Bicyclic peptide ligands with detectable moieties and uses thereof

Abstract

To provide agents that selectively bind to transmembrane proteins such as CAIX, MT1-MMP, PSMA, CD38, EphA2, or αvβ3 for the diagnosis, imaging, and treatment of cancer.SOLUTION: The present invention relates to polypeptides which are covalently bound to molecular scaffolds such that two or more peptide loops are subtended between bonding points to the scaffold and further linked to a detectable moiety. In particular, the invention describes bicyclic peptide ligands useful for selectively delivering the linked detectable moiety to cancer cells. The invention also describes peptides which are high affinity binders of CAIX, MT1-MMP, PSMA, CD38, EphA2, or αvβ3.SELECTED DRAWING: None

Description

Technical Field

[0001] Sequence Listing This application contains a Sequence Listing that has been electronically filed in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy, created on June 25, 2018, is named 392664-004WO(160781)_SL.txt and is 3,997 bytes in size. Technical Field of the Invention The present invention relates to polypeptides covalently bound to a molecular scaffold, such that two or more peptide loops are sandwiched between attachment points to the scaffold and further linked to a detectable moiety. In particular, the present invention describes bicyclic peptide ligands useful for selectively delivering a linked detectable moiety to cancer cells. The present invention also describes peptides that are high affinity binders of carbonic anhydrase IX (CAIX), membrane type 1 matrix metalloproteinase (MT1-MMP), prostate specific membrane antigen (PSMA), cluster of differentiation 38 (CD38), Eph receptor tyrosine kinase A2 (EphA2) or integrin αvβ3 (αvβ3). The present invention also includes pharmaceutical compositions comprising the peptide ligands, and the use of the peptide ligands in diagnosing, imaging, preventing, suppressing or treating disorders or diseases mediated by CAIX, MT1-MMP, PSMA, CD38, EphA2 or αvβ3.

Background Art

[0002] Background of the Invention Transmembrane proteins overexpressed in cancer cells provide potential means for selectively targeting and imaging cancer cells. Imaging of cancer cells plays an important role in cancer diagnosis and treatment. Six such transmembrane proteins are carbonic anhydrase IX (CAIX), membrane type 1 matrix metalloproteinase (MT1-MMP), prostate specific membrane antigen (PSMA), cluster of differentiation 38 (CD38), Eph receptor tyrosine kinase A2 (EphA2) and integrin αvβ3 (αvβ3).

[0003] Human carbonic anhydrase IX (hCA IX) is an isoform that binds to the outer cell membrane with its catalytic domain located in the extracellular space. Under physiological conditions, hCAIX is expressed only in specific tissues of the digestive tract. Its overexpression has been shown both in vitro and in vivo during hypoxia in cancer cells. Expression of hCAIX has been detected in carcinomas of the neck, ovary, kidney, esophagus, lung, breast and brain. In tumors, hCAIX is a critical molecule for maintaining normal levels of intracellular pH, and its expression preferentially proliferates hypoxic tumor cells under acidic conditions (Chiche et al. (2009) Cancer Res 69, 358).

[0004] MT1-MMP is a transmembrane metalloprotease that plays a major role in extracellular matrix remodeling by directly degrading some of its components and indirectly activating pro-MMP2. MT1-MMP is critical for tumor angiogenesis (Sounni et al (2002) FASEB J. 16(6), 555-564) and is overexpressed in various solid tumors.

[0005] Prostate-specific membrane antigen (PSMA) (also known as glutamate carboxypeptidase II (GCPII), N-acetyl-L-aspartyl-L-glutamate peptidase I (NAALADase I), and NAAG peptidase) is an enzyme encoded by the FOLH1 (folate hydrolase 1) gene in humans. Human GCPII contains 750 amino acids and has a weight of approximately 84 kDa. Human PSMA is highly expressed in the prostate, approximately 100-fold more than in most other tissues. In some prostate cancers, PSMA is the second most upregulated gene product, with an 8- to 12-fold increase in levels compared to non-cancerous prostate cells. Due to this high expression, PSMA has been developed as a potential biomarker for some cancer therapies and imaging. In human prostate cancer, tumors with higher expression are associated with a shorter time to progression and a greater percentage of patients suffering from recurrence.

[0006] CD38 is a 45kD type II transmembrane glycoprotein with a long C-terminal extracellular domain and a short N-terminal cytoplasmic domain. CD38 is upregulated in many hematopoietic malignancies and in cell lines derived from various hematopoietic malignancies, including non-Hodgkin lymphoma (NHL), Burkitt lymphoma (BL), multiple myeloma (MM), B chronic lymphocytic leukemia (B-CLL), B and T acute lymphocytic leukemia (ALL), T cell lymphoma (TCL), acute myeloid leukemia (AML), hairy cell leukemia (HCL), Hodgkin lymphoma (HL), and chronic myeloid leukemia (CML).

[0007] EphA2 is a 130 kDa receptor tyrosine kinase that is expressed in adult epithelia. A member of the Eph family of tyrosine kinases, known as ephrins, EphA2 is a transmembrane receptor tyrosine kinase with a cell-binding ligand. EphA2 expression has been found to be altered in many metastatic cells, including lung, breast, colon, and prostate tumors. In addition, the distribution and / or phosphorylation of EphA2 are altered in metastatic cells. Moreover, cells transformed to overexpress EphA2 demonstrate malignant growth, and stimulation of EphA2 is sufficient to reverse malignant growth and invasiveness. EphA2 is a potent oncogenic protein.

[0008] Integrin αvβ3 is a transmembrane glycoprotein receptor that plays a major role in tumor growth, invasion, metastasis, and angiogenesis. It is overexpressed in various types of tumor cells (Jin et al (2016) Mol Cancer Ther; 15(9). 2076-2085). Accordingly, there remains a high unmet need in developing agents that selectively bind to transmembrane proteins such as CAIX, MT1-MMP, PSMA, CD38, EphA2, or αvβ3 for cancer diagnosis, imaging, and treatment.

Prior Art Documents

Non-Patent Documents

[0009]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Summary of the Invention

Means for Solving the Problem

[0010] It has now been found that the compounds of the present invention and pharmaceutically acceptable compositions thereof are effective as inhibitors of CAIX, MT1-MMP, PSMA, CD38, EphA2 or αvβ3. Such compounds have the general formula I:

Chemical Formula

[0011] The compounds of the present invention and pharmaceutically acceptable compositions thereof are useful for treating various diseases, disorders or conditions associated with CAIX, MT1-MMP, PSMA, CD38, EphA2 or αvβ3. Such diseases, disorders or conditions include those described herein. The compounds provided by the present invention are also useful for the study of CAIX, MT1-MMP, PSMA, CD38, EphA2 or αvβ3 enzymes in biological and pathological phenomena; the study of these enzymes present in body tissues; and the in vitro or in vivo comparative evaluation of new CAIX, MT1-MMP, PSMA, CD38, EphA2 or αvβ3 inhibitors.

Brief Description of the Drawings

[0012]

Figure 1

Figure 2

Figure 3A

Figure 3B

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Figure 15

Figure 16

Figure 17

Figure 18

Figure 19

Figure 20

Figure 21

Figure 22A

Figure 22B

[0013] DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS 1. Overview of Certain Embodiments of the Invention: Using dedicated phage display and cyclic peptide technology (Bicycle® technology), a high-affinity binding peptide for membrane-type matrix metalloprotease 1 (MT1-MMP / MMP14) was identified. MT1-MMP (MT1) is a cell surface membrane protease that is normally involved in tissue remodeling and has been found to be overexpressed in many solid tumors. Overexpression of MT1 has been associated with cancer invasiveness and poor prognosis. Attempts to target the proteolytic activity of MT1 and other MMPs in cancer have mostly failed in clinical trials due to toxicity caused by insufficient selectivity, but MT1-MMP remains an attractive cancer target for targeted cytotoxic delivery approaches.

[0014] Using a diverse selection phage library containing unique peptide sequences from 10 11 to 10 13 that are post-translationally cyclized on a thiol-reactive scaffold, a small (1.5 - 2 kDa) constrained bicyclic peptide binder (bicycle) to the hemopexin domain of MT1 was identified. The initial binder was subjected to affinity maturation by directed screening and stabilization by chemical optimization.

[0015] The bicyclic constrained peptide binder (bicyclic) was identified to bind to the hemopexin domain of MT1 with an apparent Kd of approximately 2 nM. The bicyclic peptide (N241) binds with an affinity similar to that of the entire extracellular domain of the protease, but does not show binding to the catalytic domain. N241 does not show binding to any of the closely related MMP family members tested (MMP15, MMP16, MMP24, MMP1, Pro-MMP1, MMP2). Characterization of the pharmacological effects of N241 on MT1 in vitro indicates that the peptide has no direct effect on the catalytic activity of the protease, on the related MMP catalytic activities (MMP1, MMP2 and MMP9), or on cell migration or invasion. However, the binding of fluorescently tagged N241 to MT1 in HT1080 fibrosarcoma cells results in rapid internalization of the compound and subsequent lysosomal localization. In addition, 177 When Lu-labeled N241 is intravenously injected into mice bearing MT1-positive tumor xenografts, it demonstrates rapid tumor localization at high levels, such as a dose of 15-20% injected per gram of tumor in less than 60 minutes. In contrast, the unbound bicyclic peptide does not show tumor localization. These properties suggest that N241 could be a good delivery vehicle for a detectable moiety targeting MT1-positive tumor cells. Bicyclic detectable moiety conjugates (BDMCs) with various linkers and detectable moieties that retain binding to MT1 were prepared. The imaging activity of selected BDMCs was demonstrated in MT1-positive human tumor cell xenografts in mice as described in WO2016 / 067035, which is hereby incorporated by reference in its entirety.

[0016] A series of bicyclic detectable moiety conjugates (BDMCs) were prepared that contain a constrained bicyclic peptide that binds with high affinity and specificity to membrane-type matrix metalloprotease 1 (MT1-MMP, MMP14), which is covalently linked via a linker to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), a chelating ligand capable of binding to a radionuclide. MT1-MMP is necessarily involved in tissue remodeling, but overexpression of this cell surface protease has been linked to poor patient prognosis with respect to tumor aggressiveness and invasiveness, as well as many cancer hallmarks. A bicyclic binder for MT1-MMP (N241) was identified using a dedicated phage display peptide technology consisting of a highly diverse phage library of linear amino acid sequences constrained within two loops by a central chemical scaffold. While binding with similar affinity and specificity as observed with monoclonal antibodies, the small-sized bicyclic peptides (1.5 - 2 kDa) facilitate their rapid extravasation and tumor penetration to make this an ideal format for targeted delivery of detectable moieties for cancer imaging and therapy.

[0017] A series of bicyclic-linker-detectable moiety BDMCs with variable spacer formats were prepared to modulate the bicyclic presentation and evaluated for their ability to target and image tumors in an MT1-positive tumor xenograft model.

[0018] The bicyclic detectable moiety conjugates (BDMCs) of the present invention demonstrate selective targeting of tumor cells in a human tumor xenograft model of fibrosarcoma. Without wishing to be bound by any particular theory, it is believed that the small-sized BDMCs may offer significant advantages over other targeted imaging approaches, such as antibody-detectable moiety conjugates, by virtue of improved rapid extravasation and tumor penetration.

[0019] In certain embodiments, the present invention provides a method of treating a particular cancer in a subject, the method comprising administering to the subject an effective amount of a detectable moiety conjugate comprising a high affinity binder of MT1-MMP, or a pharmaceutically acceptable salt or composition thereof.

[0020] In certain embodiments, the present invention provides a method of imaging a particular cancer in a subject, the method comprising administering to the subject an effective amount of a detectable moiety conjugate comprising a high affinity binder of MT1-MMP, or a pharmaceutically acceptable salt or composition thereof.

[0021] In a similar fashion, dedicated phage display and cyclic peptide technology (Bicycle® technology) was utilized to identify high affinity binding peptides for carbonic anhydrase IX (CAIX), PSMA, CD38, EphA2 and integrin αvβ3.

[0022] In certain embodiments, the present invention provides a method of treating a particular cancer in a subject, the method comprising administering to the subject an effective amount of a detectable moiety conjugate comprising a high affinity binder of CAIX, or a pharmaceutically acceptable salt or composition thereof.

[0023] In certain embodiments, the present invention provides a method of imaging a particular cancer in a subject, the method comprising administering to the subject an effective amount of a detectable moiety conjugate comprising a high affinity binder of CAIX, or a pharmaceutically acceptable salt or composition thereof.

[0024] In certain embodiments, the present invention provides a method of treating a particular cancer in a subject, the method comprising administering to the subject an effective amount of a detectable moiety conjugate comprising a high affinity binder of PSMA, or a pharmaceutically acceptable salt or composition thereof.

[0025] In certain embodiments, the present invention provides a method for imaging a particular cancer in a subject, the method comprising administering to the subject an effective amount of a detectable moiety conjugate comprising a high affinity binder of PSMA, or a pharmaceutically acceptable salt or composition thereof.

[0026] In certain embodiments, the present invention provides a method for treating a particular cancer in a subject, the method comprising administering to the subject an effective amount of a detectable moiety conjugate comprising a high affinity binder of CD38, or a pharmaceutically acceptable salt or composition thereof.

[0027] In certain embodiments, the present invention provides a method for imaging a particular cancer in a subject, the method comprising administering to the subject an effective amount of a detectable moiety conjugate comprising a high affinity binder of CD38, or a pharmaceutically acceptable salt or composition thereof.

[0028] In certain embodiments, the present invention provides a method for treating a particular cancer in a subject, the method comprising administering to the subject an effective amount of a detectable moiety conjugate comprising a high affinity binder of EphA2, or a pharmaceutically acceptable salt or composition thereof.

[0029] In certain embodiments, the present invention provides a method for imaging a particular cancer in a subject, the method comprising administering to the subject an effective amount of a detectable moiety conjugate comprising a high affinity binder of EphA2, or a pharmaceutically acceptable salt or composition thereof.

[0030] In certain embodiments, the present invention provides a method for treating a particular cancer in a subject, the method comprising administering to the subject an effective amount of a detectable moiety conjugate comprising a high affinity binder of αvβ3, or a pharmaceutically acceptable salt or composition thereof.

[0031] In certain embodiments, the invention provides a method of imaging a particular cancer in a subject, the method comprising administering to the subject an effective amount of a detectable moiety conjugate comprising a high affinity binding agent for αvβ3, or a pharmaceutically acceptable salt or composition thereof.

[0032] In some embodiments, the peptide sequence is treated with a molecular scaffold reagent to form a compound of the invention.

[0033] The compounds and compositions of the invention are useful as inhibitors of CAIX, MT1-MMP, PSMA, CD38, EphA2 or αvβ3. In some embodiments, the provided compounds inhibit CAIX. In some embodiments, the provided compounds inhibit MT1-MMP. In some embodiments, the provided compounds inhibit PSMA. In some embodiments, the provided compounds inhibit CD38. In some embodiments, the provided compounds inhibit EphA2. In some embodiments, the provided compounds inhibit αvβ3.

[0034] In certain embodiments, the invention provides a compound of formula I:

Chemical formula

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

[0035] 2. Compounds and Definitions: Peptide Ligand The compounds of the present invention include those generally described herein and are further exemplified by the classes, subclasses and species disclosed herein. As used herein, unless otherwise indicated, the following definitions shall apply. For the purposes of the present invention, chemical elements are identified according to the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75 th Ed. In addition, general principles of organic chemistry are described in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito: 1999 and "March's Advanced Organic Chemistry", 5 th Ed., Ed.: Smith, M.B. and March, J., John Wiley & Sons, New York: 2001, the entire contents of which are incorporated herein by reference.

[0036] Cyclic peptides can bind to protein targets with high affinity and target specificity and are, therefore, an attractive class of molecules for the development of therapeutic agents. Indeed, some cyclic peptides are already being successfully used in the clinic, for example, as the antibacterial peptide vancomycin, the immunosuppressive drug cyclosporine, or the anticancer drug octreotide (Driggers et al. (2008), Nat Rev Drug Discov 7 (7), 608-24). The favorable binding properties result from the relatively large interaction surface formed between the peptide and the target and the reduced conformational flexibility of the cyclic structure. Typically, macrocycles bind to surfaces of hundreds of square angstroms, such as, for example, the cyclic peptide CXCR4 antagonist CVX15 (400 Å2; Wu et al. (2007), Science 330, 1066-71), the cyclic peptide with an Arg-Gly-Asp motif that binds to integrin αVβ3 (355 Å2) (Xiong et al. (2002), Science 296 (5565), 151-5), or the cyclic peptide inhibitor uPAIN-1 that binds to urokinase-type plasminogen activator (603 Å2; Zhao et al. (2007), J Struct Biol 160 (1), 1-10).

[0037] Due to their cyclic arrangement, peptide macrocycles are less flexible than linear peptides, leading to a smaller loss of entropy upon binding to the target and resulting in higher binding affinity. The reduced flexibility also leads to locking of the target-specific conformation and an increase in binding specificity compared to linear peptides. This effect has been exemplified by potent and selective inhibitors of matrix metalloproteinase 8, MMP-8, which lose their selectivity less than other MMPs when their ring is opened (Cherney et al. (1998), J Med Chem 41 (11), 1749-51). The favorable binding properties achieved through macrocyclization are even more pronounced in polycyclic peptides with more than one peptide ring, such as, for example, in vancomycin, nisin, and actinomycin.

[0038] Different research teams have previously linked polypeptides with cysteine residues to synthetic molecular structures (Kemp and McNamara (1985), J. Org. Chem; Timmerman et al. (2005), ChemBioChem). Meloen and colleagues used tris(bromomethyl)benzene and related molecules for the rapid and quantitative cyclization of multiple peptide loops to a synthetic scaffold for structural mimicry of the protein surface (Timmerman et al. (2005), ChemBioChem). Methods for the generation of candidate pharmaceutical compounds, in which the compounds are generated by linking a cysteine-containing polypeptide to a molecular scaffold such as tris(bromomethyl)benzene, are disclosed in WO2004 / 077062 and WO2006 / 078161.

[0039] Phage display-based combinatorial approaches have been developed to generate and screen large libraries of bicyclic peptides against a target of interest (Heinis et al. (2009), Nat Chem Biol 5 (7), 502-7 and WO2009 / 098450). Briefly, a combinatorial library of linear peptides containing two regions of three cysteine residues and six random amino acids (Cys-(Xaa)6-Cys-(Xaa)6-Cys) was displayed on phage and cyclized by covalently linking the cysteine side chains to a small molecule (tris-(bromomethyl)benzene).

[0040] When referred to herein, a peptide ligand refers to a peptide that is covalently bound to a molecular scaffold. Typically, such a peptide contains two or more reactive groups (e.g., cysteine residues) capable of forming a covalent bond with the scaffold, and a sequence sandwiched between said reactive groups that forms a loop when the peptide is bound to the scaffold and is thus referred to as a loop sequence. In the case of the present invention, the peptide contains at least three cysteine residues and forms at least two loops on the scaffold. Those skilled in the art will recognize that other amino acid residues capable of forming a covalent bond with the scaffold (e.g., lysine, Dap or serine) can be used to form the bicyclic peptides of the present invention.

[0041] Advantages of Peptide Ligands Certain bicyclic peptides of the present invention have several advantageous properties that allow them to be considered suitable drug-like molecules for injection, inhalation, nasal, intraocular, oral or topical administration. Without being bound by any particular theory, such advantageous properties may include:

[0042] Species cross-reactivity. This is a typical requirement for preclinical pharmacodynamic and pharmacokinetic evaluation;

[0043] Protease stability. Bicyclic peptide ligands should ideally demonstrate stability against plasma proteases, epithelial ("membrane anchor") proteases, gastric and intestinal proteases, lung surface proteases, intracellular proteases, etc. Protease stability such that bicyclic lead candidates can be developed in animal models and administered to humans with confidence should be maintained across different species;

[0044] Desirable solubility profile. This is a function of the ratio of charged and hydrophilic to hydrophobic residues and intramolecular / intermolecular H-bonds, which is important for formulation and absorption purposes;

[0045] Optimal plasma half-life in circulation. Depending on the clinical indication and treatment regimen, it may be required to develop bicyclic peptides for short-term exposure in acute disease management settings or to develop bicyclic peptides with enhanced retention in circulation, and thus optimal for the management of more chronic conditions. Other factors that drive the desired plasma half-life are the toxicological requirements associated with sustained exposure of the agent for maximum therapeutic efficiency; and

[0046] Selectivity. Certain peptide ligands of the present invention demonstrate good selectivity over other carbonic anhydrases, metalloproteases and integrins.

[0047] The term "aliphatic" or "aliphatic group", as used herein, means a straight-chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon that is completely saturated or contains one or more units of unsaturation, or a monocyclic or bicyclic hydrocarbon that is completely saturated or contains one or more units of unsaturation but is not aromatic (also referred to herein as "carbocyclic", "alicyclic", or "cycloalkyl") and has a single point of attachment to the remainder of the molecule. Unless otherwise specified, an aliphatic group contains from 1 to 6 aliphatic carbon atoms. In some embodiments, the aliphatic group contains from 1 to 5 aliphatic carbon atoms. In other embodiments, the aliphatic group contains from 1 to 4 aliphatic carbon atoms. In still other embodiments, the aliphatic group contains from 1 to 3 aliphatic carbon atoms, and in yet other embodiments, the aliphatic group contains from 1 to 2 aliphatic carbon atoms. In some embodiments, "alicyclic" (or "carbocyclic" or "cycloalkyl") refers to a monocyclic C3-C6 hydrocarbon that is completely saturated or contains one or more units of unsaturation but is not aromatic and has a single point of attachment to the remainder of the molecule. Suitable aliphatic groups include, but are not limited to, linear or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl groups, and their hybrids such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.

[0048] As used herein, the term "bridged bicyclic" refers to any bicyclic ring system, i.e., a carbocyclic or heterocyclic, saturated or partially unsaturated, having at least one bridge. As defined by IUPAC, a "bridge" is an unbranched chain of atoms or an atom or valence bond connecting two bridgeheads, where a "bridgehead" is any skeletal atom of the ring system that is bonded to three or more skeletal atoms (excluding hydrogen). In some embodiments, the bridged bicyclic group has 7 to 12 ring members and 0 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Such bridged bicyclic groups are well known in the art and include the groups specified below, where each group is bonded to the remainder of the molecule at any replaceable carbon or nitrogen atom. Unless otherwise specified, the bridged bicyclic group is optionally substituted by one or more substituents as specified for aliphatic groups. Additionally or alternatively, any replaceable nitrogen of the bridged bicyclic group is optionally substituted. Exemplary bridged bicyclics are

Chemical formula

Chemical formula

[0049] The term "lower alkyl" refers to a C 1~4 straight or branched alkyl group. Exemplary lower alkyl groups are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, and tert-butyl.

[0050] The term "lower haloalkyl" refers to a C 1~4 straight or branched alkyl group substituted by one or more halogen atoms.

[0051] The term "heteroatom" means one or more of oxygen, sulfur, nitrogen, phosphorus or silicon (any oxidized form of nitrogen, sulfur, phosphorus or silicon; any quaternized form of any basic nitrogen, or; any replaceable nitrogen of a heterocyclic ring such as N (such as 3,4-dihydro-2H-pyrrolyl), NH (such as pyrrolidinyl) or NR + (such as N-substituted pyrrolidinyl)).

[0052] As used herein, the term "unsaturated" means that a moiety has one or more units of unsaturation.

[0053] As used herein, the term "divalent C 1~8 (or C 1~6 ) saturated or unsaturated, straight-chain or branched, hydrocarbon chain" refers to a straight-chain or branched divalent alkylene, alkenylene and alkynylene chain as defined herein.

[0054] The term "alkylene" refers to a divalent alkyl group. An "alkylene chain" is a polymethylene group, i.e., -(CH2) n - where n is a positive integer, preferably from 1 to 6, from 1 to 4, from 1 to 3, from 1 to 2, or from 2 to 3. A substituted alkylene chain is a polymethylene group in which one or more methylene hydrogen atoms are replaced by substituents. Suitable substituents include those described below for substituted aliphatic groups.

[0055] The term "alkenylene" refers to a divalent alkenyl group. A substituted alkenylene chain is a polymethylene group containing at least one double bond in which one or more hydrogen atoms are replaced by substituents. Suitable substituents include those described below for substituted aliphatic groups.

[0056] As used herein, the term "cyclopropylenyl" has the following structure:

Chemical formula

[0057] The term "halogen" means F, Cl, Br or I.

[0058] The term "aryl", used alone or as part of a larger moiety such as "aralkyl", "aralkoxy" or "aryloxyalkyl", refers to a monocyclic or bicyclic ring system having a total of 5 to 14 ring members, wherein at least one ring in the system is aromatic and each ring in the system contains 3 to 7 ring members. The term "aryl" may be used interchangeably with the term "aryl ring". In certain embodiments of the present invention, "aryl" refers to an aromatic ring system including, but not limited to, phenyl, biphenyl, naphthyl, aromatic moieties, etc., which may carry one or more substituents. As used herein, groups in which an aromatic ring is fused to one or more non-aromatic rings, such as indanyl, phthalimidyl, naphthimidyl, phenanthridinyl or tetrahydronaphthyl, are also included within the scope of the term "aryl".

[0059] The terms "heteroaryl" and "heteroar-" are used alone or as part of a larger moiety, for example, "heteroaralkyl" or "heteroaralkoxy" have 5 to 10 ring atoms, preferably 5, 6 or 9 ring atoms; have 6, 10 or 14 π electrons shared in a cyclic array; and, in addition to carbon atoms, have 1 to 5 heteroatoms, and refer to a group. The term "heteroatom" refers to nitrogen, oxygen or sulfur and includes any oxidized form of nitrogen or sulfur and any quaternized form of basic nitrogen. Heteroaryl groups include, but are not limited to, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl and pteridinyl. The terms "heteroaryl" and "heteroar-" as used herein also include groups in which the heteroaromatic ring is fused to one or more aryl, cycloaliphatic or heterocyclyl rings, where the radical or point of attachment is on the heteroaromatic ring. Non-limiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl and pyrido[2,3-b]-1,4-oxazin-3(4H)-one. Heteroaryl groups may be monocyclic or bicyclic. The term "heteroaryl" may be used interchangeably with the terms "heteroaryl ring", "heteroaryl group" or "heteroaromatic", and any of these terms includes rings that are optionally substituted. The term "heteroaralkyl" refers to an alkyl group substituted by heteroaryl, where the alkyl and heteroaryl moieties are independently optionally substituted.

[0060] As used herein, the terms "heterocycle", "heterocyclyl", "heterocyclic radical" and "heterocyclic ring" are used interchangeably and are either saturated or partially unsaturated and, in addition to carbon atoms, have one or more, preferably 1 to 4, heteroatoms as defined above and refer to stable 5- to 7-membered monocyclic or 7- to 10-membered bicyclic heterocyclic moieties. When referring to the ring atoms of a heterocycle, the term "nitrogen" includes substituted nitrogen. By way of example, in a saturated or partially unsaturated ring having 0 to 3 heteroatoms selected from oxygen, sulfur or nitrogen, nitrogen may be N (such as in 3,4-dihydro-2H-pyrrolyl), NH (such as in pyrrolidinyl) or + NR (such as in N-substituted pyrrolidinyl).

[0061] A heterocyclic ring can be attached to its pendant group at any heteroatom or carbon atom, thereby providing a stable structure, and any of the ring atoms may optionally be substituted. Examples of such saturated or partially unsaturated heterocyclic radicals include, but are not limited to, tetrahydrofuranyl, tetrahydrothiophenyl, pyrrolidinyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl and quinuclidinyl. The terms "heterocycle", "heterocyclyl", "heterocyclyl ring", "heterocyclic group", "heterocyclic moiety" and "heterocyclic radical" are used interchangeably herein and also include groups in which a heterocyclyl ring is fused to one or more aryl, heteroaryl or cycloaliphatic rings, such as indolinyl, 3H-indolyl, chromanyl, phenanthridinyl or tetrahydroquinolinyl. A heterocyclyl group may be monocyclic or bicyclic. The term "heterocyclylalkyl" refers to an alkyl group substituted by a heterocyclyl, where the alkyl and heterocyclyl moieties are independently optionally substituted.

[0062] As used herein, the term "partially unsaturated" refers to a ring moiety containing at least one double or triple bond. The term "partially unsaturated" is intended to encompass rings having multiple sites of unsaturation, but is not intended to include aryl or heteroaryl moieties as defined herein.

[0063] As described herein, the compounds of the invention may contain "optionally substituted" moieties. In general, the term "substituted", whether preceded by the term "optionally" or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. Unless otherwise indicated, an "optionally substituted" group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a defined group, the substituents may be the same or different at all positions. Combinations of substituents contemplated by the present invention preferably result in the formation of stable or chemically feasible compounds. The term "stable", as used herein, refers to compounds that are not substantially modified when subjected to the conditions for their production, detection, and in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein.

[0064] Suitable monovalent substituents on a substitutable carbon atom of an "optionally substituted" group are, independently, halogen; -(CH2) 0~4 R 〇 ; -(CH2) 0~4 OR 〇 ; -O(CH2) 0~4 R o , -O-(CH2) 0~4 C(O)OR 〇 ; -(CH2) 0~4 CH(OR 〇 )2; -(CH2) 0~4 SR 〇 ; R 〇 Optionally substituted by -(CH2) 0~4 Ph; R〇 which may be replaced by -(CH2) 0~4 O(CH2) 0~1 Ph;R 〇 which may be replaced by -CH=CHPh;R 〇 which may be replaced by -(CH2) 0~4 O(CH2) 0~1 -pyridyl; -NO2; -CN; -N3; -(CH2) 0~4 N(R 〇 )2; -(CH2) 0~4 N(R 〇 )C(O)R 〇 ; -N(R 〇 )C(S)R 〇 ; -N(R 〇 )C(NR 〇 )N(R 〇 )2; -(CH2) 0~4 N(R 〇 )C(O)NR 〇 2; -N(R 〇 )C(S)NR 〇 2; -(CH2) 0~4 N(R 〇 )C(O)OR 〇 ; -N(R 〇 )N(R 〇 )C(O)R 〇 ; -N(R 〇 )N(R 〇 )C(O)NR 〇 2; -N(R 〇 )N(R 〇 )C(O)OR 〇 ; -(CH2) 0~4 C(O)R 〇 ; -C(S)R 〇 ; -(CH2) 0~4 C(O)OR 〇 ; -(CH2) 0~4 C(O)SR 〇 ; -(CH2) 0~4 C(O)OSiR 〇 3; -(CH2) 0~4 OC(O)R 〇 ; -OC(O)(CH2) 0~4 SR-; -SC(S)SR 〇 ; -(CH2) 0~4 SC(O)R 〇 ; -(CH2)0~4 C(O)NR 〇 2;-C(S)NR 〇 2;-C(S)SR°;-(CH2) 0~4 OC(O)NR 〇 2;-C(O)N(OR 〇 )R 〇 ;-C(O)C(O)R 〇 ;-C(O)CH2C(O)R 〇 ;-C(NOR 〇 )R 〇 ;-(CH2) 0~4 SSR 〇 ;-(CH2) 0~4 S(O)2R 〇 ;-(CH2) 0~4 S(O)2OR 〇 ;-(CH2) 0~4 OS(O)2R 〇 ;-S(O)2NR 〇 2;-(CH2) 0~4 S(O)R 〇 ;-N(R 〇 )S(O)2NR 〇 2;-N(R 〇 )S(O)2R 〇 ;-N(OR 〇 )R 〇 ;-C(NH)NR 〇 2;-P(O)2R 〇 ;-P(O)R 〇 2;-OP(O)R 〇 2;-OP(O)(OR 〇 )2;-SiR 〇 3;-(C 1~4 linear or branched alkylene)O-N(R 〇 )2; or -(C 1~4 linear or branched alkylene)C(O)O-N(R 〇 )2, where each R 〇 may be substituted as defined below and is independently hydrogen, C 1~6 aliphatic, -CH2Ph, -O(CH2) 0~1Ph, -CH2-(5- to 6-membered heteroaryl ring), or a 5- to 6-membered saturated, partially unsaturated or aryl ring having 0 to 4 heteroatoms independently selected from nitrogen, oxygen or sulfur, or, notwithstanding the above definition, R 〇 Two independent occurrences of 〇 together with the intervening atoms may form a 3- to 12-membered saturated, partially unsaturated or aryl mono- or bicyclic ring having 0 to 4 heteroatoms independently selected from nitrogen, oxygen or sulfur, which is substituted as defined below.

[0065] R 〇 (or a ring formed by two independent occurrences of R 〇 together with the intervening atoms) suitable monovalent substituents on are independently halogen, -(CH2) 0~2 R ● , -(haloR ● ), -(CH2) 0~2 OH, -(CH2) 0~2 OR ● , -(CH2) 0~2 CH(OR ● )2; -O(haloR ● ), -CN, -N3, -(CH2) 0~2 C(O)R ● , -(CH2) 0~2 C(O)OH, -(CH2) 0~2 C(O)OR ● , -(CH2) 0~2 SR ● , -(CH2) 0~2 SH, -(CH2) 0~2 NH2, -(CH2) 0~2 NHR ● , -(CH2) 0~2 NR ● 2, -NO2, -SiR ● 3, -OSiR ● ; -C(O)SR ● , -(C 1~4 linear or branched alkylene)C(O)OR ● or -SSR ● where each R ●is unsubstituted or, when preceded by "halo", substituted only by one or more halogens, C 1~4 aliphatic, -CH2Ph, -O(CH2) 0~1 Ph, or independently selected from a 5- to 6-membered saturated, partially unsaturated or aryl ring having 0 to 4 heteroatoms independently selected from nitrogen, oxygen or sulfur. R 〇 Suitable divalent substituents on the saturated carbon atoms of R are =O and =S.

[0066] Suitable divalent substituents on the saturated carbon atoms of a "optionally substituted" group are as follows: =O, =S, =NNR * 2, =NNHC(O)R * 、=NNHC(O)OR * 、=NNHS(O)2R * 、=NR * 、=NOR * 、-O(C(R * 2)) 2~3 O- or -S(C(R * 2)) 2~3 S-, where each independent occurrence of R * is hydrogen, C 1~6 aliphatic which may be substituted as defined below, or selected from an unsubstituted 5- to 6-membered saturated, partially unsaturated or aryl ring having 0 to 4 heteroatoms independently selected from nitrogen, oxygen or sulfur. Suitable divalent substituents bonded to the adjacent replaceable carbon of an "optionally substituted" group are -O(CR * 2) 2~3 O-, where each independent occurrence of R * is hydrogen, C 1~6 aliphatic which may be substituted as defined below, or selected from an unsubstituted 5- to 6-membered saturated, partially unsaturated or aryl ring having 0 to 4 heteroatoms independently selected from nitrogen, oxygen or sulfur.

[0067] R * Suitable substituents on the aliphatic group of R are halogen, -R ● 、-(haloR ● )、-OH、-OR ●, -O(halo R ● ), -CN, -C(O)OH, -C(O)OR ● , -NH2, -NHR ● , -NR ● 2 or -NO2, wherein each R ● is unsubstituted or, when preceded by "halo", is substituted only by one or more halogens and independently is C 1~4 aliphatic, -CH2Ph, -O(CH2) 0~1 Ph, or a 5- to 6-membered saturated, partially unsaturated or aryl ring having 0 to 4 heteroatoms independently selected from nitrogen, oxygen or sulfur.

[0068] Suitable substituents on the nitrogen of a "optionally substituted" group are -R † , -NR † 2, -C(O)R † , -C(O)OR † , -C(O)C(O)R † , -C(O)CH2C(O)R † , -S(O)2R † , -S(O)2NR † 2, -C(S)NR † 2, -C(NH)NR † 2 or -N(R † )S(O)2R † , wherein each R † is independently hydrogen, C 1~6 aliphatic which may be substituted as defined below, unsubstituted -OPh, or an unsubstituted 5- to A 6-membered saturated, partially unsaturated or aryl ring having 0 to 4 heteroatoms independently selected from nitrogen, oxygen or sulfur, or, notwithstanding the above definition, two independent occurrences of R † together with the intervening atoms form an unsubstituted 3- to 12-membered saturated, partially unsaturated or aryl mono- or bicyclic ring having 0 to 4 heteroatoms independently selected from nitrogen, oxygen or sulfur.

[0069] R † Suitable substituents on the aliphatic group of are independently halogen, -R ●, -(haloR ● ), -OH, -OR ● , -O(haloR ● ), -CN, -C(O)OH, -C(O)OR ● , -NH2, -NHR ● , -NR ● 2 or -NO2, where each R ● is unsubstituted or, when preceded by "halo", is substituted only by one or more halogens and independently is C 1~4 aliphatic, -CH2Ph, -O(CH2) 0~1 Ph, or a 5- to 6-membered saturated, partially unsaturated or aryl ring having from 0 to 4 heteroatoms independently selected from nitrogen, oxygen or sulfur.

[0070] As used herein, the term "pharmaceutically acceptable salt" refers to salts that, within the scope of sound medical judgment, are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, etc., and that have a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S.M. Berge et al., in J. Pharmaceutical Sciences, 1977, 66, 1-19, which is incorporated herein by reference, describe pharmaceutically acceptable salts in detail. Pharmaceutically acceptable salts of the compounds of the present invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts of amino groups formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid, or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid, or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphor, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, etc.

[0071] Salts derived from appropriate bases include alkali metals, alkaline earth metals, ammonium and N + (C1~4 It includes an alkyl) tetra-salt. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc. Further pharmaceutically acceptable salts include, where appropriate, non-toxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates, and aryl sulfonates.

[0072] Unless otherwise stated, the structures shown herein are all isomeric (e.g., enantiomeric, diastereomeric, and geometric (or conformational)) forms of the structure; for example, the R and S configurations for each chiral center, the Z and E double bond isomers, as well as the Z and E conformational isomers. Thus, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the present invention are within the scope of the present invention. Unless otherwise stated, all tautomeric forms of the compounds of the present invention are within the scope of the present invention. In addition, unless otherwise stated, the structures shown herein are also intended to include compounds that differ only in the presence of one or more isotope-enriched atoms. For example, replacement of hydrogen by deuterium or tritium or 13 C- or 14 Compounds having the structure of the present invention including replacement of carbon by C-enriched carbon are within the scope of the present invention. Such compounds are useful, for example, as analytical tools, as probes in biological assays, or as therapeutic agents according to the present invention. In certain embodiments, the provided compounds include one or more deuterium atoms.

[0073] As used herein, the term "inhibitor" is defined as a compound that binds to and / or inhibits CAIX, MT1-MMP, PSMA, CD38, EphA2, or αvβ3 with measurable affinity. In certain embodiments, the inhibitor has an IC of less than about 50 μM, less than about 1 μM, less than about 500 nM, less than about 100 nM, less than about 10 nM, or less than about 1 nM. 50and / or have a binding constant.

[0074] The compounds of the present invention may be linked to a detectable moiety. Such compounds will be found to be useful as imaging agents. Those skilled in the art will recognize that the detectable moiety may be attached to the provided compound via a suitable substituent. As used herein, the term "suitable substituent" refers to a moiety that can form a covalent bond with the detectable moiety. Such moieties are well known to those skilled in the art and include, for example, groups containing a carboxylate moiety, an amino moiety, a thiol moiety, or a hydroxyl moiety, among others. It will be appreciated that such moieties may be attached to the provided compound either directly or via a tethering group such as a divalent saturated or unsaturated hydrocarbon chain. In some embodiments, such moieties may be attached via click chemistry. In some embodiments, such moieties may be attached to an alkyne via 1,3-addition cyclization of an azide, optionally in the presence of a copper catalyst. Methods of using click chemistry are known in the art and include those described by Rostovtsev et al., Angew. Chem. Int. Ed. 2002, 41, 2596-99 and Sun et al., Bioconjugate Chem., 2006, 17, 52-57.

[0075] As used herein, the term "detectable moiety" is used interchangeably with the term "label" and refers to any moiety that can be detected, such as a primary label and a secondary label. Radioisotopes (e.g., tritium, 225 Ac, 227 Ac, 241 Am, 72 As, 74 As, 211 At, 198 Au, 11 B, 7 Be, 212 Bi, 213 Bi, 75 Br, 77 Br, 11 C,14 C、 48 Ca、 109 Cd 139 what 141 what 252 Cf. 55 Co. 57 Co. 60 Co. 51 Cr、 130 Cs、 131 Cs、 137 Cs、 61 Cu、 62 Cu、 64 Cu、 67 Cu、 165 Dy 152 Me、 155 Me、 18 F、 55 Fe 59 Fe 64 Ga 67 Ga 68 Ga 153 God 68 Ge 122 I 123 I 124 I 125 I 131 I 132 I 111 in 115m in 191m Ir 192 Ir 81m Cr 177 sun 51 Mn 52 Mn 99 For 13 N 95 Nb 15 Or 191 Or 194 Or 32 Q 33 Q 203 Pb、 212 Pb、 103 pd 109 pd 238 gun 223 Ra 226 Ra 82 Rb、 186 Re 188 Re 105 Rh、 97 Ru 103 Ru35 S, 46 Sc, 47 Sc, 72 Se, 75 Se, 28 Si, 145 Sm, 153 Sm, 117m Sn, 85 Sr, 89 Sr, 90 Sr, 178 Ta, 179 Ta, 182 Ta, 149 Tb, 96 Tc, 99m Tc, 228 Th, 229 Th, 201 Tl, 170 Tm, 171 Tm, 188 W, 127 Xe, 133 Xe, 88 Y, 90 Y 91 Y, 169 Yb, 62 Zn, 65 Zn, 89 Zr or 95 Zr, where the superscript m indicates the metastable state), primary labels such as mass tags and fluorescent labels are signal generating reporters that can be detected without further modification. The detectable moiety also includes luminescent and phosphorescent groups.

[0076] The term "secondary label", as used herein, refers to moieties such as biotin and various protein antigens that require the presence of a second intermediate for the production of a detectable signal. For biotin, the secondary intermediate can include a streptavidin-enzyme conjugate. For antigen labels, the secondary intermediate can include an antibody-enzyme conjugate. Some fluorescent groups act as secondary labels because they transfer energy to another group in the process of non-radiative fluorescence resonance energy transfer (FRET) and the second group produces a detection signal.

[0077] As used herein, the terms "fluorescent label", "fluorescent dye" and "fluorophore" refer to a moiety that absorbs light energy at a defined excitation wavelength and emits light energy at a different wavelength. Examples of fluorescent labels include, but are not limited to, Alexa Fluor dyes (Alexa Fluor 350, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 660 and Alexa Fluor 680), AMCA, AMCA-S, BODIPY dyes (BODIPY FL, BODIPY R6G, BODIPY TMR, BODIPY TR, BODIPY 530 / 550, BODIPY 558 / 568, BODIPY 564 / 570, BODIPY 576 / 589, BODIPY 581 / 591, BODIPY 630 / 650, BODIPY 650 / 665), carboxyrhodamine 6G, carboxy-X-rhodamine (ROX), Cascade Blue, Cascade Yellow, Coumarin 343, cyanine dyes (Cy3, Cy5, Cy3.5, Cy5.5, Cy7, Cy7.5), dansyl, dapoxyl, dialkylaminocoumarin, 4’,5’-dichloro-2’,7’-dimethoxy-fluorescein, DM-NERF, eosin, erythrosin, fluorescein, FAM, hydroxycoumarin, IR dyes (IRD40, IRD700, IRD800), JOE, Lissamine rhodamine B, Marina Blue, methoxycoumarin, naphthofluorescein, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, PyMPO, pyrene, rhodamine B, rhodamine 6G, Rhodamine Green, Rhodamine Red, Rhodol Green, 2’,4’,5’,7’-tetrabromo-sulfone-fluorescein, tetramethyl-rhodamine (TMR), carboxytetramethylrhodamine (TAMRA), Texas Red, Texas Red-X.

[0078] As used herein, the term "mass tag" refers to any moiety that can be uniquely detected based on its mass using mass spectrometry (MS) detection techniques. Examples of mass tags include electrophoretic release tags such as N-[3-[4'-[(p-methoxytetrafluorobenzyl)oxy]phenyl]-3-methylglyceronyl]isonipecotic acid, 4'-[2,3,5,6-tetrafluoro-4-(pentafluorophenoxyl)]methylacetophenone, and derivatives thereof. The synthesis and utility of these mass tags are described in U.S. Patent Nos. 4,650,750, 4,709,016, 5,360,819, 5,516,931, 5,602,273, 5,604,104, 5,610,020, and 5,650,270. Other examples of mass tags include, but are not limited to, nucleotides, dideoxynucleotides, oligonucleotides of variable length and base composition, oligopeptides, oligosaccharides, and other synthetic polymers of variable length and monomer composition. A wide variety of organic molecules, both neutral and charged (biomolecules or synthetic compounds), within an appropriate mass range (100 to 2000 daltons) may be used as mass tags.

[0079] As used herein, the term "quantum dot" refers to any moiety that is a highly luminescent semiconductor nanocrystal (e.g., cadmium selenide capped with zinc sulfide). The synthesis and utility of these quantum dots are described in U.S. Patent Nos. 6,326,144, 6,468,808, 7,192,785, 7,151,047, and in the scientific literature (see Chan and Nie (1998) Science 281(5385) 2016-2018).

[0080] As used herein, the terms "measurable affinity" and "measurably inhibit" mean a measurable change in CAIX, MT1-MMP, PSMA, CD38, EphA2 or αvβ3 activity between a sample containing a compound or composition of the invention and CAIX, MT1-MMP, PSMA, CD38, EphA2 or αvβ3 and an equivalent sample containing CAIX, MT1-MMP, PSMA, CD38, EphA2 or αvβ3 in the absence of said compound and its composition.

[0081] 3. Description of Exemplary Embodiments: As noted above, in certain embodiments, the invention provides a compound of formula I:

Chemical formula

Chemical formula

Chem.

Chem.

Chem.

Chem.

Chem.

[0082] As defined above and as described herein, L 1 , L 2 and L 3 each is a covalent bond or a C 1~8 divalent hydrocarbon chain, wherein 1, 2 or 3 methylene units of the chain are independently optionally replaced by -S-, -N(R)-, -O-, -C(O)-, -OC(O)-, -C(O)O-, -C(O)N(R)-, -N(R)C(O)-, -S(O)-, -S(O)2- or -N(R)CH2C(O)-.

[0083] In some embodiments, each of L 1 , L 2 and L 3 is a covalent bond. In some embodiments, each of L 1 , L 2 and L 3 is -CH2S-. In some embodiments, each of L 1 , L 2 and L 3 is -CH2NH-. In some embodiments, each of L 1 , L 2 and L 3 is -CH2O-. In some embodiments, each of L 1 , L 2 and L 3 is -CH2CH2O-. In some embodiments, each of L 1 , L 2 and L 3 is -CH2CH2CH2CH2NH-. In some embodiments, each of L 1 , L2 and L 3 each of which is -CH2N(CH3)-. In some embodiments, L 1 , L 2 and L 3 each of which is -CH2CH2CH2CH2N(CH3)-.

[0084] In some embodiments, L 1 is a covalent bond. In some embodiments, L 1 is -CH2S-. In some embodiments, L 1 is -CH2O-. In some embodiments, L 1 is -CH2CH2O-. In some embodiments, L 1 is -CH2NH-. In some embodiments, L 1 is -CH2CH2CH2CH2NH-. In some embodiments, L 1 is -CH2N(CH3)-. In some embodiments, L 1 is -CH2CH2CH2CH2N(CH3)-. In some embodiments, L 1 is -CH2SCH2-. In some embodiments, L 1 is -CH2OCH2-. In some embodiments, L 1 is -CH2CH2OCH2-. In some embodiments, L 1 is -CH2NHCH2-. In some embodiments, L 1 is -CH2N(CH3)CH2-. In some embodiments, L 1 is -CH2CH2CH2CH2NHCH2-. In some embodiments, L 1 is -CH2CH2CH2CH2N(CH3)CH2-. In some embodiments, L 1 is -CH2SCH2C(O)NH-. In some embodiments, L 1 is -CH2OCH2C(O)NH-. In some embodiments, L 1 is -CH2CH2OCH2C(O)NH-. In some embodiments, L 1 is -CH2NHCH2C(O)NH-. In some embodiments, L1 is -CH2N(CH3)CH2C(O)NH-. In some embodiments, L 1 is -CH2CH2CH2CH2NHCH2C(O)NH-. In some embodiments, L 1 is -CH2CH2CH2CH2N(CH3)CH2C(O)NH-. In some embodiments, L 1 is -CH2SCH2C(O)-. In some embodiments, L 1 is -CH2OCH2C(O)-. In some embodiments, L 1 is -CH2CH2OCH2C(O)-. In some embodiments, L 1 is -CH2NHCH2C(O)-. In some embodiments, L 1 is -CH2N(CH3)CH2C(O)-. In some embodiments, L 1 is -CH2CH2CH2CH2NHCH2C(O)-. In some embodiments, L 1 is -CH2CH2CH2CH2N(CH3)CH2C(O)-. In some embodiments, L 1 is -CH2SCH2CH2C(O)NH-. In some embodiments, L 1 is -CH2OCH2CH2C(O)NH-. In some embodiments, L 1 is -CH2CH2OCH2CH2C(O)NH-. In some embodiments, L 1 is -CH2NHCH2CH2C(O)NH-. In some embodiments, L 1 is -CH2N(CH3)CH2CH2C(O)NH-. In some embodiments, L 1 is -CH2CH2CH2CH2NHCH2CH2C(O)NH-. In some embodiments, L 1 is -CH2CH2CH2CH2N(CH3)CH2CH2C(O)NH-. In some embodiments, L 1 is -CH2SCH2CH2C(O)-. In some embodiments, L 1 is -CH2OCH2CH2C(O)-. In some embodiments, L 1is -CH2CH2OCH2CH2C(O)-. In some embodiments, L 1 is -CH2NHCH2CH2C(O)-. In some embodiments, L 1 is -CH2N(CH3)CH2CH2C(O)-. In some embodiments, L 1 is -CH2CH2CH2CH2NHCH2CH2C(O)-. In some embodiments, L 1 is -CH2CH2CH2CH2N(CH3)CH2CH2C(O)-. In some embodiments, L 1 is selected from those shown in Table 1 below. In some embodiments, L 1 is selected from those shown in Table 3 below.

[0085] In some embodiments, L 2 is a covalent bond. In some embodiments, L 2 is -CH2S-. In some embodiments, L 2 is -CH2O-. In some embodiments, L 2 is -CH2CH2O-. In some embodiments, L 2 is -CH2NH-. In some embodiments, L 2 is -CH2CH2CH2CH2NH-. In some embodiments, L 2 is -CH2N(CH3)-. In some embodiments, L 2 is -CH2CH2CH2CH2N(CH3)-. In some embodiments, L 2 is -CH2SCH2-. In some embodiments, L 2 is -CH2OCH2-. In some embodiments, L 2 is -CH2CH2OCH2-. In some embodiments, L 2 is -CH2NHCH2-. In some embodiments, L 2 is -CH2N(CH3)CH2-. In some embodiments, L 2 is -CH2CH2CH2CH2NHCH2-. In some embodiments, L 2is -CH2CH2CH2CH2N(CH3)CH2-. In some embodiments, L 2 is -CH2SCH2C(O)NH-. In some embodiments, L 2 is -CH2OCH2C(O)NH-. In some embodiments, L 2 is -CH2CH2OCH2C(O)NH-. In some embodiments, L 2 is -CH2NHCH2C(O)NH-. In some embodiments, L 2 is -CH2N(CH3)CH2C(O)NH-. In some embodiments, L 2 is -CH2CH2CH2CH2NHCH2C(O)NH-. In some embodiments, L 2 is -CH2CH2CH2CH2N(CH3)CH2C(O)NH-. In some embodiments, L 2 is -CH2SCH2C(O)-. In some embodiments, L 2 is -CH2OCH2C(O)-. In some embodiments, L 2 is -CH2CH2OCH2C(O)-. In some embodiments, L 2 is -CH2NHCH2C(O)-. In some embodiments, L 2 is -CH2N(CH3)CH2C(O)-. In some embodiments, L 2 is -CH2CH2CH2CH2NHCH2C(O)-. In some embodiments, L 2 is -CH2CH2CH2CH2N(CH3)CH2C(O)-. In some embodiments, L 2 is -CH2SCH2CH2C(O)NH-. In some embodiments, L 2 is -CH2OCH2CH2C(O)NH-. In some embodiments, L 2 is -CH2CH2OCH2CH2C(O)NH-. In some embodiments, L 2 is -CH2NHCH2CH2C(O)NH-. In some embodiments, L 2 is -CH2N(CH3)CH2CH2C(O)NH-. In some embodiments, L 2is -CH2CH2CH2CH2NHCH2CH2C(O)NH-. In some embodiments, L 2 is -CH2CH2CH2CH2N(CH3)CH2CH2C(O)NH-. In some embodiments, L 2 is -CH2SCH2CH2C(O)-. In some embodiments, L 2 is -CH2OCH2CH2C(O)-. In some embodiments, L 2 is -CH2CH2OCH2CH2C(O)-. In some embodiments, L 2 is -CH2NHCH2CH2C(O)-. In some embodiments, L 2 is -CH2N(CH3)CH2CH2C(O)-. In some embodiments, L 2 is -CH2CH2CH2CH2NHCH2CH2C(O)-. In some embodiments, L 2 is -CH2CH2CH2CH2N(CH3)CH2CH2C(O)-. In some embodiments, L 2 is selected from those shown in Table 1 below. In some embodiments, L 2 is selected from those shown in Table 3 below.

[0086] In some embodiments, L 3 is a covalent bond. In some embodiments, L 3 is -CH2S-. In some embodiments, L 3 is -CH2O-. In some embodiments, L 3 is -CH2CH2O-. In some embodiments, L 3 is -CH2NH-. In some embodiments, L 3 is -CH2CH2CH2CH2NH-. In some embodiments, L 3 is -CH2N(CH3)-. In some embodiments, L 3 is -CH2CH2CH2CH2N(CH3)-. In some embodiments, L 3 is -CH2SCH2-. In some embodiments, L 3 is -CH2OCH2-. In some embodiments, L3 is -CH2CH2OCH2-. In some embodiments, L 3 is -CH2NHCH2-. In some embodiments, L 3 is -CH2N(CH3)CH2-. In some embodiments, L 3 is -CH2CH2CH2CH2NHCH2-. In some embodiments, L 3 is -CH2CH2CH2CH2N(CH3)CH2-. In some embodiments, L 3 is -CH2SCH2C(O)NH-. In some embodiments, L 3 is -CH2OCH2C(O)NH-. In some embodiments, L 3 is -CH2CH2OCH2C(O)NH-. In some embodiments, L 3 is -CH2NHCH2C(O)NH-. In some embodiments, L 3 is -CH2N(CH3)CH2C(O)NH-. In some embodiments, L 3 is -CH2CH2CH2CH2NHCH2C(O)NH-. In some embodiments, L 3 is -CH2CH2CH2CH2N(CH3)CH2C(O)NH-. In some embodiments, L 3 is -CH2SCH2C(O)-. In some embodiments, L 3 is -CH2OCH2C(O)-. In some embodiments, L 3 is -CH2CH2OCH2C(O)-. In some embodiments, L 3 is -CH2NHCH2C(O)-. In some embodiments, L 3 is -CH2N(CH3)CH2C(O)-. In some embodiments, L 3 is -CH2CH2CH2CH2NHCH2C(O)-. In some embodiments, L 3 is -CH2CH2CH2CH2N(CH3)CH2C(O)-. In some embodiments, L 3 is -CH2SCH2CH2C(O)NH-. In some embodiments, L 3is -CH2OCH2CH2C(O)NH-. In some embodiments, L 3 is -CH2CH2OCH2CH2C(O)NH-. In some embodiments, L 3 is -CH2NHCH2CH2C(O)NH-. In some embodiments, L 3 is -CH2N(CH3)CH2CH2C(O)NH-. In some embodiments, L 3 is -CH2CH2CH2CH2NHCH2CH2C(O)NH-. In some embodiments, L 3 is -CH2CH2CH2CH2N(CH3)CH2CH2C(O)NH-. In some embodiments, L 3 is -CH2SCH2CH2C(O)-. In some embodiments, L 3 is -CH2OCH2CH2C(O)-. In some embodiments, L 3 is -CH2CH2OCH2CH2C(O)-. In some embodiments, L 3 is -CH2NHCH2CH2C(O)-. In some embodiments, L 3 is -CH2N(CH3)CH2CH2C(O)-. In some embodiments, L 3 is -CH2CH2CH2CH2NHCH2CH2C(O)-. In some embodiments, L 3 is -CH2CH2CH2CH2N(CH3)CH2CH2C(O)-. In some embodiments, L 3 is selected from those shown in Table 1 below. In some embodiments, L 3 is selected from those shown in Table 3 below.

[0087] As defined above and as described herein, each of R's is independently hydrogen or C 1~4 alkyl.

[0088] In some embodiments, R is hydrogen. In some embodiments, R is C 1~4 alkyl.

[0089] In some embodiments, R is methyl. In some embodiments, R is ethyl. In some embodiments, R is n-propyl. In some embodiments, R is isopropyl. In some embodiments, R is n-butyl. In some embodiments, R is isobutyl. In some embodiments, R is tert-butyl.

[0090] In some embodiments, R is selected from those shown in Table 1 below. In some embodiments, R is selected from those shown in Table 3 below.

[0091] As defined above and as described herein, each of m, n, o, and p is independently 0 or 1.

[0092] In some embodiments, m is 0. In some embodiments, m is 1. In some embodiments, m is selected from those shown in Table 1 below. In some embodiments, m is selected from those shown in Table 3 below.

[0093] In some embodiments, n is 0. In some embodiments, n is 1. In some embodiments, n is selected from those shown in Table 1 below. In some embodiments, n is selected from those shown in Table 3 below.

[0094] In some embodiments, o is 0. In some embodiments, o is 1. In some embodiments, o is selected from those shown in Table 1 below. In some embodiments, o is selected from those shown in Table 3 below.

[0095] In some embodiments, p is 0. In some embodiments, p is 1. In some embodiments, p is selected from those shown in Table 1 below. In some embodiments, p is selected from those shown in Table 3 below.

[0096] As defined above and as described herein, each of q and r is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

[0097] In some embodiments, q is 1. In some embodiments, q is 2. In some embodiments, q is 3. In some embodiments, q is 4. In some embodiments, q is 5. In some embodiments, q is 6. In some embodiments, q is 7. In some embodiments, q is 8. In some embodiments, q is 9. In some embodiments, q is 10. In some embodiments, q is 11. In some embodiments, q is 12. In some embodiments, q is 13. In some embodiments, q is 14. In some embodiments, q is 15. In some embodiments, q is selected from those shown in Table 1 below. In some embodiments, q is selected from those shown in Table 3 below.

[0098] In some embodiments, r is 1. In some embodiments, r is 2. In some embodiments, r is 3. In some embodiments, r is 4. In some embodiments, r is 5. In some embodiments, r is 6. In some embodiments, r is 7. In some embodiments, r is 8. In some embodiments, r is 9. In some embodiments, r is 10. In some embodiments, r is 11. In some embodiments, r is 12. In some embodiments, r is 13. In some embodiments, r is 14. In some embodiments, r is 15. In some embodiments, r is selected from those shown in Table 1 below. In some embodiments, r is selected from those shown in Table 3 below.

[0099] As defined above and as described herein, R 1 is R or -C(O)R.

[0100] In some embodiments, R1 is R. In some embodiments, R 1 is -C(O)R.

[0101] In some embodiments, R 1 is hydrogen. In some embodiments, R 1 is methyl. In some embodiments, R 1 is ethyl. In some embodiments, R 1 is n-propyl. In some embodiments, R 1 is isopropyl. In some embodiments, R 1 is n-butyl. In some embodiments, R 1 is isobutyl. In some embodiments, R 1 is tert-butyl.

[0102] In some embodiments, R 1 is -C(O)CH3. In some embodiments, R 1 is -C(O)CH2CH3. In some embodiments, R 1 is -C(O)CH2CH2CH3. In some embodiments, R 1 is -C(O)CH(CH3)2. In some embodiments, R 1 is -C(O)CH2CH2CH2CH3. In some embodiments, R 1 is -C(O)CH2CH(CH3)2. In some embodiments, R 1 is -C(O)C(CH3)3. In some embodiments, R 1 is selected from those shown in Table 1 below. In some embodiments, R 1 is selected from those shown in Table 3 below.

[0103] As defined above and as described herein, R 4 and R 6 each, independently, is hydrogen, or a C 1~6A group which is selected from an aliphatic, 3- to 8-membered saturated or partially unsaturated monocyclic carbocyclic ring, phenyl, an 8- to 10-membered bicyclic aromatic carbocyclic ring, a 4- to 8-membered saturated or partially unsaturated monocyclic heterocyclic ring having 1 to 2 heteroatoms independently selected from nitrogen, oxygen or sulfur, a 5- to 6-membered monocyclic heteroaromatic ring having 1 to 4 heteroatoms independently selected from nitrogen, oxygen or sulfur, or an 8- to 10-membered bicyclic heteroaromatic ring having 1 to 5 heteroatoms independently selected from nitrogen, oxygen or sulfur, and which is optionally substituted.

[0104] In some embodiments, R 4 is hydrogen. In some embodiments, R 4 is an optionally substituted C 1~6 aliphatic. In some embodiments, R 4 is an optionally substituted 3- to 8-membered saturated or partially unsaturated monocyclic carbocyclic ring. In some embodiments, R 4 is an optionally substituted phenyl. In some embodiments, R 4 is an optionally substituted 8- to 10-membered bicyclic aromatic carbocyclic ring. In some embodiments, R 4 is an optionally substituted 4- to 8-membered saturated or partially unsaturated monocyclic heterocyclic ring having 1 to 2 heteroatoms independently selected from nitrogen, oxygen or sulfur. In some embodiments, R 4 is an optionally substituted 5- to 6-membered monocyclic heteroaromatic ring having 1 to 4 heteroatoms independently selected from nitrogen, oxygen or sulfur. In some embodiments, R 4 is an optionally substituted 8- to 10-membered bicyclic heteroaromatic ring having 1 to 5 heteroatoms independently selected from nitrogen, oxygen or sulfur.

[0105] In some embodiments, R 4 is methyl. In some embodiments, R 4 is

Chemical formula

[0106] In some embodiments, R 4 is [Chemical formula] [In the formula, the bonding site has (S) stereochemistry]. In some embodiments, R 4 is [Chemical formula] [In the formula, the bonding site has (R) stereochemistry].

[0107] In some embodiments, R 4 is [Chemical formula] is. In some embodiments, R 4 is [Chemical formula] is. In some embodiments, R 4 is [Chemical formula] is. In some embodiments, R 4 is [Chemical formula] is. In some embodiments, R 4 is [Chemical formula] is. In some embodiments, R 4 is [Chemical formula] is.

[0108] In some embodiments, R 4 is [Chemical formula] is. In some embodiments, R 4is [Chemical formula] . In some embodiments, R 4 is [Chemical formula] . In some embodiments, R 4 is [Chemical formula] .

[0109] In some embodiments, R 4 is [Chemical formula] . In some embodiments, R 4 is [Chemical formula] . In some embodiments, R 4 is [Chemical formula] . In some embodiments, R 4 is [Chemical formula] . In some embodiments, R 4 is [Chemical formula] . In some embodiments, R 4 is [Chemical formula] . In some embodiments, R 4 is [Chemical formula] It is. In some embodiments, R 4 is

Chem.

[0110] In some embodiments, R 4 is selected from those shown in Table 1 below. In some embodiments, R 4 is selected from those shown in Table 3 below.

[0111] In some embodiments, R 6 is hydrogen. In some embodiments, R 6 is an optionally substituted C 1~6 aliphatic. In some embodiments, R 6 is an optionally substituted 3- to 8-membered saturated or partially unsaturated monocyclic carbocyclic ring. In some embodiments, R 6 is an optionally substituted phenyl. In some embodiments, R 6 is an optionally substituted 8- to 10-membered bicyclic aromatic carbocyclic ring. In some embodiments, R 6 is an optionally substituted 4- to 8-membered saturated or partially unsaturated monocyclic heterocyclic ring having 1 to 2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R 6 is an optionally substituted 5- to 6-membered monocyclic heteroaromatic ring having 1 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R 6 is an optionally substituted 8- to 10-membered bicyclic heteroaromatic ring having 1 to 5 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0112] In some embodiments, R 6 is methyl. In some embodiments, R 6 is

Chem.

[0113] In some embodiments, R 6 teeth, [ka] wherein the attachment site has the (S) stereochemistry. In some embodiments, R 6 teeth, [ka] wherein the attachment site has the (R) stereochemistry.

[0114] In some embodiments, R 6 teeth, [ka] In some embodiments, R 6 teeth, [ka] In some embodiments, R 6 teeth, [ka] In some embodiments, R 6is [Chem.] . In some embodiments, R 6 is [Chem.] . In some embodiments, R 6 is [Chem.] .

[0115] In some embodiments, R 6 is [Chem.] . In some embodiments, R 6 is [Chem.] . In some embodiments, R 6 is [Chem.] . In some embodiments, R 6 is [Chem.] .

[0116] In some embodiments, R 6 is [Chem.] . In some embodiments, R 6 is [Chem.] . In some embodiments, R 6 is [Chem.] is. In some embodiments, R 6 is

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

[0117] In some embodiments, R 6 is selected from those shown in Table 1 below. In some embodiments, R 6 is selected from those shown in Table 3 below.

[0118] As defined above and as described herein, each of R 4’ and R 6’ is independently hydrogen or methyl.

[0119] In some embodiments, R 4’ is hydrogen. In some embodiments, R 4’ is methyl.

[0120] In some embodiments, R 4’ is selected from those shown in Table 1 below. In some embodiments, R 4’ is selected from those shown in Table 3 below.

[0121] In some embodiments, R 6’ is hydrogen. In some embodiments, R 6’ is methyl.

[0122] In some embodiments, R 6’ is selected from those shown in Table 1 below. In some embodiments, R 6’ is selected from those shown in Table 3 below.

[0123] As defined above and as described herein, each of R 2 , R 3 , R 5 and R 7 is independently hydrogen or C 1~4 is aliphatic; or the R 5 group and its adjacent R 4 group together with their intervening atoms optionally form a 4- to 8-membered saturated or partially unsaturated monocyclic heterocyclic ring having 1 to 2 heteroatoms independently selected from nitrogen, oxygen, or sulfur; or the R 7 group and its adjacent R 6 group together with their intervening atoms optionally form a 4- to 8-membered saturated or partially unsaturated monocyclic heterocyclic ring having 1 to 2 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0124] In some embodiments, R 2 is hydrogen. In some embodiments, R 2 is C1~4 It is aliphatic. In some embodiments, R 2 is methyl. In some embodiments, R 2 is ethyl. In some embodiments, R 2 is n-propyl. In some embodiments, R 2 is isopropyl. In some embodiments, R 2 is n-butyl. In some embodiments, R 2 is isobutyl. In some embodiments, R 2 is tert-butyl.

[0125] In some embodiments, R 2 is selected from those shown in Table 1 below. In some embodiments, R 2 is selected from those shown in Table 3 below.

[0126] In some embodiments, R 3 is hydrogen. In some embodiments, R 3 is C 1~4 It is aliphatic. In some embodiments, R 3 is methyl. In some embodiments, R 3 is ethyl. In some embodiments, R 3 is n-propyl. In some embodiments, R 3 is isopropyl. In some embodiments, R 3 is n-butyl. In some embodiments, R 3 is isobutyl. In some embodiments, R 3 is tert-butyl.

[0127] In some embodiments, R 3 is selected from those shown in Table 1 below. In some embodiments, R 3 is selected from those shown in Table 3 below.

[0128] In some embodiments, R 5 is hydrogen. In some embodiments, R 5 is C1~4 It is aliphatic. In some embodiments, R 5 is methyl. In some embodiments, R 5 is ethyl. In some embodiments, R 5 is n-propyl. In some embodiments, R 5 is isopropyl. In some embodiments, R 5 is n-butyl. In some embodiments, R 5 is isobutyl. In some embodiments, R 5 is tert-butyl.

[0129] In some embodiments, R 5 group and its adjacent R 4 group, together with their intervening atoms, become

Chemical formula

Chemical formula

[0130] In some embodiments, R 5 is selected from those shown in Table 1 below. In some embodiments, R 5 is selected from those shown in Table 3 below.

[0131] In some embodiments, R 7 is hydrogen. In some embodiments, R 7 is C 1~4 is aliphatic. In some embodiments, R 7 is methyl. In some embodiments, R 7 is ethyl. In some embodiments, R 7 is n-propyl. In some embodiments, R 7is isopropyl. In some embodiments, R 7 is n-butyl. In some embodiments, R 7 is isobutyl. In some embodiments, R 7 is tert-butyl.

[0132] In some embodiments, R 7 group and its adjacent R 6 group, together with their intervening atoms,

Chemical formula

Chemical formula

[0133] In some embodiments, R 7 is selected from those shown in Table 1 below. In some embodiments, R 7 is selected from those shown in Table 3 below.

[0134] As defined above and as described herein, the scaffold is a trivalent group that connects to and orients the cyclic peptide.

[0135] In some embodiments, the scaffold is

Chemical formula

Chemical formula

Chemical formula

[0136] In some embodiments, the scaffold is

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0137] In some embodiments, the scaffold is

Chemical formula

Chemical formula

Chemical formula

[0138] In some embodiments, the scaffold is

Chemical formula

[0139] In some embodiments, the scaffold is [Chemistry] is. In some embodiments, the scaffold is [Chemistry] is. In some embodiments, the scaffold is [Chemistry] is. In some embodiments, the scaffold is [Chemistry] is. In some embodiments, the scaffold is [Chemistry] is. In some embodiments, the scaffold is [Chemistry] is. In some embodiments, the scaffold is [Chemistry] is. In some embodiments, the scaffold is [Chemistry] is. In some embodiments, the scaffold is [Chemistry] is. In some embodiments, the scaffold is [Chemistry] is.

[0140] In some embodiments, the scaffold is [Chemistry] is. In some embodiments, the scaffold is [Chemistry] is. In some embodiments, the scaffold is [Chemistry] is. In some embodiments, the scaffold is [Chemistry] is. In some embodiments, the scaffold is [Chemistry] is. In some embodiments, the scaffold is [Chemistry] In some embodiments, the scaffold is [ka] In some embodiments, the scaffold is [ka] In some embodiments, the scaffold is [ka] In some embodiments, the scaffold is [ka] In some embodiments, the scaffold is [ka] In some embodiments, the scaffold is [ka] In some embodiments, the scaffold is [ka] In some embodiments, the scaffold is [ka] In some embodiments, the scaffold is [ka] In some embodiments, the scaffold is [ka] is.

[0141] In some embodiments, the scaffold comprises: [ka] In some embodiments, the scaffold is [ka] In some embodiments, the scaffold is [ka] In some embodiments, the scaffold is [ka] In some embodiments, the scaffold is [ka] In some embodiments, the scaffold is [ka] In some embodiments, the scaffold is [ka] In some embodiments, the scaffold is [ka] In some embodiments, the scaffold is [ka] is.

[0142] In some embodiments, the scaffold comprises: [ka] In some embodiments, the scaffold is [ka] is.

[0143] In some embodiments, the scaffold comprises:

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

[0144] In some embodiments, the scaffold is

Chem.

Chem.

[0145] As defined above and as described herein, Loop A is L 2 amino acid residues linked to and L 1a divalent natural or unnatural amino acid residue or peptide that is bound to an amino acid residue linked to

Chem.

Chem.

Chem.

Chem.

[0146] In some embodiments, Loop A is

Chem.

Chem.

Chem.

Chem.

[0147] In some embodiments, loop A is

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0148] In some embodiments, loop A is

Chemical formula

Chemical formula

[0149] As defined above and as described herein, loop B is L 1 amino acid residues linked to and L3 a divalent natural or unnatural amino acid residue or peptide that is bound to an amino acid residue linked to [Chemical formula] and includes. In some embodiments, Loop B is L 1 an amino acid residue linked to and an amino acid residue linked to L 3 a divalent natural amino acid residue that is bound to an amino acid residue linked to [Chemical formula] and includes. In some embodiments, Loop B is L 1 an amino acid residue linked to and an amino acid residue linked to L 3 a divalent unnatural amino acid residue that is bound to an amino acid residue linked to [Chemical formula] and includes. In some embodiments, Loop B is L 1 an amino acid residue linked to and an amino acid residue linked to L 3 a divalent peptide that is bound to an amino acid residue linked to [Chemical formula] and includes.

[0150] In some embodiments, Loop B is [Chemical formula] is. In some embodiments, Loop B is [Chemical formula] is. In some embodiments, Loop B is [Chemical formula] is. In some embodiments, Loop B is [Chemical formula] is.

[0151] In some embodiments, loop B is

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0152] In some embodiments, loop B is

Chemical formula

Chemical formula

[0153] In some embodiments, Loop A comprises 1 to 15 amino acid residues and Loop B comprises 1 to 15 amino acid residues.

[0154] In some embodiments, Loop A comprises 5 amino acid residues and Loop B comprises 5 amino acid residues. In some embodiments, Loop A comprises 6 amino acid residues and Loop B comprises 5 amino acid residues. In some embodiments, Loop A comprises 2 amino acid residues and Loop B comprises 7 amino acid residues. In some embodiments, Loop A comprises 3 amino acid residues and Loop B comprises 7 amino acid residues. In some embodiments, Loop A comprises 3 amino acid residues and Loop B comprises 9 amino acid residues. In some embodiments, Loop A comprises 3 amino acid residues and Loop B comprises 6 amino acid residues. In some embodiments, Loop A comprises 2 amino acid residues and Loop B comprises 6 amino acid residues. In some embodiments, Loop A comprises 6 amino acid residues and Loop B comprises 5 amino acid residues.

[0155] In some embodiments, Loop A is selected from those shown in Table 1 below. In some embodiments, Loop A is selected from those shown in Table 3 below.

[0156] In some embodiments, Loop B is selected from those shown in Table 1 below. In some embodiments, Loop B is selected from those shown in Table 3 below.

[0157] As defined above and as described herein,

Chemical formula

[0158] As defined above and as described herein,

Chemical formula

[0159] The detectable moiety, as defined above and described herein, 1 is any moiety that can be detected.

[0160] In some embodiments, the detectable moiety 1 is any moiety that can be detected.

[0161] One of ordinary skill in the art will appreciate that various detectable moieties are suitable for achieving the imaging effects of the present invention.

[0162] As used herein, "M" is a metal.

[0163] In some embodiments, the detectable moiety 1 can be connected at any available position. In some embodiments, the detectable moiety 1 can be connected by any available -OH, -C(O)OH, -SH, -NH2 or -NHCH3.

[0164] In some embodiments, the detectable moiety 1 is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (also known as DOTA, DotA or tetraxetan): [Chemical formula] is. In some embodiments, the detectable moiety 1 is Bn-DOTA: [Chemical formula] is. In some embodiments, the detectable moiety 1 is Bn-oxo-DOTA: [Chemical formula] is. In some embodiments, the detectable moiety 1 is Bn-PCTA:

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0165] In some embodiments, the detectable moiety 1 is desferrioxamine (DFO):

Chemical formula

[0166] In some embodiments, the detectable moiety 1 is heptamethine dye:

Chemical formula

[0167] is a DOTA complex with a metal: 1 [Chem.] In some embodiments, the detectable moiety 1 is a Bn-DOTA complex with a metal: [Chem.] In some embodiments, the detectable moiety 1 is a Bn-oxo-DOTA complex with a metal: <000270)9>[Chem.] In some embodiments, the detectable moiety 1 is a Bn-PCTA complex with a metal: [Chem.] In some embodiments, the detectable moiety 1 is a TETA complex with a metal: [Chem.] In some embodiments, the detectable moiety 1 is a Bn-NOTA complex with a metal: [Chem.] In some embodiments, the detectable moiety 1 is a CHX-A”-DTPA complex with a metal: [Chemical formula] In some embodiments, the detectable moiety 1 is a Bn-DTPA complex with a metal: [Chemical formula] In some embodiments, the detectable moiety 1 is a 2B3M-DTPA complex with a metal: [Chemical formula] In some embodiments, the detectable moiety

[0168] In some embodiments, the detectable moiety 1 is a desferrioxamine (DFO) complex with a metal: [Chemical formula] In some embodiments, the detectable moiety

[0169] In some embodiments, the detectable moiety 1 forms a complex with a metal.

[0170] In some embodiments, the detectable moiety 1 is a scorpionate. In some embodiments, the detectable moiety 1 is [Chemical formula] In some embodiments, the detectable moiety 1 is a nanomaterial. In some embodiments, the detectable moiety 1 is a quantum dot.

[0171] In some embodiments, the detectable moiety 1 is selected from those shown in Table 1 below. In some embodiments, the detectable moiety 1is selected from those shown in Table 3 below.

[0172] The detectable moiety, as defined above and described herein 2 is any moiety that can be detected.

[0173] In some embodiments, the detectable moiety 2 is any moiety that can be detected.

[0174] In some embodiments, the detectable moiety 2 can be connected at any available position. In some embodiments, the detectable moiety 2 can be connected with any available -OH, -C(O)OH, -SH, -NH2 or -NHCH3.

[0175] In some embodiments, the detectable moiety 2 is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (also known as DOTA, DotA or tetraxetan):

Chem.

Chem.

Chem.

Chem.

Chem.

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0176] In some embodiments, the detectable moiety 2 is desferrioxamine (DFO):

Chemical formula

[0177] In some embodiments, the detectable moiety 2 is heptamethine dye:

Chemical formula

Chemical formula

Chemical formula

[0178] In some embodiments, the detectable moiety 2 is a DOTA complex with a metal: [Chemical formula] is as follows. In some embodiments, the detectable moiety 2 is a Bn-DOTA complex with a metal: [Chemical formula] is as follows. In some embodiments, the detectable moiety 2 is a Bn-oxo-DOTA complex with a metal: [Chemical formula] is as follows. In some embodiments, the detectable moiety 2 is a Bn-PCTA complex with a metal: [Chemical formula] is as follows. In some embodiments, the detectable moiety 2 is a TETA complex with a metal: [Chemical formula] is as follows. In some embodiments, the detectable moiety 2 is a Bn-NOTA complex with a metal: [Chemical formula] is as follows. In some embodiments, the detectable moiety 2 is a CHX-A”-DTPA complex with a metal: [Chemical formula] is as follows. In some embodiments, the detectable moiety 2 is a Bn-DTPA complex with a metal: [Chemical formula] is. In some embodiments, the detectable moiety 2 is a 2B3M-DTPA complex with a metal:

Chemical formula

[0179] In some embodiments, the detectable moiety 2 is a desferrioxamine (DFO) complex with a metal:

Chemical formula

[0180] In some embodiments, the detectable moiety 2 forms a complex with a metal.

[0181] In some embodiments, the detectable moiety 2 is a scorpionate. In some embodiments, the detectable moiety 2 is

Chemical formula

[0182] In some embodiments, the detectable moiety 2 is selected from those shown in Table 1 below. In some embodiments, the detectable moiety 2 is selected from those shown in Table 1 below.

[0183] In some embodiments, the metal is a radioactive metal. In some embodiments, the metal is 111 In. In some embodiments, the metal is 67 Ga. In some embodiments, the metal is 68 Ga. In some embodiments, the metal is 86 Y. In some embodiments, the metal is90 It is Y. In some embodiments, the metal is 177 Lu.

[0184] In some embodiments, the metal is 89 Zr. In some embodiments, the metal is 95 Zr.

[0185] As defined above and as described herein, the linker 1 is hydrogen, -C(O)R,

Chemical formula

Chemical formula

[0186] In some embodiments, the linker 1 is hydrogen, where n is 0. In some embodiments, the linker 1 is hydrogen, where n is 0. In some embodiments, the linker 1 is -C(O)R, where n is 0. In some embodiments, the linker 1 is -C(O)CH3, where n is 0. In some embodiments, the linker 1 is

Chemical formula

[0187] In some embodiments, the linker 1 is a covalent bond. In some embodiments, the linker 1 is [Chem.] is. In some embodiments, the linker 1 is [Chem.] is. In some embodiments, the linker 1 is [Chem.] is. In some embodiments, the linker 1 is [Chem.] is. In some embodiments, the linker 1 is [Chem.] is.

[0188] In some embodiments, the linker 1 is [Chem.] is. In some embodiments, the linker 1 is [Chem.] is. In some embodiments, the linker 1 is [Chem.] is.

[0189] In some embodiments, the linker 1 is selected from those shown in Table 1 below. In some embodiments, the linker 1 is selected from those shown in Table 3 below.

[0190] As defined above and as described herein, the linker 2 is a -NH2 or a divalent moiety that connects the bicyclic C-terminus to the detectable moiety 2 , where when p is 0, the linker 2 is -NH2.

[0191] In some embodiments, the linker 2 is -NH2, where p is 0. In some embodiments, the linker 2 is a divalent moiety that connects the bicyclic C-terminus to the detectable moiety 2 .

[0192] In some embodiments, the linker 2 is

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0193] In some embodiments, the linker 2 is [Chemical formula] is. In some embodiments, the linker 2 is [Chemical formula] is. In some embodiments, the linker 2 is [Chemical formula] is. In some embodiments, the linker 2 is [Chemical formula] is.

[0194] In some embodiments, the linker 2 is [Chemical formula] is. In some embodiments, the linker 2 is [Chemical formula] is.

[0195] In some embodiments, the linker 2 is [Chemical formula] is.

[0196] In some embodiments, the linker 2 is selected from those shown in Table 1 below. In some embodiments, the linker 2 is selected from those shown in Table 3 below.

[0197] As defined above and as described herein, ring A is 18-crown-6, 1,7,13-triaza-18-crown-6, and optionally substituted with 0 to 3 oxo, methyl, ethyl, or spiroethylene groups, and has 0 to 6 heteroatoms independently selected from nitrogen, oxygen, or sulfur, and is selected from the group consisting of 3- to 12-membered saturated, partially unsaturated, bridged bicyclic, bridged tricyclic, propellane, or aromatic rings.

[0198] In some embodiments, ring A is 18-crown-6. In some embodiments, ring A is 1,7,13-triaza-18-crown-6. In some embodiments, ring A is optionally substituted with 0 to 3 oxo, methyl, ethyl, or spiroethylene groups and has 0 to 6 heteroatoms independently selected from nitrogen, oxygen, or sulfur, and is a 3- to 12-membered saturated, partially unsaturated, bridged bicyclic, bridged tricyclic, propellane, or aromatic ring.

[0199] In some embodiments, ring A is

Chemical formula

Chemical formula

[0200] In some embodiments, ring A is

Chemical formula

Chemical formula

Chemical formula

[0201] In some embodiments, ring A is [ka] In some embodiments, ring A is [ka] In some embodiments, ring A is [ka] In some embodiments, ring A is [ka] In some embodiments, ring A is [ka] is.

[0202] In some embodiments, ring A is [ka] In some embodiments, ring A is [ka] is.

[0203] In some embodiments, ring A is [ka] In some embodiments, ring A is [ka] In some embodiments, ring A is [ka] is.

[0204] In some embodiments, ring A is [Chemical formula] is. In some embodiments, ring A is [Chemical formula] is. In some embodiments, ring A is selected from those shown in Table 1 below. In some embodiments, ring A is selected from those shown in Table 3 below.

[0205] In certain embodiments, the present invention provides a compound of formula I-a, wherein the scaffold is ring A, thereby: [Chemical formula] or a pharmaceutically acceptable salt thereof [wherein loop A, loop B, ring A, L 1 , L 2 , L 3 , linker 1 , linker 2 , detectable moiety 1 , detectable moiety 2 , R 1 , R 2 , R 3 , m, n, o, and p are each defined above, both alone and in combination, as described in the embodiments herein] forms a bicyclic ring of formula I.

[0206] In certain embodiments, the present invention provides a loop A that is [Chemical formula] and loop B is [Chemical formula] and thereby provides a bicyclic ring of formula II: [Chemical formula] or a pharmaceutically acceptable salt thereof [wherein L 1 , L 2 , L 3 , scaffold, R1 , R 2 , R 3 , R 4 , R 4’ , R 5 , R 6 , R 6’ , R 7 , linker 1 , linker 2 , detectable moiety 1 , detectable moiety 2 , each of m, n, o, p, q and r is defined above and as described in the embodiments herein, both alone and in combination, to provide a bicyclic ring of Formula I that forms

[0207] In certain embodiments, the present invention provides that p is 0, whereby a bicyclic ring of Formula II-a:

Chemical formula

[0208] In certain embodiments, the present invention provides that n is 0, whereby a bicyclic ring of Formula II-b:

Chemical formula

[0209] Exemplary compounds of the present invention are set forth in Table 1 below. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11] [Table 1-12]

Table 1-13

Table 1-14

Table 1-15

Table 1-16

Table 1-17

Table 1-18

Table 1-19

Table 1-20

[0210] In some embodiments, the present invention provides a compound as specified in Table 1 above, or a pharmaceutically acceptable salt thereof.

[0211] In some embodiments, the present invention provides a compound of Formula I that is not any of the compounds described in Table 2 below.

[0212] In some embodiments, the present invention provides a compound of Formula I that is not any of the compounds from WO2013 / 050617 described in Table 2 below.

[0213] In some embodiments, the present invention provides a compound of Formula I that is not any of the compounds from WO2016 / 067035 described in Table 2 below.

Table 2

[0214] In some embodiments, the present invention is not the compound specified in Table 2 above.

[0215] Exemplary compounds of the present invention are specified in Table 3 below.

Table 3-1

Table 3-2

Table 3-3

[0216] In some embodiments, the present invention provides a compound specified in Table 3 above, or a pharmaceutically acceptable salt thereof.

[0217] The compounds in Table 3 are useful as negative controls for the compounds in Table 1. For example, I-26 (K d for MT1-MMP greater than 5,000 nM) is an I-23 analog having an alanine residue flanking the central cysteine residue, and thus is inactive in MT1-MMP, and thus serves as a negative control for I-23 (K d for MT1-MMP of 0.52 ± 0.24 nM). I-27 (K d for MT1-MMP greater than 5,000 nM) is an I-24 analog having an alanine residue flanking the central cysteine residue, and thus is inactive in MT1-MMP, and thus serves as a negative control for I-24 (K d for MT1-MMP of 1.37 ± 0.53 nM). Such control compounds can be useful, for example, in in vivo distribution studies of active MT1-MMP binders.

[0218] 4. General methods for providing the compounds of the present invention The compounds of the present invention can generally be prepared or isolated by synthetic and / or semi-synthetic methods known to those skilled in the art for similar compounds, as well as by the methods described in detail in the examples herein.

[0219] The compounds of the present invention can be prepared by treating a peptide with a molecular scaffold reagent. The molecular scaffold reagent contains a scaffold and a reactive functional group such as a leaving group ("LG") or a Michael acceptor ("MA") that forms a covalent bond between the peptide and the molecular scaffold through replacement of the leaving group or addition to the Michael acceptor group, followed by subsequent protonation of the addition complex.

[0220] The compounds of the present invention are formed by treating a peptide with various molecular scaffold reagents to form a bicyclic intermediate, and then coupling this with a detectable moiety using standard amide formation methodology.

[0221] One such peptide has the following amino acid sequence: βAla-Sar10-A-C(D-Ala)NE(1Nal)(D-Ala)CEDFYD(tBuGLy)C (SEQ ID NO: 1) which is Peptide 1 (17-69-07-N241).

[0222] The bicyclic peptide formed by treating 17-69-07-N241 with the molecular scaffold reagent 1,3,5-tris(bromomethyl)benzene ("TBMB") as described in WO2016 / 067035 gives rise to an MT1-MMP binder having a K d of 1.2 nM.

[0223] Another such peptide contains only the amino acid residues corresponding to the bicyclic portion of Peptide 1 and has the following amino acid sequence: C(D-Ala)NE(1Nal)(D-Ala)CEDFYD(tBuGLy)C (SEQ ID NO: 2) which is Peptide 2.

[0224] Another such peptide has the following amino acid sequence: βAla-Sar10-A-CTECWVDGWVPC (SEQ ID NO: 3) which is Peptide 3.

[0225] A bicyclic peptide formed by treating 3 with the molecular scaffold reagent 1,3,5-tris(bromomethyl)benzene ("TBMB"), as described in UK Provisional Application No. P2120, gives rise to a CAIX binder with a K of 10 nM. d

[0226] Another such peptide contains only the amino acid residues corresponding to the bicyclic portion of peptide 3 and has the following amino acid sequence: CTECWVDGWVPC (SEQ ID NO: 4) and is peptide 4.

[0227] Another such peptide has the following amino acid sequence: and is peptide 5, which has Ac-CIL(HArg)PNCDLDGRCA-Sar6-(D-K) (SEQ ID NO: 5).

[0228] A bicyclic peptide formed by treating 5 with the molecular scaffold reagent 1,3,5-tris(bromomethyl)benzene ("TBMB"), as described in UK Provisional Application No. P2122, gives rise to an αvβ3 binder with a K of 15 nM. i

[0229] Another such peptide contains a subset of the amino acid residues corresponding to peptide 5, including the bicyclic portion, an acetyl residue at the N-terminus, and an alanine C-terminal extension, and has the following amino acid sequence: Ac-CIL(HArg)PNCDLDGRCA (SEQ ID NO: 6) and is peptide 6.

[0230] ​​In the following schemes, when a particular Michael acceptor (“MA”), leaving group (“LG”), or transformation condition is shown, one of ordinary skill in the art will understand that other Michael acceptors, leaving groups, and transformation conditions are also suitable and contemplated. Such acceptors, groups, and transformations are described in detail in March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, M. B. Smith and J. March, 5 th Edition, John Wiley & Sons, 2001, Comprehensive Organic Transformations, R. C. Larock, 2 nd Edition, John Wiley & Sons, 1999, and Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3 rd edition, John Wiley & Sons, 1999, the entire contents of each of which are hereby incorporated by reference.

[0231] As used herein, the term “leaving group” (LG) includes, but is not limited to, halogens (e.g., fluoride, chloride, bromide, iodide), sulfonates (e.g., mesylate, tosylate, benzenesulfonate, brosylate, nosylate, triflate), diazonium, and the like.

[0232] As used herein, the term “activated ester” (AE) includes, but is not limited to, acyl halides (e.g., acyl fluoride, acyl chloride, acyl bromide, acyl iodide), N-succinimidyl esters, uronium esters (e.g., 1-hydroxy-7-azabenzotriazole, -OAt), and the like.

[0233] In certain embodiments, the compounds of the invention of Formula I are generally prepared according to Scheme I set forth below: [Chemistry] [Chemistry] [Chemistry]

[0234] In the above Scheme I, LG, L 1 , L 2 , L 3 , support, linker 1 , linker 2 , R 1 , R 2 , R 3 , loop A, loop B, detectable moiety 1 , detectable moiety 2 , AE, m, n, o and p are each as defined above and below and are within the classes and subclasses as described herein.

[0235] In one aspect, the present invention provides a method for preparing a compound of formula I according to the steps shown in the above Scheme I. In some embodiments, step S-1 involves contacting a support reagent R-1 with a peptide P-1 to replace a leaving group LG, thereby forming an intermediate, which is further treated with an activated ester of a detectable moiety to yield a compound of formula I. In some embodiments, LG is a halogen. In some embodiments, LG is chlorine. In some embodiments, LG is a sulfonate. In some embodiments, AE is an N-succinimidyl ester. In some embodiments, a base is added to facilitate the replacement. In some embodiments, the base is ammonium carbonate. In some embodiments, the base is an amine. In some embodiments, the base is N,N-diisopropylethylamine.

[0236] In certain embodiments, step S-1 involves a compound of formula P-1 with a compound of formula [Chemistry] [wherein, LG and ring A are as defined above and below, and are within the classes and subclasses as described herein] comprises contacting with a compound of.

[0237] In some embodiments, the reaction further comprises a solvent. In some embodiments, the solvent is acetonitrile. In some embodiments, the reaction further comprises a solvent. In some embodiments, the solvent is DMSO. In some embodiments, the solvent is a mixture of water and acetonitrile.

[0238] In some embodiments, LG is a halogen. In some embodiments, LG is chlorine. In some embodiments, LG is a sulfonate. In some embodiments, a catalyst is added to facilitate the replacement. In some embodiments, the catalyst is generated from a third-generation XPhos precatalyst. In some embodiments, the solvent is tert-butanol. In some embodiments, the solvent is a mixture of water and tert-butanol.

[0239] In certain embodiments, the compounds of the invention of formula I are generally prepared according to Scheme II as set forth below: [Chemical formula] [Chemical formula] [Chemical formula] [[ID=3�]]

[0240] In Scheme II above, MA, L 1 , L 2 , L 3 , support, linker 1 , linker 2 , R 1 , R 2 , R 3, Loop A, Loop B, Detectable Portion 1 , Detectable Portion 2 , AE, m, n, o, and p are each as defined above and below and are within the classes and subclasses as described herein.

[0241] In one aspect, the present invention provides a method for preparing a compound of formula I according to the steps shown in Scheme II above. In some embodiments, step A-1 involves contacting a support reagent R-2 with a peptide P-1 to affect the Michael addition to MA, thereby forming an intermediate, which is further treated with an activated ester of a detectable moiety to yield a compound of formula I. In some embodiments, MA is an α,β-unsaturated amide. In some embodiments, MA is an α,β-unsaturated ketone. In some embodiments, MA is an α,β-unsaturated ester. In some embodiments, MA is an α,β-unsaturated sulfone. In some embodiments, MA is an α,β-unsaturated nitrile. In some embodiments, a base is added to facilitate the Michael addition. In some embodiments, AE is an N-succinimidyl ester. In some embodiments, the base is ammonium carbonate. In some embodiments, the base is an amine. In some embodiments, the base is N,N-diisopropylethylamine.

[0242] In certain embodiments, step A-1 involves contacting a compound of formula P-1 with a compound of the formula [Chemical Formula] [wherein MA and Ring A are each as defined above and below and are within the classes and subclasses as described herein].

[0243] In some embodiments, the reaction further comprises a solvent. In some embodiments, the solvent is acetonitrile. In some embodiments, the reaction further comprises a solvent. In some embodiments, the solvent is DMSO. In some embodiments, the solvent is a mixture of water and acetonitrile.

[0244] In some embodiments, MA is an α,β-unsaturated amide. In some embodiments, MA is an α,β-unsaturated ketone. In some embodiments, MA is an α,β-unsaturated ester. In some embodiments, MA is an α,β-unsaturated sulfone. In some embodiments, MA is an α,β-unsaturated nitrile. In some embodiments, a base is added to facilitate the Michael addition. In some embodiments, the base is ammonium carbonate. In some embodiments, the base is an amine. In some embodiments, the base is N,N-diisopropylethylamine.

[0245] One of ordinary skill in the art will understand that the compounds of formula I may contain one or more stereocenters and may exist as a racemic or diastereomeric mixture. One of ordinary skill in the art will also understand that there are many methods known in the art for the separation of isomers to obtain stereoenriched or stereopure isomers of those compounds, including but not limited to HPLC, chiral HPLC, fractional crystallization of diastereomeric salts, kinetic enzymatic resolution (e.g., by lipases or esterases from fungi, bacteria or animals), and formation of covalent diastereomeric derivatives using enantiomeric enrichment reagents.

[0246] One skilled in the art will understand that the various functional groups present in the compounds of the present invention, such as aliphatic groups, alcohols, carboxylic acids, esters, amides, aldehydes, halogens and nitriles, can be interconverted by techniques well known in the art, including but not limited to reduction, oxidation, esterification, hydrolysis, partial oxidation, partial reduction, halogenation, dehydration, partial hydration and hydration. "March's Advanced Organic Chemistry", 5 th Ed., Ed.: Smith, M.B. and March, J., John Wiley & Sons, New York: 2001. Such interconversions may require one or more of the aforementioned techniques, and certain methods for synthesizing the compounds of the present invention are described below by way of illustration.

[0247] 5. Use, Formulation and Administration Pharmaceutically acceptable compositions According to another embodiment, the present invention provides a composition comprising a compound of the present invention or a pharmaceutically acceptable derivative thereof and a pharmaceutically acceptable carrier, adjuvant or vehicle. The amount of the compound in the composition of the present invention is such that it is effective to measurably inhibit CAIX, MT1-MMP, PSMA, CD38, EphA2 or αvβ3 or a mutant thereof in a biological sample or in a patient. In certain embodiments, the amount of the compound in the composition of the present invention is such that it is effective to measurably inhibit CAIX, MT1-MMP, PSMA, CD38, EphA2 or αvβ3 or a mutant thereof in a biological sample or in a patient. In certain embodiments, the composition of the present invention is formulated for administration to a patient in need of such a composition. In some embodiments, the composition of the present invention is formulated for oral administration to a patient.

[0248] The term "patient" as used herein means an animal, preferably a mammal, and most preferably a human.

[0249] The term "pharmaceutically acceptable carrier, adjuvant or vehicle" refers to a non-toxic carrier, adjuvant or vehicle that does not destroy the pharmacological activity of the compound formulated therewith. Pharmaceutically acceptable carriers, adjuvants or vehicles that can be used in the compositions of the present invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffering substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and lanolin.

[0250] "Pharmaceutically acceptable derivative" means any non-toxic salt, ester, salt of an ester or other derivative of a compound of the present invention that can provide, either directly or indirectly, the compound of the present invention or its inhibitory active metabolite or residue upon administration to a recipient.

[0251] As used herein, the term "inhibitory active metabolite or residue thereof" means that the metabolite or residue is also an inhibitor of CAIX, MT1-MMP, PSMA, CD38, EphA2 or αvβ3 or a mutant thereof.

[0252] The compositions of the present invention can be administered orally, parenterally, by inhalation spray, topically, rectally, intranasally, buccally, vaginally or via an implant reservoir. The term "parenterally" as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, hepatic, intralesional and intracranial injection or infusion techniques. Preferably, the compositions are administered orally, intraperitoneally or intravenously. The sterile injectable form of the compositions of the present invention may be an aqueous or oleaginous suspension. These suspensions can be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may be a sterile injectable solution or suspension in a non-toxic pharmaceutically acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be used are water, Ringer's solution and isotonic sodium chloride solution. In addition, a sterile fixed oil can be conventionally used as a solvent or suspending medium.

[0253] For this purpose, any non-irritating fixed oil containing synthetic mono- or di-glycerides may be used. Fatty acids such as oleic acid and their glyceride derivatives are useful in the preparation of injection solutions, especially in their polyoxyethylated versions, as well as natural pharmaceutically acceptable oils such as olive oil or castor oil. These oil solutions or suspensions may contain long-chain alcohol diluents or dispersants such as carboxymethyl cellulose or similar dispersing agents commonly used in the formulation of pharmaceutically acceptable dosage forms containing emulsifying and suspending agents. Other commonly used surfactants such as Tween, Span, and other emulsifiers or bioavailability enhancers commonly used in the manufacture of pharmaceutically acceptable solid, liquid or other dosage forms may be used for the purposes of the formulation.

[0254] The pharmaceutically acceptable composition of the present invention can be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, commonly used carriers include lactose and corn starch. Lubricants such as magnesium stearate are also typically added. For oral administration in capsule form, useful diluents include lactose and dried corn starch. When an aqueous suspension is required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added.

[0255] Alternatively, the pharmaceutically acceptable composition of the present invention can be administered in the form of suppositories for rectal administration. These can be prepared by mixing the active substance with suitable non-irritating excipients that are solid at room temperature but liquid at rectal temperature and thus melt in the rectum to release the drug. Such materials include cocoa butter, beeswax and polyethylene glycol.

[0256] The pharmaceutically acceptable composition of the present invention may be administered topically, especially when the target of treatment includes areas or organs that are readily accessible by topical application, including diseases of the eye, skin or lower intestinal tract. Suitable topical formulations are readily prepared for each of these areas or organs.

[0257] Topical application for the lower intestinal tract can be achieved in rectal suppository formulations (see above) or in suitable enema formulations. Topical transdermal patches may also be used.

[0258] In topical application, the pharmaceutically acceptable compositions provided can be formulated as suitable ointments containing the active ingredient suspended or dissolved in one or more carriers. Carriers for topical administration of the compounds of the present invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compounds, emulsifying wax or water. Alternatively, the pharmaceutically acceptable compositions provided can be formulated as suitable lotions or creams containing the active ingredient suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl ester wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

[0259] For ocular use, the pharmaceutically acceptable compositions provided can be formulated as a micronized suspension in isotonic, pH-adjusted sterile saline, with or without the addition of a preservative such as benzalkonium chloride, or, preferably, as a solution in isotonic, pH-adjusted sterile saline. Alternatively, for ocular use, the pharmaceutically acceptable compositions can be formulated as an ointment such as petrolatum.

[0260] The pharmaceutically acceptable compositions of the present invention may be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well known in the pharmaceutical arts and can be prepared as solutions in saline using benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and / or other conventional solubilizing or dispersing agents.

[0261] Most preferably, the pharmaceutically acceptable compositions of the present invention are formulated for oral administration. Such formulations may be administered with or without food. In some embodiments, the pharmaceutically acceptable compositions of the present invention are administered without food. In other embodiments, the pharmaceutically acceptable compositions of the present invention are administered with food.

[0262] The amount of the compounds of the invention that may be combined with a carrier material to produce a single dosage form composition will vary depending on the host being treated and the particular mode of administration. Preferably, the provided compositions should be formulated so that an inhibitor in a dosage between 0.01 and 100 mg / kg body weight / day can be administered to a patient receiving these compositions.

[0263] It should also be understood that the specific dosage and treatment regimen for any particular patient will be determined by a variety of factors including the activity of the specific compound being used, age, body weight, general health, sex, eating habits, time of administration, rate of excretion, drug combination, as well as the judgment of the treating physician and the severity of the particular disease being treated. The amount of the compounds of the invention in the composition will also be determined by the particular compound in the composition.

[0264] Use of the Compounds and Pharmaceutically Acceptable Compositions Certain bicyclic peptides of the invention have specific utility as CAIX binders.

[0265] Various forms of the enzyme carbonic anhydrase (CA) catalyze the hydration of carbon dioxide to produce bicarbonate anions (HCO3 - ) and protons. The substrates of the reaction catalyzed by CA include several physiological processes including the formation and transport of CO2, protons and bicarbonate anions, for example, respiration, maintenance of pH levels, bone development and other processes. In the human body, 12 catalytically active CA isozymes have been identified that differ in cellular localization and expression in various tissues.

[0266] Clinical regulation of the activity of human carbonic anhydrase (hCA) by small molecule inhibitors has proven to be a reliable treatment for several human diseases and has been, for decades, still a major component of therapies for hypertension, glaucoma, hyperthyrosis and hypoglycemia (Supuran (2008) Nat. Rev. Drug Discov. 7, 168). Traditional inhibitors of carbonic anhydrase that bind within the active site of CA are aromatic or heteroaromatic sulfonamides.

[0267] Human carbonic anhydrase IX (hCAIX) is an isoform that binds to the outer cell membrane (its catalytic domain is located in the extracellular space). Under physiological conditions, hCAIX is expressed only in specific tissues of the gastrointestinal tract. Its overexpression has been shown both in vitro and in vivo during hypoxia in cancer cells. Expression of hCAIX has been detected in carcinomas of the neck, ovary, kidney, esophagus, lung, breast and brain. In tumors, hCAIX is a crucial molecule for maintaining normal levels of intracellular pH, and its expression favors the growth of hypoxic tumor cells under acidic conditions (Chiche et al. (2009) Cancer Res 69, 358). Thus, the hCAIX enzyme is a convenient target for the development of specific inhibitors to be used as anticancer therapeutics with a new mechanism of action (Neri and Supuran (2011) Nature Reviews 10, 767).

[0268] According to a further aspect of the present invention, there is provided a compound or composition as defined herein for use in preventing, suppressing or treating a disease or disorder mediated by CAIX.

[0269] According to a further aspect of the present invention, there is provided a method of preventing, suppressing or treating a disease or disorder mediated by CAIX, comprising administering to a patient in need thereof a compound or composition as defined herein.

[0270] In one embodiment, CAIX is mammalian CAIX. In a further embodiment, mammalian CAIX is human CAIX (hCAIX).

[0271] In one embodiment, the disease or disorder mediated by CAIX is selected from cancer.

[0272] In another aspect, certain bicyclic peptides of the invention have specific utility as high affinity binders of membrane type 1 matrix metalloproteinase (MT1-MMP, also known as MMP14). MT1-MMP is a transmembrane matrix metalloproteinase that plays a major role in extracellular matrix remodeling by directly degrading some of its components and indirectly activating pro-MMP2. MT1-MMP is critical for tumor angiogenesis (Sounni et al (2002) FASEB J. 16(6), 555-564), is overexpressed in a variety of solid tumors, and thus the MT1-MMP-binding bicyclic peptides of the invention are particularly useful in the targeted treatment of cancer, especially solid tumors such as non-small cell lung cancer. In one embodiment, the bicyclic peptide of the invention is specific for human MT1-MMP. In a further embodiment, the bicyclic peptide of the invention is specific for mouse MT1-MMP. In even further embodiments, the bicyclic peptide of the invention is specific for human and mouse MT1-MMP. In even further embodiments, the bicyclic peptide of the invention is specific for human, mouse and canine MT1-MMP.

[0273] In another aspect, certain bicyclic peptides of the invention have specific utility as high affinity binders of prostate specific membrane antigen (PSMA).

[0274] PSMA is a type II integral membrane protein that has abundant and restricted expression on the surface of prostate cancer, particularly in androgen-independent, progressive, and metastatic diseases (Schulke, N.; et al. Proc Natl Acad Sci USA 2003, 100, 12590-12595). The latter is important because almost all PCa becomes androgen-independent. It is also expressed in the endothelium of most solid tumors outside the prostate (Chang, S. S.; et al. Cancer Res 1999, 59, 3192-3198). PSMA meets the criteria for a promising target for therapy, namely, abundant and restricted expression (in the prostate) at all stages of the disease, presentation on the cell surface but not shed into the circulation, and association with enzymatic or signaling activity (Schulke, N.; et al. Proc Natl Acad Sci USA 2003, 100, 12590-12595). The PSMA gene is located on the short arm of chromosome 11 and functions as both a folate hydrolase and a neuropeptidase. This is a neuropeptidase function equivalent to that of glutamate carboxypeptidase II (GCPII), also known as "brain PSMA," which can modulate glutamatergic transmission by cleaving N-acetylaspartylglutamate (NAAG) into N-acetylaspartate (NAA) and glutamate (Nan, F.; et al. J Med Chem 2000, 43, 772-774). There are up to 10 6 PSMA molecules per cancer cell, further suggesting it as an ideal target for imaging and therapy by radionuclide-based techniques (Tasch, J.; et al. Crit Rev Immunol 2001, 21, 249-261).

[0275] In another aspect, certain bicyclic peptides of the invention have specific utility as CD38 binders.

[0276] CD38 is a 45kD type II transmembrane glycoprotein with a long C-terminal extracellular domain and a short N-terminal cytoplasmic domain. The CD38 protein is a bifunctional ectoenzyme that catalyzes the conversion of NAD+ to cyclic ADP-ribose (cADPR) and can also hydrolyze cADPR to ADP-ribose. During ontogeny, CD38 appears on CD34+ pluripotent stem cells and lineage-determining progenitor cells of lymphocytes, erythrocytes, and myeloid cells. CD38 expression persists mainly in the lymphoid lineage with variable expression levels at different stages of T and B cell development.

[0277] CD38 is upregulated in many hematopoietic malignancies and in cell lines derived from various hematopoietic malignancies including non-Hodgkin lymphoma (NHL), Burkitt lymphoma (BL), multiple myeloma (MM), B chronic lymphocytic leukemia (B-CLL), B and T acute lymphoblastic leukemia (ALL), T cell lymphoma (TCL), acute myeloid leukemia (AML), hairy cell leukemia (HCL), Hodgkin lymphoma (HL), and chronic myeloid leukemia (CML). On the other hand, the most primitive pluripotent stem cells of the hematopoietic system are CD38-. The correlation between CD38 expression in hematopoietic malignancies and their disease progression makes CD38 an attractive target for antibody therapy.

[0278] CD38 is Ca 2+Mobilization (Morra et al. (1998) FASEB J. 12; 581-592; Zilber et al. (2000) Proc Natl Acad Sci USA 97, 2840-2845), and signaling through tyrosine phosphorylation of a number of signaling molecules including phospholipase C-γ, ZAP-70, syk and c-cbl in lymphocytes and myeloid cells or cell lines (Funaro et al. (1993) Eur J Immunol 23, 2407-2411; Morra et al. (1998), supra; Funaro et al. (1990) J Immunol 145, 2390-2396; Zubiaur et al. (1997) J Immunol 159, 193-205; Deaglio et al. (2003) Blood 102, 2146-2155; Todisco et al. (2000) Blood 95, 535-542; Konopleva et al. (1998) J Immunol 161, 4702-4708; Zilber et al. (2000) Proc Natl Acad Sci USA 97, 2840-2845; Kitanaka et al. (1997) J Immunol 159, 184-192; Kitanaka et al. (1999) J Immunol 162, 1952-1958; Mallone et al. (2001) Int Immunol 13, 397-409) have been reported to be involved. Based on these observations, CD38 has been proposed to be an important signaling molecule during normal development in the maturation and activation of lymphocytes and myeloid cells.

[0279] The exact role of CD38 in signaling and hematopoiesis is not yet clear, because, among other reasons, most of these signaling studies have used cell lines that heterologously overexpress CD38, a non-physiological ligand, and anti-CD38 monoclonal antibodies. The CD38 protein is a Ca 2+It has an enzyme activity that produces cADPR, a molecule capable of inducing mobilization (Lee et al. (1989) J Biol Chem 264, 1608-1615; Lee and Aarhus (19) Cell Regul 2, 203-209). Therefore, CD38 ligation with monoclonal antibodies increases the production of cADPR, thereby increasing Ca in lymphocytes 2+ and has been proposed to trigger mobilization and signaling (Lee et al. (1997) Adv Exp Med Biol 419, 411-419). Contrary to this hypothesis, cleavage and point mutation analysis of the CD38 protein have shown that neither its cytoplasmic tail nor its enzyme activity is required for signaling mediated by anti-CD38 antibodies (Kitanaka et al. (1999) J Immunol 162, 1952-1958; Lund et al. (1999) J Immunol 162, 2693-2702; Hoshino et al. (1997) J Immunol 158, 741-747).

[0280] The most compelling evidence for the function of CD38 is provided by CD38− / − knockout mice, which, due to the defect in dendritic cell migration, have a defect in their innate immunity and a reduced T cell-dependent humoral response (Partida-Sanchez et al. (2004) Immunity 20, 279-291; Partida-Sanchez et al. (2001) Nat Med 7, 1209-1216). Nevertheless, because the CD38 expression pattern during hematopoiesis varies greatly between humans and mice, it is not clear whether CD38 function in mice is the same as that in humans: a) Unlike in human immature progenitor stem cells, similar progenitor stem cells in mice express high levels of CD38 (Randall et al. (1996) Blood 87, 4057-4067; Dagher et al. (1998) Biol Blood Marrow Transplant 4, 69-74), and b) During human B cell development, high levels of CD38 expression are seen in germinal center B cells and plasma cells (Uckun (1990) Blood 76, 1908-1923; Kumagai et al. (1995) J Exp Med 181, 1101-1110), whereas in mice, the CD38 expression levels in the corresponding cells are low (Oliver et al. (1997) J Immunol 158, 1108-1115; Ridderstad and Tarlinton (1998) J Immunol 160, 4688-4695).

[0281] The literature has described several anti-human CD38 antibodies with different growth characteristics in various tumor cells and cell lines. For example, the chimeric OKT10 antibody with mouse Fab and human IgG1 Fc mediates antibody-dependent cell-mediated cytotoxicity (ADCC) very efficiently against lymphoma cells in the presence of peripheral blood mononuclear effector cells derived from either MM patients or normal individuals (Stevenson et al. (1991) Blood 77, 1071-1079). The CDR-grafted humanized version of the anti-CD38 antibody AT13 / 5 has been shown to have potent ADCC activity against CD38-positive cell lines (U.S. Patent Application No. 09 / 797,941). Human monoclonal anti-CD38 antibodies have been shown to mediate the in vitro killing of CD38-positive cell lines by ADCC and / or complement-dependent cytotoxicity (CDC), and to delay tumor growth in SCID mice bearing the MM cell line RPMI-8226 (WO2005 / 103083). On the other hand, several anti-CD38 antibodies, IB4, SUN-4B7 and OKT10, except for IB6, AT1 and AT2, induced the proliferation of peripheral blood mononuclear cells (PBMC) derived from normal individuals (Ausiello et al. (2000) Tissue Antigens 56, 539-547).

[0282] Some prior art antibodies have been shown to be able to trigger apoptosis in CD38+ B cells. However, they can do so only in the presence of stromal cells or stroma-derived cytokines. The agonistic anti-CD38 antibody (IB4) has been reported to prevent apoptosis of human germinal center (GC) B cells (Zupo et al. (1994) Eur J Immunol 24, 1218-1222) and to induce the proliferation of KG-1 and HL-60 AML cells (Konopleva et al. (1998) J Immunol 161, 4702-4708), but to induce apoptosis in Jurkat T lymphoblast cells (Morra et al. (1998) FASEB J 12, 581-592). Another anti-CD38 antibody, T16, has been shown to induce apoptosis in immature lymphocytes and leukemic lymphoblasts from ALL patients (Kumagai et al. (1995) J Exp Med 181, 1101-1110) and in leukemic myeloblasts from AML patients (Todisco et al. (2000) Blood 95, 535-542), but T16 induced apoptosis only in the presence of stromal cells or stroma-derived cytokines (IL-7, IL-3, stem cell factor).

[0283] In another aspect, certain bicyclic peptides of the invention have specific utility as EphA2 binders.

[0284] Eph receptor tyrosine kinases (Ephs) belong to a large group of receptor tyrosine kinases (RTKs) that phosphorylate proteins on tyrosine residues. Ephs and their ligands, the ephrins, bind to control cell positioning and tissue organization (Poliakov et al. (2004) Dev Cell 7, 465-80). Functional and biochemical Eph responses occur in higher ligand oligomerization states (Stein et al. (1998) Genes Dev 12, 667-678).

[0285] Among a number of patterning functions, various Ephs and ephrins have been shown to play roles in blood vessel development. Knockout of EphB4 and ephrin-B2 results in the lack of the ability to remodel the capillary bed in blood vessels (Poliakov et al., supra) and fetal lethality. Constitutive expression of some Eph receptors and ephrins has also been observed in newly formed adult microvessels (Brantley-Sieders et al. (2004) Curr Pharm Des 10, 3431-42; Adams (2003) J Anat 202, 105-12).

[0286] Deregulated recurrence of some ephrins and their receptors in adults has also been observed to contribute to tumor invasion, metastasis and angiogenesis (Nakamoto et al. (2002) Microsc Res Tech 59, 58-67; Brantley-Sieders et al., supra). Furthermore, some Eph family members have been found to be overexpressed on tumor cells derived from various human tumors (Brantley-Sieders et al., supra); Marme (2002) Ann Hematol 81 Suppl 2, S66; Booth et al. (2002) Nat Med 8, 1360-1).

[0287] EPH receptor A2 (Ephrin type-A receptor 2) is a protein encoded by the EPHA2 gene in humans.

[0288] EphA2 is often upregulated in multiple human cancers, such as breast (Zelinski et al (2001) Cancer Res. 61, 2301-2306; Zhuang et al (2010) Cancer Res. 70, 299-308; Brantley-Sieders et al (2011) PLoS One 6, e24426), lung (Brannan et al (2009) Cancer Prev Res (Phila) 2, 1039-1049; Kinch et al (2003) Clin Cancer Res. 9, 613-618; Guo et al (2013) J Thorac Oncol. 8, 301-308), stomach (Nakamura et al (2005) Cancer Sci. 96, 42-47; Yuan et al (2009) Dig Dis Sci 54, 2410-2417), pancreas (Mudali et al (2006) Clin Exp Metastasis 23, 357-365), prostate (Walker-Daniels et al (1999) Prostate 41, 275-280), liver (Yang et al (2009) Hepatol Res. 39, 1169-1177) and glioblastoma (Wykosky et al (2005) Mol Cancer Res. 3, 541-551; Li et al (2010) Tumour Biol. 31, 477-488), which are often correlated with disease progression, metastasis and poor prognosis.

[0289] The overall role of EphA2 in cancer progression remains undefined, although there is evidence of interactions at multiple stages of cancer progression, including tumor cell proliferation, survival, invasion, and angiogenesis. Downregulation of EphA2 expression suppresses tumor cancer cell proliferation (Binda et al (2012) Cancer Cell 22, 765-780), whereas EphA2 blockade inhibits VEGF-induced cell migration (Hess et al (2001) Cancer Res. 61, 3250-3255), sprouting and angiogenesis (Cheng et al (2002) Mol Cancer Res. 1, 2-11; Lin et al (2007) Cancer 109, 332-40), and the progression of metastasis (Brantley-Sieders et al (2005) FASEB J. 19, 1884-1886).

[0290] Antibody-drug conjugates with EphA2 have been shown to significantly reduce tumor growth in rat and mouse xenograft models (Jackson et al (2008) Cancer Research 68, 9367-9374), and a similar approach has been tried in humans, but the treatment had to be discontinued due to treatment-related adverse events (Annunziata et al (2013) Invest New drugs 31, 77-84).

[0291] In another aspect, certain bicyclic peptides of the invention have specific utility as integrin αvβ3 binders.

[0292] Integrins are heterodimeric matrix receptors that anchor cells to the substrate and transmit externally derived signals across the plasma membrane. Integrin αvβ3 is involved in osteoclast-mediated bone resorption both in vivo and in vitro. This heterodimeric molecule recognizes the amino acid motif Arg-Gly-Asp (RGD) contained in bone matrix proteins such as osteopontin and bone sialoprotein. Integrin αvβ3 is expressed in osteoclasts, and its expression is modulated by resorptive steroids and cytokines. Based on blocking experiments, αvβ3 integrin has been identified as a major functional adhesion receptor in osteoclasts. Inhibitors of integrin αvβ3 reduce the ability of osteoclasts to bind to and resorb bone. Integrin αvβ makes a major contribution to osteoclast function, and inhibitors of this integrin are considered to treat or prevent osteoporosis, osteolytic metastases, and malignancy-induced hypercalcemia. [[ID=??]] [[ID=??]]

[0293] [[ID=??]] It seems there is some issue with the tags in the original text as they are not in a proper format for translation. I've left them as they are but noted the problem. If you can correct the tags in the original text, it would be better for a more accurate translation.There are many bone diseases associated with osteoclast-mediated bone lysis. Osteoporosis is the most common one, which is induced when bone resorption and formation do not cooperate and bone destruction invalidates bone construction. Osteoporosis can also be caused by other conditions such as hormonal imbalance, diseases or drugs (e.g., corticosteroids or anti-epileptic drugs). Bone is one of the most common sites of metastasis by human breast, prostate, lung and thyroid cancers as well as other cancers. Osteoporosis may occur due to postmenopausal estrogen deficiency. Secondary osteoporosis may be associated with rheumatoid arthritis. Bone metastasis shows a very unique step of osteoclast bone resorption that is not seen in metastasis of other organs. Cancer-related osteolysis is widely accepted to be essentially mediated by osteoclasts, which are thought to be activated and can be activated indirectly via osteoblasts or directly by tumor products. In addition, hypercalcemia (an increase in blood calcium concentration) is an important complication of osteolytic bone diseases. It appears relatively frequently in patients with extensive bone destruction and is particularly common in breast, lung, kidney, ovarian and pancreatic cancers as well as in multiple myeloma.

[0294] Disintegrins are a family of low molecular weight RGD-containing peptides that specifically bind to integrin αIIbβ3, α5β1 and αvβ3, which are expressed on platelets as well as on other cells including vascular endothelial cells and some tumor cells. In addition to their potent antiplatelet activity, the study of disintegrins has revealed new uses in the diagnosis of cardiovascular diseases and in the design of therapeutic agents in arterial thrombosis, osteoporosis and angiogenesis-related tumor growth and metastasis. Rhodostomin (Rho), a disintegrin derived from the venom of Colloselasma rhodostoma, has been found to inhibit platelet aggregation in vivo and in vitro through blockade of platelet glycoprotein αIIbβ3.

[0295] The role of αvβ3 integrin in bone disorders has been well documented (Ross et al (2006) Journal of Clinical Investigation 116(5); Rodan et al (1997) Journal of Endocrinology 154, S47-S56; Teitelbaum (2005) Journal of Clinical Endocrinology and Metabolism 90(4), 2466-2468; Teitelbaum (2000) Journal of Bone and Mineral Metabolism 18, 344-349; Nakamura et al (2007) Journal of Bone and Mineral Metabolism 25, 337-344; Duong et al (1999) Journal of Bone and Mineral Metabolism 17, 1-6; and Teti et al (2002) Calcified Tissue International 71, 293-299). In addition to bone disorders, αvβ3 integrin plays an important role in angiogenesis and tumor growth under conditions not related to bone disorders.

[0296] According to a further aspect of the invention, there is provided a peptide ligand as defined herein for use in preventing, suppressing or treating a disease or disorder mediated by integrin αvβ3.

[0297] According to a further aspect of the invention, there is provided a method of preventing, suppressing or treating a disease or disorder mediated by integrin αvβ3, the method comprising administering to a patient in need thereof a peptide ligand as defined herein.

[0298] In one embodiment, the integrin αvβ3 is mammalian integrin αvβ3. In a further embodiment, the mammalian integrin αvβ3 is human integrin αvβ3.

[0299] In one embodiment, the disease or disorder mediated by integrin αvβ3 is selected from bone diseases (such as osteoporosis), cancer, and diseases associated with angiogenesis.

[0300] In a further embodiment, the disease or disorder mediated by integrin αvβ3 is selected from cancer.

[0301] The compounds and compositions described herein are generally useful for inhibiting the activity of one or more enzymes, carbonic anhydrase, metalloprotease, carboxypeptidase, hydrolase, kinase or integrin.

[0302] The polypeptide ligands selected according to the methods of the present invention can be used in in vivo treatment and prophylactic applications, in vitro and in vivo diagnostic applications, in vitro assay and reagent applications, etc. Ligands having a selected level of specificity are useful in applications involving testing in non-human animals where cross-reactivity is desirable, or in diagnostic applications where cross-reactivity with homologs or paralogs needs to be carefully controlled. In some applications such as vaccine applications, the ability to elicit an immune response against a given range of antigens can be exploited to tailor the vaccine to specific diseases and pathogens.

[0303] Substantially pure peptide ligands with at least 90 to 95% homogeneity are preferred for administration to mammals, and 98 to 99% or greater homogeneity is most preferred for pharmaceutical use, especially when the mammal is human. Once the selected polypeptide is purified to the desired homogeneity, it may be used diagnostically or therapeutically (including in vitro), or in developing and performing assay procedures, immunofluorescent staining, etc. (Lefkovite and Pernis, (1979 and 1981) Immunological Methods, Volumes I and II, Academic Press, NY).

[0304] The activity of the compounds utilized in the present invention as inhibitors of CAIX, MT1-MMP, PSMA, CD38, EphA2 or αvβ3 or mutants thereof can be assayed in vitro, in vivo or in cell lines. Alternative in vitro assays quantify the ability of an inhibitor to bind to CAIX, MT1-MMP, PSMA, CD38, EphA2 or αvβ3. Inhibitor binding can be measured by radiolabeling the inhibitor prior to binding, isolating the inhibitor / CAIX, inhibitor / MT1-MMP, inhibitor / PSMA, inhibitor / CD38, inhibitor / EphA2, or inhibitor / αvβ3 complex, and determining the amount of bound radiolabel. Alternatively, inhibitor binding can be determined by performing a competition experiment where a novel inhibitor is incubated with CAIX, MT1-MMP, PSMA, CD38, EphA2 or αvβ3 that is bound to a known radioligand. Representative in vitro and in vivo assays useful in assaying MT1-MMP inhibitors include those described and disclosed in Pietraszek et al., (2014) FEBS Letters 588(23), 4319-4324; Cheltsov et al., (2012) Cancer Res. 72(9), 2339-49; and WO2009 / 098450, each of which is incorporated herein by reference in its entirety. Representative in vitro and in vivo assays useful in assaying CAIX inhibitors include those described and disclosed in Wind et al., (2011) Ann Clin Biochem. 48(2), 112-120; Gandhi et al., (2015) J. Urology 193(4), e870-e871; and WO2004 / 005348, each of which is incorporated herein by reference in its entirety. Representative in vitro and in vivo assays useful in assaying αvβ3 inhibitors include those described and disclosed in Wang et al (2005) Bioconjug Chem 16(3), 729-34.The detailed conditions for assaying the compounds utilized in the present invention as inhibitors of CAIX, MT1-MMP, PSMA, CD38, EphA2 or αvβ3 or mutants thereof are specified in the following examples.

[0305] As used herein, the terms “treatment,” “treat,” and “treating” refer to restoring, alleviating, delaying the onset of, or inhibiting the progression of a disease or disorder, or one or more symptoms thereof, as described herein. In some embodiments, treatment can be administered after one or more symptoms have developed. In other embodiments, treatment can be administered in the absence of symptoms. For example, treatment can be administered to a susceptible individual prior to the onset of symptoms (e.g., from the perspective of a symptom history and / or from the perspective of genetic or other susceptibility factors). Treatment may continue, for example, after symptoms have resolved, to prevent or delay their recurrence.

[0306] The provided compounds are inhibitors of CAIX, MT1-MMP, PSMA, CD38, EphA2 or αvβ3, and thus are useful for treating one or more disorders associated with the activity of CAIX, MT1-MMP, PSMA, CD38, EphA2 or αvβ3. Accordingly, in certain embodiments, the present invention provides a method for treating a CAIX-mediated, MT1-MMP-mediated, PSMA-mediated, CD38-mediated, EphA2-mediated or αvβ3-mediated disorder, the method comprising administering to a patient in need thereof a compound of the present invention or a pharmaceutically acceptable composition thereof.

[0307] As used herein, the terms “CAIX-mediated,” “MT1-MMP-mediated,” “PSMA-mediated,” “CD38-mediated,” “EphA2-mediated,” or “αvβ3-mediated” disorders, diseases, and / or conditions mean any disease or other adverse condition in which CAIX, MT1-MMP, PSMA, CD38, EphA2, or αvβ3 or a mutant thereof is known to play a role, as used herein. Accordingly, another embodiment of the invention relates to treating or reducing the severity of one or more diseases in which CAIX, MT1-MMP, PSMA, CD38, EphA2, or αvβ3 or a mutant thereof is known to play a role.

[0308] Examples of cancers that can be treated (or inhibited) include tumors of epithelial origin (various types of adenomas and carcinomas, including adenocarcinomas, squamous cell carcinomas, transitional cell carcinomas, and other carcinomas), such as carcinomas of the bladder and urinary tract, carcinomas of the breast, carcinomas of the gastrointestinal tract (including the esophagus, stomach, small intestine, colon, rectum, and anus), carcinomas of the liver (hepatocellular carcinoma), carcinomas of the gallbladder and biliary tract, carcinomas of the exocrine pancreas, carcinomas of the kidney, carcinomas of the lung (e.g., adenocarcinoma, small cell lung cancer, non-small cell lung cancer, bronchioloalveolar carcinoma, and mesothelioma), carcinomas of the head and neck (e.g., cancers of the tongue, oral cavity, larynx, pharynx, nasopharynx, tonsils, salivary glands, nasal cavity, and paranasal sinuses), carcinomas of the ovary, carcinomas of the fallopian tube, carcinomas of the peritoneum, carcinomas of the vagina, carcinomas of the vulva, carcinomas of the penis, carcinomas of the cervix, carcinomas of the myometrium, carcinomas of the endometrium, carcinomas of the thyroid (e.g., thyroid follicular carcinoma), carcinomas of the adrenal gland, carcinomas of the prostate, carcinomas of the skin, and carcinomas of the appendages (e.g., melanoma, basal cell carcinoma, squamous epithelial cell carcinoma, keratoacanthoma, dysplastic nevus); lymphocytic hematological malignancies and related conditions (e.g., acute lymphoblastic leukemia [ALL], chronic lymphocytic leukemia [CLL], B-cell lymphomas such as diffuse large B-cell lymphoma [DLBCL], follicular lymphoma, Burkitt lymphoma, mantle cell lymphoma, T-cell lymphoma and leukemia, natural killer [NK] cell lymphoma, Hodgkin lymphoma, hairy cell leukemia, monoclonal gammopathy of undetermined significance, plasmacytoma, multiple myeloma, and post-transplant lymphoproliferative disorder) as well as myeloid hematological malignancies and related conditions (e.g., acute myeloid leukemia [AML], chronic myeloid leukemia [CML], chronic myelomonocytic leukemia [CMML], eosinophilic syndrome, myeloproliferative disorders such as polycythemia vera, essential thrombocythemia, and primary myelofibrosis, myeloproliferative syndrome, myelodysplastic syndrome, and promyelocytic leukemia), including hematological malignancies (i.e., leukemia, lymphoma) and pre-malignant blood disorders and borderline malignant tumor disorders; tumors of mesenchymal origin, such as osteosarcoma, fibrosarcoma, chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma, liposarcoma, angiosarcoma, Kaposi sarcoma, Ewing sarcoma, synovial sarcoma, epitheloid sarcoma, gastrointestinal stromal tumor, benign and malignant histiocytoma, and dermatofibrosarcoma protuberans, etc., sarcomas of soft tissue, bone, or cartilage;Tumors of the central or peripheral nervous system (e.g., astrocytomas, gliomas and glioblastomas, meningiomas, ependymomas, pineal tumors and schwannomas); endocrine tumors (e.g., pituitary tumors, adrenal tumors, islet cell tumors, parathyroid tumors, carcinoid tumors and medullary carcinoma of the thyroid); tumors of the eye and its appendages (e.g., retinoblastoma); germ cell tumors and trophoblastic tumors (e.g., teratomas, seminomas, undifferentiated germ cell tumors, cystic teratomas and choriocarcinoma); and pediatric tumors and fetal tumors (e.g., medulloblastoma, neuroblastoma, Wilms' tumor and primitive neuroectodermal tumors); or congenital or other syndromes (e.g., xeroderma pigmentosum) that leave the patient susceptible to malignant tumors; are mentioned but not limited to these.

[0309] In a further embodiment, the cancer is selected from cancers of the neck, ovary, kidney, esophagus, lung, breast and brain.

[0310] References herein to the term "prevention" involve the administration of a protective composition prior to the induction of the disease. "Suppression" refers to the administration of the composition after an inciting event but prior to the clinical manifestations of the disease. "Treatment" involves the administration of a protective composition after the symptoms of the disease have become apparent.

[0311] Animal model systems are available for use in screening the effectiveness of peptide ligands in protecting against or treating a disease. The use of animal model systems is facilitated by the present invention, which enables the development of polypeptide ligands that can cross-react with human and animal targets to permit the use of animal models.

[0312] Furthermore, the present invention provides for the use of a compound as defined herein, or a pharmaceutically acceptable salt or hydrate or solvate thereof, for the preparation of a medicament for the treatment of a proliferative disease.

[0313] Combination therapy Depending on the particular condition or disease being treated, additional therapeutic agents that are normally administered to treat that condition may be administered in combination with the compounds and compositions of the present invention. As used herein, additional therapeutic agents that are normally administered to treat a particular disease or condition are known as "appropriate for the disease or condition being treated".

[0314] In certain embodiments, the provided combination or its composition is administered in combination with another therapeutic agent.

[0315] In certain embodiments, the combination therapy of the present invention or a pharmaceutically acceptable composition thereof is administered in combination with a monoclonal antibody or an siRNA therapeutic.

[0316] Those additional agents may be administered separately from the provided combination therapy as part of a multiple dosing regimen. Alternatively, those agents may be part of a single dosage form, mixed together with the compounds of the present invention in a single composition. When administered as part of a multiple dosing regimen, the two active agents may be provided simultaneously, sequentially, or within a defined period of time of each other, usually within 5 hours of each other.

[0317] As used herein, the terms "combination", "combined" and related terms refer to the simultaneous or sequential administration of therapeutic agents according to the present invention. For example, the combinations of the present invention may be administered with another therapeutic agent, either simultaneously or sequentially in separate unit dosage forms or together in a single unit dosage form.

[0318] The amount of additional therapeutic agent present in the compositions of the present invention will not exceed the amount that would normally be administered in a composition containing the therapeutic agent as the sole active agent. Preferably, the amount of additional therapeutic agent in the presently disclosed compositions will range from about 50% to 100% of the amount normally present in a composition containing the agent as the sole therapeutic active agent.

[0319] In one embodiment, the present invention provides a composition comprising a compound of Formula I and one or more additional therapeutic agents. The therapeutic agent may be administered together with the compound of Formula I, or before or after administration of the compound of Formula I. Suitable therapeutic agents are described in more detail below. In certain embodiments, the compound of Formula I may be administered up to 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours or 18 hours before the therapeutic agent. In other embodiments, the compound of Formula I may be administered up to 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours or 18 hours after the therapeutic agent.

[0320] In another embodiment, the present invention provides a method of treating a hematological malignancy, comprising administering to a patient in need thereof a compound of Formula I and one or more additional therapeutic agents selected from rituximab (Rituxan®), cyclophosphamide (Cytoxan®), doxorubicin (Hydrodaunorubicin®), vincristine (Oncovin®), prednisone, hedgehog signaling inhibitors, BTK inhibitors, JAK / pan-JAK inhibitors, TYK2 inhibitors, PI3K inhibitors, SYK inhibitors, and combinations thereof.

[0321] In another embodiment, the present invention provides a method for treating a solid tumor, comprising administering to a patient in need thereof a compound of formula I and one or more additional therapeutic agents selected from Rituxan®, Cytoxan®, Hydrodaunorubicin®, Oncovin®, prednisone, hedgehog signaling inhibitors, BTK inhibitors, JAK / pan-JAK inhibitors, TYK2 inhibitors, PI3K inhibitors, SYK inhibitors, and combinations thereof.

[0322] In another embodiment, the present invention provides a method for treating a hematological malignancy, comprising administering to a patient in need thereof a compound of formula I and a hedgehog (Hh) signaling pathway inhibitor. In some embodiments, the hematological malignancy is DLBCL (Ramirez et al "Defining causative factors contributing in the activation of hedgehog signaling in diffuse large B-cell lymphoma" Leuk. Res. (2012), which was published online on July 17 and is incorporated herein by reference in its entirety).

[0323] In another embodiment, the present invention provides a method for treating diffuse large B-cell lymphoma (DLBCL), comprising administering to a patient in need thereof a compound of formula I and one or more additional therapeutic agents selected from Rituxan®, Cytoxan®, Hydrodaunorubicin®, Oncovin®, prednisone, hedgehog signaling inhibitors, and combinations thereof.

[0324] In another embodiment, the present invention provides a method for treating multiple myeloma, which comprises administering to a patient in need thereof a compound of formula I, and one or more additional therapeutic agents selected from bortezomib (Velcade®), and dexamethasone (Decadron®), hedgehog signaling inhibitors, BTK inhibitors, JAK / pan-JAK inhibitors, TYK2 inhibitors, PI3K inhibitors, SYK inhibitors in combination with lenalidomide (Revlimid®).

[0325] In another embodiment, the present invention provides a method for treating Waldenström macroglobulinemia, which comprises administering to a patient in need thereof a compound of formula I, and one or more additional therapeutic agents selected from chlorambucil (Leukeran®), cyclophosphamide (Cytoxan®, Neosar®), fludarabine (Fludara®), cladribine (Leustatin®), rituximab (Rituxan®), hedgehog signaling inhibitors, BTK inhibitors, JAK / pan-JAK inhibitors, TYK2 inhibitors, PI3K inhibitors and SYK inhibitors.

[0326] In another embodiment, the present invention is a method of treating a disease or reducing its severity, comprising administering to a patient in need thereof a compound of formula I and a BTK inhibitor, wherein the disease is inflammatory bowel disease, arthritis, systemic lupus erythematosus (SLE), vasculitis, idiopathic thrombocytopenic purpura (ITP), rheumatoid arthritis, psoriatic arthritis, osteoarthritis, Still's disease, juvenile arthritis, diabetes, myasthenia gravis, Hashimoto's thyroiditis, Ord's thyroiditis, Graves' disease, autoimmune thyroiditis, Sjögren's syndrome, multiple sclerosis, systemic sclerosis, Lyme neuroborreliosis, Guillain - Barré syndrome, acute disseminated encephalomyelitis, Addison's disease, opsoclonus - myoclonus syndrome, ankylosing spondylitis, antiphospholipid antibody syndrome, aplastic anemia, autoimmune hepatitis, autoimmune gastritis, pernicious anemia, celiac disease, Goodpasture's syndrome, idiopathic thrombocytopenic purpura, optic neuritis, scleroderma, primary biliary cirrhosis, Reiter's syndrome, Takayasu arteritis, temporal arteritis, warm autoimmune hemolytic anemia, Wegener's granulomatosis, psoriasis, alopecia universalis, Behçet's disease, chronic fatigue, autonomic neuropathy, membranous glomerulonephritis, endometriosis, interstitial cystitis, pemphigus vulgaris, bullous pemphigoid, neurotic myotonia, scleroderma, vulvodynia, proliferative disorder, rejection of a transplanted organ or tissue, acquired immunodeficiency syndrome (AIDS, also known as HIV), type 1 diabetes, graft - versus - host disease, transplantation, transfusion, anaphylaxis, allergy (e.g., allergy to plant pollen, latex, drugs, food, insect venom, animal hair, animal dander, house dust mites or cockroach cup organs), type I hypersensitivity, allergic conjunctivitis, allergic rhinitis, and atopic dermatitis, asthma, appendicitis, atopic dermatitis, asthma, allergy, blepharitis, bronchiolitis, bronchitis, bursitis, cervicitis, cholangitis, cholecystitis, chronic graft rejection, colitis, conjunctivitis, Crohn's disease, cystitis, dacryoadenitis, dermatitis, dermatomyositis, encephalitis, endocarditis, endometritis, enteritis, enterocolitis, ethmoiditis, epididymitis, fasciitis, fibrositis, gastritis, gastroenteritis, Henoch - Schönlein purpura, hepatitis, hidradenitis suppurativa, immunoglobulin A nephropathy, interstitial lung disease, laryngitis, mastitis, meningitis, osteomyelitis myocarditis, myositis, nephritis, oophoritis, orchitis, osteitis, otitis, pancreatitis, parotitis, pericarditis, peritonitis, pharyngitis, pleuritis, phlebitis, pneumonitis, pneumonia, polymyositis,Rectitis, prostatitis, pyelonephritis, rhinitis, salpingitis, sinusitis, stomatitis, synovitis, tendinitis, tonsillitis, ulcerative colitis, uveitis, vaginitis, vasculitis, or vulvitis, B-cell proliferative disorders, such as diffuse large B-cell lymphoma, follicular lymphoma, chronic lymphocytic leukemia, chronic lymphocytic leukemia, acute lymphocytic leukemia, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma / Waldenström macroglobulinemia, splenic marginal zone lymphoma, multiple myeloma (also known as plasmacytic myeloma), non-Hodgkin lymphoma, Hodgkin lymphoma, plasmacytoma, extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, mantle cell lymphoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, Burkitt lymphoma / leukemia, or lymphomatoid granulomatosis, breast cancer, prostate cancer, or cancer of mast cells (e.g., mastocytoma, mast cell leukemia, mast cell sarcoma, systemic mastocytosis), bone cancer, colorectal cancer, pancreatic cancer, bone and joint diseases including, but not limited to, rheumatoid arthritis, seronegative spondyloarthropathies (including ankylosing spondylitis, psoriatic arthritis and Reiter's syndrome), Behçet's disease, Sjögren's syndrome, systemic sclerosis, osteoporosis, bone cancer, bone metastasis, thromboembolic disorders, (e.g., myocardial infarction, angina pectoris, restenosis after angioplasty, reocclusion after angioplasty, restenosis after coronary artery bypass grafting, reocclusion after coronary artery bypass grafting, stroke, transient ischemia, peripheral arterial occlusive disease, pulmonary embolism, deep vein thrombosis), inflammatory pelvic disease, urethritis, skin sunburn, sinusitis, pneumonitis, encephalitis, meningitis, myocarditis, nephritis, osteomyelitis, myositis, hepatitis, gastritis, enteritis, dermatitis, gingivitis, appendicitis, pancreatitis, cholecystitis, agammaglobulinemia, psoriasis, allergy, Crohn's disease, irritable bowel syndrome, ulcerative colitis, Sjögren's disease, tissue graft rejection, hyperacute rejection of transplanted organs, asthma, allergic rhinitis, chronic obstructive pulmonary disease (COPD), autoimmune polyendocrine diseases (also known as autoimmune polyendocrine syndrome), autoimmune alopecia, pernicious anemia, glomerulonephritis, dermatomyositis, multiple sclerosis, scleroderma, vasculitis, autoimmune hemolytic and thrombocytopenic states, Goodpasture syndrome, atherosclerosis, Addison's disease, Parkinson's disease, Alzheimer's disease, diabetes, septic shock, systemic lupus erythematosus (SLE),Provided is a method selected from rheumatoid arthritis, psoriatic arthritis, juvenile arthritis, osteoarthritis, chronic idiopathic thrombocytopenic purpura, Waldenström's macroglobulinemia, myasthenia gravis, Hashimoto's thyroiditis, atopic dermatitis, degenerative joint disease, vitiligo, autoimmune hypopituitarism, Guillain-Barré syndrome, Behçet's disease, scleroderma, fungating polyps, acute inflammatory responses (such as acute respiratory distress syndrome and ischemia / reperfusion injury), and Graves' disease.

[0327] In another embodiment, the present invention is a method of treating a disease or reducing its severity, comprising administering to a patient in need thereof a compound of formula I and a PI3K inhibitor, wherein the disease is selected from cancer, neurodegenerative disorders, angiogenesis disorders, viral diseases, autoimmune diseases, inflammatory disorders, hormone-related diseases, conditions related to organ transplantation, immunodeficiency disorders, destructive bone disorders, proliferative disorders, infectious diseases, conditions related to cell death, thrombin-induced platelet aggregation, chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), liver diseases, pathological immune states associated with T cell activation, cardiovascular disorders and CNS disorders.

[0328] In another embodiment, the present invention is a method for treating a disease or reducing its severity, comprising administering to a patient in need thereof a compound of formula I and a PI3K inhibitor, wherein the disease is a benign or malignant tumor, brain, kidney (e.g., renal cell carcinoma (RCC)), liver, adrenal gland, bladder, breast, stomach, gastric tumor, ovary, colon, rectum, prostate, pancreas, lung, vagina, endometrium, cervix, testis, urogenital tract, esophagus, larynx, skin, bone or thyroid cancer or solid tumor, sarcoma, glioblastoma, neuroblastoma, multiple myeloma or gastrointestinal cancer, especially colon cancer or colon adenoma or head and neck tumors, epidermal hyperplasia, psoriasis, prostatic hyperplasia, neoplasm, epithelial neoplasm, adenoma, adenocarcinoma, keratoacanthoma, squamous cell carcinoma, large cell carcinoma, non-small cell lung cancer, lymphoma (e.g., including non-Hodgkin lymphoma (NHL) and Hodgkin lymphoma (also referred to as Hodgkin or Hodgkin disease)), breast cancer, follicular cancer, undifferentiated cancer, papillary cancer, seminoma, melanoma or leukemia, wherein the disease is Cowden syndrome, Lhermitte-Duclos disease and Banayan-Zonana syndrome, or a disease in which the PI3K / PKB pathway is abnormally activated, both intrinsic (non-allergic) asthma and extrinsic (allergic) asthma, mild asthma, moderate asthma, severe asthma, bronchial asthma, exercise-induced asthma, occupational asthma and any type or origin of asthma induced after a bacterial infection, acute lung injury (ALI), adult / acute respiratory distress syndrome (ARDS), chronic obstructive pulmonary disease, chronic bronchitis or airway or lung diseases (COPD, COAD or COLD) including the associated dyspnea, emphysema, and exacerbation of airway hypersensitivity as a result of other drug therapies, especially other inhaled drug therapies, any type or origin of bronchitis including but not limited to acute, arachidonic acid, catarrhal, croupous, chronic or tuberculous bronchitis, e.g., aluminosis, anthracosis, asbestosis, chalicosis, madarosis, hemosiderosis, silicosis, tabacosis and byssinosis, any type or origin of pneumoconiosis (a lung inflammatory, generally occupational, disease caused by repeated inhalation of dust, with frequent associated airway obstruction, whether chronic or acute), Loffler's syndrome, eosinophilic, pneumonia, parasitic (especially metazoan) invasion (including tropical eosinophilia), allergic bronchopulmonary aspergillosis,Polyarteritis nodosa (including Churg-Strauss syndrome), eosinophilic granuloma, and eosinophil-related airway disorders caused by drug reactions, psoriasis, contact dermatitis, atopic dermatitis, alopecia areata, erythema multiforma, dermatitis herpetiformis, scleroderma, vitiligo, hypersensitivity vasculitis, urticaria, bullous pemphigoid, dermatitis herpetiformis, pemphigus, acquired epidermolysis bullosa, conjunctivitis, keratoconjunctivitis sicca, and vernal conjunctivitis, nasal diseases including allergic rhinitis, and inflammatory diseases with an autoimmune component or etiology related to an autoimmune reaction or including autoimmune blood diseases (e.g., hemolytic anemia, aplastic anemia, erythroleukemia, and idiopathic thrombocytopenia), systemic lupus erythematosus, rheumatoid arthritis, polychondritis, scleroderma, Wegener's granulomatosis, dermatomyositis, chronic active hepatitis, myasthenia gravis, Stevens-Johnson syndrome, idiopathic sprue, autoimmune inflammatory bowel diseases (e.g., ulcerative colitis and Crohn's disease), endocrine ophthalmopathy, Graves' disease, sarcoidosis, alveolitis, chronic hypersensitivity pneumonitis, multiple sclerosis, primary biliary cirrhosis, uveitis (anterior and posterior), keratoconjunctivitis sicca and vernal catarrh, interstitial pulmonary fibrosis, psoriatic arthritis and glomerulonephritis (including nephrotic syndrome with and without nephrotic syndrome including idiopathic nephrotic syndrome or minimal change nephropathy), restenosis, cardiac hypertrophy, atherosclerosis, myocardial infarction, ischemic stroke and congestive heart failure, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease, and cerebral ischemia, and neurodegenerative diseases caused by trauma, glutamate neurotoxicity, and hypoxia are provided.,

[0329] Compounds and compositions according to the methods of the present invention may be administered in any amount and by any route of administration effective to treat or reduce the severity of cancer, autoimmune disorders, proliferative disorders, inflammatory disorders, neurodegenerative or neuropathic disorders, schizophrenia, bone-related disorders, liver disease or heart disorders. The exact amount required will vary from subject to subject depending on the species, age and general condition of the subject, the severity of the infection, the particular agent, the mode of its administration, etc. The compounds of the present invention are preferably formulated in dosage unit form for ease of administration and uniformity of dosage. The expression "dosage unit form" as used herein refers to physically discrete units of the agent appropriate to the patient to be treated. However, it will be understood that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific effective dosage level for any particular patient or organism will depend on a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound used; the specific composition used; the age, weight, general health, sex and eating habits of the patient; the time of administration, the route of administration, and the excretion rate of the specific compound used; the duration of the treatment; drugs used in combination with or concurrently with the specific compound used, as well as similar factors well known in the medical arts. The term "patient" as used herein means an animal, preferably a mammal, and most preferably a human.

[0330] The pharmaceutically acceptable compositions of the present invention can be administered to humans and other animals orally, rectally, parenterally, intracisternally, vaginally, intraperitoneally, topically (such as in powders, ointments or drops), buccally, as an oral or nasal spray, etc., depending on the severity of the infection being treated. In certain embodiments, the compounds of the present invention may be administered orally or parenterally once or more times a day at dosage levels of from about 0.01 mg / kg to about 50 mg / kg and preferably from about 1 mg / kg to about 25 mg / kg of the subject's body weight per day to achieve the desired therapeutic effect.

[0331] Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active compound, the liquid dosage forms may contain inert diluents commonly used in the art, such as water, or ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3 - butylene glycol, dimethylformamide, oils (especially cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof and other solvents, solubilizers and emulsifiers, etc. In addition to the inert diluent, the oral composition may also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents.

[0332] Injectable preparations, for example, sterile aqueous or oleaginous suspensions, may be formulated according to known techniques using suitable dispersing or wetting agents and suspending agents. Sterile injectable preparations may be sterile injectable solutions, suspensions or emulsions in a non - toxic parenterally acceptable diluent or solvent such as, for example, a solution in 1,3 - butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil containing synthetic mono - or diglycerides can be used. In addition, fatty acids such as oleic acid are used in the preparation of injectable solutions.

[0333] Injectable formulations may be sterilized, for example, by filtration through a bacteria - retaining filter or by incorporating a sterilizing agent in the form of a sterile solid composition that can be dissolved or dispersed in sterile water or other sterile injectable medium before use.

[0334] In order to sustain the effects of the compounds of the present invention, it is often desirable to slow the absorption of the compounds from subcutaneous or intramuscular injections. This can be accomplished by use of a liquid suspension of a crystalline or amorphous material having poor water solubility. The absorption rate of the compound then depends on its dissolution rate, which in turn can depend on crystal size and crystal form. Alternatively, delayed absorption of parenterally administered compound forms is accomplished by dissolving or suspending the compound in an oily vehicle. Depot injection forms are prepared by forming a microencapsulated matrix of the compound in a biodegradable polymer such as polylactide - polyglycolide. The rate of compound release can be controlled depending on the ratio of the compound to the polymer and the nature of the particular polymer used. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injection formulations are also prepared by encapsulating the compound in liposomes or microemulsions that are compatible with living tissue.

[0335] Compositions for rectal or vaginal administration are preferably suppositories prepared by mixing a compound of the present invention with a suitable non - irritating excipient or carrier such as cocoa butter, polyethylene glycol or suppository wax, which is solid at ambient temperature but liquid at body temperature and thus melts within the rectal or vaginal cavity to release the active compound.

[0336] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is combined with at least one inert pharmaceutically acceptable excipient or carrier such as sodium citrate or dibasic calcium phosphate, and / or a) fillers or extenders such as starch, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrants such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarders such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may also include buffering agents.

[0337] Solid compositions of the same type may be used as fillers in soft and hard gelatin capsules using excipients such as lactose or milk sugar and high molecular weight polyethylene glycol. Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared using coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical field. These may optionally contain opacifying agents and may be compositions that release only the active ingredient or preferentially release it in a delayed manner in certain parts of the intestinal tract, if necessary. Examples of embedding compositions that can be used include polymeric substances and waxes. Solid compositions of the same type may be used as fillers in soft and hard gelatin capsules using excipients such as lactose or milk sugar and high molecular weight polyethylene (polethylene) glycol.

[0338] The active compound may be in microencapsulated form, with one or more excipients as noted above. Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared using coatings and shells such as enteric coatings, release control coatings, and other coatings well-known in the pharmaceutical formulation art. In such solid dosage forms, the active compound may be admixed with at least one inert diluent such as sucrose, lactose, or starch. Such dosage forms may, as is normal practice, also contain additional substances other than inert diluents, such as tableting lubricants and other tableting aids such as magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets, and pills, the dosage forms may also contain buffering agents. These may optionally contain opacifying agents and may be compositions that release only the active ingredient or preferentially release it in a delayed manner in certain parts of the intestinal tract, as required. Examples of embedding compositions that can be used include polymeric substances and waxes.

[0339] Dosage forms for topical or transdermal administration of the compounds of the present invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, or patches. The active ingredient is admixed as required under sterile conditions with a pharmaceutically acceptable carrier and any required preservatives or buffer solutions. Ophthalmic formulations, ear drops, and eye drops are also contemplated as being within the scope of the present invention. In addition, the present invention contemplates the use of transdermal patches, which have the additional advantage of providing controlled delivery of the compound into the body. Such dosage forms can be prepared by dissolving or dispersing the compound in a suitable medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled either by providing a rate-controlling membrane or by dispersing the compound in a polymer matrix or gel.

[0340] According to one embodiment, the present invention relates to a method for inhibiting carbonic anhydrase activity in a biological sample, the method comprising contacting the biological sample with a compound of the present invention or a composition comprising the compound.

[0341] According to another embodiment, the present invention relates to a method for inhibiting metalloprotease activity in a biological sample, the method comprising contacting the biological sample with a compound of the present invention or a composition comprising the compound.

[0342] According to another embodiment, the present invention relates to a method for inhibiting integrin activity in a biological sample, the method comprising contacting the biological sample with a compound of the present invention or a composition comprising the compound.

[0343] According to another embodiment, the present invention relates to a method for inhibiting CAIX, MT1-MMP, PSMA, CD38, EphA2 or αvβ3 or mutant activity thereof in a biological sample, the method comprising contacting the biological sample with a compound of the present invention or a composition comprising the compound.

[0344] As used herein, the term "biological sample" includes, but is not limited to, cell cultures or extracts thereof, biopsy materials or extracts thereof obtained from mammals, and blood, saliva, urine, feces, semen, tears or other body fluids or extracts thereof.

[0345] Inhibition of CAIX, MT1-MMP, PSMA, CD38, EphA2 or αvβ3 or mutant activity thereof in a biological sample is useful for a variety of purposes known to those skilled in the art. Examples of such purposes include, but are not limited to, biological assays.

[0346] Another embodiment of the present invention relates to a method of inhibiting carbonic anhydrase, metalloprotease or integrin activity in a patient, comprising administering to the patient a compound of the present invention or a composition comprising said compound.

[0347] According to another embodiment, the present invention relates to a method of inhibiting CAIX, MT1-MMP, PSMA, CD38, EphA2 or αvβ3 or mutant activity thereof in a patient, comprising administering to the patient a compound of the present invention or a composition comprising said compound. In other embodiments, the present invention provides a method for treating a disorder mediated by CAIX, MT1-MMP, PSMA, CD38, EphA2 or αvβ3 or a mutant thereof in a patient in need thereof, comprising administering to the patient a compound according to the present invention or a pharmaceutically acceptable composition thereof. Such disorders are described in detail herein.

[0348] Depending on the particular condition or disease being treated, additional therapeutic agents normally administered to treat that condition may be present in the compositions of the present invention. As used herein, additional therapeutic agents normally administered to treat a particular disease or condition are known as "appropriate for the disease or condition being treated".

[0349] The compounds of the present invention may be advantageously used in combination with other anti-proliferative compounds. Such anti-proliferative compounds include aromatase inhibitors; anti-estrogens; topoisomerase I inhibitors; topoisomerase II inhibitors; microtubule-active compounds; alkylating compounds; histone deacetylase inhibitors; compounds that induce the cell differentiation process; cyclooxygenase inhibitors; MMP inhibitors; mTOR inhibitors; antitumor antimetabolites; platinum compounds; compounds that target / reduce protein or lipid kinase activity and further anti-angiogenic compounds; compounds that target, reduce or inhibit the activity of protein or lipid phosphatases; gonadorelin agonists; anti-androgens; methionine aminopeptidase inhibitors; matrix metalloproteinase inhibitors; bisphosphonates; biological response modifiers; anti-proliferative antibodies; heparanase inhibitors; inhibitors of Ras oncogenic isoforms; telomerase inhibitors; proteasome inhibitors; compounds used in the treatment of hematological malignancies; compounds that target, reduce or inhibit the activity of Flt-3; 17-AAG (17-allylaminogeldanamycin, NSC330507), 17-DMAG (17-dimethylaminoethylamino-17-demethoxy-geldanamycin, NSC707545), IPI-504, CNF1010, CNF2024, CNF1010 and other Hsp90 inhibitors manufactured by Conforma Therapeutics; temozolomide (Temodal®); kinesin spindle protein inhibitors such as SB715992 or SB743921 manufactured by GlaxoSmithKline or pentamidine / chlorpromazine manufactured by CombinatoRx; MEK inhibitors such as ARRY142886 manufactured by Array BioPharma, AZD6244 manufactured by AstraZeneca, PD181461 manufactured by Pfizer, and leucovorin, but are not limited thereto. The term "aromatase inhibitor", as used herein, refers to a compound that inhibits the production of estrogen, for example, the conversion of the substrates androstenedione and testosterone to estrone and estradiol, respectively.The terms include, but are not limited to, steroids, especially atamestane, exemestane and formestane, and, in particular, non-steroids, especially aminoglutethimide, logretimide, pyridoglutethimide, trilostane, testolactone, ketoconazole, vorozole, fadrozole, anastrozole and letrozole. Exemestane is marketed under the trade name Aromasin™. Formestane is marketed under the trade name Lentaron™. Fadrozole is marketed under the trade name Afema™. Anastrozole is marketed under the trade name Arimidex™. Letrozole is marketed under the trade name Femara™ or Femar™. Aminoglutethimide is marketed under the trade name Orimeten™. The combinations of the present invention comprising chemotherapeutic agents that are aromatase inhibitors are particularly useful for the treatment of hormone receptor positive tumors such as breast tumors.

[0350] As used herein, the term "antiestrogenic agent" refers to a compound that antagonizes the effects of estrogen at the estrogen receptor level. The term includes, but is not limited to, tamoxifen, fulvestrant, raloxifene and raloxifene hydrochloride. Tamoxifen is marketed under the trade name Nolvadex™. Raloxifene hydrochloride is marketed under the trade name Evista™. Fulvestrant can be administered under the trade name Faslodex™. The combinations of the present invention comprising chemotherapeutic agents that are antiestrogenic agents are particularly useful for the treatment of estrogen receptor positive tumors such as breast tumors.

[0351] As used herein, the term "antiandrogenic agent" relates to any substance capable of inhibiting the biological effects of male hormones and includes, but is not limited to, bicalutamide (Casodex™). As used herein, the term "gonadorelin agonist" includes, but is not limited to, abarelix, goserelin and goserelin acetate. Goserelin can be administered under the trade name Zoladex™.

[0352] As used herein, the term "topoisomerase I inhibitor" includes, but is not limited to, topotecan, gimatecan, irinotecan, camptothecin and its analogs, 9-nitrocamptothecin, and the macromolecular camptothecin conjugate PNU-166148. Irinotecan can be administered, for example, in the form in which it is commercially available, e.g., under the trademark Camptosar™. Topotecan is commercially available under the trade name Hycamptin™.

[0353] As used herein, the term "topoisomerase II inhibitor" includes, but is not limited to, anthracyclines such as doxorubicin (including liposomal formulations such as Caelyx™), daunorubicin, epirubicin, idarubicin, and nemorubicin, anthraquinones such as mitoxantrone and losoxantrone, and podophyllotoxins such as etoposide and teniposide. Etoposide is commercially available under the trade name Etopophos™. Teniposide is commercially available under the trade name VM26-Bristol. Doxorubicin is commercially available under the trade name Acriblastin™ or Adriamycin™. Epirubicin is commercially available under the trade name Farmorubicin™. Idarubicin is commercially available under the trade name Zavedos™. Mitoxantrone is commercially available under the trade name Novantron.

[0354] The term "microtubule agent" relates to microtubule stabilizers, microtubule destabilizers, and microtubule polymerization inhibitors, including but not limited to taxanes such as paclitaxel and docetaxel; vinca alkaloids such as vinblastine or vinblastine sulfate, vincristine or vincristine sulfate, and vinorelbine; discodermolide; colchicine and epothilone and their derivatives, etc. Paclitaxel is commercially available under the trade name Taxol™. Docetaxel is commercially available under the trade name Taxotere™. Vinblastine sulfate is commercially available under the trade name Vinblastin R.P™. Vincristine sulfate is commercially available under the trade name Farmistin™.

[0355] The term "alkylating agent", as used herein, includes but is not limited to cyclophosphamide, ifosfamide, melphalan or nitrosoureas (BCNU or Gliadel). Cyclophosphamide is commercially available under the trade name Cyclostin™. Ifosfamide is commercially available under the trade name Holoxan™.

[0356] The term "histone deacetylase inhibitor" or "HDAC inhibitor" relates to compounds that inhibit histone deacetylases and possess anti-proliferative activity. This includes but is not limited to suberoylanilide hydroxamic acid (SAHA).

[0357] The term "antineoplastic antimetabolite" includes but is not limited to DNA demethylating compounds such as 5-fluorouracil or 5-FU, capecitabine, gemcitabine, 5-azacitidine and decitabine; folic acid antagonists such as methotrexate and edatrexate, and pemetrexed, etc. Capecitabine is commercially available under the trade name Xeloda™. Gemcitabine is commercially available under the trade name Gemzar™.

[0358] The term "platinum compound", as used herein, includes but is not limited to carboplatin, cisplatin, cisplatinum and oxaliplatin. Carboplatin can be administered, for example, in the form in which it is commercially available, for example under the trademark Carboplat™. Oxaliplatin can be administered, for example, in the form in which it is commercially available, for example under the trademark Eloxatin™.

[0359] The term "compound that targets / reduces protein or lipid kinase activity; or protein or lipid phosphatase activity; or further anti-angiogenic compound", as used herein, refers to protein tyrosine kinase and / or serine and / or threonine kinase inhibitors or lipid kinase inhibitors, e.g., a) compounds that target, reduce or inhibit the activity of platelet-derived growth factor receptor (PDGFR), e.g., compounds that target, reduce or inhibit the activity of PDGFR, especially compounds that inhibit the PDGF receptor such as imatinib, SU101, SU6668 and GFB-111, etc., N-phenyl-2-pyrimidine-amine derivatives, etc.; b) compounds that target, reduce or inhibit the activity of fibroblast growth factor receptor (FGFR); c) compounds that target, reduce or inhibit the activity of insulin-like growth factor receptor I (IGF-IR), e.g., compounds that target, reduce or inhibit the activity of IGF-IR, especially compounds that inhibit the kinase activity of the IGF-I receptor or antibodies that target the extracellular domain of the IGF-I receptor or its growth factor; d) compounds that target, reduce or inhibit the activity of the Trk receptor tyrosine kinase family or ephrin B4 inhibitors; e) compounds that target, reduce or inhibit the activity of the Axl receptor tyrosine kinase family; f) compounds that target, reduce or inhibit the activity of the Ret receptor tyrosine kinase; g) compounds that target, reduce or inhibit the activity of the Kit / SCFR receptor tyrosine kinase such as imatinib, etc.; h) compounds that target, reduce or inhibit the activity of the C-kit receptor tyrosine kinase, which is part of the PDGFR family, e.g., compounds that target, reduce or inhibit the activity of the c-Kit receptor tyrosine kinase family, especially compounds that inhibit the c-Kit receptor such as imatinib, etc.; i) compounds that target, reduce or inhibit the activity of members of the c-Abl family, their gene fusion products (e.g., BCR-Abl kinase) and mutants, e.g., c-Abl family members, and imatinib or nilotinib (AMN107); PD180970;Compounds that target and reduce or inhibit the activity of their gene fusion products such as AG957; NSC680410; PD173955 manufactured by Parke Davis; or dasatinib (BMS-354825), N-phenyl-2-pyrimidine-amine derivatives, etc.; j) Compounds that target and reduce or inhibit the activity of members of the Raf family of protein kinase C (PKC) and serine / threonine kinases, MEK, SRC, JAK / pan-JAK, FAK, PDK1, PKB / Akt, Ras / MAPK, PI3K, SYK, TYK2, BTK and TEC families, and / or members of the cyclin-dependent kinase family (CDK) including staurosporine derivatives such as midostaurin; Further examples of compounds include UCN-01, safingol, BAY43-9006, bryostatin 1, perifosine; ilmofosine; RO318220 and RO320432; GO6976; AISIS 3521; LY333531 / LY379196; isochinoline compounds; FTI; PD184352 or QAN697 (PI3K inhibitor) or AT7519 (CDK inhibitor); k) Compounds that target and reduce or inhibit the activity of protein tyrosine kinase inhibitors, for example, compounds that target and reduce or inhibit the activity of protein tyrosine kinase inhibitors include imatinib mesylate (Gleevec (trademark)), or tyrphostin A23 / RG-50810; AG99; tyrphostin AG213; tyrphostin AG1748; tyrphostin AG490; tyrphostin B44; tyrphostin B44 (+) enantiomer; tyrphostin AG555; AG494; tyrphostin AG556, AG957 and other tyrphostins, and adafostin (4-{[(2,5-dihydroxyphenyl)methyl]amino}-benzoic acid adamantyl ester; NSC680410, adafostin).l) Compounds that target, reduce or inhibit the activity of receptor tyrosine kinases of the epidermal growth factor family (EGFR1, ErbB2, ErbB3, ErbB4 as homo - or heterodimers) and their mutants, for example, compounds that target, reduce or inhibit the activity of the epidermal growth factor receptor family, in particular, compounds that inhibit members of the EGF receptor tyrosine kinase family such as EGF receptor, ErbB2, ErbB3 and ErbB4, or compounds, proteins or antibodies that bind to EGF or EGF - related ligands, CP358774, ZD1839, ZM105180; trastuzumab (Herceptin™), cetuximab (Erbitux™), Iressa, Tarceva, OSI - 774, Cl - 1033, EKB - 569, GW - 2016, E1.1, E2.4, E2.5, E6.2, E6.4, E2.11, E6.3 or E7.6.3 and 7H - pyrrolo - [2,3 - d] pyrimidine derivatives; m) Compounds that target, reduce or inhibit the activity of the c - Met receptor, for example, compounds that target, reduce or inhibit the activity of c - Met, in particular, compounds that inhibit the kinase activity of the c - Met receptor, or antibodies that target the extracellular domain of c - Met or bind to HGF; n) Compounds that target, reduce or inhibit the kinase activity of one or more JAK family members (JAK1 / JAK2 / JAK3 / TYK2 and / or pan - JAK), including but not limited to PRT - 062070, SB - 1578, baricitinib, pacritinib, momelotinib, VX - 509, AZD - 1480, TG - 101348, tofacitinib and ruxolitinib; o) Compounds that target, reduce or inhibit the kinase activity of PI3 kinase (PI3K), including but not limited to ATU - 027, SF - 1126, DS - 7423, PBI - 05204, GSK - 2126458, ZSTK - 474, buparlisib, pictilisib, PF - 4691502, BYL - 719, dactolisib, XL - 147, XL - 765 and idelalisib.and compounds that target, reduce or inhibit the signaling effects of the Hedgehog protein (Hh) or smoothened receptor (SMO) pathway, including but not limited to cyclopamine, vismodegib, itraconazole, erismodegib and IPI-926 (salidegib).;

[0360] As used herein, the term "PI3K inhibitor" includes, but is not limited to, compounds having inhibitory activity against one or more enzymes within the phosphatidylinositol-3-kinase family, including PI3Kα, PI3Kγ, PI3Kδ, PI3Kβ, PI3K-C2α, PI3K-C2β, PI3K-C2γ, Vps34, p110-α, p110-β, p110-γ, p110-δ, p85-α, p85-β, p55-γ, p150, p101 and p87. Examples of PI3K inhibitors useful in the present invention include, but are not limited to, ATU-027, SF-1126, DS-7423, PBI-05204, GSK-2126458, ZSTK-474, buparlisib, pictilisib, PF-4691502, BYL-719, dactolisib, XL-147, XL-765 and idelalisib.

[0361] As used herein, the term "BTK inhibitor" includes, but is not limited to, compounds having inhibitory activity against Bruton's tyrosine kinase (BTK), including but not limited to AVL-292 and ibrutinib.

[0362] As used herein, the term "SYK inhibitor" includes, but is not limited to, compounds having inhibitory activity against spleen tyrosine kinase (SYK), including but not limited to PRT-062070, R-343, R-333, Excellair, PRT-062607 and fostamatinib.

[0363] Further examples of conditions treatable by BTK inhibiting compounds and such compounds in combination with the compounds of the present invention can be found in WO2008039218 and WO2011090760, which are hereby incorporated by reference in their entirety.

[0364] Further examples of conditions treatable by SYK inhibiting compounds and such compounds in combination with the compounds of the present invention can be found in WO2003063794, WO2005007623 and WO2006078846, which are hereby incorporated by reference in their entirety.

[0365] Further examples of conditions treatable by PI3K inhibiting compounds and such compounds in combination with the compounds of the present invention can be found in WO2004019973, WO2004089925, WO2007016176, US8138347, WO2002088112, WO2007084786, WO2007129161, WO2006122806, WO2005 / 113554 and WO2007044729, which are hereby incorporated by reference in their entirety.

[0366] Further examples of conditions treatable by JAK inhibiting compounds and such compounds in combination with the compounds of the present invention can be found in WO2009114512, WO2008109943, WO2007053452, WO2000142246 and WO2007070514, which are hereby incorporated by reference in their entirety.

[0367] Further anti - angiogenic compounds include those having a different mechanism for their activity, e.g., compounds unrelated to protein inhibition or lipid kinase inhibition, such as thalidomide (Thalomid™) and TNP - 470.

[0368] Examples of proteasome inhibitors useful for use in combination with the compounds of the present invention include, but are not limited to, bortezomib, disulfiram, epigallocatechin-3-gallate (EGCG), salinosporamide A, carfilzomib, ONX-0912, CEP-18770, and MLN9708.

[0369] Compounds that target, decrease, or inhibit the activity of a protein or lipid phosphatase include, for example, inhibitors of phosphatase 1, phosphatase 2A, or CDC25 such as okadaic acid or its derivatives.

[0370] Compounds that induce a cell differentiation process include, but are not limited to, retinoic acid, α-, γ-, or δ-tocopherol, or α-, γ-, or δ-tocotrienol.

[0371] As used herein, the term cyclooxygenase inhibitor includes, but is not limited to, cox-2 inhibitors, 5-alkyl-substituted 2-arylaminophenylacetic acids and derivatives such as celecoxib (Celebrex™), rofecoxib (Vioxx™), etoricoxib, valdecoxib, or 5-alkyl-2-arylaminophenylacetic acids such as 5-methyl-2-(2'-chloro-6'-fluoroanilino)phenylacetic acid, lumiracoxib.

[0372] The term "bisphosphonates" as used herein includes, but is not limited to, etidronic acid, clodronic acid, tiludronic acid, pamidronic acid, alendronic acid, ibandronic acid, risedronic acid, and zoledronic acid. Etidronic acid is commercially available under the trade name Didronel™. Clodronic acid is commercially available under the trade name Bonefos™. Tiludronic acid is commercially available under the trade name Skelid™. Pamidronic acid is commercially available under the trade name Aredia™. Alendronic acid is commercially available under the trade name Fosamax™. Ibandronic acid is commercially available under the trade name Bondranat™. Risedronic acid is commercially available under the trade name Actonel™. Zoledronic acid is commercially available under the trade name Zometa™. The term "mTOR inhibitor" relates to compounds that inhibit the mammalian target of rapamycin (mTOR), such as sirolimus (Rapamune®), everolimus (Certican™), CCI-779, and ABT578, and possess antiproliferative activity.

[0373] The term "heparanase inhibitor" as used herein refers to a compound that targets, decreases, or inhibits heparan sulfate degradation. The term includes, but is not limited to, PI-88. The term "biological response modifier" as used herein refers to a lymphokine or interferon.

[0374] The term "inhibitor of Ras oncogenic isoforms" such as H-Ras, K-Ras, or N-Ras as used herein refers to a compound that targets, decreases, or inhibits the oncogenic activity of Ras, for example, a "farnesyltransferase inhibitor" such as L-744832, DK8G557, or R115777 (Zarnestra™). The term "telomerase inhibitor" as used herein refers to a compound that targets, decreases, or inhibits the activity of telomerase. Compounds that target, decrease, or inhibit the activity of telomerase are, inter alia, compounds that inhibit the telomerase receptor such as telomestatin.

[0375] As used herein, the term "methionine aminopeptidase inhibitor" refers to a compound that targets, reduces, or inhibits the activity of methionine aminopeptidase. Compounds that target, reduce, or inhibit the activity of methionine aminopeptidase include, but are not limited to, bengamides or derivatives thereof.

[0376] As used herein, the term "proteasome inhibitor" refers to a compound that targets, reduces, or inhibits the activity of the proteasome. Compounds that target, reduce, or inhibit the activity of the proteasome include, but are not limited to, bortezomib (Velcade™) and MLN341.

[0377] As used herein, the term "matrix metalloproteinase inhibitor" or ("MMP" inhibitor) includes, but is not limited to, collagen peptide mimetic and non-peptide mimetic inhibitors, tetracycline derivatives, e.g., hydroxamate peptide mimetic inhibitor batimastat and its orally bioavailable analog marimastat (BB-2516), prinomastat (AG3340), metastat (NSC683551) BMS-279251, BAY12-9566, TAA211, MMI270B or AAJ996.

[0378] As used herein, the term "compound used in the treatment of hematological malignancies" refers to an FMS-like tyrosine kinase receptor (Flt-3R) inhibitor, which is a compound that targets, reduces, or inhibits the activity of FMS-like tyrosine kinase receptor; interferon, 1-β-D-arabinofuranosylcytosine (ara-c) and busulfan; and an ALK inhibitor, which is a compound that targets, reduces, or inhibits the activity of anaplastic lymphoma kinase, including, but not limited to, these.

[0379] Compounds that target, reduce or inhibit the activity of FMS-like tyrosine kinase receptor (Flt-3R) are, inter alia, compounds, proteins or antibodies that inhibit members of the Flt-3R receptor kinase family such as PKC412, midostaurin, staurosporine derivatives, SU11248 and MLN518.

[0380] As used herein, the term "HSP90 inhibitor" targets, reduces or inhibits the endogenous ATPase activity of HSP90; and includes, but is not limited to, compounds that target, reduce or inhibit the degradation of HSP90 client proteins via the ubiquitin proteasome pathway. Compounds that target, reduce or inhibit the endogenous ATPase activity of HSP90 are, inter alia, 17-allylamino,17-demethoxygeldanamycin (17AAG), geldanamycin derivatives; other geldanamycin-related compounds; compounds, proteins or antibodies that inhibit the ATPase activity of HSP90 such as radicicol and HDAC inhibitors.

[0381] As used herein, the term "antiproliferative antibody" includes, but is not limited to, trastuzumab (Herceptin™), trastuzumab-DM1, arcitumomab, bevacizumab (Avastin™), rituximab (Rituxan®), PRO64553 (anti-CD40) and 2C4 antibody. By antibody is meant intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies formed from at least two intact antibodies, and antibody fragments, so long as they exhibit the desired biological activity.

[0382] For the treatment of acute myeloid leukemia (AML), the compounds of the present invention can be used in combination with standard leukemia therapies, and in particular, in combination with therapies used for the treatment of AML. In particular, the compounds of the present invention can be administered in combination with, for example, farnesyltransferase inhibitors and / or other drugs useful for the treatment of AML such as daunorubicin, adriamycin, Ara-C, VP-16, teniposide, mitoxantrone, idarubicin, carboplatinum and PKC412.

[0383] Other anti-leukemia compounds include, for example, pyrimidine analogs such as Ara-C, a 2'-alpha-hydroxyribose (arabinoside) derivative of deoxycytidine. Also included are purine analogs of hypoxanthine, 6-mercaptopurine (6-MP) and fludarabine phosphate. Compounds that target, reduce or inhibit the activity of histone deacetylase (HDAC) inhibitors such as sodium butyrate and suberoylanilide hydroxamic acid (SAHA) inhibit the activity of enzymes known as histone deacetylases. Specific HDAC inhibitors include MS275, SAHA, FK228 (formerly FR901228), trichostatin A, and N-hydroxy-3-[4-[[[2-(2-methyl-1H-indol-3-yl)-ethyl]-amino]methyl]phenyl]-2E-2-propenamide or a pharmaceutically acceptable salt thereof and N-hydroxy-3-[4-[(2-hydroxyethyl){2-(1H-indol-3-yl)ethyl]-amino]methyl]phenyl]-2E-2-propenamide or a pharmaceutically acceptable salt thereof, especially the lactate, and include compounds disclosed in US 6,552,065. Somatostatin receptor antagonists, as used herein, refer to compounds that target, treat or inhibit somatostatin receptors such as octreotide and SOM230. The tumor cell damage approach refers to approaches such as ionizing radiation. The term "ionizing radiation" as referred to above and hereinafter means ionizing radiation that appears either as electromagnetic rays (such as X-rays and gamma rays) or particles (such as alpha and beta particles). Ionizing radiation is provided in, but not limited to, radiation therapy and is known in the art. See Hellman, Principles of Radiation Therapy, Cancer, in Principles and Practice of Oncology, Devita et al., Eds., 4 th See Edition, Vol. 1, pp. 248-275 (1993).

[0384] Also included are EDG binders and ribonucleotide reductase inhibitors. As used herein, the term "EDG binder" refers to a class of immunosuppressive agents that modulate lymphocyte recirculation, such as FTY720. The term "ribonucleotide reductase inhibitor" refers to pyrimidine or purine nucleoside analogs, including but not limited to fludarabine and / or cytosine arabinoside (ara-C), 6-thioguanine, 5-fluorouracil, cladribine, 6-mercaptopurine (especially in combination with ara-C for ALL), and / or pentostatin. Ribonucleotide reductase inhibitors are, inter alia, hydroxyurea or 2-hydroxy-1H-isoindole-1,3-dione derivatives.

[0385] In particular, 1-(4-chloroanilino)-4-(4-pyridylmethyl)phthalazine or a pharmaceutically acceptable salt thereof, 1-(4-chloroanilino)-4-(4-pyridylmethyl) succinate phthalazine; Angiostatin (trademark); Endostatin (trademark); anthranilic acid amide; ZD4190; ZD6474; SU5416; SU6668; bevacizumab; or anti-VEGF antibodies or anti-VEGF receptor antibodies such as rhuMAb and RHUFab, VEGF aptamers such as Macugon; compounds, proteins or monoclonal antibodies against VEGF such as FLT-4 inhibitors, FLT-3 inhibitors, VEGFR-2 IgGI antibodies, angiostatin (RPI4610) and bevacizumab (Avastin (trademark)) are also included.

[0386] As used herein, photodynamic therapy refers to a therapy for treating or preventing cancer using certain chemicals known as photosensitizing compounds. Examples of photodynamic therapy include treatment with compounds such as Visudyne (trademark) and porfimer sodium.

[0387] Angiogenesis-inhibiting steroids, as used herein, refer to compounds that block or inhibit angiogenesis, such as anecortave, triamcinolone, hydrocortisone, 11-α-epihydrocortisol, cortisone, 17α-hydroxyprogesterone, corticosterone, deoxycorticosterone, testosterone, estrone, and dexamethasone.

[0388] Implants containing corticosteroids refer to compounds such as fluocinolone and dexamethasone.

[0389] Other chemotherapeutic compounds include, but are not limited to, plant alkaloids, hormone compounds and antagonists; biological response modifiers, preferably lymphokines or interferons; antisense oligonucleotides or oligonucleotide derivatives; shRNA or siRNA; or various miscellaneous compounds or compounds with other or unknown mechanisms of action.

[0390] The structures of the active compounds identified by code number, common name, or trade name can be obtained from the current edition of the standard compendium "The Merck Index", or from databases such as international patents (e.g., IMS World Publications).

[0391] The compounds of the present invention may be used in combination with known therapeutic processes, such as the administration of hormones or radiation. In certain embodiments, the provided compounds may be used as radiosensitizers, particularly for the treatment of tumors that exhibit poor sensitivity to radiotherapy.

[0392] The compounds of the present invention can be administered alone or in combination with one or more other therapeutic compounds. Possible combination therapies can take the form of a fixed combination, or the administration of the compounds of the present invention and one or more other therapeutic compounds can be alternating, or given independently of each other, or a combination of a fixed combination and one or more other therapeutic compounds. The compounds of the present invention can be administered, inter alia, for tumor therapy, in combination with chemotherapy, radiotherapy, immunotherapy, phototherapy, surgical intervention or combinations thereof. Long-term therapy is possible, as described above, in the context of other treatment strategies, as well as adjuvant therapy. Other possible treatments are therapies for maintaining the condition of patients after tumor regression, or even chemoprevention, for example, in at-risk patients.

[0393] Those additional active substances may be administered separately from the composition containing the compound of the present invention as part of a multiple dosing regimen. Alternatively, those active substances may be part of a single dosage form mixed together with the compound of the present invention in a single composition. When administered as part of a multiple dosing regimen, the two active agents may be provided simultaneously, sequentially, or within a certain period of time relative to each other, usually within 5 hours of each other.

[0394] As used herein, the terms "combination", "combined" and related terms refer to the simultaneous or sequential administration of therapeutic agents according to the present invention. For example, the compounds of the present invention may be administered with another therapeutic agent, either simultaneously or sequentially in separate unit dosage forms or together in a single unit dosage form. Accordingly, the present invention provides a single unit dosage form comprising a compound of the present invention, an additional therapeutic agent, and a pharmaceutically acceptable carrier, adjuvant or vehicle.

[0395] The amounts of both the compounds of the present invention and the additional therapeutic agents (in compositions containing additional therapeutic agents as described above) that can be combined with a carrier material to produce a single dosage form will vary depending on the host being treated and the particular mode of administration. Preferably, the compositions of the present invention should be formulated such that the compound of the present invention can be administered at a dosage between 0.01 and 100 mg / kg body weight / day.

[0396] In compositions containing an additional therapeutic agent, the additional therapeutic agent and the compound of the present invention can act synergistically. Thus, the amount of the additional therapeutic agent in such compositions will be less than the amount required in a monotherapy utilizing only that therapeutic agent. In such compositions, an additional therapeutic agent can be administered at a dosage between 0.01 and 1,000 μg / kg body weight / day.

[0397] The amount of the additional therapeutic agent present in the compositions of the present invention will not exceed the amount that would normally be administered in a composition containing that therapeutic agent as the sole active agent. Preferably, the amount of the additional therapeutic agent in the presently disclosed compositions will range from about 50% to 100% of the amount normally present in a composition containing that agent as the sole therapeutic active agent.

Examples

[0398] [[ID=I4]] Illustration As shown in the following examples, in certain exemplary embodiments, the compounds are prepared according to the following general procedure. The general method shows the synthesis of certain specific compounds of the present invention, but it will be understood that the following general method and other methods known to those skilled in the art can be applied to all compounds and their respective subclasses and species as described herein.

[0399] Materials and Methods (Example 1) Peptide Synthesis - Solid Phase Reagents with Leaving Groups Peptide synthesis was based on Fmoc chemistry using a Symphony peptide synthesizer manufactured by Peptide Instruments and a Syro II synthesizer by MultiSynTech. Standard Fmoc amino acids were used (Sigma, Merck) with appropriate side-chain protecting groups, and standard coupling conditions applicable in each case were used, followed by deprotection using standard methodology. Peptides were purified using HPLC and, after isolation, were modified with a molecular scaffold reagent bearing a leaving group. For this, the linear peptides were diluted to approximately 35 mL maximum with H2O, 100 mM molecular scaffold reagent in approximately 500 μL of acetonitrile was added, and the reaction was initiated with 1 M NH4HCO3 in 5 mL of H2O. The reaction was allowed to proceed at room temperature for approximately 30 - 60 minutes and, once the reaction was complete (as judged by MALDI), was lyophilized. After lyophilization, the reaction mixture was loaded onto a Gemini C18 column (Phenomenex). The solvents (H2O, acetonitrile) were acidified with 0.1% trifluoroacetic acid. The gradient was in the range of 30 - 70% acetonitrile over 15 minutes at a flow rate of 15 - 20 mL / min using a Gilson preparative HPLC system. The pure fractions containing the desired product were pooled, lyophilized, and stored at -20 °C for storage.

[0400] (Example 2) Peptide synthesis - Molecular scaffold reagents containing Michael acceptors Alternatively, peptides were purified using HPLC and, after isolation, were modified with a molecular scaffold reagent containing a Michael acceptor. For this, the linear peptides were diluted to approximately 35 mL maximum with 50:50 MeCN:H2O, 100 mM molecular scaffold reagent containing a Michael acceptor in approximately 500 μL of acetonitrile was added, and the reaction was initiated with 1 M NH4HCO3 in 5 mL of H2O. The reaction was allowed to proceed at room temperature for approximately 30 - 60 minutes and, once the reaction was complete (as judged by MALDI), was lyophilized. Once complete, 1 M L-cysteine hydrochloride monohydrate (Sigma) in 1 mL of H2O was added to the reaction at room temperature over approximately 60 minutes to quench any excess molecular scaffold reagent containing a Michael acceptor.

[0401] After lyophilization, while purifying the modified peptide as described above, Luna C8 was replaced with a Gemini C18 column (Phenomenex), and the acid was changed to 0.1% trifluoroacetic acid. The pure fractions containing the correct desired product were pooled, lyophilized, and kept at -20 °C for storage.

[0402] All amino acids were used in the L-configuration unless otherwise noted.

[0403] (Example 3) Conjugation of the bicyclic peptide with DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) is a chelating agent that can be conjugated to a bicyclic to obtain constructs that are used in biodistribution studies in animal models after being labeled with a radioisotope (i.e., Lu 3+ ) or an MRI contrast agent (i.e., Gd 3+ ). The commercially available N-succinimidyl ester of DOTA is coupled to the amino group of the bicyclic peptide, generally the N-terminal amino group. [Table 4]

[0404] Materials and methods Equipment LCMS

[0405] MALDI mass spectrometer Solvents and compounds and consumables

[0406] Dry dimethyl sulfoxide (DMSO)

[0407] DOTA N-succinimidyl ester

[0408] N,N-Diisopropylethylamine (DIPEA)

[0409] 100 mM Tris(hydroxymethyl)aminomethane (TRIS), pH 9

[0410] Complete procedure Prepare a 20 mM solution of the bicyclic peptide in dry DMSO.

[0411] Prepare a 100 mM solution of DOTA N-succinimidyl ester in dry DMSO. Since the N-succinimidyl ester is moisture-sensitive, keep it under nitrogen in a desiccator in a -20 °C freezer.

[0412] Add the DOTA N-succinimidyl ester solution to the peptide solution in a 5-fold excess over the peptide. Mix the resulting reaction mixture well and sediment any water droplets on the walls.

[0413] Add undiluted DIPEA to the mixture in a 20-fold excess over the peptide. Mix the resulting solution well and sediment any water droplets on the walls. Thus, the approximate initial concentrations of the reactants are Bicyclic peptide 15 mM DOTA N-succinimidyl ester 19 mM DIPEA 300 mM are as follows.

[0414] Stir or shake the reaction mixture at room temperature and monitor the progress of the reaction using LC / MS or MALDI-TOF.

[0415] Once the reaction is complete, quench the mixture with 100 mM TRIS pH 9, dilute to 10 mL with 6 M guanidine hydrochloride, and purify the mixture by RP-HPLC.

[0416] (Example 4) Conjugation of the bicyclic peptide with 5(6)-carboxyfluorescein The commercially available N-succinimidyl ester of 5(6)-carboxyfluorescein (CAS number: 92557-80-7) is coupled to the amino group of the bicyclic peptide, generally the N-terminal amino group.

[0417] Materials and Methods

[0418] Equipment MALDI mass spectrometer. Procedure Prepare a 20 mM solution of the bicyclic peptide in dry DMSO in an Eppendorf tube. Prepare a 100 mM solution of 5(6)-carboxyfluorescein N-succinimidyl ester in dry DMSO in an Eppendorf tube. Since the N-succinimidyl ester is moisture-sensitive, keep it under nitrogen in a desiccator in a -20 °C freezer. Since 5(6)-carboxyfluorescein is photosensitive, handle it and the peptide labeled with it in amber glass / plastic products and keep light exposure as low as possible. Add the fluorescent dye solution to the peptide solution such that it has 5-fold excess dye over the peptide. Mix the resulting reaction mixture well and sediment any water droplets on the walls. Add undiluted DIPEA to the mixture such that it has 20-fold excess DIPEA over the peptide. Mix the resulting solution well and sediment any water droplets on the walls. Thus, the approximate initial concentrations of the reactants are bicyclic peptide (10 mM), fluorescent dye N-succinimidyl ester (50 mM), and DIPEA (200 mM). Stir or shake the reaction mixture at room temperature and use MALDI-TOF to follow the progress of the reaction. Once the reaction is complete, quench the mixture with 100 mM TRIS pH 9, transfer the mixture to a 50 mL Falcon tube, dilute it to 10 mL with 6 M guanidine hydrochloride, and purify the mixture by RP-HPLC.

[0419] (Example 5) Conjugation of bicyclic peptides with Alexa Fluor® fluorescent dyes Alexa Fluor® dyes are a family of fluorescent dyes produced by Molecular Probes, Inc. These are commercially available as N-succinimidyl esters and can be coupled to the amino groups of bicyclic peptides, generally the N-terminal amino group.

[0420] The Alexa Fluor® N-succinimidyl esters used were as follows: Alexa Fluor 488 N-succinimidyl ester and Alexa Fluor 680 N-succinimidyl ester.

[0421] Materials and Methods Equipment MALDI mass spectrometer. Procedure

[0422] Prepare a 20 mM solution of the bicyclic peptide in dry DMSO in an Eppendorf tube. Prepare a 15 mM solution of Alexa Fluor® N-succinimidyl ester in dry DMSO in an Eppendorf tube. Since the N-succinimidyl ester is moisture-sensitive, keep it under nitrogen in a desiccator in a -20 °C freezer. Since the Alexa Fluor® dyes are photosensitive, handle them and the peptides labeled with them in amber glass / plastic products and keep light exposure as low as possible. Add the fluorescent dye solution to the peptide solution such that the dye is 2.5-fold in excess of the peptide. Mix the resulting reaction mixture well and sediment any water droplets on the walls. Add undiluted DIPEA to the mixture such that the DIPEA is 20-fold in excess of the peptide. Mix the resulting solution well and sediment any water droplets on the walls. Thus, the approximate initial concentrations of the reactants are bicyclic peptide (5 mM), fluorescent dye N-succinimidyl ester (12.5 mM), and DIPEA (100 mM). Stir or shake the reaction mixture at room temperature and use MALDI-TOF to track the progress of the reaction. When the reaction is complete, quench the mixture with 100 mM TRIS pH 9, transfer the mixture to a 50 mL Falcon tube, dilute it to 10 mL with 6 M guanidine hydrochloride, and purify the mixture by RP-HPLC.

[0423] (Example 6) CAIX assay The CAIX competitive binding assay affinity (Ki) of the peptides of the present invention for human CAIX was determined using a competitive fluorescence polarization assay similar to that described in Dubois et al. (2011) Radiotherapy and Oncology 99(3), 424-43, using A-(CAECWIDGWVPC)-A-Sar6-K(Fl) (SEQ ID NO: 7) as the fluorescent ligand.

[0424] (Example 7) Determination of the dissociation rate constant of the bicyclic binder with MT1-MMP Direct binding fluorescence polarization (anisotropy) assay Direct binding fluorescence polarization or anisotropy assays are performed by titrating a fixed concentration of a fluorescent tracer (here, the fluorescent bicyclic peptide under study) with its binding partner (here, the MT1-MMP hemopexin domain). As the concentration of the binding partner increases during the titration, the polarization signal changes in proportion to the ratio of bound and unbound material. This enables the determination of the dissociation rate (K d ). The assay data can be fitted using standard ligand binding equations.

[0425] Typically, the concentration of the tracer is far below the K d of the tracer:titrant pair ideally, and the concentration selected is usually about 1 nM or less. The titrant (binding partner) concentration varies from 0.1 nM to typically up to 5 μM maximum. The range is selected such that the maximum change in fluorescence polarization can be observed. The buffer used is phosphate buffered saline in the presence of 0.01% Tween. The experiments are performed in black 384 well low binding / low volume plates (Corning 3820), and the fluorescence polarization signal is measured using a BMG Ferastaar FS plate reader. The fluorescent tracer referred to in the text is a bicyclic peptide fluorescently labeled using 5,6-carboxyfluorescein. Fluoresceination may be performed on the N-terminal amino group of the peptide, which is separated from the bicyclic core sequence by a sarcosine spacer (usually Sar10). This can be done during or after Fmoc solid phase synthesis (after cyclization and purification with a molecular scaffold reagent) if the N-terminal amino group is unique to the peptide. Fluoresceination may also be performed on the C-terminal, usually on a lysine introduced as the first C-terminal residue, which is then separated from the bicyclic core sequence by a sarcosine spacer (usually Sar6). Thus, the N-terminal tracer can have a molecular format described as Fluo-Ala-Sar10-A (bicyclic core sequence) and (bicyclic core sequence)-A-Sar6-K(Fluo) for constructs with a C-terminal fluorescently labeled.

[0426] The fluorescent tracers used in the examples were A-(17-69)-A-Sar6-K(Fluo), A-(17-69-07)-A-Sar6-K(Fluo), and A-(17-69-12)-A-Sar6-K(Fluo). Due to the acidic nature of the 17-69 fluorescent peptides, they were typically prepared as concentrated DMSO stocks from which dilutions were made in 100 mM Tris pH 8 buffer.

[0427] (Example 8) Competitive assay using fluorescence polarization (anisotropy) Due to their high affinity for the MT1-MMP hemopexin domain (PEX), the fluorescent derivatives of 17-69-07 and 17-69-12 (designated as 17-69-07-N040 and 17-69-12-N005, respectively) can be used in competition experiments (using FP for detection). Here, a pre-formed complex of PEX with the fluorescent PEX-binding tracer is titrated with the free non-fluorescent bicyclic peptide. Since all 17-69-based peptides are expected to bind at the same site, the titrant will displace the fluorescent tracer from the PEX. The dissociation of the complex can be measured quantitatively, and the K d for the competitor (titrant) against the target protein can be determined. The advantage of the competition method is that the affinity of the non-fluorescent bicyclic peptide can be determined accurately and rapidly.

[0428] The concentration of the tracer is usually K d or less (here, 1 nM), and the binding protein (here, the hemopexin of MT1-MMP) is present at a 15-fold excess such that more than 90% of the tracer binds. Subsequently, the non-fluorescent competitor bicyclic peptide (usually just the bicyclic core sequence) is titrated to displace the fluorescent tracer from the target protein. The replacement of the tracer is measured and correlated with the decrease in fluorescence polarization. The decrease in fluorescence polarization is proportional to the ratio of the target protein bound to the non-fluorescent titrant and is thus a measure of the affinity of the titrant for the target protein.

[0429] The raw data is fitted to an analytical solution of a cubic equation that describes the equilibrium among the fluorescent tracer, the titrant, and the binding protein. The fitting requires a value for the affinity of the fluorescent tracer for the target protein, which can be determined separately by direct binding FP experiments (see previous section). Curve fitting was performed using SigmaPlot 12.0 and an adapted version of the equation described by Zhi-Xin Wang (FEBS Letters 360 (1995) 111-114).

[0430] (Example 9) Integrin αvβ3 competitive binding assay The affinity (Ki) of the peptides of the invention for integrin αvβ3 was determined using a competitive fluorescence polarization assay similar to that described by Wang et al (2005) Bioconjug Chem 16(3), 729-34, using a 5 nM peptide with the sequence: FITC-LC-GRGDSP (SEQ ID NO: 8) as the ligand.

[0431] (Example 10) I-25 (palmitoylated MT1-MMP specific bicyclic peptide) organ distribution and dosing study General procedure for radiolabeling with Lu-177

[0432] Labeling reaction reactants:

[0433] 50 μL of 0.25 M Na acetate buffer pH 5.2 + 0.1% Tween 20;

[0434] 10 μL of [Lu-177]LuCl3 in 0.05 M HCl; and

[0435] 2 μL of diluted peptide (2 nanomoles).

[0436] The reaction mixture was incubated at 98 °C for 2, 10, 30, 45 minutes and 5 hours. Heating was stopped at each time point and 5 μL of the labeled reaction was diluted in 100 μL of injection buffer (0.25 M Na acetate pH 7 + 0.05% Tween 20) and then analyzed by radio-HPLC. TLC was carried out in parallel to confirm the results of radio-HPLC.

[0437] Organ distribution study, general method: For organ distribution, each dose is prepared by dilution in the injection buffer (0.25 M Na acetate pH 7 + 0.05% Tween 20) of the labeled reaction mixture. 177 The Lu-radiolabeled peptide was injected via the tail vein of nu / nu mice (50 kBq per mouse) transplanted with each cell line. At the indicated time points after injection, the animals were sacrificed. Blood, heart, lung, spleen, liver, kidney, muscle, brain, femur and tumor were excised, bottle dried and weighed. Radioactivity was measured with a γ-counter (Cobra II; Canberra Packard) together with a sample of the injection solution to calculate the percentage of injected dose per gram of tissue (%ID / g). Exemplary procedure for radiolabeling with Lu-177

[0438] 6.7 days t 1 / 2 with γ- and β-emitters 177 LuCl3 (NEZ307D, Perkin Elmer, in 0.04 M HCl) was used for both in vivo biodistribution and internalization studies. The labeling reaction was in 10 μL of 50 mM HCl 177LuCl3 (approximately 10 MBq), 2 μL of diluted peptide (1 mM solution in 50 mM HEPES buffer pH 7 + 0.1% Tween 20 diluted from a 10 mM DMSO stock) was added to 50 μL of 50 mM HEPES buffer pH 7 supplemented with 0.1% Tween 20. The reaction mixture was incubated at 98 °C for 10 minutes. Radiochemical yield (RCY) was determined using a high-performance liquid chromatography (HPLC) system equipped with a Latek P402 (Latek, Eppelheim, Germany), a HITACHI variable UV detector (absorbance measured at 214 and 254 nm), and a gamma detector (Bioscan, WA, USA), using a Chromolith RP-18 column (100 × 4.6 mm; Merck). Analytical HPLC runs were performed at 4 mL / min using a linear gradient from A (0.1% trifluoroacetic acid (TFA) in water) to B (0.1% TFA in acetonitrile) (0% B to 100% B in 6 minutes). Thin-layer chromatography was performed in parallel to confirm the results of radio-HPLC. The RCY generally exceeded 95%, and thus the product of the labeling reaction was directly diluted with the appropriate buffer and additional unlabeled peptide if required, and used in subsequent experiments.

[0439] Exemplary procedure for organ distribution studies 5 × 10 of HT1080 6 cells were subcutaneously inoculated into the right flank of male 6-week-old BALB / c nu / nu mice (Charles River Laboratories). Tumors were grown for approximately 1 week until a maximum size of about 200 mm 3 was reached. Each dose was a fixed volume of 5 picomoles 177The Lu-labeled peptide (approximately 50 kBq) was prepared by mixing it with different amounts of unlabeled peptide (derived from a 1 mM stock solution in 0.25 M sodium acetate pH 7 + 0.05% Tween 20) in 100 μL of injection buffer (0.25 M sodium acetate pH 7 + 0.05% Tween 20). Three animals per dose and time point were used. The radiolabeled peptide solution was injected via the tail vein and the animals were subsequently sacrificed at various time points. The organs of interest (blood, heart, lung, spleen, liver, kidney, muscle, small intestine, brain, HT1080 tumor) were excised, bottle-dried and weighed. Radioactivity was measured using a gamma counter and calculated as %ID / g.

[0440] Results: Lipidation of biomolecules is known to increase serum half-life via serum albumin binding in circulation (Di, L. (2015) Strategic approaches to optimizing peptide ADME properties. AAPS J 17, 134-43.), and potential modifications were provided to the MT1 bicyclic peptide I-23 to further increase tumor-specific signals in vivo through prolonged exposure. The palmitoylated bicyclic peptide derivative I-25 was generated based on stabilized I-23, where its N-terminus was modified with an extended sarcosine oligomer spacer terminated with palmitoic acid, and lysine modified with DOTA was placed within the sarcosine spacer. Oligosarcosine was selected as a spacer due to its extended non-globular structure, high water solubility, and ease of synthesis (Teufel, D. P., Johnson, C. M., Lum, J. K., and Neuweiler, H. (2011) Backbone-driven collapse in unfolded protein chains. J Mol Biol 409, 250-62). To ensure that the bicyclic peptide and its MT1-MMP binding activity were spatially well separated from the albumin-binding palmitic acid, the total spacer length was set to 15 sarcosines. The molecular design should, in principle, enable the formation of a ternary complex among albumin, the fatty acid-modified radiolabeled bicyclic peptide, and MT1-MMP. Indeed, despite the extended modification on the N-terminus of the peptide, I-25 retained its affinity for MT1-MMP (Kd = 2.25 ± 0.5 nM).

[0441] By administering a range of dose levels (10, 100, 750, 1000, 5000 picomoles / mouse; Figure 1), the 48-hour time point post-injection was selected to assess the optimal dose for high tumor-to-organ contrast in order to 177A biodistribution study using Lu-labeled I-25 was conducted. A significant tumor signal of approximately 26% ID / g was observed at 750 - 1000 picomoles / mouse. Less than 4.5% additional signal was observed in the spleen, liver, and small intestine.

[0442] A dose of 750 pmol of I-25 was found to be optimal. As shown in Figure 1, when using 750 picomoles as the injection dose, the background activity was very low in all organs except the kidney, while the tumor accumulation in the HT1080 xenograft was extremely high (24.1 ± 2.5% ID / g) even at 48 hours after injection.

[0443] (Example 11) Organ distribution and dose study of I-23 (a bicyclic peptide specifically proteolytically stabilized against MT1-MMP) Non-PA and stabilized derivative I-23 was produced to increase the resistance to proteolysis in vivo. The I-23 sequence differs from the 17-69-07-N144 sequence in that the I-23 sequence contains four modifications: D-Ala1, 1NAl4, D-Ala5, and tBuGly11. Variable amounts of I-23 and a fixed amount of 177 Lu (approximately 50 kBq / mouse) was used to evaluate various doses in a biodistribution study using HT1080 xenograft mice. The biodistribution was performed 1 hour after injection.

[0444] As shown in Figure 2, a dose of 50 to 150 pmol / mouse was found to be optimal. Compared to the molecule 17-69-07-N144, all doses of the stabilized I-23 showed significantly higher tumor uptake, as shown in Figures 3A and 3B. The tumor uptake was slightly blocked at a dose of 500 picomoles and almost completely blocked using 5 nanomoles of I-23.

[0445] The results demonstrate that protein stabilization of the molecule leads to an improvement in the tumor signal for I-23 compared to 17-69-07-N144, making the molecule a suitable probe for molecular imaging (12.4% ID / g at the 1-hour time point for I-23, Figure 3).

[0446] (Example 12) I-24 Organ Distribution and Dosage Study I-24 contains I-23 and the Sar10 spacer. Variable amounts of I-24 and a fixed amount of 177 Lu (approximately 50 kBq / mouse) were used to evaluate various dosages in an in vivo distribution study using HT1080 xenograft mice. The in vivo distribution was performed 1 hour after injection.

[0447] A dosage of 150 pmol was found to be optimal. The signal in the tumor was slightly lower than that of I-23 but significantly greater than that of the non-stabilized 17-69-07-N144 (Figure 4). Tumor uptake was slightly blocked at a dosage of 500 picomol and almost completely blocked using 5 nanomol of I-24.

[0448] The results of these studies are shown in Figures 5 and 6.

[0449] (Example 13) Specificity of Tumor Uptake of Stabilized Non-PA Variants I-27 represents the MT1-MMP inactive variant of I-24. The organ distribution of I-27 showed that tumor uptake was clearly reduced to one twenty-fifth, from 7.04 ± 2.83% ID / g tumor to 0.28 ± 0.05% ID / g tumor, as shown in Figure 7.

[0450] In addition, two different inactive variants of I-23, I-26, and 17-69-07-N246 without a spacer were characterized in terms of organ distribution. Again, in both cases, tumor uptake was shown to be essentially absent compared to the binding variant I-23, as shown in Figure 8.

[0451] (Example 14) Organ distribution of I-23 at different time points after injection The distribution of I-23 molecules at different time points was performed. Tumor localization was shown to decrease significantly at 1 hour after injection. As shown in Figure 9, at 24 hours after injection, tumor uptake was reduced from 12.02 ± 2.37% ID / g (1 hour after injection) to 1.54 ± 0.06% ID / g.

[0452] (Example 15) PET imaging study of I-23 General method for μPET imaging: Mice were anesthetized (2% sevoflurane, Abbott), placed in a small animal PET scanner (Inveon PET, Siemens), and injected with 68Ga-labeled bicyclic peptide. A 20-minute transmission scan, a 50-minute dynamic scan, and a static scan from 100 to 120 minutes after injection were performed. Images were iteratively reconstructed using the space alternating generalized expectation maximization method (SAGE, 16 subsets, 4 iterations) with pre-correction of the central line and converted to standardized uptake value (SUV) images. Quantification was performed using the ROI (region of interest) technique and expressed as the SUV mean.

[0453] General method for radiolabeling with Ga-68: 68 Ga (half-life 68 minutes; β + 89%; Eβ+ max 1.9 MeV) was obtained from a self-made 68 Ge / 68 Ga generator based on a pyrogallol resin support. 1 GBq of 68 Ga was eluted using 5.5 M HCl. The activity was captured on a small anion exchanger cartridge (AG1X8, Biorad, Richmond, CA, USA) as 68 Ga]GaCl4 - and eluted from the cartridge as 68 Ga]GaCl3 in ultrapure water (Merck, Darmstadt, Germany) with a final volume of 300 μL.

[0454] 1 nmol of the bicyclic peptide in 1 μL of DMSO was added to a mixture of 50 μL of 0.05 M HEPES, 0.1% Tween-20 and 80 μL of 68 Ga]Ga 3+ eluent (about 200 MBq). 30% NaOH was used to adjust the pH of the labeling solution to 4.2. The reaction mixture was incubated at 98 °C for 10 minutes. The radiochemical yield (RCY) was determined using reversed-phase high-performance liquid chromatography (RP-HPLC; Chromolith RP-18e, 100×4.6 mm; Merck, Darmstadt, Germany). Analytical HPLC runs were performed at a flow rate of 4 mL / min using a linear A-B gradient (from 0% B to 100% B in 6 minutes). Solvent A consisted of 0.1% aqueous TFA and solvent B was 0.1% TFA in CH3CN.

[0455] For μPET imaging, the labeled reaction mixture was diluted six-fold in injection buffer (0.25 M sodium acetate 0.05% Tween-20 pH 7) to obtain 150 picomoles in 100 μL of injection buffer. Anesthetized mice (2% sevoflurane, Abbott) were injected with 100 μL of 68 Ga-labeled I-23 (about 25 MBq) and placed in a small animal PET scanner (Inveon PET, Siemens). Imaging was performed 60 minutes after injection and included a 15-minute transmission scan before injection and a 60-minute emission scan after injection. Scans from μPET imaging were iteratively reconstructed using the spatial alternating generalized expectation maximization method (SAGE, 16 subsets, 4 iterations) with pre-correction of the central line and converted to standardized uptake value (SUV) images. Quantification was performed using the ROI (region of interest) technique and expressed as the mean SUV. The results from this study are shown in Figure 10.

[0456] μPET imaging revealed selective tumor uptake of MMP-14 and rapid clearance from non-target major organs in a xenograft model, resulting in high imaging contrast as early as 20 minutes after injection. The compound represents a highly promising radiopharmaceutical candidate for future clinical PET imaging.

[0457] (Example 16) PET Imaging Study of I-24 Radioactive labeling with Ga-68 was performed as described previously. The labeling reaction mixture was diluted six-fold in injection buffer (0.25 M sodium acetate 0.05% Tween-20 pH 7) to obtain 150 picomoles in 100 μL of injection buffer. Anesthetized mice (2% sevoflurane, Abbott) were injected with 100 μL of 68 Ga-labeled I-24 (approximately 19 MBq) and placed in a small animal PET scanner (Inveon PET, Siemens). Imaging was performed 60 minutes after injection and included a 15-minute transmission scan before injection and a 60-minute emission scan after injection. The scans from μPET imaging were iteratively reconstructed using the spatial alternating generalized expectation maximization method (SAGE, 16 subsets, 4 iterations) with pre-correction of the central line and converted to standardized uptake value (SUV) images. Quantification was performed using the ROI (region of interest) technique and expressed as the SUV mean. The results from this study are shown in Figure 11.

[0458] (Example 17) PET Imaging of 17-69-07-N144 Compared with Inactive Variant 17-69-07-N246 Radioactive labeling with Ga-68 was performed as described previously. The labeling reaction mixture was diluted six-fold in injection buffer (0.25 M sodium acetate 0.05% Tween-20 pH 7) to obtain 150 picomoles in 100 μL of injection buffer. Anesthetized mice (2% sevoflurane, Abbott) were injected with 100 μL of 68Injected the Ga labels 17-69-07-N144 and 17-69-07-N246 and placed them in a small animal PET scanner (Inveon PET, Siemens). Imaging was performed 60 minutes after injection and included a 15-minute transmission scan before injection and a 60-minute emission scan after injection. Repeatedly reconstructed scans from μPET imaging using the spatial alternating generalized expectation maximization method (SAGE, 16 subsets, 4 iterations) that applies pre-correction of the central root, and converted them into standardized uptake value (SUV) images. Quantification was performed using ROI (region of interest) technology and expressed as the SUV mean. The results from this study are shown in Figure 12.

[0459] (Example 18) Determine the binding to EphA2 and the pharmacokinetics of the bicyclic peptide-DOTA conjugate in a xenograft model: I-17 organ distribution and dose study Variable amounts of I-17 and a fixed amount of 177 Lu (approximately 50 kBq / mouse) were used to evaluate various doses in an in vivo distribution study using HT1080 xenograft mice. The in vivo distribution was performed 1 hour after injection. The results of this study are shown in Figures 13 and 14.

[0460] Doses from 50 to 150 pmol were found to be optimal. Tumor uptake was slightly blocked at a dose of 500 picomoles and almost completely blocked using 5 nanomoles of I-17.

[0461] (Example 19) Organ distribution of different EphA2 variants (stabilized and higher affinity) I-13 to I-15 represent DOTA-labeled bicyclics with higher affinity and higher stability targeting EphA2. I-17 represents a stabilized candidate-like molecule for EphA2. I-18 contains additional stabilization in loop 1 at the expense of a slightly lower affinity. I-28 is an inactive variant of I-17.

[0462] The results of this study are shown in Figs. 15 to 17.

[0463] (Example 20) μPET imaging of I-13 and I-15 Radioactive labeling with Ga-68 was performed as described above. The labeling reaction mixture was diluted six-fold in injection buffer (0.25 M sodium acetate 0.05% Tween-20 pH 7) to obtain 150 picomoles in 100 μL of injection buffer. Anesthetized mice (2% sevoflurane, Abbott) were injected with 100 μL of 68 Ga-labeled I-13 and I-15 and placed in a small animal PET scanner (Inveon PET, Siemens). Imaging was performed 60 minutes after injection and included a 15-minute transmission scan before injection and a 60-minute emission scan after injection. Scans from μPET imaging were iteratively reconstructed using the spatial alternating generalized expectation maximization method (SAGE, 16 subsets, 4 iterations) with pre-correction of the central line and converted to standardized uptake value (SUV) images. Quantification was performed using ROI (region of interest) technology and expressed as the SUV mean.

[0464] The results of this study are shown in Fig. 18.

[0465] The high liver uptake of I-13 already shown in the organ distribution (Fig. 1) could also be confirmed in PET imaging. I-15 presented clearly reduced liver uptake. Tumor signals were extremely high for both compounds.

[0466] (Example 21) Specific cellular uptake and internalization of I-19 To determine the specific cellular uptake and internalization of I-19, 10 5Cells were seeded into 24-well cell culture plates coated with poly-L-lysine 24 hours prior to incubation. After washing, the cells were incubated with 30 nM of the radiolabeled compound for 45 minutes each at 37 °C and 4 °C. Specific cellular uptake was determined by competitive blocking with 100 μM of each cold compound (non-DOTA). Cellular uptake was terminated by washing 4 times with 1 mL of ice-cold PBS. The cells were then incubated twice for 5 minutes with glycine-HCl (50 mM, pH = 2.8) in 0.5 mL of PBS to remove the surface-bound fraction. The cells were washed with 1 mL of ice-cold PBS and lysed using 0.3 N NaOH (0.5 mL). The surface-bound and internalized fractions were measured with a gamma counter. Cellular uptake was calculated as the percentage [%ID / 10 6 of the radioactivity initially added that was bound to the cells] 6 Specific cell surface binding was observed and no internalization was observed.

[0467] These results are shown in Figure 19.

[0468] (Example 22) Determine the binding to CD38 and the pharmacokinetics of the bicyclic peptide-DOTA conjugate in a xenograft model: Internalization experiment using monomeric bicyclic I-21 Compound I-21 was labeled with Ga-68 and used at a concentration of 30 nM in the assay. The exposure time was 45 minutes at 37 °C. Blocking was performed using the same compound but unlabeled (the concentration was 110 μM).

[0469] These results are shown in Figures 20 and 21. Although the inventors have described several embodiments of the present invention, it will be apparent that other embodiments can be provided by modifying the inventors' basic examples and utilizing the compounds and methods of the present invention. Thus, it will be seen that the scope of the present invention is defined by the appended claims rather than the specific embodiments that have been presented by way of example.

[0470] (Example 23) Organ distribution and dose study of I-25 (palmitoylated MT1-MMP specific bicyclic peptide) Applying the exemplary procedure as described in Example 10, the palmitoylated bicyclic peptide derivative I-25 was generated based on stabilized I-23, where its N-terminus was modified with an extended sarcosine oligomer spacer terminated with palmitic acid and a lysine modified with DOTA was placed within the sarcosine spacer. An organ distribution study (Figure 22A) investigating additional time points (2, 6, and 24 hours) at an optimal dose of 750 picomoles / mouse showed high tumor accumulation as early as 25.03 ± 1.61% ID / g at 2 hours post-injection. At 6 hours post-injection, 177 the tumor uptake of Lu-labeled BCY-C5 reached a maximum of 35.88 ± 1.11% ID / g, while most of the activity in background organs was removed. At later time points, 177 Lu-labeled BCY-C5 was further removed from circulation and thus presented a very high tumor-to-organ ratio (the signal ratio of tumor to blood was 19 at 6 hours and 97 at 48 hours), making this molecule an attractive option for molecular diagnostic imaging.

[0471] a non-MT1-MMP binding "mutant" of I-25 in which D-Ala5 is replaced with L-Ala5 and Glu6 is replaced with L-Ala5 177In vivo distribution studies were performed using the Lu-labeled I-29. This mutant essentially modifies / removes the side chain that is crucial for the interaction with MT1-MMP and serves as an inactive control peptide. The molecule is essentially the palmitoylated inactive version of I-27 described herein. This inactive palmitoylated control peptide was evaluated in a mouse xenograft tumor model and compared with the active I-25 comparator at the 6-hour time point when tumor uptake was maximal (750 picomoles per mouse). The tumor uptake measured for I-29 was 4.8%, which is approximately one-eighth of that of active I-25 (Figure 22B), indicating that significant target-selective uptake occurs in palmitoylated I-25 and that I-25 is a potent imaging reagent for MT1-MMP-expressing tumors in vivo.

Claims

1. A compound of formula I: 【Chemical 1】 or a pharmaceutically acceptable salt thereof, wherein L １ , L ２ and L ３ each of which is independently a covalent bond or a C １～８ divalent hydrocarbon chain, wherein one, two or three methylene units of said chain are optionally and independently replaced by -S-, -N(R)-, -O-, -C(O)-, -OC(O)-, -C(O)O-, -C(O)N(R)-, -N(R)C(O)-, -S(O)-, -S(O) ２ -, or -N(R)CH ２ C(O)-, and optionally independently replaced by Each of R is, independently, hydrogen or C １～４ alkyl, each of m, n, o and p is independently 0 or 1, each of q and r is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, R １ is R or -C(O)R, R ４ and R ６ each of which is independently hydrogen, or a group optionally substituted with selected independently from C １～６ aliphatic, a 3- to 8-membered saturated or partially unsaturated monocyclic carbocyclic ring, phenyl, an 8- to 10-membered bicyclic aromatic carbocyclic ring, a 4- to 8-membered saturated or partially unsaturated monocyclic heterocyclic ring having 1 to 2 heteroatoms selected independently from nitrogen, oxygen or sulfur, a 5- to 6-membered monocyclic heteroaromatic ring having 1 to 4 heteroatoms selected independently from nitrogen, oxygen or sulfur, or an 8- to 10-membered bicyclic heteroaromatic ring having 1 to 5 heteroatoms selected independently from nitrogen, oxygen or sulfur R ４’ and R ６’ each of which is independently hydrogen or methyl, R ２ , R ３ , R ５ and R ７ Each of is independently hydrogen or C １～４ aliphatic or R ５ group and its adjacent R ４ groups, together with their intervening atoms if necessary, form a 4- to 8-membered saturated or partially unsaturated monocyclic heterocyclic ring having 1 to 2 heteroatoms independently selected from nitrogen, oxygen or sulfur, or R ７ The group and its adjacent R ６ groups, together with their intervening atoms if necessary, form a 4- to 8-membered saturated or partially unsaturated monocyclic heterocyclic ring having 1 to 2 heteroatoms independently selected from nitrogen, oxygen or sulfur, the scaffold is a trivalent group that connects to and orients the cyclic peptide, Loop A is L ２ and the amino acid residue linked to １ and binds to the amino acid residue or peptide, natural or non-natural, divalent, linked to 【Chemical Formula 2】 comprising Loop B is L １ linked amino acid residues and L ３ linked amino acid residues and is a divalent natural or unnatural amino acid residue or peptide, where Loop B is 【Chemical Formula 3】 comprising 【Chemical Formula 4】 refers to the binding site with the bicyclic N-terminus, 【Chemical Formula 5】 refers to the binding site with the bicyclic C-terminus, Detectable part １ is any part that can be detected, Detectable part ２ is any part that can be detected, Linker １ is hydrogen, -C(O)R, [Chemical Formula 6] 、 or a detectable moiety at the N-terminus of said bicyclic ring １ and is a divalent moiety that connects thereto, where when n is 0, the linker １ is hydrogen, -C(O)R, or 【Chemical Formula 7】 and Linker ２ is -NH ２ or a divalent moiety that connects the C-terminus of the bicyclic to a detectable moiety ２ wherein when p is 0, the linker ２ is -NH ２ and ring A is 18-crown-6, 1,7,13-triaza-18-crown-6, and 0 to 3 oxo, methyl, ethyl or spiroethylene groups optionally substituted therewith, independently selected from nitrogen, oxygen or sulfur A compound or a pharmaceutically acceptable salt thereof selected from the group consisting of 3- to 12-membered saturated, partially unsaturated, bridged bicyclic, bridged tricyclic, propellane or aromatic rings having 0 to 6 heteroatoms.

2. L １ 、 L ２ and L ３ each is a C １～８ divalent hydrocarbon chain, wherein one, two or three methylene units of said chain are optionally and independently replaced by -S-, -N(R)-, -O-, -C(O)-, -OC(O)-, -C(O)O-, -C(O)N(R)-, -N(R)C(O)-, -S(O)-, -S(O) ２ -, or -N(R)CH ２ C(O)-, and the compound according to claim 1

3. R １ is hydrogen or -C(O)CH ３ The compound according to claim 1, wherein

4. Linker １ is a covalent bond, 【Chemical Formula 8】 【Chemical Formula 9】 The compound according to claim 1, wherein

5. p is 0 and the linker ２ is -NH ２ The compound according to claim 1

6. the scaffold is 【Chemical Formula 10】 The compound according to claim 1, wherein

7. Detectable part １ is 【Chemical 11】 【Chemical Formula 12】 The compound according to claim 1, wherein

8. Detectable part １ is 【Chemical 13】 【Chemical 14】 and in the formula, M is ６７ Ga, ６８ Ga, １１１ In, １７７ Lu, ８６ Y, ９０ Y, ８９ Zr and ９５ the compound according to claim 1, selected from the group consisting of Zr.

9. loop A is 【Chemical Formula 15】 【Chemical 16】 The compound according to claim 1, wherein

10. loop B is 【Chemical 17】 【Chemical Formula 18】 【Chemical 19】 The compound according to claim 1, wherein

11. Detectable moiety １ The compound according to claim 1, wherein the detectable moiety is a fluorophore selected from the group consisting of Alexa Fluor 350, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 660, Alexa Fluor 680, AMCA, AMCA-S, BODIPY FL, BODIPY R6G, BODIPY TMR, BODIPY TR, BODIPY 530 / 550, BODIPY 558 / 568, BODIPY 564 / 570, BODIPY 576 / 589, BODIPY 581 / 591, BODIPY 630 / 650, BODIPY 650 / 665, carboxyrhodamine 6G, carboxy-X-rhodamine, Cascade Blue, Cascade Yellow, Coumarin 343, Cy3, Cy5, Cy3.5, Cy5.5, Cy7, Cy7.5, dansyl, dapoxyl, dialkylaminocoumarin, 4',5'-dichloro-2',7'-dimethoxy-fluorescein, DM-NERF, eosin, erythrosin, fluorescein, FAM, IRD40, IRD700, IRD800, JOE, Lissamine rhodamine B, Marina Blue, methoxycoumarin, naphthofluorescein, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, PyMPO, pyrene, rhodamine B, rhodamine 6G, Rhodamine Green, Rhodamine Red, Rhodol Green, 2',4',5',7'-tetra-bromo-sulfone-fluorescein, tetramethyl-rhodamine, carboxytetramethylrhodamine, Texas Red and Texas Red-X.

12. The compound according to claim 1, selected from those shown in Table 1.

13. A pharmaceutical composition comprising a compound according to any one of claims 1 to 12 and a pharmaceutically acceptable carrier, adjuvant or vehicle.

14. A method of inhibiting CAIX in a patient or biological sample, comprising administering to the patient a compound according to any one of claims 1 to 12 or a pharmaceutical composition thereof, or contacting the compound or a pharmaceutical composition thereof with the biological sample.

15. A method of inhibiting MT1-MMP in a patient or biological sample, comprising administering to the patient a compound according to any one of claims 1 to 12 or a pharmaceutical composition thereof, or contacting the compound or a pharmaceutical composition thereof with the biological sample.

16. A method for inhibiting ανβ3 in a patient or a biological sample, comprising administering to the patient the compound or its pharmaceutical composition according to any one of claims 1 to 12, or contacting the compound or its pharmaceutical composition with the biological sample.

17. A method for inhibiting PSMA in a patient or a biological sample, comprising administering to the patient the compound or its pharmaceutical composition according to any one of claims 1 to 12, or contacting the compound or its pharmaceutical composition with the biological sample.

18. A method for inhibiting CD38 in a patient or a biological sample, comprising administering to the patient the compound or its pharmaceutical composition according to any one of claims 1 to 12, or contacting the compound or its pharmaceutical composition with the biological sample.

19. A method for inhibiting EphA2 in a patient or a biological sample, comprising administering to the patient the compound or its pharmaceutical composition according to any one of claims 1 to 12, or contacting the compound or its pharmaceutical composition with the biological sample.

20. A method for treating a CAIX-mediated disorder, disease or condition in a patient, comprising administering to the patient the compound or its pharmaceutical composition according to any one of claims 1 to 12.

21. A method for treating a MT1-MMP-mediated disorder, disease or condition in a patient, comprising administering to the patient the compound or its pharmaceutical composition according to any one of claims 1 to 12.

22. A method for treating an ανβ3-mediated disorder, disease or condition in a patient, comprising administering to the patient the compound or its pharmaceutical composition according to any one of claims 1 to 12.

23. A method for treating a PSMA-mediated disorder, disease or condition in a patient, comprising administering to the patient the compound or its pharmaceutical composition according to any one of claims 1 to 12.

24. A method for treating a CD38-mediated disorder, disease or condition in a patient, comprising administering to the patient the compound or its pharmaceutical composition according to any one of claims 1 to 12.

25. A method of treating an EphA2-mediated disorder, disease or condition in a patient, comprising administering to the patient a compound or a pharmaceutical composition thereof according to any one of claims 1 to 12.

26. The method according to claim 20, wherein the CAIX-mediated disorder, disease or condition is selected from the group consisting of cancer or a proliferative disorder.

27. The method according to claim 21, wherein the MT1-MMP-mediated disorder, disease or condition is selected from the group consisting of cancer or a proliferative disorder.

28. The method according to claim 22, wherein the αvβ3-mediated disorder, disease or condition is selected from the group consisting of cancer or a proliferative disorder.

29. The method according to claim 23, wherein the PSMA-mediated disorder, disease or condition is selected from the group consisting of cancer or a proliferative disorder.

30. The method according to claim 24, wherein the CD38-mediated disorder, disease or condition is selected from the group consisting of cancer or a proliferative disorder.

31. The method according to claim 25, wherein the EphA2-mediated disorder, disease or condition is selected from the group consisting of cancer or a proliferative disorder.

32. The cancer or proliferative disorder is a tumor of epithelial origin (including various types of adenomas and carcinomas such as adenocarcinoma, squamous cell carcinoma, transitional cell carcinoma, and other carcinomas), for example, carcinomas of the bladder and urinary tract, carcinomas of the breast, carcinomas of the gastrointestinal tract (including the esophagus, stomach, small intestine, colon, rectum, and anus), carcinomas of the liver (hepatocellular carcinoma), carcinomas of the gallbladder and biliary tract, carcinomas of the exocrine pancreas, carcinomas of the kidney, carcinomas of the lung (e.g., adenocarcinoma, small cell lung cancer, non-small cell lung cancer, bronchioloalveolar carcinoma, and mesothelioma), carcinomas of the head and neck (e.g., cancers of the tongue, oral cavity, larynx, pharynx, nasopharynx, tonsils, salivary glands, nasal cavity, and paranasal sinuses), carcinomas of the ovary, carcinomas of the fallopian tube, carcinomas of the peritoneum, carcinomas of the vagina, carcinomas of the vulva, carcinomas of the penis, carcinomas of the cervix, carcinomas of the myometrium, carcinomas of the endometrium, carcinomas of the thyroid (e.g., thyroid follicular carcinoma), carcinomas of the adrenal gland, carcinomas of the prostate, carcinomas of the skin, and carcinomas of the appendages (e.g., melanoma, basal cell carcinoma, squamous epithelial cell carcinoma, keratoacanthoma, dysplastic nevus); lymphocytic hematological malignancies and related conditions (e.g., B-cell lymphomas such as acute lymphoblastic leukemia [ALL], chronic lymphocytic leukemia [CLL], diffuse large B-cell lymphoma [DLBCL], etc., follicular lymphoma, Burkitt lymphoma, mantle cell lymphoma, T-cell lymphoma and leukemia, natural killer [NK] cell lymphoma, Hodgkin lymphoma, hairy cell leukemia, monoclonal gammopathy of undetermined significance, plasmacytoma, multiple myeloma, and post-transplant lymphoproliferative disorder) as well as myeloid hematological malignancies and related conditions (e.g., acute myeloid leukemia [AML], chronic myeloid leukemia [CML], chronic myelomonocytic leukemia [CMML], eosinophilic syndrome, myeloproliferative disorders such as polycythemia vera, essential thrombocythemia, and primary myelofibrosis, myeloproliferative syndrome, myelodysplastic syndrome, and promyelocytic leukemia), including hematological malignancies (i.e., leukemia, lymphoma) as well as pre-malignant blood disorders and borderline malignant tumor disorders; tumors of mesenchymal origin, for example, osteosarcoma, fibrosarcoma, chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma, liposarcoma, angiosarcoma, Kaposi sarcoma, Ewing sarcoma, synovial sarcoma, epitheloid sarcoma, gastrointestinal stromal tumor, benign and malignant histiocytoma, and dermatofibrosarcoma protuberans, etc., sarcomas of soft tissue, bone, or cartilage; tumors of the central nervous system or peripheral nervous system (e.g., astrocytoma, glioma, and glioblastoma, meningioma, ependymoma, pineal tumor, and schwannoma);Endocrine tumors (e.g., pituitary tumors, adrenal tumors, islet cell tumors, parathyroid tumors, carcinoid tumors and medullary carcinoma of the thyroid); tumors of the eye and its appendages (e.g., retinoblastoma); germ cell tumors and trophoblastic tumors (e.g., teratoma, seminoma, undifferentiated germ cell tumor, cystic teratoma and choriocarcinoma); and pediatric tumors and fetal tumors (e.g., medulloblastoma, neuroblastoma, Wilms tumor and primitive neuroectodermal tumor); or a method according to any one of claims 26 to 31, selected from the group consisting of congenital or other syndromes (e.g., xeroderma pigmentosum) which leave the patient susceptible to malignant tumors.

33. A method of diagnosing or imaging a CAIX-mediated disorder, disease or condition in a patient, comprising administering to the patient a compound or a pharmaceutical composition thereof according to any one of claims 1 to 12.

34. A method of diagnosing or imaging an MT1-MMP-mediated disorder, disease or condition in a patient, comprising administering to the patient a compound or a pharmaceutical composition thereof according to any one of claims 1 to 12.

35. A method of diagnosing or imaging an αvβ3-mediated disorder, disease or condition in a patient, comprising administering to the patient a compound or a pharmaceutical composition thereof according to any one of claims 1 to 12.

36. A method of diagnosing or imaging a PSMA-mediated disorder, disease or condition in a patient, comprising administering to the patient a compound or a pharmaceutical composition thereof according to any one of claims 1 to 12.

37. A method of diagnosing or imaging a CD38-mediated disorder, disease or condition in a patient, comprising administering to the patient a compound or a pharmaceutical composition thereof according to any one of claims 1 to 12. Claim 38 A method for diagnosing or imaging an EphA2-mediated disorder, disease or condition in a patient, the method comprising administering to the patient a compound or a pharmaceutical composition thereof according to any one of claims 1 to 12.

Images