WO2017082924A1

WO2017082924A1 - Peptide and peptide mimetic binding antagonists of polo-like kinase 1 polo box domain and methods of use

Info

Publication number: WO2017082924A1
Application number: PCT/US2015/060629
Authority: WO
Inventors: Terrence R. Burke, Jr.; David HYMEL
Original assignee: The United States Of America, As Represented By The Secretary, Department Of Health & Human Services
Priority date: 2015-11-13
Filing date: 2015-11-13
Publication date: 2017-05-18

Abstract

The description provides novel compounds that may serve as anticancer therapeutics. The compounds of the description bind to polo-like kinases through the polo-box domain. The peptide derivatives of the description have achieved improved efficacy in biochemical assays against Plk1. The description also provides methods of use, methods of preparation, compositions, and kits thereof. Further, the description provides a novel method of design and/or synthesis of phosphoryl-derived peptide derivatives useful as therapeutic agents.

Description

PEPTIDE AND PEPTIDE MIMETIC BINDING ANTAGONISTS OF POLO-LIKE KINASE 1 POLO BOX DOMAIN AND METHODS OF USE

STATEMENT OF FEDERALLY SPONSORED RESEARCH

This work was supported by the Intramural Research Program of the National Institutes of Health. The U.S. government has certain rights in the description.

BACKGROUND

Phosphorylated amino acids are responsible for numerous binding interactions within cells that mediate protein-protein interactions and biochemical pathways. As such, synthetic phosphopeptides and peptide mimetics have received interest as competitive inhibitors of these interactions.

For example, polo-like kinases (collectively, Plks) are a conserved subfamily of Ser/Thr protein kinases that play critical roles in cell proliferation. Plks are characterized by the presence of a highly conserved C-terminal polo-box domain (PBD) composed of two structurally-related PBl (residues 411-489 in Plkl) and PB2 (residues 511-592) motifs.

Multiple forms of Plks, designated Plkl, Plk2/Snk, Plk3/Prk/Fnk, and Plk4/Sak, exist in mammals. Plk4 is the most distantly related member of the Plk subfamily and one of the two Plk4 variants, Sak-a, contains only the PBl motif near the end of an unusually long C-terminal extension. Among the Plks, Plkl has been studied most extensively because of its ability to override cellular checkpoints and induce genetic instability, leading to oncogenic

transformation of human cells. Not surprisingly, Plkl is overexpressed in a broad spectrum of human cancers and has been proposed as a new prognostic marker for many types of malignancies.

Furthermore, interference with Plkl function induces apoptotic cell death in most tumor cells, but not in normal cells, and reduces tumor growth in mouse xenograft models. A Plkl inhibitor known as BI 6727 (volasertib) is presently undergoing clinical trials for the treatment of various human cancers, including acute myeloid leukemia. In contrast to the role of Plkl in cell proliferation and tumorigenesis, the two most closely related kinases, Plk2 and Plk3, appear to play a role in checkpoint-mediated cell cycle arrest to ensure genetic stability and prevent oncogenic transformation. Thus, specific inhibition of Plkl, but not Plk2 or Plk3, is critically important for anti-Plkl cancer therapy.

The PBD of Plkl plays a critical role in proper subcellular localization and mitotic functions of Plkl by interacting with phosphorylated Ser/Thr peptides with the invariable Ser residue at the -1 position (S-p-S/T motif). Crystal structures of the Plkl PBD in complex with artificial phosphopeptides optimized for PBD binding have revealed that the PB1 and PB2 motifs have identical folds described as β6α (a six-stranded anti-parallel β- sheet and an a-helix) and form a hetero-dimeric phosphopeptide-binding module.

The phosphopeptide binds to a cleft formed between PB 1 and PB2 and interacts with key amino acid residues from both polo-boxes. His538 and Lys540 from PB2 are pivotal for electrostatic interactions with the negatively charged phosphate group of phospho- Ser/Thr (p-Ser/Thr) residue, whereas Trp414 from PB 1 is critical for the selection of Ser at the -1 position by engaging in two hydrogen bonding interactions and van der Waals interactions with the Ser-1 residue. These residues are conserved in the PBDs of Plkl, Plk2, and Plk3 (in short, Plkl-3), attesting to their importance (Plk4 has a distinct binding module and forms a homodimer through a motif called cryptic polo-box).

By examining PBD-binding phosphopeptides, the phosphopeptide "PLHSpT" was identified that specifically interacts with the Plkl PBD with a high affinity, but not with the two closely-related Plk2 and Plk3 PBDs. Based on this peptide sequence, peptides with high PBD-binding affinity may be designed and prepared; however, even with high PBD- binding affinity, it is difficult for the peptides to achieve activity in whole-cell systems, possibly due to poor bioavailability arising from poor solubility or limited membrane transport (or both). Therefore, there is a need in the art to design and prepare PBD-binding peptides with improved pharmaceutical properties, including increased bioavailability.

SUMMARY

In certain aspects, the description provides peptido-mimetic compounds comprising an amino acid analog selected from the group consisting of a phospho-(p)Thr analog, pSer analog, Pmab or C-3 substituted Pmab derivative as described herein, and combinations thereof. In certain embodiments, the peptide-mimetic compound comprises at least one natural (i.e., alpha) amino acid and a Pmab-derivative amino acid analog as described herein. In certain embodiments, the description provides peptido-mimetic compounds comprising at least one C- 3 substituted Pmab derivative, phosphatase stable C-3 substituted Pmab derivative phospho- amino acid analog or a combination thereof.

In additional embodiments, the description provides peptido-mimetic ligands of polo box domains (PBD) comprising an amino acid analog as described herein, e.g., a phosphatase stable phospho-amino acid analog. In certain embodiments, the peptide-mimetic ligand of PBD comprises a dipeptide having the structure: Ser-[Z], wherein Z is a phosphatase stable phospho- amino acid analog as described herein, e.g., a C-3 substituted Pmab derivative as described herein.

In certain embodiments, the peptide-mimetic ligand of PBD comprises, consists or consists essentially of the structure: Xo-6-Ser-[Z]-X'o-8, wherein X is any amino acid, and Z is phosphatase stable phospho-amino acid analog as described herein, and wherein only one of X or X' can be zero.

In certain embodiments, the peptide-mimetic ligand of PBD comprises, consists or consists essentially of the structure: Xo-3-Ser-[Z]-X'o-2, wherein X is any amino acid , and Z is phosphatase stable phospho-amino acid analog as described herein, and wherein only one of X or X' can be zero.

In certain additional embodiments, the description provides a peptido-mimetic ligand of PBD comprising, consisting or consisting essentially of the structure Xo-3-His-Ser-[Z]-X'o-2, wherein X is any amino acid, and Z is phosphatase stable phospho-amino acid analog as described herein

In certain embodiments, the PBD is that of polo-like kinase 1 (Plkl), which is a critical regulator of mitotic events and cellular proliferative potential, and includes methods synthesis and use of the same. In particular, the description provides novel compounds that inhibit polo- like kinases by binding to the polo-box domain.

In one aspect, the description provides novel PBD-binding peptides (also referred to as "peptide derivatives") that may serve as anti-cancer therapeutics. The description also provides methods of use and kits thereof. In a further aspect, the description provides a novel method of design or synthesis (or both) of phosphoryl-derived peptide derivatives useful as therapeutic agents.

In certain embodiments, the novel compounds are peptide derivatives that contain 4-5 residue peptides, comprising, such as, a pThr analog, pSer analog, Pmab (i.e.,

phosphonomethylamino butyric acid) or C-3 substituted Pmab residue as described herein.

The peptide derivatives in accordance with the description demonstrate good cellular uptake. Certain peptide derivatives of the description demonstrate good cellular efficacy. In certain embodiments, the peptide derivatives in accordance with the description demonstrate high PBD-binding affinity. The description also provides the compounds as pharmaceutically acceptable salts, solvates, hydrates, or stereoisomers. In another aspect, the description provides the compounds in pharmaceutically acceptable carriers and the use of the compounds for the preparation of a medicament.

In any of the aspects or embodiments described herein, the peptido-mimetic compound is from 3 to 500 residues, or from 3 to 250 residues, or from 3 to 150 residues, or from 3 to 50 residues, including all values in between. In certain embodiments, the peptido-mimetic compound as described herein is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 ,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more residues.

The description further provides kits containing the compounds of the description, and kits for synthesizing the compounds of the description.

In one aspect, the description provides a compound of Formula II, or salt, solvate, or hydrate thereof:

Formula II

wherein R₂ is optionally substituted C2-C4 alkyl, optionally substituted cycloalkyl, optionally substituted aralkyl, optionally substituted heteroarylalkyl, haloalkyl, optionally substituted morpholinomethyl, O , or optionally substituted indolylalkyl; Y is CH₂, NH, or O; and R₄ is optionally substituted aralkyl. In another aspect, R₂ is Et, Pr, /-Pr,

each X is independently H, Me, Et, CF₃, F, CI, Br, OMe, or N(Me)₂; and Y is CH₂, NH, or O.

In another aspect, R4 is -( H₂)s-Ph. In another aspect, R₂ is Et, Pr, j-Pr, Bu, ₅

₅

; each o is independently 1-3; each X is independently H, Me, Et, CF₃, F, CI, Br, OMe, or N(Me)₂; Y is CH₂, NH, or O; and R₄ is - (CH₂)₈-Ph.

Exemplified compounds of the above formulae include, but are not limited to, the compounds provided infra.

The description provides compositions including any of the compounds of the above formulae (hereinafter "the compounds of the description") in a pharmaceutically acceptable carrier, for use, for example, for the preparation of a medicament. The medicament can be, for example, a medicament for the prevention, amelioration, or treatment of a hyperproliferative disorder such as cancer.

The compounds of the description can be used in methods for the prevention, amelioration, or treatment of a subject for a hyperproliferative disorder. Such methods can further include identification of a subject suffering from or suspected of suffering from a hyperproliferative disorder and/or monitoring the subject for prevention, amelioration, or treatment of a hyperproliferative disorder.

In certain embodiments, the hyperproliferative disorder is cancer. Cancers can be characterized as solid tumors and non-solid tumors. Cancers include, but are not limited to Acute Lymphoblastic Leukemia, Acute Myeloid Leukemia, Adrenocortical Carcinoma, Basal Cell Carcinoma, Bladder Cancer, Bone Cancer, Brain Tumor, Breast Cancer, Cervical Cancer, Chronic Lymphocytic Leukemia, Chronic Myelogenous Leukemia, Chronic Myeloproliferative Disorders, Colon Cancer, Colorectal Cancer, Cutaneous T-Cell Lymphoma, Esophageal Cancer, Ewing Family of Tumors, Retinoblastoma, Gastric (Stomach) Cancer, Gastrointestinal Tumors, Glioma, Head and Neck Cancer, Hepatocellular (Liver) Cancer, Hodgkin Lymphoma, Islet Cell Tumors (Endocrine Pancreas), Kidney (Renal Cell) Cancer, Laryngeal Cancer, Non- small Cell Lung Cancer, Small Cell Lung Cancer, Lymphoma, Medulloblastoma, Melanoma, Pancreatic Cancer, Prostate Cancer, Renal Cancer, Rectal cancer, Thyroid Cancer.

The description provides kits containing at least one compound of the descriptions and instructions for use.

The description also provides a compound (including a peptide derivative) prepared according to any preparation method of the description.

The description also includes methods of designing, synthesizing, and/or using the compounds of the description. In certain embodiments, the description provides compounds made according to any synthetic method disclosed herein.

The preceding general areas of utility are given by way of example only and are not intended to be limiting on the scope of the present disclosure and appended claims. Additional objects and advantages associated with the compositions, methods, and processes of the present invention will be appreciated by one of ordinary skill in the art in light of the instant claims, description, and examples. For example, the various aspects and embodiments of the invention may be utilized in numerous combinations, all of which are expressly contemplated by the present description. These additional advantages objects and embodiments are expressly included within the scope of the present invention. The publications and other materials used herein to illuminate the background of the invention, and in particular cases, to provide additional details respecting the practice, are incorporated by reference. Other aspects of the description are discussed infra.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated into and form a part of the specification, illustrate several embodiments of the present invention and, together with the description, serve to explain the principles of the invention. The drawings are only for the purpose of illustrating an embodiment of the invention and are not to be construed as limiting the invention. Further objects, features and advantages of the invention will become apparent from the following detailed description taken in conjunction with the accompanying figures showing illustrative embodiments of the invention, in which:

Figure 1. Peptide mimetics as described herein afford enhanced interactions with Arg557 and Leu491 of Plkl-PBD, which are located proximal to the 3-position of Pmab in the pThr-binding pocket.

Figure 2. ELISA-based inhibition of full-length Polo-like kinase 1 by Compounds W- 64, W-66, and W-2. DETAILED DESCRIPTION

Presently described are high affinity peptide mimetic ligands of the polo-like kinase 1 (Plkl) that contain a phospho-threonine (pThr) analog residue. This pThr residue is critical to maintain high affinity binding, but it is also a substrate of cellular phosphotases that hydrolyze the phosphate group and render the compound(s) inactive. Thus, the description provides novel compounds that inhibit polo-like kinases by binding to the polo-box domain. The use of the phosphonate analog, (2S,3R) 2-amino-3-methyl-4-phosphonobutanoic acid (Pmab), prevents inactivation by cellular phosphatases.

In certain aspects, the orthogonally protected amino acid, (N-Fmoc, 0,0-(bis-(tert- butyl))-Pmab, must be custom synthesized to allow for its use in solid-phase peptide synthesis (SPPS). The previous synthetic routes, developed in our lab, required either 15 or 20 steps with 12% and 14% overall yields, respectively. The work reported here provides a more efficient synthetic route to a key intermediate in the synthesis of Pmab. Using this method, along with a 7-step transformation previously reported by our group, this critical building block can now be produced on gram-scale in 9 steps and at least about 23% overall yield.

An important aspect of this new metholdology is that it allows for the efficient synthesis of Pmab analogs that differ at the C-3 position. Furthermore, these analogs can be synthesized with orthogonal protecting groups that render them suitable for facile incorporation into peptides or peptide mimetics using both solution-phase and solid-phase peptide synthesis. These analogs can be easily accessed by reacting diverse aldehydes using the anti- selective Mannich reaction followed by the 7-step transformation to the corresponding phosphonate. Very few of these C-3 analogs of phospho-threonine have been reported in the literature due to the previous requirement of a lengthy and impractical synthetic method. Therefore, the methodology allows for efficient preparation of orthogonally protected phosphonate-containing reagents that are compatible with the SPPS of new genres of phosphatase-stable pThr analogs, which may yield peptide mimetics having significantly improved biological properties.

The following is a detailed description provided to aid those skilled in the art in practicing the present invention. Those of ordinary skill in the art may make modifications and variations in the embodiments described herein without departing from the spirit or scope of the present disclosure. All publications, patent applications, patents, figures and other references mentioned herein are expressly incorporated by reference in their entirety.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description is for describing particular embodiments only and is not intended to be limiting of the invention.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise (such as in the case of a group containing a number of carbon atoms in which case each carbon atom number falling within the range is provided), between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the invention.

The following terms are used to describe the present invention. In instances where a term is not specifically defined herein, that term is given an art-recognized meaning by those of ordinary skill applying that term in context to its use in describing the present invention.

The articles "a" and "an" as used herein and in the appended claims are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article unless the context clearly indicates otherwise. By way of example, "an element" means one element or more than one element.

The phrase "and/or," as used herein in the specification and in the claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with "and/or" should be construed in the same fashion, i.e., "one or more" of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the "and/or" clause, whether related or unrelated to those elements specifically identified. Thus, as a non- limiting example, a reference to "A and/or B", when used in conjunction with open-ended language such as "comprising" can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and/or" as defined above. For example, when separating items in a list, "or" or "and/or" shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of or "exactly one of," or, when used in the claims, "consisting of," will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e., "one or the other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of."

In the claims, as well as in the specification above, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding,"

"composed of," and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases "consisting of and "consisting essentially of shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

As used herein in the specification and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from anyone or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified. Thus, as a nonlimiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently "at least one of A and/or B") can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

It should also be understood that, in certain methods described herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited unless the context indicates otherwise.

The terms "co-administration" and "co-administering" or "combination therapy" refer to both concurrent administration (administration of two or more therapeutic agents at the same time) and time varied administration (administration of one or more therapeutic agents at a time different from that of the administration of an additional therapeutic agent or agents), as long as the therapeutic agents are present in the patient to some extent, preferably at effective amounts, at the same time. In certain preferred aspects, one or more of the present compounds described herein, are coadministered in combination with at least one additional bioactive agent, especially including an anticancer agent. In particularly preferred aspects, the co-administration of compounds results in synergistic activity and/or therapy, including anticancer activity.

The term "compound", as used herein, unless otherwise indicated, refers to any specific chemical compound disclosed herein and includes tautomers, regioisomers, geometric isomers, and where applicable, stereoisomers, including optical isomers (enantiomers) and other steroisomers (diastereomers) thereof, as well as pharmaceutically acceptable salts and derivatives (including prodrug forms) thereof where applicable, in context. Within its use in context, the term compound generally refers to a single compound, but also may include other compounds such as stereoisomers, regioisomers and/or optical isomers (including racemic mixtures) as well as specific enantiomers or enantiomerically enriched mixtures of disclosed compounds. The term also refers, in context to prodrug forms of compounds which have been modified to facilitate the administration and delivery of compounds to a site of activity. It is noted that in describing the present compounds, numerous substituents and variables associated with same, among others, are described. It is understood by those of ordinary skill that molecules which are described herein are stable compounds as generally described hereunder. When the bond is shown, both a double bond and single bond are represented within the context of the compound shown.

The term "patient" or "subject" is used throughout the specification to describe an animal, preferably a human or a domesticated animal, to whom treatment, including prophylactic treatment, with the compositions according to the present invention is provided. For treatment of those infections, conditions or disease states which are specific for a specific animal such as a human patient, the term patient refers to that specific animal, including a domesticated animal such as a dog or cat or a farm animal such as a horse, cow, sheep, etc. In general, in the present invention, the term patient refers to a human patient unless otherwise stated or implied from the context of the use of the term.

An "agent" is understood herein to include a therapeutically active compound or a potentially therapeutically active compound. An agent can be a previously known or unknown compound. As used herein, an agent is typically a non-cell based compound, however, an agent can include a biological therapeutic agent, e.g., peptide or nucleic acid therapeutic, cytokine, antibody, etc.

An "agonist" is understood herein as a chemical substance capable of initiating the same reaction or activity typically produced by the binding of an endogenous substance or ligand to its target. An "antagonist" is understood herein as a chemical substance capable of inhibiting the reaction or activity typically produced by the binding of an endogenous substance (e.g., an endogenous agonist) to its target to prevent signaling through a receptor, to prevent downstream signaling, or to prevent cellular events (e.g., progression through cell cycle) that are the normal result of activation of the target. The antagonist can bind directly to the target or can act through other proteins or factors required for signaling. Agonists and antagonists can modulate some or all of the activities of the endogenous substance or ligand that binds to the target. Antagonists are typically characterized by determining the amount of the antagonist is required to inhibit the activity of the endogenous agonist. For example, an inhibitor at 0.01-, 0.1-, 1-, 5-, 10-, 50-, 100-, 200-, 500-, or 1000-fold molar concentration relative to the agonist can inhibit the activity of the agonist by at least 10%, 50%, 90%, or more.

As used herein "amelioration" or "treatment" is understood as meaning to lessen or decrease at least one sign, symptom, indication, or effect of a specific disease or condition. For example, amelioration or treatment of cancer can be determined using the standard RECIST (Response Evaluation Criteria in Solid Tumors) criteria including the assessment of tumor burden, by survival time, reduced presence of tumor markers (e.g., prostate specific antigen), or any other clinically acceptable indicators of disease state or progression. Amelioration and treatment can require the administration of more than one dose of an agent or therapeutic. As used herein, "prevention" is understood as to limit, reduce the rate or degree of onset, or inhibit the development of at least one sign or symptom of a disease or condition. For example, a subject having a genetic predisposition to develop a disease may develop disease later in life, e.g., delay of BRCA1 or BRCA2 related breast cancer development from third or fourth decade of life to fifth or beyond. Prevention can require the administration of more than one dose of an agent or therapeutic.

Chemical classes and groups are provided herein and referred to by chemical names, common names, and/or chemical structures. In the absence of an explicit definition herein, definitions of chemical structures can be found in chemical dictionaries, science textbooks, such as organic chemistry textbooks, or in databases. Chemical classes and groups commonly referred to herein are provided as follows. The term "alkoxy," as used herein, refers to an alkyl group which is linked to another moiety though an oxygen atom. Alkoxy groups can be optionally substituted with one or more substituents. As used herein, "Coalkoxy" refers to a hydroxyl (-OH) group.

The terms "alkoxyalkyl," "polyaminoalkyl" and "thioalkoxyalkyl" refer to alkyl groups, as described above, which further include oxygen, nitrogen or sulfur atoms replacing one or more carbons of the hydrocarbon backbone, e.g., oxygen, nitrogen or sulfur atoms.

The terms "alkenyl" and "alkynyl" refer to unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double or triple bond, respectively. For example, the description contemplates cyano and propargyl groups.

The term "alkyl" refers to the radical of saturated aliphatic groups, including straight- chain alkyl groups, branched-chain alkyl groups, and cycloalkyl substituted alkyl groups. In certain embodiments, the term "alkyl" refers to a group having two radical groups, such as "- CH₂-". The term alkyl further includes alkyl groups, which can further include oxygen, nitrogen, sulfur or phosphorous atoms replacing one or more carbons of the hydrocarbon backbone, e.g., oxygen, nitrogen, sulfur or phosphorous atoms. In preferred embodiments, a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., Q- C30 for straight chain, C3-C30 for branched chain), or 20 or fewer, even 10 or fewer.

Moreover, the term alkyl as used throughout the specification and claims is intended to include both "unsubstituted alkyls" and "substituted alkyls," the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents can include, for example, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety. It will be understood by those skilled in the art that the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate. An "alkylaryl" moiety is an alkyl substituted with an aryl (e.g., phenylmethyl (benzyl)). The term "alkyl" also includes unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double or triple bond. The terms "cycloalkyl" and "cycloalkenyl" as employed herein includes saturated and partially unsaturated cyclic, respectively, hydrocarbon groups having 3 to 12 carbons, preferably 3 to 8 carbons, and more preferably 3 to 6 carbon.

As used herein, an amide is understood as a derivative of an oxoacids in which an acidic hydroxyl group has been replaced by an amino or substituted amino group. Compounds having one, two or three acyl groups on a given nitrogen are generically included and may be designated as primary, secondary and tertiary amides, respectively, e.g. PhC(=0)NH₂ benzamide, CH₃S(=0)₂NMe₂ Ν,Ν-dimethylmethanesulfonamide, [RC(=0)]₂NH secondary amides (see imides), [RC(=0)]3N tertiary amides, PhP(=0)(OH)NH₂ phenylphosphonamidic acid. An amide group as used herein is understood as a group with -NH₂, NHR and NR_2. Amide groups should not be distinguished by means of the terms primary, secondary and tertiary.

As used herein, an "allyl" group is understood as a structure containing a carbon-carbon double bond. For example, it includes a structural formula H₂C=CH-CH₂R, where R is the connection to the rest of the molecule.

As used herein, "amine" or "amino" is understood as Compounds formally derived from ammonia by replacing one, two or three hydrogen atoms by hydrocarbon groups, and having the general structures RNH₂ (primary amines), R₂NH (secondary amines), R₃N (tertiary amines). An amino group is understood as having the structure -NH₂, -NHR, or -NR₂.

As used herein, "aryl group" is understood as refers to any functional group or substituent derived from a simple aromatic ring, may it be phenyl, thiophene, indolyl, etc (see IUPAC nomenclature, goldbook.iupac.org/A00464.html). Aryl groups derived from arenes by removal of a hydrogen atom from a ring carbon atom. Groups similarly derived from heteroarenes are sometimes subsumed in this definition. "Aryl" is used for the sake of abbreviation or generalization. For example, a simple aryl group is phenyl, C₆Hs; it is derived from benzene. The tolyl group, CH₃C₆H₄, is derived from toluene (methylbenzene). The xylyl group, (CH₃)₂C₆H₃, is derived from xylene (dimethylbenzene). The class of heterocyclyl groups derived from heteroarenes by removal of a hydrogen atom from any ring atom is referred to as heteroaryl.

As used herein, "carboxylic acid" is understood as a group having the structure

RC(=0)OH. A carboxylic acid group is understood to denote the -C(=0)OH group including its carbon atom. As used herein, "carbonyl group" is understood as a group containing the carbonyl group, C=0. The term is commonly used in the restricted sense of aldehydes and ketones, however as used herein it includes carboxylic acids and derivatives.

As used herein, "carboxyl" or "carboxy" group is understood as a structure containing - COOH or -COOR. The term includes carboxylic acids and derivatives.

The term "enantiomers" refers to two stereoisomers of a compound which are non- superimposable mirror images of one another. An equimolar mixture of two enantiomers is called a "racemic mixture" or a "racemate."

As used herein, a "halogen" or "halo" is understood as an element located in Group VIIA of the periodic table. Halogens are reactive nonmetals having seven valence electrons. Halogen groups include -F, -CI, -Br, and -I.

As used herein, modification of a class of chemical group with the term "hetero" is understood as the class of functional groups derived from the particular class of the functional group by removal of a hydrogen atom from any carbon atom.

"Heterocyclyl" groups as used herein are univalent groups formed by removing a hydrogen atom from any ring atom of a heterocyclic compound.

The term "heterocyclic" as used herein, refers to organic compounds that contain at least at least one atom other than carbon (e.g., S, O, N) within a ring structure. The ring structure in these organic compounds can be either aromatic or non-aromatic. Some examples of heterocyclic moieties include, are not limited to, pyridine, pyrimidine, pyrrolidine, furan, tetrahydrofuran, tetrahydrothiophene, and dioxane.

As used herein, "olefin group" is understood as an acyclic and or cyclic hydrocarbon having one or more carbon-carbon double bonds, apart from the formal ones in aromatic compounds. The class olefins subsumes alkenes and cycloalkenes and the corresponding polyenes.

In compounds, amino acid positions are determined relative to the phosphothreonine which is arbitrarily defined as position zero (0). Amino acids to the C-terminus of the peptide (to the right) are indicated as positions +1 (adjacent to the phosphothreonine), +2 (adjacent to the + 1 position, but not the phosphothrenine), etc. Similarly, amino acids towards the N- terminus are defined as positions -1 (adjacent to the phosphothreonine), -2 (adjacent to the -1 position, but not the phosphothrenine), etc.

"Contacting a cell" is understood herein as providing an agent to a test cell e.g., a cell to be treated in culture or in an animal, such that the agent or isolated cell can interact with the test cell or cell to be treated, potentially be taken up by the test cell or cell to be treated, and have an effect on the test cell or cell to be treated. The agent or isolated cell can be delivered to the cell directly (e.g., by addition of the agent to culture medium or by injection into the cell or tissue of interest), or by delivery to the organism by an enteral or parenteral route of administration for delivery to the cell by circulation, lymphatic, or other means.

As used herein, "changed as compared to a control" sample or subject is understood as having a level of the analyte or diagnostic or therapeutic indicator to be detected at a level that is statistically different than a sample from a normal, untreated, or control sample. Control samples include, for example, cells in culture, one or more laboratory test animals, or one or more human subjects. Methods to select and test control samples are within the ability of those in the art. An analyte can be a naturally occurring substance that is characteristically expressed or produced by the cell or organism (e.g., PSA) or a substance produced by a reporter construct (e.g, β-galactosidase or luciferase). Depending on the method used for detection the amount and measurement of the change can vary. For example, a change in the amount of cleavage of analyte present will depend on the exact reaction conditions and the amount of time after exposure to the agent the sample is collected. Changed as compared to a control reference sample can also include decreased binding of a ligand to a receptor in the presence of an antagonist or other inhibitor. Determination of statistical significance is within the ability of those skilled in the art.

As used herein, "detecting", "detection" and the like are understood that an assay performed for identification of a specific analyte in a sample or a product from a reporter construct in a sample. Detection can also include identification of activation of a kinase or other enzyme. Detection can include the identification of a mutation in a gene sequence, such as a point mutation, a deletion of all or part of the coding sequence or transcriptional/ translational regulatory sequences of the gene, a truncation of the gene sequence, or any other alteration that can alter the expression level or the sequence of the protein expressed by the gene, particularly when the alteration of the sequence results in a phenotypic change in the subject. Detection can include the determination of the size of a tumor, the presence or absence of metastases, the presence or absence of angiogenesis, etc. The amount of analyte detected in the sample can be none or below the level of detection of the assay or method.

By "diagnosing" as used herein refers to a clinical or other assessment of the condition of a subject based on observation, testing, or circumstances for identifying a subject having a disease, disorder, or condition based on the presence of at least one sign or symptom of the disease, disorder, or condition. Typically, diagnosing using the method of the description includes the observation of the subject for other signs or symptoms of the disease, disorder, or condition by physical examination, imaging, further laboratory tests, etc.

As used herein, a "diagnostic marker" is understood as one or more signs or symptoms of a disease or condition that can be assessed, preferably quantitatively to monitor the progress or efficacy of a disease treatment or prophylactic treatment or method. A diagnostic marker can be a substance that is released by a tumor (e.g., antigens such as PSA or enzymes). A diagnostic marker can be tumor size and/or grade of tumor and/or growth rate of tumor. A diagnostic marker can be the presence or absence of angiogenesis. A diagnostic marker can be a change in blood counts or cellular function measured in an in vitro assay, or the presence and characteristics of metastases (number and size).

The term "effective" can mean an amount of a compound, composition or component which, upon single or multiple dose administration to the cell or subject, when used within the context of its intended use, effects an intended result. The term effective subsumes all other effective amount or effective concentration terms, which are otherwise described or used in the present application. The terms "effective" and "effectiveness" can include both

pharmacological effectiveness and physiological safety. Pharmacological effectiveness refers to the ability of the treatment to result in a desired biological effect in the patient. Physiological safety refers to the level of toxicity, or other adverse physiological effects at the cellular, organ and/or organism level (often referred to as side-effects) resulting from administration of the treatment. On the other hand, the term "ineffective" indicates that a treatment does not provide sufficient pharmacological effect to be therapeutically useful, even in the absence of deleterious effects, at least in the unstratified population. (Such a treatment may be ineffective in a subgroup that can be identified by the expression profile or profiles.) "Less effective" means that the treatment results in a therapeutically significant lower level of pharmacological effectiveness and/or a therapeutically greater level of adverse physiological effects, e.g., greater liver toxicity.

Thus, in connection with the administration of a drug, a drug which is "effective against" a disease or condition indicates that administration in a clinically appropriate manner results in a beneficial effect for at least a statistically significant fraction of patients, such as an improvement of symptoms, a cure, a reduction in disease signs or symptoms, extension of life, improvement in quality of life, or other effect generally recognized as positive by medical doctors familiar with treating the particular type of disease or condition. "Therapeutically effective amount," as used herein refers to an amount of an agent which is effective, upon single or multiple dose administration to the cell or subject, in ameliorating or treating or preventing a symptom of a disease or disorder, including prolonging the survivability of a patient with such a disorder beyond that expected in the absence of such treatment.

An agent can be administered to a subject, either alone or in combination with one or more therapeutic agents, as a pharmaceutical composition in mixture with conventional excipient, e.g., pharmaceutically acceptable carrier, or therapeutic treatments such as radiation.

It will be appreciated that the actual preferred amounts of active compounds used in a given therapy will vary according to e.g., the specific compound being utilized, the particular composition formulated, the mode of administration and characteristics of the subject, e.g., the species, sex, weight, general health and age of the subject. Optimal administration rates for a given protocol of administration can be readily ascertained by those skilled in the art using conventional dosage determination tests conducted with regard to the foregoing guidelines.

As used herein, "Fmoc" is understood as 9-Fluorenylmethyloxycarbonyl having the molecular formula C15H11CIO2. The structure of this protective group is well known.

As used herein, "heterologous" as in "heterologous protein" is understood as a protein not natively expressed in the cell in which it is expressed. The heterologous protein may be, but need not be, from a different species.

The term "hyperproliferative disorder" or "neoplasia" includes malignancies characterized by excess cell proliferation or growth, or reduced cell death. In specific embodiments, the term "cancer" includes but is not limited to carcinomas, sarcomas, leukemias, and lymphomas. The term "cancer" also includes primary malignant tumors, e.g., those whose cells have not migrated to sites in the subject's body other than the site of the original tumor, and secondary malignant tumors, e.g., those arising from metastasis, the migration of tumor cells to secondary sites that are different from the site of the original tumor. Tumors include solid tumors (i.e., non-blood tumors) and blood tumors. Cancers include, but are not limited to, Acute Lymphoblastic Leukemia, Acute Myeloid Leukemia, Adrenocortical Carcinoma, Basal Cell Carcinoma, Bladder Cancer, Bone Cancer, Brain Tumor, Breast Cancer, Cervical Cancer, Chronic Lymphocytic Leukemia, Chronic Myelogenous Leukemia, Chronic Myeloproliferative Disorders, Colon Cancer, Colorectal Cancer, Cutaneous T-Cell Lymphoma, Esophageal Cancer, Ewing Family of Tumors, Retinoblastoma, Gastric (Stomach) Cancer, Gastrointestinal Tumors, Glioma, Head and Neck Cancer, Hepatocellular (Liver) Cancer, Hodgkin Lymphoma, Islet Cell Tumors (Endocrine Pancreas), Kidney (Renal Cell) Cancer, Laryngeal Cancer, Lung Cancer, Non-Small Cell, Lung Cancer, Small Cell, Lymphoma, Medulloblastoma, Melanoma, Pancreatic Cancer, Prostate Cancer, Renal Cancer, Rectal cancer, Thyroid Cancer.

As used herein, "isolated" or "purified" when used in reference to a polypeptide means that a naturally polypeptide or protein has been removed from its normal physiological environment (e.g., protein isolated from plasma or tissue) or is synthesized in a non-natural environment (e.g., artificially synthesized in an in vitro translation system). Thus, an "isolated" or "purified" polypeptide can be in a cell-free solution or placed in a different cellular environment (e.g., expressed in a heterologous cell type). The term "purified" does not imply that the polypeptide is the only polypeptide present, but that it is essentially free (about 90- 95%, up to 99-100% pure) of cellular or organismal material naturally associated with it, and thus is distinguished from naturally occurring polypeptide. Similarly, an isolated nucleic acid is removed from its normal physiological environment. "Isolated" when used in reference to a cell means the cell is in culture (i.e., not in an animal), either cell culture or organ culture, of a primary cell or cell line. Cells can be isolated from a normal animal, a transgenic animal, an animal having spontaneously occurring genetic changes, and/or an animal having a genetic and/or induced disease or condition.

The term "stereoisomers" as used herein refers to isomeric molecules that have the same molecular formula and sequence of bonded atoms (constitution), but that differ only in the three-dimensional orientations of their atoms in space. The structural isomers share the same molecular formula, but the bond connections and/or their order between different atoms/groups differs. In certain embodiments of the description, stereoisomers refer to the compounds having the same order and bond connections of the constituent atoms, but different orientation in space (such as, enantiomers, and diastereomers).

The term "prodrug" includes compounds with moieties which can be metabolized in vivo. Generally, the prodrugs are metabolized in vivo by esterases or by other mechanisms to active drugs. Examples of prodrugs and their uses are well known in the art (See, e.g. , Berge et al. (1977) "Pharmaceutical Salts", /. Pharm. Sci. 66: 1-19). The prodrugs can be prepared in situ during the final isolation and purification of the compounds, or by separately reacting the purified compound in its free acid form or hydroxyl with a suitable esterifying agent. Hydroxyl groups can be converted into esters via treatment with a carboxylic acid. Examples of prodrug moieties include substituted and unsubstituted, branch or unbranched lower alkyl ester moieties, (e.g. , propionoic acid esters), lower alkenyl esters, di-lower alkyl-amino lower-alkyl esters (e.g. , dimethylaminoethyl ester), acylamino lower alkyl esters (e.g. , acetyloxymethyl ester), acyloxy lower alkyl esters (e.g. , pivaloyloxymethyl ester), aryl esters (phenyl ester), aryl-lower alkyl esters (e.g. , benzyl ester), substituted (e.g. , with methyl, halo, or methoxy substituents) aryl and aryl-lower alkyl esters, amides, lower-alkyl amides, di-lower alkyl amides, and hydroxy amides. Preferred prodrug moieties are propionoic acid esters and acyl esters. Prodrugs which are converted to active forms through other mechanisms in vivo are also included.

The term "label" or "detectable label" as used herein refers to any atom or molecule which can be used to provide a detectable (preferably quantifiable) signal, and which can be attached to a chemical compound, a nucleic acid or protein. Labels may provide signals detectable by fluorescence, radioactivity, colorimetry, gravimetry, X-ray diffraction or absorption, magnetism, enzymatic activity, and the like. Various methods of labeling polypeptides and glycoproteins are known in the art and may be used. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes (e.g., ³H), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase), biotinyl groups,

predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance. In others, the label is part of the fusion protein, e.g. Green Fluorescent Protein (GFP), Yellow Fluorescent Protein (YFP).

"Library" as used herein is understood to be a chemical library. Chemical libraries include two or more compounds (10 or more, 50 or more, 100 or more, 200 or more, 500 or more, 1000 or more, 5000 or more, 10,000 or more, etc.; or any range bracketed by the noted values), preferably that have structural and/or potential functional properties. Libraries can be used, for example for screening assays to select compounds with desired activities, e.g., kinase binding, kinase stimulating, kinase inhibiting activity.

"Obtaining" is understood herein as manufacturing, purchasing, or otherwise coming into possession of.

A "peptide" or "peptide derivative" as used herein is understood as two or more independently selected natural or non-natural amino acids joined by a peptide bond. A peptide can include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more natural or non-natural amino acids joined by peptide bonds.

As used herein, pharmaceutically acceptable salts include, without limitation, the tartrate, succinate, tartarate, bitartarate, dihydrochloride, salicylate, hemisuccinate, citrate, maleate, hydrochloride, carbamate, sulfate, nitrate, and benzoate salt forms thereof, as well as combinations thereof and the like. Any form of peptide mimetic is suitable for use in the methods of the present description, e.g., a pharmaceutically acceptable salt of a peptide mimetic, a free base of a peptide mimetic, or a mixture thereof.

As used herein, "plurality" is understood to mean more than one. For example, a plurality refers to at least two, three, four, five, or more.

A "polo-like kinase" or "Plk" as used herein collectively refers to the proteins called

Plk-1, (human sequence available as under Accession No. P53350.1 GI:1709658; mouse sequence available under Accession No. Q07832.2 GI: 1709659; rat sequence available under Accession No. Q62673.1 GI: 12230396; Pan troglodytes sequence available under Accession No. XP_001163585.1 GI: 114661620); Plk-2 (human sequence available under Accession No. Q9NYY3.3 GL22096374); Plk-3 (human sequence available under Accession No. Q9H4B4.2 01:51338822); and Plk-4 (human sequence available under Accession No. O00444.3

GI: 160113150), from any organism, preferably a mammalian organism, preferably from a human organism. Such proteins can be encoded by any nucleic acid that provides the appropriate translation product; however, in certain embodiments, the polo-like kinases are encoded by the native genes which can easily be identified using GenBank or any of a number of publicly available databases. All GenBank Nos. incorporated herein by reference as of the filing date of the instant application.

A "sample" as used herein refers to a biological material that is isolated from its environment (e.g., blood or tissue from an animal, cells, or conditioned media from tissue culture) and is suspected of containing, or known to contain an analyte, such as a tumor cell or a product from a reporter construct. A sample can also be a partially purified fraction of a tissue or bodily fluid. A reference sample can be a "normal" sample, from a donor not having the disease or condition, or from a normal tissue in a subject having the disease or condition (e.g., normal tissue vs. tumor tissue). A reference sample can also be from an untreated donor or cell culture not treated with an active agent (e.g., no treatment or administration of vehicle only) and/or stimulus. A reference sample can also be taken at a "zero time point" prior to contacting the cell or subject with the agent or cell to be tested.

An agent, antibody, polypeptide, nucleic acid, or other compound "specifically binds" a target molecule, e.g., antigen, polypeptide, nucleic acid, or other compound, when the target molecule is bound with at least 100-fold, preferably at least 500-fold, preferably at least 1000- fold, preferably at least a 5000-fold, preferably at least a 10,000-fold preference as compared to a non-specific compounds, or a pool of non-specific compounds. "Specifically binds" can be used in relation to binding one of two or more related compounds that have physically related structures, e.g., two kinases, particularly 2 polo-like kinases. For example, an agent, antibody, polypeptide, nucleic acid, or other compound can "specifically bind" one polo-like kinase (e.g., Plkl) with at least a 10-fold, 50-fold, 100-fold, 500-fold, 1000-fold, 5000-fold, 10,000-fold or more preference over another polo-like kinase, e.g., Plk2, Plk3, or Plk4. Binding preferences and affinities, absolute or relative, can be determined, for example by determining the affinity for each pair separately or by the use of competition assays or other methods well known to those of skill in the art.

A "subject" as used herein refers to living organisms. In certain embodiments, the living organism is an animal. In certain preferred embodiments, the subject is a mammal. In certain embodiments, the subject is a domesticated mammal. Examples of subjects include humans, monkeys, dogs, cats, mice, rats, cows, horses, goats, and sheep. A human subject may also be referred to as a patient.

A subject "suffering from or suspected of suffering from" a specific disease, condition, or syndrome has a sufficient number of risk factors or presents with a sufficient number or combination of signs or symptoms of the disease, condition, or syndrome such that a competent individual would diagnose or suspect that the subject was suffering from the disease, condition, or syndrome. Methods for identification of subjects suffering from or suspected of suffering from conditions such as cancer is within the ability of those in the art. Subjects suffering from, and suspected of suffering from, a specific disease, condition, or syndrome are not necessarily two distinct groups.

As used herein, "susceptible to" or "prone to" or "predisposed to" a specific disease or condition and the like refers to an individual who based on genetic, environmental, health, and/or other risk factors is more likely to develop a disease or condition than the general population. An increase in likelihood of developing a disease may be an increase of about 10%, 20%, 50%, 100%, 150%, 200%, or more.

Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50.

The term "disubstituted histidine" refers to a histidine residue substituted on the imidazole ring with at least two moieties such as aryl-(Ci_2o)alkyl (including aryl-(Ci_6)alkyl-), heteroaryl-(Ci_2o)alkyl (including heteroaryl-(Ci_6)alkyl), (Ci_2o)alkyl (including (Ci_6)alkyl), allyl-(Ci_2o)alkyl (including allyl-(Ci_6)alkyl), (Co-2o)alkoxy-C(0)-(Ci_2o)alkyl, or amino(Ci_ 2o)alkyl; wherein each of the said alkyl, aryl, and heteroaryl moieties is further optionally substituted by one or more same or different subtituents selected from the group of (Ci_6)alkyl, carboxyl, halo, hydroxyl, amine, and (Ci_6)alkoxy groups. In certain embodiments, the at least two moieties on the imidazole ring are (Ci-io)alkyl, aryl-(Ci_io)alkyl (including aryl-(Ci_6)alkyl- ), or heteroaryl-(Ci_io)alkyl, allyl-(Ci_6)alkyl, or (Ci_6)alkyl optionally substituted by one or more carboxyl, (Ci_8)alkoxyl, or hydroxyl groups. In certain embodiments, the two moieties are attached to the two nitrogen atoms of the imidazole ring.

Unless specifically stated or obvious from context, as used herein, the term "about" is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value.

All oligonucleotide sequences are written from the 5 '-end to the 3 '-end unless otherwise specifically noted.

The polo-like kinase 1 (Plkl) represents a new target for anticancer therapeutic development. Plkl contains a C-terminal polo-box domain (PBD) that recognizes phospho- Ser(pSer)/phospho-Thr (pThr)-containing motifs, which provides sub-cellular localization that is critical for proper Plkl function. Spatial disruption of Plkl distribution by blocking PBD- dependent protein-protein interactions may afford an attractive alternative to kinase-directed inhibitors for the down-regulation of Plkl function and Plkl PBD-binding antagonists and may serve as a new class of anticancer agents.

One aspect of the description provides a novel class of compounds (or peptide derivatives) that are useful as anticancer therapeutics. In another aspect, the description provides novel compounds used as intermediates in the synthetic preparation of the anticancer compounds of the present description. In another aspect, the description provides methods for the preparation of the anticancer compounds of the present description. In another aspect, the description provides methods for the preparation of intermediates used in the preparation of the anticancer compounds of the present description. The compounds, compositions and methods provided herein represent new approaches to the design and synthesis of peptides or peptide derivatives. The description can lead to the development of additional therapeutically relevant PBD-directed agents.

The description can also lead to peptide derivatives that are useful in unrelated therapeutic areas.

COMPOUNDS

The description provides high affinity compounds bearing non-natural amino acid. The compounds of the description contain a phosphoryl amino acid residue. In certain

embodiments, the compound of the description is a peptide/peptide derivative comprising a pThr analog, pSer analog, Pmab, C-3 substituted Pmab residue.

In certain embodiments, the PBD is that of polo-like kinase 1 (Plkl), which is a critical regulator of mitotic events and cellular proliferative potential, and includes methods synthesis and use of the same. In particular, the description provides novel compounds that inhibit pololike kinases by binding to the polo-box domain.

In certain embodiments, the compounds of the description have achieved good inhibition of Plkl in biochemical assays.

Formula II

wherein R₂ is optionally substituted C₂-C₄ alkyl, optionally substituted cycloalkyl, optionally substituted aralkyl, optionally substituted heteroarylalkyl, haloalkyl, optionally substituted morpholinomethyl, O , or optionally substituted indolylalkyl; Y

-Pr,

; each o is independently 1-3 ; each X is independently H, Me, Et, CF₃, F, CI, Br, OMe, or N(Me)₂; Y is CH₂, NH, or O. In another aspect, R4 is -(CH -Ph. In another aspect, R₂ is Et, Pr, -Pr, Bu,

or ; each o is independently 1-3 ; each X is independently H, Me, Et, CF₃, F, CI, Br, OMe, or N(Me)₂; Y is CH₂, NH, or O; and R₄ is - (CH₂)₈-Ph.

In another aspect, the description provides a compound of Formula 2, or salt, solvate, or hydrate thereof:

Formula 2

wherein, R is optionally substituted cycloalkyl, optionally substituted phenylethyl, optionally substituted phenylpropyl, optionally substituted heteroarylalkyl, haloalkyl, optionally substituted morpholinomethyl, lylalkyl; and Y is CH₂, NH, or O. In another aspect, R is

n is 1-3; each o is independently 1-3; each X is independently H, Me, Et, CF₃, F, CI, Br, OMe, or N(Me)₂; and Y is CH₂, NH, or O.

In certain aspects, the description provides peptide-mimetic compounds comprising a peptide derivative selected from the group consisting of pThr analong, pSer analog, Pmab, C-3 substituted Pmab and combinations thereof. In certain embodiments, the peptide-mimetic compound comprises a peptide analog of Formula 2. In certain additional embodiments, the peptide-mimetic compounds comprise at least one natural (i.e., alpha) amino acid and a peptide analog of Formula 2. In certain embodiments, the peptide analog is a C-3 substituted Pmab derived phosphatase-stable analog of phospho-threonine or phosphor-serine.

In certain embodiments, the peptide-mimetic ligand of PBD comprises a dipeptide having the structure: Ser-[Y], wherein Y is a phosphatase stable phospho-amino acid analog of Formula 2.

In certain embodiments, the peptide-mimetic ligand of PBD comprises, consists or consists essentially of the structure: Xo-6-Ser-[Z]-X'o-8, wherein X is any amino acid , and Z is phosphatase stable phospho-amino acid analog of Formula 2, and wherein only one of X or X' can be zero.

In certain additional embodiments, the peptide-mimetic ligand of PBD comprises, consists or consists essentially of the structure: X₀-3-Ser-[Z]-X'o-₂, wherein X is any amino acid , and Z is phosphatase stable phospho-amino acid analog of Formula 2, and wherein only one of X or X' can be zero.

In certain additional embodiments, the description provides a peptido-mimetic ligand of PBD comprising, consisting or consisting essentially of the structure Xo-3-His-Ser-[Z]-X'o-₂, wherein X is any amino acid , and Z is phosphatase stable phospho-amino acid analog of Formula 2.

In any of the aspects or embodiments described herein, Z can be a C-3 substituted Pmab derived phosphatase-stable analog of phospho-threonine or phosphor-serine.

In additional embodiments, the description provides peptide-mimetic ligands of polo box domains (PBD) comprising an amino acid analog of Formula 2 as described herein. In certain embodiments, the peptide-mimetic ligand of PBD comprises the structure: X-X-X-Ser- [ZJ-X-X, wherein X is any amino acid (or no amino acid), and Z refers to an amino acid analog of Formula 2 as described herein, e.g., a C-3 substituted Pmab derived phosphatase stable analog of phospho-threonine or phosphor-serine. In certain additional embodiments, X is a naturally occurring amino acid.

In certain embodiments, the ligand of the PBD comprises or consists or consists essentially of a structure selected from the group consisting of:

FSQHKTS(Z)I,

HS(Z),

N-terminal modified HS(Z) peptidomimetic,

GVLS(Z)LI,

VLS(Z)L,

N-terminal modified PLHS(Z)M and LHS(Z)M peptidomimetic,

Cyclic GLH(oct-Ph)S(Y)C thioether peptidomimetic,

FDPPLHS(Z)A,

XDPPLHS(Z)A peptidomimetic ( X = natural or non-natural amino acid),

PLHS(Z)A,

MQS(Z)PL,

FMPPPMS(Z)M,

LLCS(Z)PNGL,

MQS(Z)PL,

PMQS(Z)PLN,

and MAGPMQS(Z)PLNGAYKK, wherein Z is an amino acid analog of Formula 2 as described herein. In certain embodiments, Z is a C-3 substituted Pmab derived phosphatase stable analog of phospho-threonine or phosphor-serine.

In another aspect, the description provides a compound of Formula 1, or salt, solvate, or hydrate thereof:

Formula 1 wherein R₂ is optionally substituted C2-C4 alkyl, optionally substituted cycloalkyl, optionally substituted aralkyl, optionally substituted heteroarylalkyl, haloalkyl, optionally substituted morpholinomethyl, , or optionally subs

; each o is independently 1-3; each X is independently H, Me, Et, CF₃, F, CI,

Br, OMe, or N(Me)₂; and Y is CH₂, NH, or O.

Certain exemplified compounds of the description include, but are not limited to, the compounds of Table 1 as follows:

Formula 2

Certain exemplified compounds of the description include, but are not limited to, the compounds of Table 2 as follows:

Table 2

Formula 1

1-29 1-63

1-30 1-64

1-31 1-65

1-32 1-66

1-33 1-67

1-34 1-68

Certain exemplified compounds of the description include, but are not limited to, the compounds of Table 3 as follows:

Table 3

W-22 W-52 W-82

0

W-23 W-53 W-83

0

W-24 W-54 W-84

W-25 ^'Ό W-55 W-85

W-26 W-56 W-86

/

W-27 W-57 W-87

W-28 W-58 W-88

/

W-29 i\/^CF3 W-59 W-89

W-30 W-60

In another aspect, the description relates to a pharmaceutical composition comprising any compound of the description in a pharmaceutically acceptable carrier.

In another aspect, the description provides a method for the prevention, amelioration, or treatment of a subject for a hyperproliferative disorder comprising administration of a composition comprising any of the compounds according to the description.

In certain embodiments, the method further includes identification of a subject suffering from or suspected of suffering from a hyperproliferative disorder.

In certain embodiments, the method further comprises monitoring the subject for prevention, amelioration, or treatment of a hyperproliferative disorder. In certain embodiments, the hyperproliferative disorder comprises cancer. In certain embodiments, the cancer is selected from the group consisting of Acute Lymphoblastic Leukemia, Acute Myeloid Leukemia, Adrenocortical Carcinoma, Basal Cell Carcinoma, Bladder Cancer, Bone Cancer, Brain Tumor, Breast Cancer, Cervical Cancer, Chronic Lymphocytic Leukemia, Chronic Myelogenous Leukemia, Chronic Myeloproliferative Disorders, Colon Cancer, Colorectal Cancer, Cutaneous T-Cell Lymphoma, Esophageal Cancer, Ewing Family of Tumors, Retinoblastoma, Gastric (Stomach) Cancer, Gastrointestinal Tumors, Glioma, Head and Neck Cancer, Hepatocellular (Liver) Cancer, Hodgkin Lymphoma, Islet Cell Tumors (Endocrine Pancreas), Kidney (Renal Cell) Cancer, Laryngeal Cancer, Non-Small Cell Lung Cancer , Small Cell Lung Cancer, Lymphoma, Medulloblastoma, Melanoma, Pancreatic Cancer, Prostate Cancer, Renal Cancer, Rectal Cancer, and Thyroid Cancer.

In another aspect, the description provides a kit comprising at least one compound of the description and instructions for use.

In certain aspect, the description provides a chemical library including two or more compounds of the description.

In another embodiment, the description provides a process to prepare of a compound of Formula 2, or salt, solvate, or hydrate thereof:

Formula 2

the pro

a) to

b) reacting c

, with a compound of

Formula, O ₅ to afford a compound of Formula 2, R δ wherein R is optionally substituted cycloalkyl, optionally substituted phenylethyl, optionally substituted phenylpropyl, optionally substituted heteroarylalkyl, haloalkyl,

, or ; n is 1-3; each o is independently 1-3; each X is independently H, Me, Et, CF₃, F, CI, Br, OMe, or N(Me)₂; and Y is CH₂, NH, or O. In another aspect, the compound of Formula 2 is any of compounds 2-1 to 2-74.

In another embodiment, the description provides a process to prepare of a compound of Formula 1, or salt, sol

the process comprising:

a) phosphorylating a compound of Formula 3,

a compound of Formula 4,

b) activating the alcohol within the compound of Formula 4,

with O-phenyl thiochloroformate to afford a

c) reducing a compound of to afford a

compound of Formula 6,

d) hydrolyzing the ethyl ester of a compound of Formula 6,

, to afford a compound of Formula 7,

e) arbamate of a compound of Formula 7,

fford a compound of Formula 8,

f) protecting the amino functionality of a compound of Formula 8,

afford a compound of Formula 1 ,

wherein R₂ is optionally substituted C₂-C4 alkyl, optionally substituted cycloalkyl, optionally substituted aralkyl, optionally substituted heteroarylalkyl, haloalkyl, optionally substituted morpholinomethyl, -3. In anoth

er aspect, R₂ is Me, Et, Pr, j-Pr, Bu,

; each o is independently 1-3; each X is independently H, Me, Et, CF₃, F, CI, Br, OMe, or N(Me)₂; and Y is CH₂, NH, or O. In another aspect, the compound of Formula 1 is any of compounds 1-1 to 1-87.

In another embodiment, the description provides a process to prepare a compound of Formula 4, or salt, solvate, or hydrate thereof:

the process comprising:

a) phosphorylating a compound of Formula 3, ^R2 , with di-tert- a compound of Formula 4,

wherein R₂ is optionally substituted C₂-C₄ alkyl, optionally substituted cycloalkyl, optionally substituted aralkyl, optionally substituted heteroarylalkyl, haloalkyl, optionally substituted morpholinomethyl, O , or optionally substituted indolylalkyl; Y is CH₂, NH, or O; and o is 1-3.

In certain embodiments, the method further comprises the step of activating the alcohol within the compound of Formula 4,

afford a compound of Formula 5,

as above..

In certain additional embodiments, the method further comprises the step of reducing a compound of

, to afford a compound of Formula 6,

wherein R₂ is as above..

In an additional embodiment, the method further comprises the step of hydrolyzing the ethyl ester of a

compound of Formula 6,

to afford a compound of Formula 7,

, wherein R₂ is as above..

In another embodiment, the method further comprises the step of deprotecting the

benzyl carbamate of a compound of Formula 7,

, to afford a compound of

Formula 8,

, wherein R₂ is as above..

In certain embodiments, the method further comprises the step of protecting the

functionalit 8, , to afford a compound of

Formula 1,

rein R₂ is as above.

In another embodiment, the description provides a process to prepare of a compound of Formula II, or salt, solvate, or hydrate thereof:

Formula II

the process comprising:

a) phosphorylating a compound of Formula 9,

, with di-tert- a compound of Formula 10,

b) activating the alcohol within the compound of Formula 10,

with O-phenyl thiochloroformate to afford a

compound of Formula 11, c) reducing a compound of Formula

compound of Formula 12,

d) hydrolyzing the ethyl ester of a compound of Formula 12,

, to afford a compound of Formula 13,

e) arbamate of a compound of Formula 13,

fford a compound of Formula 14,

f) protecting the amino functionality of a compound of Formula 14,

afford a compound of Formula 15,

g) coupling an amino acid, analog or derivative thereof to the compound of Formula 15;

wherein R₃ is optionally substituted C₁-C4 alkyl, optionally substituted cycloalkyl, optionally substituted aralkyl, optionally substituted heteroarylalkyl, haloalkyl, optionally substituted morpholinomethyl, O , or optionally substituted indolylalkyl; R₄ is

ect, R₂ is Me, Et,

; each o is independently 1-3; each X is independently H, Me, Et, CF₃, F, CI, Br, OMe, or N(Me)₂; and Y

-(CH₂)₈-Ph. In another aspect, R₂ is Me, Et, Pr, -Pr,

each X is independently H, Me, Et, CF₃, F, CI, Br, OMe, or N(Me)₂; Y is CH₂, NH, or O; and R s -(CH₂)s-Ph. In another aspect the compound of Formula II is one of W-l to W-89.

In certain aspects, the description relates to a peptide derivative prepared from any method described herein, including the methods described herein.

The description also provides an isotopically labeled compound of any of the formulae delineated herein. Such compounds have one or more isotope atoms which may or may not be radioactive (e.g., ¾ ²H, 14C, ¹³C, ³⁵S, ³²P, ¹²⁵I, and ^mI) introduced into the compound. Such compounds are useful for drug metabolism studies and diagnostics, as well as therapeutic applications.

The structures of the compounds of the description may include asymmetric carbon atoms. Accordingly, the isomers arising from such asymmetry (e.g., racemates, racemic mixtures, single enantiomers, individual diastereomers, diastereomeric mixtures) are included within the scope of this description, unless indicated otherwise. Other stereoisomeric forms may be defined, in terms of absolute stereochemistry, as (R)- or (S)- , or as (D)- or (L)- for amino acids.

Such isomers can be obtained in substantially pure form by classical separation techniques and/or by stereochemically controlled synthesis. For example, optical isomers may be prepared from their respective optically active precursors by the procedures described above, or by resolving the racemic mixtures. The resolution can be carried out in the presence of a resolving agent, by chromatography or by repeated crystallization or by some combination of these techniques which are known to those skilled in the art. Further details regarding resolutions can be found in Jacques, et al., Enantiomers, Racemates, and Resolutions (John Wiley & Sons, 1981). The compounds of this description may also be represented in multiple tautomeric forms, in such instances, the description expressly includes all tautomeric forms of the compounds described herein (e.g., alkylation of a ring system may result in alkylation at multiple sites, the description expressly includes all such reaction products). In addition, some of the compounds of this description may have one or more double or triple bonds. Such compounds can occur as cis- or trans- or E- or Z- double isomeric forms, which are included within the scope of this description. Further, the configuration of any carbon-carbon double bond appearing herein is selected for convenience only and is not intended to designate a particular configuration unless the text so states; thus a carbon-carbon double bond depicted arbitrarily herein as trans may be cis, trans, or a mixture of the two in any proportion.

All crystal forms of the compounds described herein are also expressly included in the present description.

A compound of the description can be prepared as a pharmaceutically acceptable acid addition salt by reacting the free base form of the compound with a pharmaceutically acceptable inorganic or organic acid. Alternatively, a pharmaceutically acceptable base addition salt of a compound of the description can be prepared by reacting the free acid form of the compound with a pharmaceutically acceptable inorganic or organic base.

Alternatively, the salt forms of the compounds of the description can be prepared using salts of the starting materials or intermediates.

The free acid or free base forms of the compounds of the description can be prepared from the corresponding base addition salt or acid addition salt from, respectively. For example, a compound of the description in an acid addition salt form can be converted to the

corresponding free base by treating with a suitable base (e.g., ammonium hydroxide solution, sodium hydroxide, and the like). A compound of the description in a base addition salt form can be converted to the corresponding free acid by treating with a suitable acid (e.g., hydrochloric acid, etc.).

Prodrug derivatives of the compounds of the description can be prepared by methods known to those of ordinary skill in the art (e.g., for further details see Saulnier et al., (1994), Bioorganic and Medicinal Chemistry Letters, Vol. 4, p. 1985). For example, appropriate prodrugs can be prepared by reacting a non-derivatized compound of the description with a suitable carbamylating agent (e.g., 1,1-acyloxyalkylcarbanochloridate, para-nitrophenyl carbonate, or the like).

Protected derivatives of the compounds of the description can be made by means known to those of ordinary skill in the art. A detailed description of techniques applicable to the creation of protecting groups and their removal can be found in T. W. Greene, "Protecting Groups in Organic Chemistry", 3rd edition, John Wiley and Sons, Inc., 1999. Compounds of the present description can be conveniently prepared, or formed during the process of the description, as solvates (e.g., hydrates). Hydrates of compounds of the present description can be conveniently prepared by recrystallization from an aqueous/organic solvent mixture, using organic solvents such as dioxane, tetrahydrofuran or methanol.

Acids and bases useful in the methods herein are known in the art. Acid catalysts are any acidic chemical, which can be inorganic (e.g., hydrochloric, sulfuric, nitric acids, aluminum trichloride) or organic (e.g., camphor sulfonic acid, p-toluenesulfonic acid, acetic acid, ytterbium triflate) in nature. Acids are useful in either catalytic or stoichiometric amounts to facilitate chemical reactions. Bases are any basic chemical, which can be inorganic (e.g., sodium bicarbonate, potassium hydroxide) or organic (e.g., triethylamine, pyridine) in nature. Bases are useful in either catalytic or stoichiometric amounts to facilitate chemical reactions.

The synthesized compounds can be separated from a reaction mixture and further purified by a method such as column chromatography, high pressure liquid chromatography, or recrystallization. As can be appreciated by the skilled artisan, further methods of synthesizing the compounds of the formulae herein will be evident to those of ordinary skill in the art.

Additionally, the various synthetic steps may be performed in an alternate sequence or order to give the desired compounds. In addition, the solvents, temperatures, reaction durations, etc. delineated herein are for purposes of illustration only and one of ordinary skill in the art will recognize that variation of the reaction conditions can produce the desired bridged macrocyclic products of the present description. Synthetic chemistry transformations and protecting group methodologies (protection and deprotection) useful in synthesizing the compounds described herein are known in the art and include, for example, those such as described in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T.W. Greene and P.G.M. Wuts, Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), and subsequent editions thereof.

The compounds of this description may be modified by appending various

functionalities via any synthetic means delineated herein to enhance selective biological properties. Such modifications are known in the art and include those which increase biological penetration into a given biological system (e.g., blood, lymphatic system, central nervous system), increase oral availability, increase solubility to allow administration by injection, alter metabolism and alter rate of excretion.

The compounds of the description are defined herein by their chemical structures and/or chemical names. Where a compound is referred to by both a chemical structure and a chemical name, and the chemical structure and chemical name conflict, the chemical structure is determinative of the compound's identity.

The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof. The recitation of an embodiment for a variable herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

DESIGN OF THE COMPOUNDS OF THE DESCRIPTION

The description also provides methods of design and/or synthesis of a novel class of compounds that act as kinase-directed inhibitors. It is contemplated that these compounds down-regulate Plkl function and Plkl PBD-binding antagonists and may serve as anticancer agents. The description also provides methods of use thereof. In certain embodiments, the compounds of the description achieve enhanced efficacy in biochemical and/or cellular studies.

The polo-like kinase 1 (Plkl) represents a new target for anticancer therapeutic development. Plkl contains a C-terminal polo-box domain (PBD) that recognizes phospho-Ser (pSer)/phospho-Thr (pThr) -containing motifs, which recruits Plkl to specific sub-cellular sites. This event is critical for proper Plkl function.

Over-expression of Plkl induces neoplastic transformation of human cells, whereas interference with Plkl function induces apoptosis in tumor cells but not in normal cells.

Moreover, Plkl over-expression is associated with aggressive disease stage and poor patient survival in various types of cancers (Elia et ah , Modular Protein Domains, 2005, 163-179). Over the years, efforts have been made to generate anti-Plkl inhibitors, resulting in several compounds (BI 2536, GSK Compound 1, Cyclapolin 1, DAP81, and TAL) developed to competitively inhibit the kinase activity or substrate recognition of Plkl (Strebhardt, K. et al., Nat. Rev. Cancer 6, 321-330. (2006)). However, largely because of the structural similarities among the catalytic domains of all Plks and other related kinases, it has been difficult to generate Plkl-specific inhibitors. Thus, since the non-catalytic PBD is found only in the members of the Plk subfamily, development of novel inhibitors that target the PBD of Plkl may prove to be an alternative strategy for selectively targeting Plkl .

While conducting studies on the interaction between Plkl and its physiological binding target PBIP1, a minimal phosphopeptide derived from the Thr78 region of PBIP1 was identified that exhibits a high level of affinity and specificity for the Plkl PBD. Testing of a non-hydrolyzable p-T78 mimetic peptide demonstrated that inhibition of the Plkl PBD function results in a chromosome congression defect that leads to mitotic arrest and apoptotic cell death, as observed previously in cells expressing a dominant-negative PBD (Seong, Y.S. et al. J. Biol. Chem. 277, 32282-32293 (2002); & Hanisch, A. et al., Mol. Biol. Cell 17, 448-459 (2006)). Since interference with Plkl function induces apoptosis in most tumor cells but not in normal cells, these findings demonstrate that inhibition of the PBD function is sufficient to interfere with cell proliferation activity of tumor cells.

It has been demonstrated that SpT-dependent electrostatic interactions with His538 and Lys540 residues are critical for the interaction between optimal peptides (PMQSpTPL and MQSpTPL) and the Plkl PBD12,13. Comparative in vitro binding studies and analyses of the phosphopeptide-binding pockets of PBDS+G and PBDS with PBDPL, PBDPP, and PBDLH revealed that, in addition to the SpT motif of the phosphopeptide that acts as a high affinity anchor, the N-terminal residues provide additional binding affinity and specificity to the Plkl PBD through three distinct interactions. First, the polar contact between the carbonyl oxygen N-terminal to the Leu-3 of PLHSpT or LHSpTA and the guanidinium moiety of Arg516 of Plkl PBD provides a molecular basis for a high affinity and specificity interaction. Second, docking of the N-terminal Pro-4 side chain into the pocket generated by the surrounding Trp414 and Phe535 offers additional affinity and likely another level of specificity to the interaction. Notably, the PBDs from both Plk2 and Plk3 possess Lys and Tyr residues at positions analogous to the Plkl Arg516 and Phe535 residues, respectively, in Plkl, and, as a consequence, may fail to generate as favorable an environment to accommodate the N-terminal Pro residue. Third, peptide pull-down assays demonstrate that the His-2 residue adds another layer of Plkl PBD specificity.

Besides each amino acid residue of the p-T78 peptide involved in defining the Plkl binding affinity and specificity, the positions of the phosphopeptide and glycerol in the pocket, along with the network of water molecules that mediate contacts between the phosphopeptide and the PBD, suggest that both the glycerol and the network of water molecules surrounding the phosphopeptide could be important elements of the PBD recognition by phosphopeptides.

S+G S PL

Furthermore, the structures of the PBD , PBD , and PBD were remarkably similar, hinting that the other glycerol molecule and the sulfate anion occupying the phosphopeptide -binding cleft may substitute the role of the SpT dipeptide.

The collected data demonstrate that the Plkl PBD-binding pocket accommodates (i) the core SpT motif, (ii) the N-terminal hydrophobic residue, (iii) glycerol, and (iv) a network of contacting water molecules. A combination of some or all of these four elements could be potentially used for targeted drug design. Better understanding of the PBD interaction as well as further isolation and development of PBD-binding agents would greatly facilitate the discovery of a new class of Plkl -specific anti-cancer therapeutic agents.

The description provides the design, synthesis and biological evaluation of anticancer therapeutics, which act through down-regulation of oncogenic Plkl through spatial dis- regulation achieved by blocking the function of its PBD. It has been observed that the compounds of the description achieve enhanced efficacy in cellular studies.

Compositions, Methods, and Kits

The description provides compositions including any of the compounds of the description in a pharmaceutically acceptable carrier, for use, for example, for the preparation of a medicament. The medicament can be, for example, a medicament for the prevention, amelioration, or treatment of a hyperproliferative disorder such as cancer.

In still other embodiments, such compositions are labeled for the treatment of a hyperproliferative disorder such as cancer. In a further embodiment, the effective amount is effective to treat or prevent a hyperproliferative disorder such as cancer in a subject, as described herein.

In certain embodiments, the hyperproliferative disorder is cancer. Cancers can be characterized as solid tumors and non-solid tumors. Cancers include, but are not limited to Acute Lymphoblastic Leukemia, Acute Myeloid Leukemia, Adrenocortical Carcinoma, Basal Cell Carcinoma, Bladder Cancer, Bone Cancer, Brain Tumor, Breast Cancer, Cervical Cancer, Chronic Lymphocytic Leukemia, Chronic Myelogenous Leukemia, Chronic Myeloproliferative Disorders, Colon Cancer, Colorectal Cancer, Cutaneous T-Cell Lymphoma, Esophageal Cancer, Ewing Family of Tumors, Retinoblastoma, Gastric (Stomach) Cancer, Gastrointestinal Tumors, Glioma, Head and Neck Cancer, Hepatocellular (Liver) Cancer, Hodgkin Lymphoma, Islet Cell Tumors (Endocrine Pancreas), Kidney (Renal Cell) Cancer, Laryngeal Cancer, Non- small Cell Lung Cancer, Small Cell Lung Cancer, Lymphoma, Medulloblastoma, Melanoma, Pancreatic Cancer, Prostate Cancer, Renal Cancer, Rectal cancer, Thyroid Cancer. In an embodiment, the compound is administered to the subject using a pharmaceutically-acceptable formulation. In certain embodiments, these pharmaceutical compositions are suitable for oral or parenteral administration to a subject. In still other embodiments, as described in detail below, the pharmaceutical compositions of the present description may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules, pastes; (2) parenteral administration, for example, by subcutaneous, intramuscular or intravenous injection as, for example, a sterile solution or suspension; (3) topical application, for example, as a cream, ointment or spray applied to the skin; (4) intravaginally or intrarectally, for example, as a pessary, cream or foam; or (5) aerosol, for example, as an aqueous aerosol, liposomal preparation or solid particles containing the compound. In certain embodiments, the subject is a mammal, e.g., a primate, e.g., a human.

The methods of the description further include administering to a subject a

therapeutically effective amount of a compound in combination with a pharmaceutically acceptable excipient. The phrase "pharmaceutically acceptable" refers to those compounds of the description, compositions containing such compounds, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The phrase "pharmaceutically-acceptable excipient" includes pharmaceutically- acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, carrier, solvent or encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.

Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically-acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

Compositions containing a compound(s) include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, aerosol and/or parenteral administration. The compositions may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred per cent, this amount will range from about 1 per cent to about ninety-nine percent of active ingredient, preferably from about 5 per cent to about 70 per cent, most preferably from about 10 per cent to about 30 per cent.

Methods of preparing these compositions include the step of bringing into association a compound(s) with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

Compositions of the description suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in- water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a compound(s) as an active ingredient. A compound may also be administered as a bolus, electuary or paste.

In solid dosage forms of the description for oral administration (capsules, tablets, pills, dragees, powders, granules and the like), the active ingredient is mixed with one or more pharmaceutically-acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4)

disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, acetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface- active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered active ingredient moistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceutical compositions of the present description, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. The active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration of the compound(s) include

pharmaceutically-acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

In addition to inert diluents, the oral compositions can include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compound(s) may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

Pharmaceutical compositions of the description for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more compound(s) with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active agent.

Compositions of the present description which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate. Dosage forms for the topical or transdermal administration of a compound(s) include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound(s) may be mixed under sterile conditions with a pharmaceutically-acceptable carrier, and with any preservatives, buffers, or propellants which may be required.

The ointments, pastes, creams and gels may contain, in addition to compound(s) of the present description, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a compound(s), excipients, such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

The compound(s) can be alternatively administered by aerosol. This is accomplished by preparing an aqueous aerosol, liposomal preparation or solid particles containing the compound. A nonaqueous (e.g., fluorocarbon propellant) suspension could be used. Sonic nebulizers are preferred because they minimize exposing the agent to shear, which can result in degradation of the compound.

Ordinarily, an aqueous aerosol is made by formulating an aqueous solution or suspension of the agent together with conventional pharmaceutically-acceptable carriers and stabilizers. The carriers and stabilizers vary with the requirements of the particular compound, but typically include nonionic surfactants (T weens, Pluronics, or polyethylene glycol), innocuous proteins like serum albumin, sorbitan esters, oleic acid, lecithin, amino acids, such as glycine, buffers, salts, sugars or sugar alcohols. Aerosols generally are prepared from isotonic solutions.

Transdermal patches have the added advantage of providing controlled delivery of a compound(s) to the body. Such dosage forms can be made by dissolving or dispersing the agent in the proper medium. Absorption enhancers can also be used to increase the flux of the active ingredient across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the active ingredient in a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutions and the like, are also contemplated as being within the scope of this description. Pharmaceutical compositions of this description suitable for parenteral administration comprise one or more compound(s) in combination with one or more pharmaceutically- acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the description include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants, such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be

accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally-administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.

Injectable depot forms are made by forming microencapsule matrices of compound(s) in biodegradable polymers, such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue.

When the compound(s) are administered as pharmaceuticals, to humans and animals, they can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a

pharmaceutically-acceptable carrier.

Regardless of the route of administration selected, the compound(s), which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present description, are formulated into pharmaceutically-acceptable dosage forms by conventional methods known to those of skill in the art.

Actual dosage levels and time course of administration of the active ingredients in the pharmaceutical compositions of this description may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. An exemplary dose range is from about 0. ^g to 20 milligram per kilogram of body weight per day (mg/kg/day) (e.g., 0.^g/kg to 2mg/kg, 0.3-3μg/kg, 0.18-0.54mg/kg). In other embodiments, the amount varies from about 0.1 mg/kg/day to about 100 mg/kg/day. In still other embodiments, the amount varies from about 0.001 μg to about 100 μg/kg (e.g., of body weight). Ranges intermediate to the above-recited values are also intended to be part of the description.

The description also provides methods including identification of a subject suffering from or suspected of suffering from a hyperproliferative disorder and/or monitoring the subject for prevention, amelioration, or treatment of a hyperproliferative disorder.

The description provides kits for the treatment or prevention of a hyperproliferative disorder such as cancer. The kits contain at least one compound of the descriptions and instructions for use. In one embodiment, the kit includes a therapeutic or prophylactic composition containing an effective amount of a compound of the description in unit dosage form. The description also provides kits having 2, 3, 4, 5, 6, 7, 8, 9, or 10 compounds of the description.

As used herein, "kits" are generally understood to contain at least the non-standard laboratory reagents for use in the methods of the description. For example, a kit can include at least one of, preferably at least two of at least one peptide for modification, one or more aldehyde molecules for modification of peptides, and instructions for use, all in appropriate packaging. The kit can further include any other components required to practice the method of the description, as dry powders, concentrated solutions, or ready to use solutions. In some embodiments, the kit comprises one or more containers that contain reagents for use in the methods of the description; such containers can be boxes, ampules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding reagents.

In some embodiments, a compound of the description is provided in combination with a conventional therapeutic agent. In other embodiments, the kit comprises a sterile container which contains a therapeutic or prophylactic composition; such containers can be boxes, ampoules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.

If desired a compound of the description is provided together with instructions for administering the compound to a subject having or at risk of developing neoplasia. The instructions will generally include information about the use of the composition for the treatment or prevention of neoplasia. In other embodiments, the instructions include at least one of the following: description of the therapeutic agent; dosage schedule and administration for treatment or prevention of ischemia or symptoms thereof; precautions; warnings;

indications; counter-indications; overdosage information; adverse reactions; animal pharmacology; clinical studies; and/or references. The instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.

The description further provides libraries including at least two compounds of the description."Library" as used herein is understood to be a chemical library. Chemical libraries include two or more compounds (10 or more, 50 or more, 100 or more, 200 or more, 500 or more, 1000 or more, 5000 or more, 10,000 or more, etc.; or any range bracketed by the noted values), preferably that have structural and/or potential functional properties. Libraries can be used, for example for screening assays to select compounds with desired activities, e.g., kinase binding, kinase stimulating, kinase inhibiting activity.

Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.

The practice of the present description employs, unless otherwise indicated, conventional techniques that are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, "Molecular Cloning: A Laboratory Manual", second edition (Sambrook, 1989); "Oligonucleotide Synthesis" (Gait, 1984); "Animal Cell Culture" (Freshney, 1987); "Methods in Enzymology" "Handbook of Experimental

Immunology" (Weir, 1996); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Current Protocols in Molecular Biology" (Ausubel, 1987); "PCR: The Polymerase Chain Reaction", (Mullis, 1994); "Current Protocols in Immunology" (Coligan, 1991).

Particularly useful techniques for particular embodiments will be discussed in the sections that follow.

The following non-limiting examples are illustrative of the description.

I. SYNTHESIS AND CHEMICAL ANALYSIS OF THE COMPOUNDS OF THE DESCRIPTION

A. SYNTHESIS AND PREPARATION

Compounds of the description can be synthesized and/or prepared by methods described in this section, the examples, and the chemical literature. For example, Scheme 1 illustrates the synthesis of the compounds of the description, beginning with reacting compounds of Formula 2 with di-tert-butyl phosphonate to afford compounds of Formula 16. Deoxygenation of compounds of Formula 16 affords compounds of Formula 18, which after ester hydrolysis, Fmoc-protection, and benzyloxycarbonyl (Cbz) removal affords compounds of Formula 1. Compounds of Formula 1 are then converted to the Final Compounds via primary amide formation, and peptide synthesis.

Scheme 1

In an exemplary scheme, Compound 2 is treated with (a) di-tert-butyl phosphite (1.5 equiv.), chlorotrimethylsilane (TMS-C1, 1.5 equiv.), triethylamine (TEA, 2.0 equiv.), dichloromethane (DCM), 6 h, rt; and (b) 20% aq. citric acid (w/v), MeOH, 16 h at RT to afford in compound 16. Compound 16 is then treated with O-phenyl thiochloroformate (1.5 equiv.), N, N-dimethylaminopyridine (DMAP, 3.0 equiv.), acetonitrile (MeCN) at RT to result in Compound 17. Compound 17 is treated with tributyltin hydride (3.0 equiv.), azoisobutylnitrile (AIBN, 1.0 equiv.), toluene, reflux, 20 min., to result in Compound 18. Compound 18 is treated with (a) LiOH (2.0 equiv.), tetrahydrofuran (THF)/H20 (3: 1), 16 h, rt; (b) 1 atm H₂, Pd/C (10% w/w, 0.1 equiv.), MeOH, 3 h, RT; (c) Fmoc-OSu (1.5 equiv.), NaHC0₃ (2.0 equiv.), THF/H20 (1 : 1), 16 h, rt, to yield Compound 1. As indicated above, the description provides a synthetic scheme capable of producing Compound 1 from Compound 2 in seven steps.

Phosphatase- stable analogs of phospho-threonine containing a variety of orthogonal protecting groups, which can be efficiently accessed via the key (2S)-,(3R)-alkyl intermediate and the synthetic route described herein. The new analogs are intended to afford enhanced interactions with Arg557 and Leu491 (See Figure 1), which are located proximal to the 3- position of Pmab in the pThr-binding pocket.

1. General Procedures:

General Methods.

All experiments involving moisture-sensitive compounds were conducted under anhydrous conditions (positive argon pressure) using standard syringe, cannula, and septa apparatus. Commercial reagents were purchased from Sigma, TCI America, Acros, or Chem-Impex. Fmoc-Ser(Trt)-OH, Fmoc-His(Mtt)-OH, Fmoc-Leu-OH, Fmoc-Pro-OH and Fmoc- Thr[PO(OH)((OBn)]-OH were purchased from Chem-Impex. Fmoc-His[N i)-(CH₂)₈-Ph]-OH (His*) was synthesized as previously described (39). All solvents were purchased in anhydrous form (Aldrich) and used directly. High-performance liquid chromatography (HPLC)-grade hexanes, ethyl acetate (EtOAc), dichloromethane (DCM), and methanol (MeOH) were used in chromatography purifications. Analytical thin-layer chromatography (TLC) was performed using Analtech precoated plates (Uniplate, silica gel GHLF, 250 nm) containing a fluorescence indicator. Silica column chromatography employed a Telodyne CombiFlash Rf 200i insturment with either hexane/EtOAc or DCM/MeOH gradients. Nuclear magnetic resonance (NMR) spectra were recorded using a Varian Inova 400 MHz spectrometer. Coupling constants are reported in Hertz, and peak shifts are reported in δ (ppm) relative to CDCI₃. Infrared (IR) spectra were measured on a Jasco FT/IR-4100 spectrometer. Optical rotation was measured on a Jasco P-1010 polarimeter. Low-resolution mass spectra (LRMS) were measured with either an Agilent 260 1200 LC/MSD-SL system or a Shimadzu 2020 LC/MS system. High resolution mass spectra (HRMS) were obtained by positive ion, ESI analysis on a Thermo Scientific LTQ- XL Orbitrap mass spectrometer with HPLC sample introduction using a short narrow-bore Cls reversed-phase column with CH₃CN - H₂0 gradients. Preparative HPLC purification of final peptides was performed using a Waters 2545 binary pump (MeCN/water gradient) with a Phenomenex Gemini-C18 (5 μιη, 250 x 21 mm) preparative column and ultraviolet (UV) detection. Analytical HPLC of final peptides was performed using an Agilent 1200 series quaternary pump (MeCN/water gradient) with a Phenomenex Kinetix-C18 (5 μιη, 250 x 4 mm) analytical column and UV detection.

Synthetic Procedures

General Procedure A. Compounds 2 were prepared using a similar procedure as was previously disclosed. (40, 41) The aldehyde (R₃CH₂-CHO, 1.2 - 2 equiv.) was dissolved in chloroform (0.2 M) and stirred at room temperature (RT). (S)-2-(bis(3,5- bis(trifluoromethyl)phenyl)((trimethylsilyl)oxy)methyl) pyrrolidine (0.1 equiv., Sigma) was added and the reaction was allowed to stir for 5 minutes. Ethyl ((benzyloxy) carbonyl)- tosylglycinate (1 equiv.; synthesized by a literature procedure (42)) and potassium fluoride (5 equiv.) were then added and the reaction was allowed to stir at RT for 24 - 96 hours (h).

Following completion, the reaction mixture was filtered through Celite and the eluent was concentrated. Purification by silica column chromatography (hexane/EtOAc gradient) afforded the corresponding product. ^]H NMR was used to determine anti/syn diastereomeric ratio. General Procedure B. Di-tert-butyl phosphite (1.5 equiv.) and triethylamine (2 equiv.) were added to DCM (0.2 M) in a round bottom flask at 0 °C. Chlorotrimethylsilane (1.5 equiv.) was then added and was allowed to stir at 0 °C for an additional 5 minutes to generate a white precipitate. The corresponding aldehyde 2 (1 equiv.) from General Procedure A was added and the reaction was further stirred for 3 - 6 h at RT. Following completion, the reaction was diluted with DCM, washed with brine, and the aqueous layer extracted with additional DCM. The combined organic layers were dried over Na₂S0₄ and concentrated. The resulting residue was re-dissolved in MeOH (0.05 M), followed by the addition of 20% aqueous citric acid (30% v/v) and the reaction was stirred at RT overnight. Following removal of the TMS-group by TLC, the reaction was diluted with EtOAc and washed with saturated (sat.) aqueous (aq.) NaHCC>3. The aqueous layer was extracted with EtOAc and the combined organic layers were dried over Na₂S0₄ and concentrated. Purification by silica column chromatography

(hexane/EtOAc gradient) afforded the corresponding product as a mixture of diastereomers. General Procedure C. Secondary alcohol 16 from General Procedure B was dissolved in DCM (0.1 - 0.2 M) and placed in a round-bottom flask with stirring at RT. Diisopropylethylamine (DIEA, 4 equiv.) and Ν,Ν-dimethylaminopyridine (DMAP, 0.2 equiv.) were added, followed by O-phenyl chlorothionoformate (3 equiv.). The reaction was allowed to stir for 3 - 16 h and the reaction progress was followed by TLC. Following completion, water was added and the reaction was allowed to quench for 30 minutes with stirring at RT. The mixture was then diluted with DCM and water and the aqueous layer was extracted with DCM. The combined organic layers were dried over Na₂S0₄, filtered, and concentrated. Purification by silica column chromatography (hexane/EtOAc gradient) afforded the corresponding phenylthiocarbonate as a mixture of diastereomers. The phenylthiocarbonate was dissolved in toluene (0.1 M) and placed in a round-bottom flask with stirring. Azobis-isobutylnitrile (AIBN, 1 equiv.) and tributyltin hydride (3 equiv.) were added and the reaction was heated to 110 °C for 20 minutes. Upon reaction completion, the reaction was cooled to RT and the toluene was removed under vacuum. The resulting residue was directly purified by silica column chromatography

(hexane/EtOAc gradient) to afford the corresponding product.

General Procedure D. Benzyloxycarbonyl (Cbz)-protected ethyl ester 18 from General Procedure D was dissolved in tetrahydrofuran (THF, 0.1 M) and added to a round-bottom flask at RT. Lithium hydroxide (3 equiv.) was dissolved in water (25% v/v) and added to the THF solution. The reaction was allowed to stir overnight at RT. Following saponification, the THF was removed under vacuum and the aqueous solution was diluted with 0.5 M HC1 and extracted with EtOAc twice. The combined organic layers were dried over Na₂S0₄ and concentrated. The resulting residue was dissolved in MeOH (0.05 M) and degassed with argon for several minutes. Palladium on carbon (0.2 equiv., 10% w/w) was added with stirring. The reaction was placed under a blanket of hydrogen gas and stirred at RT for 3 h to remove the Cbz group. Once completed, the reaction mixture was filtered through Celite and concentrated. The resulting residue was dissolved in 1: 1 THF/water (0.05 M). Sodium bicarbonate (5 equiv.) was added, followed by 9-Fluorenylmethyl-N-succinimidyl carbonate (Fmoc-Osu, 1.5 equiv.) and the reaction was allowed to stir overnight. Once completed, the THF was removed under vacuum and the aqueous mixture was diluted with 0.5 M HC1. The aqueous solution was extracted twice with EtOAc and the combined organic layers were dried over Na₂S0₄ and concentrated. Purification by silica column chromatography (DCM/MeOH gradient) afforded compound 1.

General Solid-Phase Peptide Synthesis (SPPS) procedure. NovaSyn TG Sieber resin was pre- swollen in DMF (4 mL) for 1 h with shaking. The resin was Fmoc- deprotected using 20% piperidine in DMF (4 mL) twice for 10 minutes each. Fmoc-protected amino acids (2 - 4 equivalents based on resin) were dissolved in DMF (3-4 mL) containing 4% DIEA and pre- activated by the addition of (l-[bis(dimethylamino)methylene]-lH-l,2,3-triazolo[4,5- bjpyridinium 3-oxid hexafluorophosphate) (HATU, 0.95 mol equivatents relative to the amino acid) for 5 minutes with gentle agitation. The resin was washed 4 times with DMF (6-8 mL), and the HATU-activated amino acid solution was added to the washed resin. Coupling reactions were shaken at room temperature and allowed to proceed from 3-16 hours depending on the equivalents used and steric bulk of each amino acid. Coupling reactions were routinely checked for completion using the Kaiser test. Once completed, the resin was filtered and washed 4 times with DMF (6-8 mL), followed by Fmoc-deprotection using 20% piperidine in DMF (4 mL, 2x 10 minutes each). Cleavage from Sieber resin and global deprotection was performed using 33% TFA with 2% triisopropylsilane (TIPS) in DCM. Crude peptides were purified using preparative reverse-phase HPLC with gradient elution (89.9/10/0.1

water/acetonitrile/trifluoroacetic acid (TFA) to 99.9/0.1 acetonitrile/TFA over 30 minutes). Example 1: Preparation of (3S,6S,9S,12R,13S)-l-((S)-l-acetylpyrrolidin-2-yl)-12- carbamoyl-9-(hvdroxymethyl)-3-isobutyl-13-methyl-l,4,7,10-tetraoxo-6-((l-(8- phenyloctyl)-lH-imidazol-5-yl)methyl)-2,5,8,ll-tetraazatetradecan-14-ylphosphonic acid (W-64)

Preparation of Ethyl (2S,3R)-2-(((benzyloxy)carbonyl)amino)-3-methyl-4-oxobutanoate (2b).

Propionaldehyde (1.3 mL, 17.9 mmol, 2 equiv.) was reacted with ethyl ((benzyloxy)carbonyl)- tosylglycinate (3.4 g, 8.94 mmol) using a similar procedure as outlined in General Procedure A to provide ethyl (2S,3R)-2-(((benzyloxy)carbonyl)amino)-3-methyl-4-oxobutanoate (2b, 2.26 g, 86%, 12: 1 anti/syn) as a white foam. [a]_D ²⁰ = +27.6 (c 1.00, CHC1₃); ^]H NMR (400 MHz, Chloroform-d) δ 9.64 (s, 1H), 7.45 - 7.28 (m, 5H), 5.58 (d, J = 8.3 Hz, 1H), 5.13 (s, 2H), 4.70 (dd, J = 8.4, 3.8 Hz, 1H), 4.19 (q, J = 7.1 Hz, 2H), 3.18 (dd, J = 7.3, 3.8 Hz, 1H), 1.24 (t, J = 7.1 Hz, 3H), 1.18 (d, J = 7.4 Hz, 3H); ¹³C NMR (101 MHz, CDC1₃) δ 201.58, 170.34, 156.39, 136.21, 128.68, 128.37, 128.18, 67.33, 62.15, 54.20, 48.84, 14.16, 9.61; IR (film) 2980, 1724, 1513, 1338, 1061, 1027, 920, 859 cm^"1; LR-MS (ESI⁺) calculated for Ci₅H₁₉N0₅: 294.1

[M+H⁺]; found: 294.3.

Preparation of Ethyl (2S,3R)-2-(((benzyloxy)carbonyl)amino)-4-(di-tert-butoxyphosphoryl)-4- hydroxy-3-methylbutanoate (16b).

Ethyl (2S,3R)-2-(((benzyloxy)carbonyl)amino)-3-methyl-4-oxobutanoate (2b, 2.1 g, 7.16 mmol) was reacted with di-tert-butyl phosphite using a similar procedure as outlined in General Procedure B to provide ethyl (2S,3R)-2-(((benzyloxy)carbonyl)amino)-4-(di-tert- butoxyphosphoryl)-4-hydroxy-3-methylbutanoate (16b, 2.88 g, 83%) as a mixture of diastereomers. [a]_D ²° = -1.6 (c 1.00, CHC1₃); ^]H NMR (400 MHz, Chloroform-d) δ 7.41 - 7.28 (m, 5H), 6.73 (d, J = 7.5 Hz, 0.5H), 6.35 (d, J = 9.8 Hz, 0.5H), 5.89 (d, J = 8.2 Hz, 0.5H), 5.11 (s, 2H), 4.34 - 4.10 (m, 3H), 3.88 (d, J = 8.1 Hz, 0.5H), 2.85 - 2.77 (m, 0.5H), 2.53 - 2.42 (m, 0.5H), 1.59 - 1.37 (m, 18H), 1.26 (t, J = 6.9 Hz, 3H), 1.13 (d, J = 7.0 Hz, 3H); ¹³C NMR (101 MHz, CDCI₃) δ 172.05, 156.92, 136.56, 128.63, 128.59, 128.19, 83.71, 70.67, 69.02, 67.04, 61.51, 59.70, 59.53, 30.62, 30.47, 14.32, 11.55; IR (film) 2980, 1725, 1507, 1394, 1370, 1321, 1255, 1164, 1096, 1065, 1038, 980, 919, 697 cm^"1; LR-MS (ESI⁺) calculated for C₂₃H₃₈N0₈P: 488.2 [M+H⁺]; found: 488.4.

Preparation of Ethyl (2S,3R)-2-(((benzyloxy)carbonyl)amino)-4-(di-tert-butoxyphosphoryl)-3- methylbutanoate (18b).

Ethyl (2S,3R)-2-(((benzyloxy)carbonyl)amino)-4-(di-tert-butoxyphosphoryl)-4-hydroxy-3- methylbutanoate (16b, 2.18 g, 5.13 mmol) was prepared using a similar procedure as outlined in General Procedure C to provide ethyl (2S,3R)-2-(((benzyloxy)carbonyl)amino)-4-(di-tert- butoxyphosphoryl)-3-methylbutanoate (18b, 1.3 g, 61% over 2 steps) as a white foam. [α]ο = +6.6 (c 1.00, CHCI₃); ^]H NMR (400 MHz, Chloroform-d) δ 7.39 - 7.27 (m, 5H), 5.78 (d, J = 8.5 Hz, 1H), 5.10 (s, 2H), 4.27 (dd, J = 8.1, 5.3 Hz, 1H), 4.19 (q, J = 7.1 Hz, 2H), 2.47 - 2.31 (m, 1H), 1.82 - 1.69 (m, 1H), 1.56 - 1.52 (m, 1H), 1.52 - 1.43 (m, 18H), 1.27 (t, J = 7.9 Hz, 3H), 1.12 (d, J = 6.8 Hz, 3H); ¹³C NMR (101 MHz, CDC1₃) δ 171.72, 156.35, 136.48, 128.61, 128.24, 128.21, 82.12, 67.08, 61.55, 59.70, 59.56, 33.89, 32.44, 30.56, 30.54, 30.52, 30.50,

30.46, 17.62, 17.58, 14.34; IR (film) 2979, 1721, 1538, 1370, 1259, 1038, 1007, 980, 919, 698 cm^"1; LR-MS (ESI⁺) calculated for C2₃H₃₈NO7P: 472.2 [M+H⁺]; found: 472.4. Preparation of(2S,3R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(di-tert- cid (lb).

Ethyl (2S,3R)-2-(((benzyloxy)carbonyl)amino)-4-(di-tert-butoxyphosphoryl)-4-hydroxy-3- methylbutanoate (18b, 1.28 g, 2.71 mmol) was prepared using a similar procedure as outlined in General Procedure D to provide (2S,3R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4- (di-tert-butoxyphosphoryl)-3-methylbutanoic acid (lb, 1.25 g, 87%) as a white foam. [α]ο²° = +30.3 (c 1.00, CHC1₃); ^]H NMR (400 MHz, Chloroform-d) δ 7.76 (d, J = 7.5 Hz, 2H), 7.60 (dd, J = 7.1, 4.3 Hz, 2H), 7.39 (t, J = 7.5 Hz, 2H), 7.31 (t, J = 7.1 Hz, 2H), 5.85 (d, J = 7.3 Hz, 1H), 4.76 (dd, J = 7.0, 4.7 Hz, 1H), 4.37 (d, J = 7.1 Hz, 2H), 4.22 (t, J = 7.1 Hz, 1H), 2.61 - 2.48 (m, 1H), 1.93 - 1.83 (m, 1H), 1.74 - 1.64 (m, 1H), 1.54 (d, J = 22.8 Hz, 18H), 1.04 (d, J = 6.9 Hz, 3H). ¹³C NMR (101 MHz, CDC1₃) δ 171.83, 155.51, 143.91, 141.43, 127.82, 127.17, 125.32, 120.10, 83.96, 67.11, 56.97, 47.28, 30.55, 25.52, 16.98; IR (film) 2981, 1714, 1510, 1450, 1395, 1371, 1263, 1247, 1153, 1077, 1038, 989, 754, 703, 667, 621 cm^"1; LR-MS (ESI⁺) calculated for C2₈H₃₈NO7P: 532.2 [M+H⁺]; found: 532.5.

Preparation of(3S,6S,9S,12R,13S)-l-((S)-l-acetylpyrrolidin-2-yl)-12-carbamoyl-9- (hydroxymethyl)-3 sobutyl-13-methyl-l,4, 7,10-tetraoxo-6-((l-(8-phenyloctyl)-lH-imidazol-5- ic acid (W-64).

The peptide 3S,6S,9S,12R,13S)-l-((S)-l-acetylpyrrolidin-2-yl)-12-carbamoyl-9-

(hydroxymethyl)-3-isobutyl-13-methyl-l,4,7,10-tetraoxo-6-((l-(8-phenyloctyl)-lH-imidazol-5- yl)methyl)-2,5,8,l l-tetraazatetradecan-14-ylphosphonic acid (W-64) was synthesized on a 0.1 mmol scale using the general SPPS procedure. Purification by preparative Reverse Phase (RP)- HPLC afforded the final peptide (W-64, 49 mg, 57% overall) with >95% purity by analytical HPLC. Low Resolution (LR)-MS (ESr) calculated for C₄iH65N₈OioP: 861.5 [M+H⁺]; found: 861.7.

Example 2: Preparation of (3S,6S,9S,12R,13S)-l-((S)-l-acetylpyrrolidin-2-yl)-13-benzyl- 12-carbamoyl-9-(hvdroxymethyl)-3-isobutyl-l,4,7,10-tetraoxo-6-((l-(8-phenyloctyl)-lH- imidazol-5-yl)methyl)-2,5,8,ll-tetraazatetradecan-14-ylphosphonic acid (W-66)

Preparation of Ethyl (2S,3R)-3-benzyl-2-(((benzyloxy)carbonyl)amino)-4-oxobutanoate (2e,

Hydrocinnamaldehyde (539 mg, 4.02 mmol, 2 equiv.) was reacted with ethyl ((benzyloxy)- carbonyl)-tosylglycinate (786 mg, 2.01 mmol) using a similar procedure as outlined in General Procedure A to provide ethyl (2S,3R)-3-benzyl-2-(((benzyloxy)carbonyl)amino)-4- oxobutanoate (2e, n=l, X=H; 425 mg, 57%; 12: 1 anti/syn) as a white foam. [a]_D ²⁰ = +32.2 (c 1.00, CHC1₃); ^]H NMR (400 MHz, Chloroform-d) δ 9.65 (s, 1H), 7.44 - 7.12 (m, 10H), 5.55 (d, J = 9.4 Hz, 1H), 5.15 (s, 2H), 4.60 (dd, J = 9.5, 3.1 Hz, 1H), 4.17 (q, J = 7.1 Hz, 2H), 3.49 (td, J = 8.1, 3.5 Hz, 1H), 3.10 (dd, J = 14.1, 6.8 Hz, 1H), 2.80 (dd, J = 14.1, 8.4 Hz, 1H), 1.23 (t, J = 7.1 Hz, 3H); ¹³C NMR (101 MHz, CDC1₃) δ 201.97, 170.73, 156.62, 137.77, 136.34, 129.17, 129.00, 128.69, 128.38, 128.19, 127.08, 67.35, 62.17, 55.59, 52.98, 31.79, 14.17; IR (film) 2935, 1721, 1498, 1455, 1370, 1331, 1255, 1050, 1028, 966, 699 cm^"1; LR-MS (ESI⁺) calculated for C21H2₃NO5: 370.2 [M+H⁺]; found: 370.3.

Preparation of Ethyl (2S,3R)-3-benzyl-2-(((benzyloxy)carbonyl)amino)-4-(di-tert- butoxyphosphoryl)-4-hydroxybutanoate (16e, 11= 1 , X=H).

Ethyl (2S,3R)-3-benzyl-2-(((benzyloxy)carbonyl)amino)-4-oxobutanoate (2e, n=l, X=H; 400 mg; 1.083 mmol) was reacted with di-tert-butyl phosphite using a similar procedure as outlined in General Procedure B to provide ethyl (2S,3R)-3-benzyl-2-(((benzyloxy)carbonyl)amino)-4- (di-tert-butoxyphosphoryl)-4-hydroxybutanoate (16e, n=l, X=H; 400 mg; 66%) as a mixture of diastereomers. [a]_D ²⁰ = -14.8 (c 1.00, CHC1₃); ^]H NMR (400 MHz, Chloroform-d) δ 7.44 - 7.27 (m, 5H), 7.25 - 7.11 (m, 5H), 6.20 (d, J = 8.3 Hz, 0.7H), 5.97 (d, J = 8.1 Hz, 0.3H), 5.17 - 5.10 (m, 2H), 4.21 - 4.08 (m, 2H), 3.99 (d, J = 8.9 Hz, 1H), 3.37 - 3.30 (m, 1H), 2.91 - 2.44 (m, 3H), 1.53 (d, J = 4.4 Hz, 18H), 1.19 (t, J = 7.1 Hz, 3H); ¹³C NMR (101 MHz, CDC1₃) δ 172.39, 156.66, 139.52, 136.87, 129.28, 128.75, 128.54, 128.24, 128.12, 126.51, 84.02, 71.32, 66.85, 61.42, 56.19, 42.43, 31.17, 30.72, 14.28; IR (film) 2980, 1506, 1498, 1395, 1370, 1256, 1189, 1162, 1095, 1082, 1039, 982, 919, 699 cm^"1; LR-MS (ESI⁺) calculated for C29H42NO₈P: 564.3 [M+H⁺]; found: 564.5.

Preparation of Ethyl (2S,3R)-3-benzyl-2-(((benzyloxy)carbonyl)amino)-4-(di-tert- 1, X=H).

Ethyl (2S,3R)-3-benzyl-2-(((benzyloxy)carbonyl)amino)-4-(di-tert-butoxyphosphoryl)-4- hydroxybutanoate (16e, n=l, X=H; 380 mg; 0.674 mmol) was reacted using a similar procedure as outlined in General Procedure C to provide ethyl (2S,3R)-3-benzyl-2-

(((benzyloxy)carbonyl)amino)-4-(di-tert-butoxyphosphoryl)butanoate (18e, n=l, X=H; 216 mg; 58% over 2 steps) as a white foam. [a]_D ²⁰ = -2.3 (c 1.00, CHCI₃); ^]H NMR (400 MHz, Chloroform-if) δ 7.45 - 7.11 (m, 10H), 6.21 (d, J = 9.0 Hz, 1H), 5.28 - 4.99 (m, 2H), 4.40 (dd, J = 9.0, 3.9 Hz, 1H), 4.15 (q, J = 7.2 Hz, 2H), 3.01 - 2.84 (m, 1H), 2.78 - 2.54 (m, 2H), 1.75 - 1.57 (m, 2H), 1.46 (s, 18H), 1.24 (t, J = 7.1 Hz, 3H); ¹³C NMR (101 MHz, CDC1₃) δ 172.07, 156.53, 139.44, 136.66, 129.53, 128.60, 128.56, 128.17, 128.16, 126.51, 82.57, 66.97, 61.56, 56.51, 38.97, 37.77, 30.50, 29.85, 14.31; IR (film) 2979, 1723, 1541, 1370, 1259, 1038, 1006, 979, 918, 699 cm^"1; LR-MS (ESI⁺) calculated for C29H42NO7P: 548.3 [M+H⁺]; found: 548.5. Preparation of(2S,3R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-benzyl-4-(di-tert- butoxyphosphoryl)butanoic acid (1-64).

Ethyl (2S,3R)-3-benzyl-2-(((benzyloxy)carbonyl)amino)-4-(di-tert- butoxyphosphoryl)butanoate (18e, n=l, X=H; 200 mg; 0.365 mmol) was reacted using a similar procedure as outlined in General Procedure D to provide (2S,3R)-2-((((9H-fluoren-9- yl)methoxy)carbonyl)amino)-3-benzyl-4-(di-tert-butoxyphosphoryl)butanoic acid (1-64, 180 mg, 81%) as a white foam. [a]_D ²° = +40.3 (c 1.00, CHC1₃); ^]H NMR (400 MHz, Chloroform-d) δ 7.76 (d, J = 7.5 Hz, 2H), 7.62 (t, J = 6.5 Hz, 2H), 7.39 (t, J = 7.5 Hz, 2H), 7.34 - 7.07 (m, 7H), 5.98 (d, J = 7.0 Hz, 1H), 4.97 - 4.92 (m, 1H), 4.39 (d, J = 7.3 Hz, 2H), 4.23 (t, J = 7.3 Hz, 1H), 3.10 - 3.04 (m, 1H), 2.82 - 2.69 (m, 1H), 2.31 (dd, J = 13.8, 10.1 Hz, 1H), 1.88 - 1.75 (m, 1H), 1.62 - 1.40 (m, 19H). ¹³C NMR (101 MHz, CDC1₃) δ 171.82, 155.23, 143.76, 141.30, 139.05, 129.10, 128.65, 127.70, 127.08, 126.46, 125.18, 119.96, 83.91, 67.04, 55.76, 47.12, 38.28, 37.03, 30.39, 25.38; IR (film) 2980, 1714, 1509, 1450, 1395, 1371, 1247, 1153, 1077, 1039, 984, 918, 754, 700, 666, 621 cm^"1; LR-MS (ESI⁺) calculated for C₃4H42NO7P: 608.3 [M+H⁺]; found: 608.5.

Preparation of(3S,6S,9S,12R,13S)-l-((S)-l-acetylpyrrolidin-2-yl)-13-benzyl-12-carbamoyl-9- (hydroxymethyl)-3-isobutyl-l,4, 7,10-tetraoxo-6-( ( l-(8-phenyloctyl)-lH-imidazol-5-yl)methyl)- -tetraazatetradecan-14-ylphosphonic acid (W-66).

The peptide 3S,6S,9S,12R,13S)-l-((S)-l-acetylpyrrolidin-2-yl)-13-benzyl-12-carbamoyl-9-

(hydroxymethyl)-3-isobutyl-l,4,7,10-tetraoxo-6-((l-(8-phenyloctyl)-lH-imidazol-5-yl)methyl)- 2,5,8,1 l-tetraazatetradecan-14-ylphosphonic acid (W-66) was synthesized on a 0.05 mmol scale using the general SPPS procedure. Purification by preparative RP-HPLC afforded the final peptide (W-66, 14 mg, 30% overall) with >95% purity by analytical HPLC. LR-MS

(ESI⁺) calculated for C47H69N₈O1₀P: 937.5 [M+H⁺]; found: 937.8.

Example 3: Preparation of (3S,6S,9S,12R,13S)-l-((S)-l-acetylpyrrolidin-2-yl)-12- carbamoyl-9-(hvdroxymethyl)-3-isobutyl-l,4,7,10-tetraoxo-13-phenethyl-6-((l-(8- phenyloctyl)-lH-imidazol-5-yl)methyl)-2,5,8,ll-tetraazatetradecan-14-ylphosphonic acid iw^i Preparation of Ethyl (2S,3R)-2-(((benzyloxy)carbonyl)amino)-3-formyl-5-phenylpentanoate (2-

4-Phenyl butanal (320 mg, 2.159 mmol, 2 equiv.) was reacted with ethyl

((benzyloxy)carbonyl)-tosylglycinate (786 mg, 2.01 mmol) using a similar procedure as outlined in General Procedure A to provide ethyl (2S,3R)-2-(((benzyloxy)carbonyl)amino)-3- formyl-5-phenylpentanoate (2-2, 365 mg, 88%, 11 : 1 anti/syn) as a white foam. [α]ο²° = +45.3 (c 1.00, CHC1₃); ^]H NMR (400 MHz, Chloroform-d) δ 9.57 (s, 1H), 7.47 - 7.08 (m, 10H), 5.55 (d, J = 9.4 Hz, 1H), 5.15 (s, 2H), 4.74 (dd, J = 9.5, 3.7 Hz, 1H), 4.18 (q, J = 6.4, 5.7 Hz, 2H), 3.19 - 3.12 (m, 1H), 2.94 - 2.85 (m, 1H), 2.80 - 2.70 (m, 1H), 2.14 - 1.97 (m, 1H), 1.83 - 1.71 (m, 1H), 1.24 (t, J = 7.1 Hz, 3H); ¹³C NMR (101 MHz, CDC1₃) δ 202.09, 170.82, 159.54, 140.75, 136.31, 128.77, 128.72, 128.64, 128.38, 128.17, 126.50, 77.36, 77.36, 67.38, 62.15, 53.30, 52.44, 33.50, 26.99, 14.19; IR (film) 2933, 1720, 1498, 1455, 1370, 1083, 1061, 1028, 698 cm^"1; LR-MS (ESI⁺) calculated for C22H25NO5: 384.2 [M+H⁺]; found: 384.3.

Preparation of Ethyl (2S,3R)-2-(((benzyloxy)carbonyl)amino)-3-((di-tert- 5 -phenylpentanoate (16e, n=2, X=H).

Ethyl (2S,3R)-2-(((benzyloxy)carbonyl)amino)-3-formyl-5-phenylpentanoate (2-2, 260 mg. 0.678 mmol) was reacted with di-tert-butyl phosphite using a similar procedure as outlined General Procedure B to provide ethyl (2S,3R)-2-(((benzyloxy)carbonyl)amino)-3-((di-tert- butoxyphosphoryl) (hydroxy) methyl)-5-phenylpentanoate (16e, n=2, X=H; 302 mg; 77%) as a mixture of diastereomers. [a]_D ²° = -7.7 (c 1.00, CHCI₃); ^]H NMR (400 MHz, Chloroform-if) δ 7.41 - 7.27 (m, 5H), 7.26 - 7.09 (m, 5H), 6.44 (d, J = 8.6 Hz, 0.7H), 5.71 (d, J = 9.9 Hz, 0.3H), 5.17 - 5.05 (m, 2H), 4.70 - 4.61 (m, 0.7H), 4.26 - 4.21 (m, 0.3H), 4.21 - 4.05 (m, 2H), 3.88 - 3.77 (m, 0.7H), 3.77 - 3.67 (m, 0.7H), 3.51 - 3.40 (m, 0.3H), 3.27 - 3.19 (m, 0.3H), 2.86 - 2.51 (m, 2H), 2.26 - 2.13 (m, 0.6H), 2.02 - 1.80 (m, 1.4H), 1.56 - 1.38 (m, 18H), 1.26 (t, J = 7.2, 3H); ¹³C NMR (101 MHz, CDC1₃) δ 172.42, 156.95, 140.94, 136.68, 128.65, 128.57, 128.40, 128.30, 128.13, 126.28, 83.93, 66.96, 61.51, 58.55, 50.48, 39.75, 33.30, 30.51, 26.39, 14.30; IR (film) 2979, 1746, 1723, 1507, 1498, 1395, 1370, 1257, 1166, 1038, 982, 919, 698, 686 cm^"1; LR-MS (ESI⁺) calculated for C₃₀H44NO₈P: 578.3 [M+H⁺]; found: 578.5.

Preparation of Ethyl (2S,3R)-2-( ((benzyloxy)carbonyl)amino)-3-( ( di-tert- lpentanoate (16e, n=2, X=H).

Ethyl (2S,3R)-2-(((benzyloxy)carbonyl)amino)-3-((di-tert-butoxyphosphoryl)(hydroxy) methyl)-5-phenylpentanoate (16e, n=2, X=H; 240 mg; 0.415 mmol) was reacted via General Procedure C to provide ethyl (2S,3R)-2-(((benzyloxy)carbonyl)amino)-3-((di-tert- butoxyphosphoryl)methyl)-5-phenylpentanoate (18e, n=2, X=H; 105 mg; 45% over 2 steps) as a white foam. [a]_D ²° = -4.8 (c 1.00, CHCI₃); ^]H NMR (400 MHz, Chloroform-d) δ 7.44 - 7.04 (m, 10H), 6.23 (d, J = 8.9 Hz, 1H), 5.23 - 5.02 (m, 2H), 4.60 (dd, J = 8.7, 4.5 Hz, 1H), 4.28 - 4.07 (m, 2H), 2.87 - 2.57 (m, 2H), 2.49 - 2.27 (m, 1H), 2.05 - 1.92 (m, 1H), 1.85 - 1.52 (m, 4H), 1.44 (d, J = 9.0 Hz, 18H), 1.27 (t, J = 7.1 Hz, 3H); ¹³C NMR (101 MHz, CDC1₃) δ 172.22, 156.73, 141.84, 136.63, 128.64, 128.58, 128.54, 128.45, 128.17, 125.92, 82.58, 67.01, 61.62, 56.52, 36.50, 33.41, 33.12, 31.59, 30.49, 14.30; IR (film) 2981, 1718, 1541, 1370, 1259, 1038, 1008, 982, 673 cm^"1; LR-MS (ESI⁺) calculated for C₃₀H44NO7P: 562.3 [M+H⁺]; found: 562.5. Preparation of(2S,3R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-((di-tert- ntanoic acid (1-2).

Ethyl (2S,3R)-2-(((benzyloxy)carbonyl)amino)-3-((di-tert-butoxyphosphoryl)methyl)-5- phenylpentanoate (18e, n=2, X=H; 100 mg; 0.178 mmol) was reacted using a similar procedure as outlined in General Procedure D to provide (2S,3R)-2-((((9H-fluoren-9- yl)methoxy)carbonyl)amino)-3-((di-tert-butoxyphosphoryl) methyl)- 5 -phenylpentanoic acid (1- 2, 82 mg, 74%) as a white foam. [a]_D ²⁰ = +30.4 (c 1.00, CHC1₃); ^]H NMR (400 MHz, Chloroform-d) δ 7.75 (d, J = 7.2 Hz, 2H), 7.61 (t, J = 7.7 Hz, 2H), 7.38 (t, J = 7.3 Hz, 2H), 7.32 - 7.12 (m, 7H), 5.95 (d, J = 7.3 Hz, 1H), 4.87 (dd, J = 7.1, 4.2 Hz, 1H), 4.35 (d, J = 7.2 Hz, 2H), 4.21 (t, J = 7.1 Hz, 1H), 2.76 - 2.56 (m, 3H), 2.50 - 2.36 (m, 1H), 2.06 - 1.86 (m, 2H), 1.72 - 1.58 (m, 1H), 1.57 - 1.47 (m, 18H). ¹³C NMR (101 MHz, CDC1₃) δ 172.31, 155.60, 144.11, 143.92, 141.42, 128.55, 128.51, 127.82, 127.18, 126.12, 125.33, 120.10, 84.02, 67.17, 55.75, 47.26, 36.42, 33.51, 32.50, 30.53, 25.52; IR (film) 2979, 1716, 1509, 1450, 1395, 1371, 1319, 1255, 1248, 1219, 1156, 1078, 1040, 988, 758, 700, 667 cm^"1; LR-MS (ESI⁺) calculated for C₃5H44NO7P: 622.3 [M+H⁺]; found: 622.6.

Preparation of3S,6S,9S,12R,13S)-l-((S)-l-acetylpyrrolidin-2-yl)-12-carbamoyl-9- (hydroxymethyl)-3 sobutyl-l,4, 7 0-tetraoxo-13^henethyl-6-((l-(8-phenyloctyl)-lH-imidazol- -yl )methyl)-2, 5, 8, 1 l-tetraazatetradecan-14-ylphosphonic acid (W-2).

The peptide 3S,6S,9S,12R,13S)-l-((S)-l-acetylpyrrolidin-2-yl)-12-carbamoyl-9- (hydroxymethyl)-3-isobutyl-l,4,7,10-tetraoxo-13-phenethyl-6-((l-(8-phenyloctyl)-lH- imidazol-5-yl)methyl)-2,5,8,l l-tetraazatetradecan-14-ylphosphonic acid (W-2) was synthesized on a 0.05 mmol scale using the general SPPS procedure. Purification by preparative RP-HPLC afforded the final peptide (W-2, 22 mg, 46% overall) with >95% purity by analytical HPLC. LR-MS (ESI⁺) calculated for C₄₈H7iN₈OioP: 951.5 [M+H⁺]; found: 951.8. II. BIOLOGICAL EXAMPLES

Example 4: Enzyme-Linked Immunosorbent Assay (ELISA)-based Inhibition Assay Against Full length Plkl and Plkl PBD (43)

Transfection and Protein Lysate Production. Plasmids encoding full length Plkl (Plasmid #41160) (44) and Plkl PBD (Plasmid #41162) (45) linked to a 3x myc tagged were purchased from Addgene (Deposited by Prof. Erich Nigg). HEK293T cells were plated on 10 cm culture dishes at 4M cells per plate. Following 24 h, the cells were transfected with 10 μg of plasmid DNA using 20 μL· of TurboFect transfection reagent (Pierce Biotechnology) according to the manufacturer's instructions. Following 24 h, the cells were harvested using trypsin, washed with phosphate-buffered saline (PBS) 7.4 buffer, and lysed in lysis buffer (PBS 7.4 + 0.5% NP-40 + protease/phosphatase inhibitor cocktail (Pierce Biotechnology)) using 3 freeze/thaw cycles. The lysed suspension was centrifuged at 10,000 xG for 10 minutes to pellet membrane proteins and nuclei. The supernatant was removed to provide a crude cytosolic lysate containing expressed myc-tagged Plkl or Plkl PBD. The total protein concetration was determined using a Bicinchoninic Acid (BCA) assay kit (Pierce Biotechnology).

ELISA Inhibition Assay.

All assay steps were performed at room temperature with gentle shaking. A

biotinylated phosphopeptide (sequence: Biotin-Ahx-PMQS(pT)PLN-NH₂) (46-48) was diluted with PBS 7.4 to 1 μΜ (from a 10 mM dimethylsulfoxide (DMSO) stock solution) and loaded onto the wells of a 96-well Neutravidin-coated plate (Pierce Biotechnology) at 100 μL· per well for 1 h. The wells were washed once with 150 μΐ. PBST (PBS 7.4 + 0.05% Tween-20), and then 100 μL· of 1% bovine serum albumin (BSA) in PBS 7.4 (blocking buffer) were added for 1 h. The cytosolic lysate containing myc-tagged protein was diluted to 300 μg/mL in PBS 7.4 containing protease/phosphatase inhibitors (Pierce Biotechnology), mixed with competitive inhibitor (from a 10X stock in 5% DMSO/PBS), and allowed to pre-incubate for 1 h (100 μΐ, per well in a 96-well plate, 30 μg total protein). The blocked ELISA plate was washed 2x with PBST (150 μΕ) and the pre-incubated lysates were added to the plate to incubate for 1 h. The wells were washed 4x with PBST (150 μΕ), then probed with anti-myc primary antibody (1:3000 dilution, mouse monoclonal, Pierce Biotechnology) for 1 h. The wells were then washed 4x with PBST (150 μΕ), and incubated with rabbit anti-mouse horseradish peroxidase (HRP) conjugate (1: 10,000 dilution, Pierce Biotechnology) for 1 h. The wells were then washed 5x with PBST (150 μί) and incubated with Turbo TMB -ELISA solution (Pierce Biotechnology) until the desired absorbance is reached (5-10 minutes). The reaction was quenched by the addition of 2M aq. H₂SO₄ and the absorbance was measured at 450 nm using a BioTek Synergy 2 96-well plate reader. Absorbance was plotted versus concentration (log M) and fit to a non-linear regression using GraphPad Prism 6 software (model: log(inhibitor) vs. response— Variable slope (four parameters)) to provide IC₅₀ values. IC₅₀ values from multiple independent experiments were normalized and averaged to provide values + standard deviation. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the description described herein. Such equivalents are intended to be encompassed by the claims.

INCORPORATION BY REFERENCE

The entire contents of all patents, published patent applications and other references cited herein are hereby expressly incorporated herein in their entireties by reference.

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Claims

We claim:

1. A compound of

Formula 2

wherein, R is optionally substituted cycloalkyl, optionally substituted phenylethyl, optionally substituted phenylpropyl, optionally substituted heteroarylalkyl, haloalkyl, optionally substituted morpholinomethyl, 0 , or optionally substituted indolylalkyl; and

Y is CH₂, NH, or O;

or salt, solvate, or hydrate thereof.

n is 2-3;

each o is independently 1-3;

each X is independently H, Me, Et, CF₃, F, CI, Br, OMe, or N(Me)₂; and

Y is CH₂, NH, or O.

3. A compound of

wherein R₂ is optionally substituted C2-C4 alkyl, optionally substituted cycloalkyl, optionally substituted aralkyl, optionally substituted heteroarylalkyl, haloalkyl, optionally substituted morpholinomethyl, O ; and

Y is CH₂, NH, or O;

or optionally substituted indolylalkyl, or salt, solvate, or hydrate thereof.

4.

each o is independently 1-3;

each X is independently H, Me, Et, CF₃, F, CI, Br, OMe, or N(Me)₂; and

Y is CH₂, NH, or O.

5. A compound of Formula II:

Formula II

wherein R₂ is optionally substituted C₂-C₄ alkyl, optionally substituted cycloalkyl, optionally substituted aralkyl, optionally substituted heteroarylalkyl, haloalkyl, optionally substituted morpholinomethyl,

, or optionally substituted indolylalkyl;

Y is CH₂, NH, or O; and

R₄ is optionally substituted aralkyl, or salt, solvate, or hydrate thereof.

6. The compound of claim 5, wherein R₂ is Et, Pr, /-Pr, Bu,

each ο is independently 1-3 ;

each X is independently H, Me, Et, CF₃, F, CI, Br, OMe, or N(Me)₂;

Y is CH₂, NH, or O; and

R₄ is -(CH₂)₈-Ph.

7. A process to prepare of a compound of Formula 2, or salt, solvate, or hydrate thereof:

HN^O

R O

Formula 2

the process comprising:

reacting to

afford c

b) reacting c

ompound 3, , with a compound of Formula, O , to afford a compound of Formula 2,

wherein R is optionally substituted cycloalkyl, optionally substituted phenylethyl, optionally substituted phenylpropyl, optionally substituted heteroarylalkyl, haloalkyl, optionally substituted morpholinomethyl, O , or optionally substituted indolylalkyl; and Y is CH₂, NH, or O, and o is 1-3.

n is 2-3;

each o is independently 1-3;

Y is CH₂, NH, or O; and

each X is independently H, Me, Et, CF₃, F, CI, Br, OMe, or N(Me)₂.

9. A process to prepare a compound of Formula 4, or salt, solvate, or hydrate thereof:

the process comprising: b) phosphorylating a compound of Formula 3,

with di-tert- butylphosphite to afford a compound of Formula 4,

wherein R₂ is optionally substituted C2-C4 alkyl, optionally substituted cycloalkyl, optionally substituted aralkyl, optionally substituted heteroarylalkyl, haloalkyl, optionally substituted morpholinomethyl, O , or optionally substituted indolylalkyl;

Y is CH₂, NH, or O; and o is 1-3.

10. The process of claim 9, further comprising the step of activating the alcohol within the

compound of Formula 4,

-phenyl thiochloroformate to

afford a compound of Formula 5,

wherein R₂ is defined as above.

11. The process of claim 10, further comprising the step of reducing a compound of

to afford a compound of Formula 6,

, wherein R₂ is defined as above.

12. The process of claim 11, further comprising the step of hydrolyzing the ethyl ester of a

compound of Formula 6, to afford a compound of Formula 7,

, wherein R₂ is defined as above.

13. The process of claim 12, further compris of deprotecting the benzyl

carbamate of a compound of Formula 77,,

, to afford a compound of Formula 8,

, wherein R₂ is defined as above.

The process of claim 13, further comprising the step of protecting the

, to afford a compound

s above.

14. The rocess of claim 9 wherein R is Me, Et, Pr, -Pr, Bu, V , "° x , ··— y ,

each o is independently 1-3;

Y is CH₂, NH, or O; and

each X is independently H, Me, Et, CF₃, F, CI, Br, OMe, or N(Me)₂.

15. A process to prepare a compound of Formula II, or salt, solvate, or hydrate thereof:

Formula II

the process comprising: a) phosphorylating a compound of Formula 9,

, with di-tert- butylphosphite to afford a compound of Formula 10,

b) activating the alcohol within the compound of Formula 10,

with O-phenyl thiochloroformate to afford a

compound of Formula

c) reducing a compound of Formula 11,

to afford a compound of Formula 12,

d) hydrolyzing the ethyl ester of a compound of Formula 12,

to afford a compound of Formula 13,

e) deprotecting the benzyl carbamate of a compound of Formula 13, fford a compound of Formula 14,

f) protecting the amino functionality of a compound of Formula 14,

, to afford a compound of Formula 15,

wherein R₃ is optionally substituted C C₄ alkyl, optionally substituted cycloalkyl, optionally substituted aralkyl, optionally substituted heteroarylalkyl, haloalkyl, optionally

; each o is independently 1-3; each X is independently H, Me, Et, CF₃, F, CI, Br, OMe, or N(Me)₂; and Y is CH₂, NH, or O;

Y is CH₂, NH, or O;

each R₄ is independently optionally substituted aralkyl; and

o is 1-3.

16.

each o is independently 1-3; Y is CH₂, NH, or O; and

each X is independently H, Me, Et, CF₃, F, CI, Br, OMe, or N(Me)₂.

each o is independently 1-3;

each X is independently H, Me, Et, CF₃, F, CI, Br, OMe, or N(Me)₂;

Y is CH₂, NH, or O; and

each R₄ is -(CH₂)₈-Ph.

18. A peptido-mimetic compound comprising the compound of claim 1.

19. The peptido-mimetic compound of claim 19, wherein the peptido-mimetic is a polo box domain (PBD) ligand.

20. The peptido-mimetic compound of claim 20, wherein the ligand has the structure: Xo-₆ -Ser-[Z]-X'o-8, wherein X is any amino acid, and Z refers to an amino acid analog of claim 1, and only one of X or X' can be zero.

21. The peptido-mimetic compound of claim 21, wherein Z is a C-3 substituted Pmab derived phosphatase stable analog of phospho-threonine or phospho-serine.

22. The peptido-mimetic of claim 21, wherein X is a naturally occurring amino acid.

23. The peptido-mimetic compound of claim 20, wherein the PBD ligand is a member selected from the group consisting of:

FSQHKTS(Z)I, HS(Z),

N-terminal modified HS(Z) peptidomimetic,

GVLS(Z)LI,

VLS(Z)L,

N-terminal modified PLHS(Z)M and LHS(Z)M peptidomimetic, Cyclic GLH(oct-Ph)S(Y)C thioether peptidomimetic,

FDPPLHS(Z)A,

XDPPLHS(Z)A peptidomimetic ( X = natural or non-natural amino acid), PLHS(Z)A,

MQS(Z)PL,

FMPPPMS(Z)M,

LLCS(Z)PNGL,

MQS(Z)PL,

PMQS(Z)PLN,

and MAGPMQS(Z)PLNGAYKK, wherein Z is a C-3 substituted Pmab derived phosphatase stable analog of phospho-threonine or phosphor-serine.