;


Y = OH, NH2, OMe, amino acid


bold residues indicate key residues for binding.













TABLE 3







Exemplary Oligooxopiperazines and their Helical Targets












Wild Type Sequence



Target
Model Type
Helical Domain*
Oligooxopiperazine Structure*





 


p53/MDM2
Trimer
p5317_28   


 (SEQ ID NO: 186)






 


p53/MDM2
Dimer A
p5317-28   


 (SEQ ID NO: 186)






 


Hifl/p300
Dimer B
Hifl140-147   


 (SEQ ID NO: 187)






 


cMyb/KIX
Dimer C
cMyb91-103   


 (SEQ ID NO: 188)









*bold residues are critical for binding of the helix to the protein partner






Example 9
Biological Potential of Oligooxopiperazines
The potential of the oligooxopiperazine molecules of the present invention to inhibit protein-protein interactions in which helices play key roles at the interfaces will be tested using the Bcl-xL/Bak-BH3 (Sattler et al., Science 275:983-986 (1997), which is hereby incorporated by reference in its entirety) and p53/Mdm2 (Kussie et al., Science 274:948-953 (1996), which is hereby incorporated by reference in its entirety) complexes as targets. Both of these complexes are intimately involved in regulating the crucial process of programmed cell death. These complexes have been chosen for the initial foray into the control of protein-protein interactions with oligooxopiperazines because these protein complexes have been targeted with several different strategies, including small molecules, allowing the evaluation of the suitability of this approach (Murray et al., Biopolymers 88:657-686 (2007); Ernst et al., Angew. Chem. Int. Ed. Engl. 42: 535-539 (2003); Walensky et al., Science 305:1466-70 (2004); Gemperli et al., J. Am. Chem. Soc. 127:1596-7 (2005); Sadowsky et al., J. Am. Chem. Soc. 129: 139-154 (2007); Davis et al., Chem. Soc. Rev. 36:326-334 (2007), which are hereby incorporated by reference in their entirety).
Oligooxopiperazine 38 (FIG. 10C) has been designed and synthesized to mimic the p53 helix. This helix features three hydrophobic residues phenylalanine, tryptophan, and leucine on the same face (at positions i, i+4, and i+7) and it binds in a deep hydrophobic cleft of Mdm2 (FIG. 10A). Modeling studies suggest that oligooxopiperazine trimer positions 1, 2, and 5, respectively, would overlay well onto i, i+4, and i+7 positions of an α-helix (FIG. 10B). Accordingly, oligooxopiperazine 38 was designed to display phenylalanine, tryptophan and leucine side chains at position 1, 2, and 5 of the trimer, respectively (FIG. 10C). For these preliminary studies, oligooxopiperazine trimer 39, which lacks the key tryptophan residue at position 2, has also been synthesized. This negative control will allow assessment of the specificity of oligooxopiperazines for their targets. A oxopiperazine trimer has the potential to display six residues and mimic a 10-mer helix. In this first generation study only three key residues from p53 will be imported into the oligoxopiperazine scaffold; in subsequent studies the other residues from the p53 sequence will also be introduced and studied in an iterative manner.
In summary, through rational design and synthesis, a new class of nonpeptidic α-helix mimetics have been developed. NMR and circular dichroism spectroscopies provide compelling evidence that oligooxopiperazine dimers adopt stable conformations that reproduce the arrangement of i, i+4, and i+7 residues on an α-helix. Given the importance of the helix conformation in protein-protein interactions, and the potential of nonpeptidic scaffolds that mimic this conformation, these oxopiperazine scaffolds will offer attractive new tools for chemical biology (Jochim and Arora, Mol. BioSyst. 5:924-926 (2009); Jones and Thornton, Proc. Natl. Acad. Sci U.S.A. 93:13-20(1996), which are hereby incorporated by reference in their entirety). Oxopiperazine helix mimetics have the potential to disrupt chosen protein-protein interactions.

## Claims

1. A pharmaceutical formulation comprising:
(i) an oligooxopiperazine of Formula I:





wherein:
each of R1, R2, R3, and R4 is independently an amino acid side chain, H, N(R)2, OR, halogen, an alkyl, or an aryl; wherein each R is independently H, an alkyl, or an aryl;
each R6 is independently H, N(R)2, OR, halogen, an alkyl, or an aryl; wherein each R is independently H, an alkyl, or an aryl;
A is X1 or C, wherein:
X1 is H, COR′, CO2R′, CONHR′, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a protecting group for protection of an amine, a targeting moiety, or a tag; wherein R′ is H, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety, or a tag; and
C is a moiety of the formula






 wherein:
each X′ is independently H, COR′, CO2R′, CONHR′, N(R′′)2, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety, or a tag; wherein:
R′ is H, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety, or a tag; and
each R′′ is independently H, CO2R′, CONHR′, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety, or a tag;

R0 is an amino acid side chain, H, N(R)2, OR, halogen, an alkyl, or an aryl;
wherein each R is independently H, an alkyl, or an aryl; and

R6 is H, N(R)2, OR, halogen, an alkyl, or an aryl; wherein each R is independently H, an alkyl, or an aryl; and


B is Y or D, wherein:
Y is OR′, COR′, N(R′′′)2, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a protecting group for protection of a carboxylic acid, a targeting moiety, or a tag; wherein:
R′ is H, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety, or a tag; and
each R′′′ is independently H, CO2R′, CONHR′, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety, or a tag; and

D is a moiety of the formula






 wherein:
R5 is an amino acid side chain, H, N(R)2, OR, halogen, an alkyl, or an aryl; wherein each R is independently H, an alkyl, or an aryl;
R6 is H, N(R)2, OR, halogen, an alkyl, or an aryl; wherein each R is independently H, an alkyl, or an aryl; and
E is X2 or F, wherein:
X2 is H, COR′, CO2R′, CONHR′, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a protecting group for protection of an amine, a targeting moiety, or a tag; wherein R′ is H, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety, or a tag; and
F is a moiety of the formula










 wherein:
 R6 is H, N(R)2, OR, halogen, an alkyl, or an aryl; wherein each R is independently H, an alkyl, or an aryl;
 R7 is an amino acid side chain; and
 Y is OR′, COR′, N(R′′′)2, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a protecting group for protection of a carboxylic acid, a targeting moiety, or a tag; wherein:
 R′ is H, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety, or a tag; and
 each R′′′ is independently H, CO2R′, CONHR′, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety, or a tag;



with the proviso that A and B are not both, respectively, C and D, and
(ii) a pharmaceutically acceptable vehicle.

2. The pharmaceutical formulation according to claim 1, wherein the oligooxopiperazine is an oligooxopiperazine of Formula IA:

3. The pharmaceutical formulation according to claim 2, wherein the oligooxopiperazine is selected from the group consisting of




wherein X is H, COCH3, or any amino acid, and Y is OH, NH2, OMe, or any amino acid.

4. The pharmaceutical formulation according to claim 1, wherein the oligooxopiperazine is an oligooxopiperazine of Formula IB:

5. The pharmaceutical formulation according to claim 4, wherein the oligooxopiperazine is selected from the group consisting of




wherein X is H, COCH3, or any amino acid, and Y is OH, NH2, OMe, or any amino acid.

6. The pharmaceutical formulation according to claim 1, wherein the oligooxopiperazine is an oligooxopiperazine of Formula IC:

7. The pharmaceutical formulation according to claim 6, wherein the oligooxopiperazine is selected from the group consisting of




wherein X is H, COCH3, or any amino acid, and Y is OH, NH2, OMe, or any amino acid.

8. The pharmaceutical formulation according to claim 1, wherein the oligooxopiperazine mimics an α-helix involved in a protein-protein interaction.

9. The pharmaceutical formulation according to claim 2, wherein R1, R2, R4, and R5 mimic the amino acid side chain of, respectively, residues i, i+4, i+6, and i+7 of an α-helix involved in a protein-protein interaction.

10. The pharmaceutical formulation according to claim 4, wherein R1, R2, and R4 mimic the amino acid side chain of, respectively, residues i, i+4, and i+7 of an α-helix involved in a protein-protein interaction.

11. The pharmaceutical formulation according to claim 4, wherein R1, R2, and R4 mimic the amino acid side chain of, respectively, residues i, i+4, and i+6 of an α-helix involved in a protein-protein interaction.

12. The pharmaceutical formulation according to claim 6, wherein R0, R1, R2, R3, and R4 mimic the amino acid side chain of, respectively, residues i, i+2, i+3, i+4, and i+7 of an α-helix involved in a protein-protein interaction.

13. The pharmaceutical formulation according to claim 8, wherein the α-helix is selected from the group consisting of those identified in FIGS. 11A-11I.

14. A method of making an oligooxopiperazine of Formula IA:




wherein:
each of R1, R2, R3, R4, and R5 is independently an amino acid side chain, H, N(R)2, OR, halogen, an alkyl, or an aryl; wherein each R is independently H, an alkyl, or an aryl;
each R6 is independently H, N(R)2, OR, halogen, an alkyl, or an aryl; wherein each R is independently H, an alkyl, or an aryl;
X1 is H, COR′, CO2R′, CONHR′, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a protecting group for protection of an amine, a targeting moiety, or a tag; wherein R′ is H, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety, or a tag; and
E is X2 or F, wherein:
X2 is H, COR′, CO2R′, CONHR′, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a protecting group for protection of an amine, a targeting moiety, or a tag; wherein R′ is H, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety, or a tag; and
F is a moiety of the formula






 wherein:
R6 is H, N(R)2, OR, halogen, an alkyl, or an aryl; wherein each R is independently H, an alkyl, or an aryl;
R7 is an amino acid side chain; and
Y is OR′, COR′, N(R′′′)2, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a protecting group for protection of a carboxylic acid, a targeting moiety, or a tag; wherein:
R′ is H, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety, or a tag; and
each R′′′ is independently H, CO2R′, CONHR′, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety, or a tag;




(i) wherein said method comprises:
providing a compound of Formula III:




 wherein:
PG is a protecting group for the protection of an amine;
R8 is an amino acid side chain, H, N(R)2, OR, halogen, an alkyl, or an aryl; wherein each R is independently H, an alkyl, or an aryl; and
R9 is —O-Res or —NH-Res, wherein Res is a solid phase peptide synthesis resin;

providing a compound of Formula IV1:




 wherein R10 is —OH or a halide; and
reacting the compound of Formula III with a first alkylating agent and the compound of Formula IV1 under conditions effective to produce a compound of Formula V:




wherein:
if E is not —CR6R7—CO—Y, where R7 is the same as R8 and Y is the same as R9, said method further comprises converting —CR6R8—CO—R9 in the compound of Formula V to E; and
if X1 is not hydrogen, said method further comprises converting the N-terminal hydrogen in the compound of Formula V to X1;


or (ii) wherein said method comprises:
providing a compound of Formula X:




 wherein:
PG1 is a protecting group for the protection of an amine, and
R10 is —OH or a halide;

providing a compound of Formula XI5/8:




 wherein:
PG2 is a protecting group for the protection of a carboxylic acid; and
R8 is an amino acid side chain, H, N(R)2, OR, halogen, an alkyl, or an aryl; wherein each R is independently H, an alkyl, or an aryl; and

reacting the compound of Formula X with the compound of Formula XI5/8 under conditions effective to produce a compound of Formula XII:




wherein:
if E is not —CR6R7—CO—Y, where R7 is the same as R8 and Y is the same as PG2, said method further comprises converting —CR6R8—CO—PG2 in the compound of Formula XII to E; and
wherein if X1 is not PG1, said method further comprises converting PG1 in the compound of Formula XII to X1.

15. The method according to claim 14, wherein the first alkylating agent is selected from the group consisting of X—CH2—CH═CH, X—CH2—CH(OR11)2, X—(CH2)2—X, and X—(CH2)2—OH, wherein each X is independently a leaving group and each R11 is independently an alkyl.

16. The method according to claim 14, wherein said providing a compound of Formula III comprises:
providing a compound of Formula VI:




providing a compound of Formula IV2:




 and
reacting the compound of Formula VI with the compound of Formula IV2 under conditions effective to produce a compound of Formula III.

17. The method according to claim 16, wherein said providing a compound of Formula VI comprises:
providing a compound of Formula VII:




providing a compound of Formula IV3:




 and
reacting the compound of Formula VII with a second alkylating agent and the compound of Formula IV3 under conditions effective to produce a compound of Formula VI.

18. The method according to claim 17, wherein the second alkylating agent is selected from the group consisting of X—CH2—CH═CH, X—CH2—CH(OR11)2, X—(CH2)2—X, and X—(CH2)2—OH, wherein each X is independently a leaving group and each R11 is independently an alkyl.

19. The method according to claim 17, wherein said providing a compound of Formula VII comprises:
providing a compound of Formula VIII:




providing a compound of Formula IV4:




 and
reacting the compound of Formula XIII with the compound of Formula IV4 under conditions effective to produce a compound of Formula VII.

20. The method according to claim 19, wherein said providing a compound of Formula VIII comprises:
providing a compound of Formula IX:




providing a compound of Formula IV5:




 and
reacting the compound of Formula IX with a third alkylating agent and the compound of Formula IV5 under conditions effective to produce a compound of Formula VIII.

21. The method according to claim 20, wherein the third alkylating agent is selected from the group consisting of X—CH2—CH═CH, X—CH2—CH(OR11)2, X—(CH2)2—X, and X—(CH2)2—OH, wherein each X is independently a leaving group and each Rif is independently an alkyl.

22. A method of making an oligooxopiperazine of Formula IB:




wherein:
each of R1, R2, R3, and R4 is independently an amino acid side chain, H, N(R)2, OR, halogen, an alkyl, or an aryl; wherein each R is independently H, an alkyl, or an aryl;
each R6 is independently H, N(R)2, OR, halogen, an alkyl, or an aryl; wherein each R is independently H, an alkyl, or an aryl;
X1 is H, COR′, CO2R′, CONHR′, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a protecting group for protection of an amine, a targeting moiety, or a tag; wherein R′ is H, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety, or a tag; and
Y is OR′, COR′, N(R′′′)2, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a protecting group for protection of a carboxylic acid, a targeting moiety, or a tag; wherein:
R′ is H, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety, or a tag; and
each R′′′ is independently H, CO2R′, CONHR′, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety, or a tag;


(i) wherein said method comprises:
providing a compound of Formula VII′:




 wherein:
PG is a protecting group for the protection of an amine; and
R9 is —O-Res or —NH-Res, wherein Res is a solid phase peptide synthesis resin;

providing a compound of Formula IV1:




 wherein R10 is —OH or a halide;
reacting the compound of Formula VII′ with a first alkylating agent and the compound of Formula IV1 under conditions effective to produce a compound of Formula VI′:




 and
converting —R9 in the compound of Formula VI′ to Y;
wherein if X1 is not hydrogen, said method further comprises converting the N-terminal hydrogen in the compound of Formula VI′ to X1;

or (ii) wherein said method comprises:
providing a compound of Formula XIII:




 wherein:
PG1 is a protecting group for the protection of an amine, and
R10 is —OH or a halide;

providing a compound of Formula XI3/4:




 wherein PG2 is a protecting group for the protection of a carboxylic acid; and
reacting the compound of Formula XIII with the compound of Formula XI3/4 under conditions effective to produce a compound of Formula X′:




 and
converting —PG2 in the compound of Formula X′ to Y;
wherein if X1 is not PG1, said method further comprises converting PG1 in the compound of Formula X′ to X1.

23. The method according to claim 22, wherein the first alkylating agent is selected from the group consisting of X—CH2—CH═CH, X—CH2—CH(OR11)2, X—(CH2)2—X, and X—(CH2)2—OH, wherein each X is independently a leaving group and each Rif is independently an alkyl.

24. The method according to claim 22, wherein said providing a compound of Formula VII′ comprises:
providing a compound of Formula VIII′:




providing a compound of Formula IV2:




 and
reacting the compound of Formula VIII′ with the compound of Formula IV2 under conditions effective to produce a compound of Formula VII′.

25. The method according to claim 24, wherein said providing a compound of Formula VIII′ comprises:
providing a compound of Formula IX′:




providing a compound of Formula IV3:




 and
reacting the compound of Formula IX′ with a second alkylating agent and the compound of Formula IV3 under conditions effective to produce a compound of Formula VIII′.

26. The method according to claim 25, wherein the second alkylating agent is selected from the group consisting of X—CH2—CH═CH, X—CH2—CH(OR11)2, X—(CH2)2—X, and X—(CH2)2—OH, wherein each X is independently a leaving group and each Rii is independently an alkyl.

27. A method of making an oligooxopiperazine of Formula IC:




wherein:
each of R0, R1, R2, R3, and R4 is independently an amino acid side chain, H, N(R)2, OR, halogen, an alkyl, or an aryl; wherein each R is independently H, an alkyl, or an aryl;
each R6 is independently H, N(R)2, OR, halogen, an alkyl, or an aryl; wherein each R is independently H, an alkyl, or an aryl;
X′ is H, COR′, CO2R′, CONHR′, N(R′′)2, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety, or a tag; wherein:
R′ is H, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety, or a tag; and
each R′′ is independently H, CO2R′, CONHR′, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety, or a tag; and

Y is OR′, COR′, N(R′′′)2, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a protecting group for protection of a carboxylic acid, a targeting moiety, or a tag; wherein:
R′ is H, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety, or a tag; and
each R′′′ is independently H, CO2R′, CONHR′, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety, or a tag;


(i) wherein said method comprises:
providing a compound of Formula VII′:




 wherein:
PG is a protecting group for the protection of an amine; and
R9 is —O-Res or —NH-Res, wherein Res is a solid phase peptide synthesis resin;

providing a compound of Formula IV1:




 wherein R10 is —OH or a halide;
reacting the compound of Formula VII′ with a first alkylating agent and the compound of Formula IV1 under conditions effective to produce a compound of Formula VI′:




converting —R9 in the compound of Formula VI′ to Y; and
converting the N-terminal hydrogen in the compound of Formula VI′ to a moiety of formula





or (ii) wherein said method comprises:
providing a compound of Formula XIII:




 wherein:
PG1 is a protecting group for the protection of an amine, and
R10 is —OH or a halide;

providing a compound of Formula XI3/4:




 wherein PG2 is a protecting group for the protection of a carboxylic acid; and
reacting the compound of Formula XIII with the compound of Formula XI3/4 under conditions effective to produce a compound of Formula X′:




converting —PG2 in the compound of Formula X′ to Y; and
converting PG1 in the compound of Formula X′ to a moiety of formula

28. The method according to claim 27, wherein the first alkylating agent is selected from the group consisting of X—CH2—CH═CH, X—CH2—CH(OR11)2, X—(CH2)2—X, and X—(CH2)2—OH, wherein each X is independently a leaving group and each R11 is independently an alkyl.

29. The method according to claim 27, wherein said providing a compound of Formula VII′ comprises:
providing a compound of Formula VIII′:




providing a compound of Formula IV2:




 and
reacting the compound of Formula VIII′ with the compound of Formula IV2 under conditions effective to produce a compound of Formula VII′.

30. The method according to claim 29, wherein said providing a compound of Formula VIII′ comprises:
providing a compound of Formula IX′:




providing a compound of Formula IV3:




 and
reacting the compound of Formula IX′ with a second alkylating agent and the compound of Formula IV3 under conditions effective to produce a compound of Formula VIII′.

31. The method according to claim 14, wherein said providing a compound of Formula X comprises:
providing a compound of Formula X′:




 and
converting PG1 in the compound of Formula X′ to hydrogen and converting PG2 in the compound of Formula X′ to R10 to produce a compound of Formula X.

32. The method according to claim 31, wherein said providing a compound of Formula X′ comprises:
providing a compound of Formula XIII:




providing a compound of Formula XI3/4:




 and
reacting the compound of Formula XIII with the compound of Formula XI3/4 under conditions effective to produce a compound of Formula X′.

33. The method according to claim 32, wherein said providing a compound of Formula XIII comprises:
providing a compound of Formula XI1/2:




 and
reacting the compound of Formula XI1/2 with a protecting group under conditions effective to produce a compound of Formula XIII.

34. The method according to claim 33, wherein said providing a compound of Formula XI1/2 comprises:
providing a compound of Formula XIV:




providing a compound of Formula XV:




 and
reacting the compound of Formula XIV with an alkylating agent and the compound of Formula XV under conditions effective to produce a compound of Formula XI1/2.

35. The method according to claim 34, wherein the alkylating agent is selected from the group consisting of X—CH2—CH═CH, X—CH2—CH(OR11)2, X—(CH2)2—X, and X—(CH2)2—OH, wherein each X is independently a leaving group and each R11 is independently an alkyl.

36. The method according to claim 22, wherein said providing a compound of Formula XIII comprises:
providing a compound of Formula XI1/2:




 and
reacting the compound of Formula XI1/2 with a protecting group under conditions effective to produce a compound of Formula XIII.

37. The method according to claim 36, wherein said providing a compound of Formula XI1/2 comprises:
providing a compound of Formula XIV:




providing a compound of Formula XV:




 and
reacting the compound of Formula XIV with an alkylating agent and the compound of Formula XV under conditions effective to produce a compound of Formula XI1/2.

38. The method according to claim 37, wherein the alkylating agent is selected from the group consisting of X—CH2—CH═CH, X—CH2—CH(OR11)2, X—(CH2)2—X, and X—(CH2)2—OH, wherein each X is independently a leaving group and each R11 is independently an alkyl.

39. The method according to claim 27, wherein said providing a compound of Formula XIII comprises:
providing a compound of Formula XI1/2:




 and
reacting the compound of Formula XI1/2 with a protecting group under conditions effective to produce a compound of Formula XIII.

40. The method according to claim 39, wherein said providing a compound of Formula XI1/2 comprises:
providing a compound of Formula XIV:




providing a compound of Formula XV:




 and
reacting the compound of Formula XIV with an alkylating agent and the compound of Formula XV under conditions effective to produce a compound of Formula XI1/2.

41. The method according to claim 40, wherein the alkylating agent is selected from the group consisting of X—CH2—CH═CH, X—CH2—CH(OR11)2, X—(CH2)2—X, and X—(CH2)2—OH, wherein each X is independently a leaving group and each R11 is independently an alkyl.