Compounds involved in cooperative binding and methods of use thereof

Abstract

To provide a new molecular modality capable of modulating the function of an undruggable target.SOLUTION: A compound (e.g., a macrocyclic compound comprising 14 to 40 ring atoms as shown below) capable of modulating a biological process by binding to presenter proteins (e.g., members of the FKBP family, members of the cyclophilin family, or PIN1) and target proteins (e.g., eukaryotic target proteins such as mammalian target proteins or fungal target proteins or prokaryotic target proteins such as bacterial target proteins).SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to compounds involved in cooperative binding and uses thereof. [Background technology]

[0002] The vast majority of small molecule drugs act by binding to functionally important pockets on target proteins, thereby modulating the activity of those proteins. For example, cholesterol-lowering drugs such as statins bind to the enzyme active site of HMG-CoA reductase, thereby preventing the enzyme from engaging its substrate. The fact that many such drug / target interaction pairs are known may lead some to mistakenly believe that, with the appropriate amount of time, effort, and resources, it is possible to discover small molecule modulators for most, if not all, proteins. This is far from the truth. Current estimates suggest that only about 10% of all human proteins are potential small molecule targets. The remaining 90% are currently considered too cumbersome or difficult for small molecule drug discovery. Such targets are commonly referred to as "undruggable." These undruggable targets comprise a vast and largely untapped reservoir of medically important human proteins. Therefore, there is significant interest in discovering new molecular modalities that can modulate the function of such undruggable targets. Summary of the Invention [Means for solving the problem]

[0003] The invention features compounds (e.g., macrocyclic compounds) that can modulate biological processes, e.g., by binding to a presenter protein (e.g., a member of the FKBP family, a member of the cyclophilin family, or PIN1) and a target protein. In some embodiments, the target protein and / or presenter protein are intracellular proteins. In some embodiments, the target protein and / or presenter protein are mammalian proteins. In some embodiments, provided compounds participate in a tripartite presenter protein / compound / target protein complex within a cell, e.g., a mammalian cell. In some embodiments, provided compounds may be useful in the treatment of diseases and disorders, such as cancer, inflammation, and infectious diseases.

[0004] In one aspect, the invention features a compound (e.g., a macrocyclic compound containing 14 to 40 ring atoms) that includes: (a) a target protein interacting portion (e.g., a eukaryotic target protein interacting portion, such as a mammalian target protein interacting portion or a fungal target protein interacting portion, or a prokaryotic target protein interacting portion, such as a bacterial target protein interacting portion), and (b) a presenter protein binding portion, wherein the compound and the presenter protein form a complex that specifically binds to the target protein, and wherein the compound and the presenter protein each do not substantially bind to the target protein in the absence of complex formation, or the compound and the presenter protein form a complex that binds to the target protein with an affinity that is at least 5-fold greater than the affinity of the compound and the presenter protein, respectively, for the target protein in the absence of complex formation, or a pharmaceutically acceptable salt thereof.

[0005] In some embodiments, the compound is a macrocyclic compound. In certain embodiments, the compound is an acyclic compound. In some embodiments, provided compounds comprise one or more linker moieties, hi some embodiments, the linker moiety connects the presenter protein binding moiety (or a portion thereof) and the target protein interacting moiety (or a portion thereof).

[0006] In some embodiments, the compound has the structure:

[0007] [ka]

[0008] wherein A comprises a mammalian target protein interacting moiety; B comprises a presenter protein binding moiety, and L 1 and L 2 are independently selected from a bond and a linear chain of up to 10 atoms independently selected from carbon, nitrogen, oxygen, sulfur, or phosphorus atoms, each atom in the chain optionally substituted with one or more substituents independently selected from alkyl, alkenyl, alkynyl, aryl, heteroaryl, chloro, iodo, bromo, fluoro, hydroxyl, alkoxy, aryloxy, carboxy, amino, alkylamino, dialkylamino, acylamino, carboxamido, cyano, oxo, thio, alkylthio, arylthio, acylthio, alkylsulfonate, arylsulfonate, phosphoryl, and sulfonyl, and any two atoms in the chain together with the substituents attached thereto can form a ring, which can be further substituted and / or fused to one or more optionally substituted carbocyclic, heterocyclic, aryl, or heteroaryl rings.

[0009] In some embodiments, the compound has the structure:

[0010] [ka]

[0011] (In the formula, Z1 and Z 2 each of which is independently hydrogen or hydroxyl. In other embodiments, L 1 , L 2 , Z 1 , and / or Z 2 At least one atom of L is involved in binding to the presenter protein and the target protein. 1 , L 2 , Z 1 , and / or Z 2 At least one atom of is not involved in binding to the presenter protein or the target protein.

[0012] In some embodiments, L 1 , L 2 , Z 1 , and / or Z 2 comprises one or more atoms involved in binding to the presenter protein and / or the target protein. 1 , L 2 , Z 1 , and / or Z 2 At least one atom of one or more of L is involved in binding to the presenter protein and / or the target protein. 1 , L 2 , Z 1 , and / or Z 2 At least one atom of one or more of is not involved in binding to the presenter protein and / or the target protein.

[0013] In some embodiments, the presenter protein binding moiety comprises between 5 and 20 ring atoms (eg, between 5 and 10, between 7 and 12, between 10 and 15, between 12 and 17, or between 15 and 20 ring atoms).

[0014] In some embodiments, the compound comprises 14 to 20 ring atoms (e.g., 14 to 16, 14 to 17, 15 to 18, 16 to 19, or 17 to 20 ring atoms, or 14, 15, 16, 17, 18, 19, or 20 ring atoms). In certain embodiments, the compound comprises 21 to 26 ring atoms (e.g., 21 to 23, 22 to 24, 23 to 25, or 24 to 26 ring atoms, or 21, 22, 23, 24, 25, 26 ring atoms). In some embodiments, the compound comprises 27 to 40 ring atoms (e.g., 27 to 30, 29 to 34, 33 to 38, 37 to 40 ring atoms, or 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 ring atoms).

[0015] In some embodiments, the presenter protein binding moiety has formula I:

[0016] [ka]

[0017] (wherein n is 0 or 1, X 1 and X 3 are independently O, S, and CR 3 R 4 , or NR 5 and X 2 is O, S, or NR 5 and R 1 , R 2 , R 3 , and R 4 are each independently hydrogen, hydroxyl, optionally substituted amino, halogen, thiol, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C1-C6 heteroalkyl, optionally substituted C2-C6 heteroalkenyl, optionally substituted C2-C6 heteroalkynyl, optionally substituted C3-C6 heteroalkyl ... 10Carbocyclyl, optionally substituted C-C 10 Aryl, optionally substituted C-C 10 arylC1-C6 alkyl, optionally substituted C2-C9 heteroaryl, optionally substituted C2-C9 heteroarylC1-C6 alkyl, optionally substituted C2-C9 heterocyclyl, or optionally substituted C2-C9 heterocyclylC1-C6 alkyl, or R 1 , R 2 , R 3 , or R 4 any two of, taken together with the atom or atoms to which they are attached, form an optionally substituted carbocyclyl, an optionally substituted heterocyclyl, an optionally substituted aryl, or an optionally substituted heteroaryl; and Each R 5 are independently hydrogen, hydroxyl, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C1-C6 heteroalkyl, optionally substituted C2-C6 heteroalkenyl, optionally substituted C2-C6 heteroalkynyl, optionally substituted C3-C6 heteroalkyl ...alkynyl, optionally substituted C3-C6 heteroalkynyl, optionally substituted C3 10 Carbocyclyl, optionally substituted C-C 10 Aryl, optionally substituted C-C 10 arylC1-C6 alkyl, optionally substituted C2-C9 heteroaryl, optionally substituted C2-C9 heteroarylC1-C6 alkyl, optionally substituted C2-C9 heterocyclyl, or optionally substituted C2-C9 heterocyclylC1-C6 alkyl, or R 5 and R 1 , R 2 , R 3 , or R 4 when taken together with the atom or atoms to which they are attached form an optionally substituted heterocyclyl or an optionally substituted heteroaryl.

[0018] In some embodiments, X 1 is L 1 connected to and X 3 is L 2 In some embodiments, X 1 is L 2 connected to and X 3 is L 1 is connected to. In some embodiments, the presenter protein binding moiety has formula Ia:

[0019] [ka]

[0020] It includes the structure shown in In some embodiments, the presenter protein binding moiety has formula II-IV:

[0021] [ka]

[0022] wherein o and p are independently 0, 1, or 2; q is an integer from 0 to 7, r is an integer from 0 to 4, X 4 and X 5 are each independently absent, CH, O, S, SO, SO, or NR 13 and Each R 6 and R 7 are independently hydrogen, hydroxyl, optionally substituted amino, halogen, thiol, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C1-C6 heteroalkyl, optionally substituted C2-C6 heteroalkenyl, optionally substituted C2-C6 heteroalkynyl, optionally substituted C3-C6 heteroalkyl ...alkynyl, optionally substituted C3-C6 heteroalkyl, optionally substituted C2-C6 heteroalkynyl, optionally substituted C3-C6 heteroalkynyl, optionally substituted C3-C6 heteroalkynyl, optionally substituted C3-C6 heteroalkynyl, optionally substituted C3-C6 heteroalkynyl, optionally substituted C3-C6 heteroalkynyl, optionally substituted C3-C6 heteroalkynyl, optionally substituted C3-C6 heteroalkynyl, optionally substituted C3-C6 heteroalkynyl, optionally substituted C3-C6 heteroalkynyl, optionally substituted C3-C6 heteroalkynyl, optionally substituted C3-C6 heteroalkynyl, optionally substituted C3-C6 heteroalkynyl, optionally substituted C3-C6 heteroalkynyl, optionally substituted C3-C6 heteroalkynyl, optionally substituted C3-C6 hetero 10Carbocyclyl, optionally substituted C-C 10 Aryl, optionally substituted C-C 10 arylC1-C6 alkyl, optionally substituted C2-C9 heteroaryl, optionally substituted C2-C9 heteroarylC1-C6 alkyl, optionally substituted C2-C9 heterocyclyl, optionally substituted C2-C9 heterocyclylC1-C6 alkyl, or R 6 and R 7 combine with the carbon atom to which they are attached to form C=O, or R 6 and R 7 in combination with optionally substituted C3 to C 10 forming a carbocyclyl or an optionally substituted C2-C9 heterocyclyl; Each R 8 are independently selected from hydroxyl, optionally substituted amino, halogen, thiol, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C1-C6 heteroalkyl, optionally substituted C2-C6 heteroalkenyl, optionally substituted C2-C6 heteroalkynyl, optionally substituted C3-C6 heteroalkyl ... 10 Carbocyclyl, optionally substituted C-C 10 Aryl, optionally substituted C-C 10 arylC1-C6 alkyl, optionally substituted C2-C9 heteroaryl, optionally substituted C2-C9 heteroarylC1-C6 alkyl, optionally substituted C2-C9 heterocyclyl, or optionally substituted C2-C9 heterocyclylC1-C6 alkyl, or two R 8 in combination with optionally substituted C3 to C 10 Carbocyclyl, optionally substituted C-C 10 forming an aryl or an optionally substituted C2-C9 heteroaryl; R 9is an optionally substituted C1-C6 alkyl, an optionally substituted C2-C6 alkenyl, an optionally substituted C2-C6 alkynyl, an optionally substituted C1-C6 heteroalkyl, an optionally substituted C2-C6 heteroalkenyl, an optionally substituted C2-C6 heteroalkynyl, an optionally substituted C3-C6 10 Carbocyclyl, optionally substituted C-C 10 Aryl, optionally substituted C-C 10 arylC1-C6 alkyl, optionally substituted C2-C9 heteroaryl, optionally substituted C2-C9 heteroarylC1-C6 alkyl, optionally substituted C2-C9 heterocyclyl, or optionally substituted C2-C9 heterocyclylC1-C6 alkyl; R 10 is an optionally substituted C1-C6 alkyl; Each R 11 are independently selected from hydroxyl, cyano, optionally substituted amino, halogen, thiol, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C1-C6 heteroalkyl, optionally substituted C2-C6 heteroalkenyl, optionally substituted C2-C6 heteroalkynyl, optionally substituted C3-C6 heteroalkyl, optionally substituted C4-C6 heteroalkynyl, optionally substituted C5-C6 heteroalkyl, optionally substituted C6-C6 heteroalkynyl, optionally substituted C7-C6 heteroalkyl, optionally substituted C8-C6 heteroalkynyl, optionally substituted C9-C6 heteroalkynyl, optionally substituted C10-C6 heteroalkynyl, optionally substituted C11-C6 heteroalkynyl, optionally substituted C12-C6 heteroalkynyl, optionally substituted C13-C6 heteroalkynyl, optionally substituted C14-C6 heteroalkynyl, optionally substituted C15-C6 heteroalkynyl, optionally substituted C16-C6 heteroalkynyl, optionally substituted C17-C6 heteroalkynyl, optionally substituted C18-C6 heteroalkynyl, optionally substituted C19 ... 10 Carbocyclyl, optionally substituted C-C 10 Aryl, optionally substituted C-C 10 arylC1-C6 alkyl, optionally substituted C2-C9 heteroaryl, optionally substituted C2-C9 heteroarylC1-C6 alkyl, optionally substituted C2-C9 heterocyclyl, or optionally substituted C2-C9 heterocyclylC1-C6 alkyl, or two R 11 in combination with optionally substituted C3 to C 10 Carbocyclyl, optionally substituted C-C 10 aryl or optionally substituted C2-C9 heteroaryl; and R 12 and R 13 are each independently hydrogen, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted aryl, C3-C7 carbocyclyl, optionally substituted C6-C 10 arylC1-C6 alkyl, and optionally substituted C3-C7 carbocyclylC1-C6 alkyl).

[0023] In some embodiments, L 1 is attached to the left side of the presenter protein binding moiety, and L 2 is attached to the right of the presenter protein binding moiety. In some embodiments, L 1 is connected to the right side of the presenter protein binding moiety, and L 2 is attached to the left side of the presenter protein binding moiety.

[0024] In some embodiments, the presenter protein binding moiety has the formula IIa-IVa:

[0025] [ka]

[0026] The structure is or includes any one of the structures shown below. In some embodiments, the presenter protein binding moiety has formula V:

[0027] [ka]

[0028] (In the formula, R 14is hydrogen, hydroxyl, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C1-C6 heteroalkyl, optionally substituted C2-C6 heteroalkenyl, optionally substituted C2-C6 heteroalkynyl, optionally substituted C3-C6 10 Carbocyclyl, optionally substituted C-C 10 Aryl, optionally substituted C-C 10 arylC1-C6 alkyl, optionally substituted C2-C9 heteroaryl, optionally substituted C2-C9 heteroarylC1-C6 alkyl, optionally substituted C2-C9 heterocyclyl, optionally substituted C2-C9 heterocyclylC1-C6 alkyl).

[0029] In certain embodiments, the presenter protein binding moiety has formula VI, VII, or VIII:

[0030] [ka]

[0031] (In the formula, s and t each independently represent an integer of 0 to 7, X 6 and X 7 , each independently O, S, SO, SO2, or NR 19 and R 15 and R 17 are each independently hydrogen, hydroxyl, or optionally substituted C1-C6 alkyl; R 16 and R 18are each independently selected from hydroxyl, optionally substituted amino, halogen, thiol, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C1-C6 heteroalkyl, optionally substituted C2-C6 heteroalkenyl, optionally substituted C2-C6 heteroalkynyl, optionally substituted C3-C6 heteroalkyl ... 10 Carbocyclyl, optionally substituted C-C 10 Aryl, optionally substituted C-C 10 arylC1-C6 alkyl, optionally substituted C2-C9 heteroaryl, optionally substituted C2-C9 heteroarylC1-C6 alkyl, optionally substituted C2-C9 heterocyclyl, or optionally substituted C2-C9 heterocyclylC1-C6 alkyl; R 19 is an optionally substituted C1-C6 alkyl, an optionally substituted C2-C6 alkenyl, an optionally substituted C2-C6 alkynyl, an optionally substituted aryl, a C3-C7 carbocyclyl, an optionally substituted C6-C 10 arylC1-C6 alkyl, and optionally substituted C3-C7 carbocyclylC1-C6 alkyl; and Ar is an optionally substituted C-C 10 aryl or optionally substituted C2-C9 heteroaryl).

[0032] In certain embodiments, the presenter protein binding moiety has the structure:

[0033] [ka]

[0034] JPEG2026015411000010.jpg211170

[0035] JPEG2026015411000011.jpg202170

[0036] or includes the structure thereof. In certain embodiments, the presenter protein binding moiety has the structure:

[0037] [ka]

[0038] or includes the structure thereof. In some embodiments, the presenter protein binding moiety has the structure:

[0039] [ka]

[0040] JPEG2026015411000014.jpg138170

[0041] or includes the structure thereof. In some embodiments, the presenter protein binding moiety has the structure:

[0042] [ka]

[0043] or includes the structure thereof. In certain embodiments, the target interacting moiety has formula IX:

[0044] [ka]

[0045] (wherein u is an integer of 1 to 20, and Each Y is independently any amino acid, O, NR 20 , S, S(O), SO2, or of formula X-XIII:

[0046] [ka]

[0047] (where each R 20 are independently selected from hydrogen, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted aryl, C3-C7 carbocyclyl, optionally substituted C6-C 10 arylC1-C6 alkyl, and optionally substituted C3-C7 carbocyclylC1-C6 alkyl, or R 20 is any R 20 , R 21 , R 22 , R 23 , R 24 , R 25 , R 26 , R 27 , R 28 , R 29 , or R 30 in combination with an optionally substituted C3-C 10 Carbocyclyl, optionally substituted C-C 10 forming an aryl or an optionally substituted C2-C9 heteroaryl; Each R 21 and R 22 are independently hydrogen, halogen, optionally substituted hydroxyl, optionally substituted amino, or R 21 and R 22 are, in combination, ═O, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C3-C 10 Carbocyclyl, optionally substituted C-C 10 Aryl, optionally substituted C-C 10arylC1-C6 alkyl, optionally substituted C2-C9 heteroaryl, optionally substituted C2-C9 heteroarylC1-C6 alkyl, optionally substituted C2-C9 heterocyclyl, or optionally substituted C2-C9 heterocyclylC1-C6 alkyl, or R 21 Or R 22 is any R 20 , R 21 , R 22 , R 23 , R 24 , R 25 , R 26 , R 27 , R 28 , R 29 , or R 30 in combination with an optionally substituted C3-C 10 Carbocyclyl, optionally substituted C-C 10 forming an aryl or an optionally substituted C2-C9 heteroaryl; Each R 23 , R 24 , R 25 , or R 26 are independently hydrogen, hydroxyl, or R 23 and R 24 combine to form =O or R 23 , R 24 , R 25 , or R 26 is any R 20 , R 21 , R 22 , R 23 , R 24 , R 25 , R 26 , R 27 , R 28 , R 29 , or R 30 in combination with an optionally substituted C3-C 10 Carbocyclyl, optionally substituted C-C 10 aryl or optionally substituted C2-C9 heteroaryl; and Each R 27 , R 28 , R29 , and R 30 are independently hydrogen, halogen, optionally substituted hydroxyl, optionally substituted amino, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C3-C6 alkyl, optionally substituted C4-C6 alkyl, optionally substituted C5-C6 alkyl, optionally substituted C6-C6 alkyl, optionally substituted C7-C6 alkyl, optionally substituted C8-C6 alkyl, optionally substituted C9-C6 alkyl, optionally substituted C10-C6 alkyl, optionally substituted C11-C6 alkyl, optionally substituted C12-C6 alkyl, optionally substituted C13-C6 alkyl, optionally substituted C14-C6 alkyl, optionally substituted C15-C6 alkyl, optionally substituted C16-C6 alkyl, optionally substituted C17-C6 alkyl, optionally substituted C18-C6 alkyl, optionally substituted C19 ... 10 Carbocyclyl, optionally substituted C-C 10 Aryl, optionally substituted C-C 10 arylC1-C6 alkyl, optionally substituted C2-C9 heteroaryl, optionally substituted C2-C9 heteroarylC1-C6 alkyl, optionally substituted C2-C9 heterocyclyl, or optionally substituted C2-C9 heterocyclylC1-C6 alkyl, or R 26 , R 27 , R 28 , R 29 , or R 30 is any R 20 , R 21 , R 22 , R 23 , R 24 , R 25 , R 26 , R 27 , R 28 , R 29 , or R 30 in combination with an optionally substituted C3-C 10 Carbocyclyl, optionally substituted C-C 10 aryl, or optionally substituted C2-C9 heteroaryl) or a structure represented by any one of the following:

[0048] In certain embodiments, the target interacting moiety has formula IX:

[0049] [ka]

[0050] The structure is or includes the structure shown in In some embodiments, the compound has Formulas XIV-XVIII:

[0051] [ka]

[0052] It has a structure represented by any one of the following: In some embodiments, the compound has the structure:

[0053] [ka]

[0054] Does not include. In some embodiments, the target interacting moiety has the structure:

[0055] [ka]

[0056] JPEG2026015411000022.jpg109170

[0057] or includes the structure thereof. In some embodiments, at least one Y, is an N-alkylated amino acid (e.g., an N-methyl amino acid). In some embodiments, at least one Y, is a D-amino acid. In some embodiments, at least one Y, is an unnatural amino acid. In certain embodiments, at least one Y, comprises a depsi bond.

[0058] In some embodiments, the compound has the structure:

[0059] [ka]

[0060] Does not include. In some embodiments, the portion of the molecule comprising each ring atom involved in binding to the target protein has a cLogP of greater than 2 (e.g., greater than 3, greater than 4, greater than 5, greater than 6). In certain embodiments, the portion of the molecule comprising each ring atom involved in binding to the target protein has a cLogP of greater than 350 Å. 2 Less than (e.g., 300 Å 2 Less than 250Å 2 Less than 200Å 2 Less than 150Å 2 Less than 125Å 2 In some embodiments, the portion of the molecule that includes each ring atom that participates in binding to the target protein includes at least one atom of the linker.

[0061] In some embodiments, the compound has a molecular weight of 400-2000 daltons (e.g., 400-600, 500-700, 600-800, 700-900, 800-1000, 900-1100, 1000-1200, 1100-1300, 1200-1400, 1300-1500, 1400-1600, 1500-1700, 1600-1800, 1700-1900, or 1800-2000 daltons). In certain embodiments, the compound has an even number of ring atoms (e.g., 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40 ring atoms). In some embodiments, the compound is cell-permeable. In some embodiments, the compound is substantially pure (e.g., the compound is provided in a preparation that is substantially free of contaminants, such as other compounds and / or components of a cell lysate). In certain embodiments, the compound is isolated. In some embodiments, the compound is an engineered compound. In some embodiments, the compound is not naturally occurring.

[0062] In certain embodiments, the complex binds to a target protein (e.g., a eukaryotic target protein, such as a mammalian target protein or a fungal target protein, or a prokaryotic target protein, such as a bacterial target protein) with an affinity that is at least 5-fold (e.g., at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold) greater than the affinity of the complex for mTOR and / or calcineurin. In some embodiments, the complex binds to a target protein with an affinity that is at least 5-fold (e.g., at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold) greater than the affinity of the compound for the target protein when the compound is not bound in complex with the presenter protein. In some embodiments, the complex binds to a target protein with an affinity that is at least 5-fold (e.g., at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold) greater than the affinity of the presenter protein for the target protein when the presenter protein is not bound in complex with the compound. In certain embodiments, the complex inhibits a naturally occurring interaction between a target protein and a ligand that specifically binds to the target protein.

[0063] In some embodiments, the presenter protein is a prolyl isomerase (e.g., a member of the FKBP family such as FKBP12, FKBP12.6, FKBP25, FKBP52, or a member of the cyclophilin family such as PP1A, CYPB, CYPC, CYP40, CYPE, CYPD, NKTR, SRCyp, CYPH, CWC27, CYPL1, CYP60, CYPJ, PPIL4, PPIL6, RANBP2, PPWD1, or PIN1).

[0064] In certain embodiments, the target protein is a eukaryotic target protein. In some embodiments, the eukaryotic target protein is a mammalian target protein such as a GTPase, a GTPase-activating protein, a guanine nucleotide exchange factor, a heat shock protein, an ion channel, a coiled-coil protein, a kinase, a phosphatase, a ubiquitin ligase, a transcription factor, a chromatin modifier / remodeler, a protein with classical protein-protein interaction domains and motifs, or any other protein involved in a biological pathway associated with a disease, disorder, or pathology. In some embodiments, the eukaryotic target protein is a fungal target protein. In certain embodiments, the target protein is a prokaryotic target protein, such as a bacterial target protein.

[0065] In some embodiments, the compound is any one of the compounds of Figure 1 or a stereoisomer thereof or a pharmaceutically acceptable salt thereof. In certain embodiments, the compound is any one of the compounds of Figure 2 or a stereoisomer thereof or a pharmaceutically acceptable salt thereof.

[0066] In some aspects, the invention features a presenter protein / compound complex comprising any of the compounds of the invention and a presenter protein. In some embodiments of the presenter protein / compound complex, the presenter protein is a protein encoded by any one of the genes in Table 1, or a homolog thereof. In some embodiments of the presenter protein / compound complex, the presenter protein is a prolyl isomerase (e.g., a member of the FKBP family, such as FKBP12, FKBP12.6, FKBP25, FKBP52, or a member of the cyclophilin family, such as PP1A, CYPB, CYPC, CYP40, CYPE, CYPD, NKTR, SRCyp, CYPH, CWC27, CYPL1, CYP60, CYPJ, PPIL4, PPIL6, RANBP2, PPWD1, or PIN1).

[0067] In some aspects, the invention features a pharmaceutical composition including any of the compounds or conjugates of the invention and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is a unit dosage form.

[0068] In some aspects, the invention features methods for modulating a target protein (e.g., a eukaryotic target protein, such as a mammalian target protein or a fungal target protein, or a prokaryotic target protein, such as a bacterial target protein). In some embodiments, such methods include contacting the target protein with a modulating (e.g., positively or negatively modulating) amount of a compound (e.g., in the presence of a presenter protein), presenter protein / compound complex, or composition of the invention.

[0069] In some aspects, the invention features methods for modulating (e.g., positively or negatively modulating) a target protein (e.g., a eukaryotic target protein, such as a mammalian target protein or a fungal target protein, or a prokaryotic target protein, such as a bacterial target protein). In some embodiments, such methods include modulating (e.g., positively or negatively modulating) the target protein by contacting a cell expressing the target protein and a presenter protein with an effective amount of a compound or composition of the invention under conditions that allow the compound to form a complex with the presenter protein and allow the resulting complex to bind to the target protein.

[0070] In some aspects, the invention features methods for modulating (e.g., positively or negatively modulating) a target protein (e.g., a eukaryotic target protein, such as a mammalian target protein or a fungal target protein, or a prokaryotic target protein, such as a bacterial target protein). In some embodiments, such methods include modulating the target protein by contacting the target protein with a presenter protein / compound complex of the invention.

[0071] In some aspects, the invention features methods for inhibiting prolyl isomerase activity. In some embodiments, such methods include inhibiting prolyl isomerase activity by contacting a cell expressing prolyl isomerase with a compound or composition of the invention under conditions that allow for the formation of a complex between the compound and prolyl isomerase.

[0072] In some aspects, the invention features methods for forming a presenter protein / compound complex in a cell. In some embodiments, such methods include contacting a cell expressing a presenter protein with a compound or composition of the invention under conditions that allow for the formation of a complex between the compound and the presenter protein.

[0073] In some aspects, the invention features methods for preparing compounds of the invention. In some embodiments, such methods include culturing a bacterial strain of the genus Streptomyces and isolating the compound from the fermentation broth. In some embodiments, the bacterial strain is an engineered strain. In some embodiments, the bacterial strain is engineered in that it has been modified to produce the compound and / or secrete the compound into the broth.

[0074] In some embodiments, the disclosure provides a method of preparing a compound described herein, the method comprising culturing a bacterial strain of the genus Streptomyces under conditions wherein the strain produces the compound and releases it into a fermentation broth, and isolating the compound from the fermentation broth.

[0075] In some embodiments, the methods provided include isolating a compound described herein from the fermentation broth. In some aspects, the invention features a tripartite complex that includes (i) a target protein (e.g., a eukaryotic target protein, such as a mammalian target protein or a fungal target protein, or a prokaryotic target protein, such as a bacterial target protein), and (ii) a presenter protein / compound complex, where the presenter protein / compound complex includes the presenter protein and any of the compounds of the invention.

[0076] In some embodiments of the tripartite complex, the target protein (e.g., a eukaryotic target protein such as a mammalian target protein or a fungal target protein, or a prokaryotic target protein such as a bacterial target protein) does not have a conventional binding pocket. In some embodiments of the tripartite complex, the presenter protein / compound complex binds to a flat surface site on the target protein. In certain embodiments of the tripartite complex, the compound (e.g., a macrocyclic compound) in the presenter protein / compound complex binds to a hydrophobic surface site on the target protein (e.g., a hydrophobic surface site on the target protein that comprises at least 30%, e.g., at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% hydrophobic residues). In some embodiments of the tripartite complex, the presenter protein / compound complex binds to the target protein at a naturally occurring protein-protein interaction site between the target protein and a protein that specifically binds to said target protein. In some embodiments of the tripartite complex, the presenter protein / compound complex does not bind to an active site on the target protein. In certain embodiments of the tripartite complex, the presenter protein / compound complex binds to the active site of the target protein. In some embodiments of the tripartite complex, the target protein is an undruggable target.

[0077] In some embodiments of the tripartite complex, the structural organization of the compound (e.g., macrocyclic compound) is substantially unchanged in the tripartite complex compared to the compound (e.g., macrocyclic compound) in the presenter protein / compound complex but not in the tripartite complex.

[0078] In certain embodiments of a tripartite complex, at least 10% (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%) of the total buried surface area of ​​the target protein in the tripartite complex comprises one or more atoms involved in binding to a compound (e.g., a macrocyclic compound). In some embodiments of a tripartite complex, at least 10% (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%) of the total buried surface area of ​​the target protein in the tripartite complex comprises one or more atoms involved in binding to a presenter protein.

[0079] In some embodiments of the tripartite complex, the compound (e.g., the macrocyclic compound) contributes at least 10% (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%) of the total binding free energy of the tripartite complex. In certain embodiments of the tripartite complex, the presenter protein contributes at least 10% (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%) of the total binding free energy of the tripartite complex.

[0080] In some embodiments of the tripartite complex, at least 70% (e.g., at least 80%, at least 90%, at least 95%) of the binding interactions between one or more atoms of the compound (e.g., the macrocyclic compound) and one or more atoms of the target protein (e.g., a eukaryotic target protein, such as a mammalian target protein or a fungal target protein, or a prokaryotic target protein, such as a bacterial target protein) are van der Waals interactions and / or π-effect interactions.

[0081] In another aspect, the invention features a compound collection that includes a plurality of compounds (e.g., of the macrocyclic compounds described herein). In some embodiments, the compound collection includes a plurality of compounds that are variants of one another. chemical terms It will be appreciated by those of skill in the art that certain compounds described herein can exist in one or more different isomeric (e.g., stereoisomers, geometric isomers, tautomers) and / or isotopic (replacement of one or more atoms with a different isotope of that atom, e.g., replacement of hydrogen with deuterium) forms. Unless otherwise specified or apparent from context, depicted structures can be understood to represent any such isomeric or isotopic forms, either alone or in combination.

[0082] The compounds described herein can be asymmetric (e.g., having one or more stereocenters). Unless otherwise specified, all stereoisomers, such as enantiomers and diastereomers, are intended. Compounds of the present disclosure that contain asymmetrically substituted carbon atoms can be isolated in optically active or racemic forms. Methods on how to prepare optically active forms from optically active starting materials are known in the art, for example, by resolution of racemic mixtures or by stereoselective synthesis. Many geometric isomers of olefins, C=N double bonds, and the like can also be present in the compounds described herein, and all such stable isomers are contemplated in the present disclosure. Cis and trans geometric isomers of the compounds of the present disclosure are described and may be isolated as a mixture of isomers or as separated isomeric forms.

[0083] In some embodiments, one or more compounds depicted herein may exist in various tautomeric forms. Unless explicitly excluded, as will be clear from the context, reference to such a compound encompasses all such tautomeric forms. In some embodiments, tautomeric forms result from the swapping of a single bond with an adjacent double bond and the accompanying migration of a proton. In certain embodiments, a tautomeric form may be a prototropic tautomer of an isomeric protonation state having the same empirical formula and total charge as the referenced form. Examples of prototropic tautomeric moieties are ketone-enol pairs, amide-imidic acid pairs, lactam-lactim pairs, amide-imidic acid pairs, enamine-imine pairs, and cyclic forms in which protons can occupy more than one position on a heterocyclic ring system, such as 1H- and 3H-imidazole, 1H-, 2H-, and 4H-1,2,4-triazole, 1H- and 2H-isoindole, and 1H- and 2H-pyrazole. In some embodiments, tautomeric forms may be in equilibrium or sterically locked to one form by appropriate substitution. In certain embodiments, tautomeric forms may undergo acetal interconversion, for example, as shown in the following scheme:

[0084] [ka]

[0085] This arises from interconversions exemplified by: Those skilled in the art will appreciate that in some embodiments, isotopes of the compounds described herein may be prepared and / or utilized in accordance with the present invention. "Isotopes" refer to atoms having the same atomic number but different mass numbers resulting from different numbers of neutrons in the nuclei. For example, isotopes of hydrogen include tritium and deuterium. In some embodiments, isotopic substitution (e.g., replacement of hydrogen with deuterium) may alter the physicochemical properties of a molecule, such as metabolism and / or racemization rate of a chiral center.

[0086] As is known in the art, many chemical entities (particularly many organic molecules and / or many small molecules) can exist in a variety of solid forms, e.g., amorphous and / or crystalline forms (e.g., polymorphs, hydrates, solvates, etc.). In some embodiments, such entities can be utilized in any form, including any solid form. In some embodiments, such entities are utilized in a particular form, e.g., a particular solid form.

[0087] In some embodiments, the compounds described and / or represented herein may be provided and / or utilized in salt form. In certain embodiments, the compounds described and / or represented herein may be provided and / or available in hydrated or solvated form.

[0088] At various places in the present specification, substituents of compounds of the present disclosure are disclosed in groups or in ranges. It is specifically intended that the present disclosure include all individual subcombinations of the members of such groups and ranges. For example, "C 1~6 The term "alkyl" is specifically intended to individually disclose methyl, ethyl, C alkyl, C alkyl, C alkyl, and C alkyl. Furthermore, where a compound contains multiple positions where substitution is disclosed in a group or range, unless otherwise specified, the disclosure is intended to cover individual compounds and groups of compounds (e.g., families and subfamily) containing all individual subcombinations of members at each position.

[0089] As used herein, phrases of the form "optionally substituted X" (e.g., optionally substituted alkyl) are intended to be equivalent to "X when X is optionally substituted" (e.g., "alkyl when alkyl is optionally substituted"). It is not intended to imply that the feature "X" (e.g., alkyl) itself is optional.

[0090] The term "alkyl," as used herein, refers to a saturated hydrocarbon group containing 1 to 20 (e.g., 1 to 10 or 1 to 6) carbons. In some embodiments, the alkyl group is unbranched (i.e., linear). In some embodiments, the alkyl group is branched. Alkyl groups are exemplified by methyl, ethyl, n- and iso-propyl, n-, sec-, iso-, and tert-butyl, neopentyl, and the like, and include (1) alkoxy, (2) alkylsulfinyl, (3) amino, as defined herein (e.g., unsubstituted amino (i.e., -NH) or substituted amino (i.e., -N(R N1 )2(wherein, R N1 is as defined for amino), (4) C 6~10 Aryl C 1~6 Alkoxy, (5) Azido, (6) Halo, (7) (C 2~9 heterocyclyl)oxy, (8) hydroxyl optionally substituted with an O-protecting group, (9) nitro, (10) oxo (e.g., carboxaldehyde or acyl), (11) C 1~7 Spirocyclyl, (12) thioalkoxy, (13) thiol, (14) —COR optionally substituted with an O-protecting group A’ (In the formula, R A’ is (a)C 1~20 Alkyl (e.g., C 1~6 alkyl), (b) C 2~20 Alkenyl (e.g., C 2~6 alkenyl), (c) C 6~10 Aryl, (d) hydrogen, (e) C 1~6 ALC-C 6~10 Aryl, (f) Amino-C 1~20 Alkyl, (g)-(CH2) s2 (OCH2CH2) s1 (CH2) s3 OR' (wherein s1 is an integer of 1 to 10 (e.g., 1 to 6 or 1 to 4), s2 and s3 are each independently an integer of 0 to 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and R' is H or C 1~20 (h) -NR N1 (CH2)s2 (CH2CH2O) s1 (CH2) s3 NR N1 (wherein s1 is an integer of 1 to 10 (e.g., 1 to 6 or 1 to 4), s2 and s3 are each independently an integer of 0 to 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and each R N1 are independently hydrogen or optionally substituted C 1~6 (15) —C(O)NR B’ R C’ (In the formula, R B’ and R C’ each of which is independently (a) hydrogen, (b) C 1~6 Alkyl, (c) C 6~10 aryl, and (d) C 1~6 ALC-C 6~10 aryl), (16)-SO2R D’ (In the formula, R D’ is (a)C 1~6 Alkyl, (b) C 6~10 Aryl, (c) C 1~6 ALC-C 6~10 (17) -SO2NR E’ R F’ (In the formula, R E’ and R F’ each of which is independently (a) hydrogen, (b) C 1~6 Alkyl, (c) C 6~10 aryl, and (d) C 1~6 ALC-C 6~10 aryl), (18)—C(O)R G’ (In the formula, R G’ is (a)C 1~20 Alkyl (e.g., C 1~6 alkyl), (b) C 2~20 Alkenyl (e.g., C 2~6 alkenyl), (c) C 6~10 Aryl, (d) hydrogen, (e) C 1~6 ALC-C 6~10 Aryl, (f) Amino-C1~20 Alkyl, (g)-(CH2) s2 (OCH2CH2) s1 (CH2) s3 OR' (wherein s1 is an integer of 1 to 10 (e.g., 1 to 6 or 1 to 4), s2 and s3 are each independently an integer of 0 to 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and R' is H or C 1~20 (h) -NR N1 (CH2) s2 (CH2CH2O) s1 (CH2) s3 NR N1 (wherein s1 is an integer of 1 to 10 (e.g., 1 to 6 or 1 to 4), s2 and s3 are each independently an integer of 0 to 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and each R N1 are independently hydrogen or optionally substituted C 1~6 (19)-NR H’ C(O)R I’ (In the formula, R H’ is (a1) hydrogen and (b1) C 1~6 alkyl, and R I’ is (a2)C 1~20 Alkyl (e.g., C 1~6 alkyl), (b2) C 2~20 Alkenyl (e.g., C 2~6 alkenyl), (c2) C 6~10 Aryl, (d2) hydrogen, (e2) C 1~6 ALC-C 6~10 Aryl, (f2) Amino-C 1~20 Alkyl, (g2)-(CH2) s2 (OCH2CH2) s1 (CH2) s3 OR' (wherein s1 is an integer of 1 to 10 (e.g., 1 to 6 or 1 to 4), s2 and s3 are each independently an integer of 0 to 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and R' is H or C 1~20(h2)-NR N1 (CH2) s2 (CH2CH2O) s1 (CH2) s3 NR N1 (wherein s1 is an integer of 1 to 10 (e.g., 1 to 6 or 1 to 4), s2 and s3 are each independently an integer of 0 to 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and each R N1 are independently hydrogen or optionally substituted C 1~6 (20)-NR J’ C(O)OR K’ (In the formula, R J’ is (a1) hydrogen and (b1) C 1~6 alkyl, and R K’ is (a2)C 1~20 Alkyl (e.g., C 1~6 alkyl), (b2) C 2~20 Alkenyl (e.g., C 2~6 alkenyl), (c2) C 6~10 Aryl, (d2) hydrogen, (e2) C 1~6 ALC-C 6~10 Aryl, (f2) Amino-C 1~20 Alkyl, (g2)-(CH2) s2 (OCH2CH2) s1 (CH2) s3 OR' (wherein s1 is an integer of 1 to 10 (e.g., 1 to 6 or 1 to 4), s2 and s3 are each independently an integer of 0 to 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and R' is H or C 1~20 (h2)-NR N1 (CH2) s2 (CH2CH2O) s1 (CH2) s3 NR N1(wherein s1 is an integer of 1 to 10 (e.g., 1 to 6 or 1 to 4), s2 and s3 are each independently an integer of 0 to 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and each R N1 are independently hydrogen or optionally substituted C 1~6 (21) amidine; and (22) silyl groups, such as trimethylsilyl, t-butyldimethylsilyl, triisopropylsilyl, and the like. In some embodiments, each of these groups can be further substituted as described herein. For example, the alkylene group of a C1-alkaryl can be further substituted with an oxo group to provide the respective aryloyl substituent.

[0091] The terms "alkylene" and the prefix "alk-" as used herein refer to a saturated divalent hydrocarbon group derived from a straight or branched chain saturated hydrocarbon by removing two hydrogen atoms, and are exemplified by methylene, ethylene, isopropylene, and the like. x~y The terms "alkylene" and "C x~y The prefix "alk-" denotes an alkylene group having x to y carbons. Exemplary values ​​of x are 1, 2, 3, 4, 5, and 6, and exemplary values ​​of y are 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, or 20 (e.g., C 1~6 , C 1~10 , C 2~20 , C 2~6 , C 2~10 , or C 2~20 In some embodiments, the alkylene can be further substituted with 1, 2, 3, or 4 substituents, as defined herein for an alkyl group.

[0092] The term "alkenyl," as used herein, unless otherwise specified, represents a monovalent straight- or branched-chain group of 2 to 20 carbons (e.g., 2 to 6 or 2 to 10 carbons) containing one or more carbon-carbon double bonds and is exemplified by ethenyl, 1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, and the like. Alkenyl includes both cis and trans isomers. Alkenyl groups can be optionally substituted with 1, 2, 3, or 4 substituents independently selected from amino, aryl, cycloalkyl, or heterocyclyl (e.g., heteroaryl), as defined herein, or any of the exemplary alkyl substituents described herein.

[0093] The term "alkynyl," as used herein, refers to a monovalent straight- or branched-chain group of 2 to 20 carbon atoms (e.g., 2 to 4, 2 to 6, or 2 to 10 carbons) containing a carbon-carbon triple bond, and is exemplified by ethynyl, 1-propynyl, and the like. Alkynyl groups can be optionally substituted with 1, 2, 3, or 4 substituents independently selected from aryl, cycloalkyl, or heterocyclyl (e.g., heteroaryl), as defined herein, or any of the exemplary alkyl substituents described herein.

[0094] As used herein, the term "amino" refers to -N(R N1 )2(in the formula, each R N1 are independently H, OH, NO2, N(R N2 )2, SO2OR N2 , SO2R N2 , SOR N2, an N-protecting group, alkyl, alkenyl, alkynyl, alkoxy, aryl, alkaryl, cycloalkyl, alkcycloalkyl, carboxyalkyl (e.g., optionally substituted with an O-protecting group such as an optionally substituted arylalkoxycarbonyl group or any described herein), sulfoalkyl, acyl (e.g., acetyl, trifluoroacetyl, or others described herein), alkoxycarbonylalkyl (e.g., optionally substituted with an O-protecting group such as an optionally substituted arylalkoxycarbonyl group or any described herein), heterocyclyl (e.g., heteroaryl), or alkheterocyclyl (e.g., alkheteroaryl), and these enumerated R N1 Each of the groups can be optionally substituted as defined herein for each group, or two R N1 are combined to form a heterocyclyl or N-protecting group, and each R N2 are independently H, alkyl, or aryl). The amino group of the present invention can be unsubstituted amino (i.e., —NH) or substituted amino (i.e., —N(R N1 )2). In a preferred embodiment, amino can be -NH2 or -NHR N1 (In the formula, R N1 are independently OH, NO2, NH2, and NR N2 2. SO2OR N2 , SO2R N2 , SOR N2 , alkyl, carboxyalkyl, sulfoalkyl, acyl (e.g., acetyl, trifluoroacetyl, or others described herein), alkoxycarbonylalkyl (e.g., t-butoxycarbonylalkyl), or aryl, and each R N2 is H, C 1~20 Alkyl (e.g., C 1~6 alkyl), or C 6~10 aryl).

[0095] As used herein, the term "amino acid" refers to a molecule having a side chain, an amino group, and an acid group (e.g., a carboxy group of -COH or a sulfo group of -SOH), where the amino acid is attached to the parent molecular moiety by the side chain, the amino group, or the acid group (e.g., the side chain). As used herein, the term "amino acid" in its broadest sense refers to any compound and / or substance that can be incorporated into a polypeptide chain, for example, by the formation of one or more peptide bonds. In some embodiments, an amino acid has the general structure HN-C(H)(R)-COOH. In some embodiments, an amino acid is a naturally occurring amino acid. In some embodiments, an amino acid is a synthetic amino acid. In some embodiments, an amino acid is a D-amino acid. In some embodiments, an amino acid is an L-amino acid. A "standard amino acid" refers to any of the 20 standard L-amino acids commonly found in naturally occurring peptides. A "non-standard amino acid" refers to any amino acid other than the standard amino acids, whether prepared synthetically or obtained from a natural source. In some embodiments, amino acids, including those at the carboxy and / or amino termini in a polypeptide, may contain structural modifications relative to the above general structure. For example, in some embodiments, amino acids may be modified by methylation, amidation, acetylation, and / or substitution relative to the general structure. In some embodiments, such modifications may, for example, alter the circulating half-life of a polypeptide containing the modified amino acid compared to one containing the otherwise identical unmodified amino acid. In some embodiments, such modifications do not significantly alter the relevant activity of a polypeptide containing the modified amino acid compared to one containing the otherwise identical unmodified amino acid. As will be clear from the context, in some embodiments, the term "amino acid" is used to refer to a free amino acid. In some embodiments, it is used to refer to an amino acid residue of a polypeptide. In some embodiments, an amino acid is attached to a parent molecular moiety by a carbonyl group, with the side chain or amino group attached to the carbonyl group. In some embodiments, an amino acid is an α-amino acid. In certain embodiments, an amino acid is a β-amino acid.In some embodiments, the amino acid is a γ-amino acid. Exemplary side chains include optionally substituted alkyl, aryl, heterocyclyl, alkaryl, alkheterocyclyl, aminoalkyl, carbamoylalkyl, and carboxyalkyl. Exemplary amino acids include alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, hydroxynorvaline, isoleucine, leucine, lysine, methionine, norvaline, ornithine, phenylalanine, proline, pyrrolysine, selenocystine, serine, taurine, threonine, tryptophan, tyrosine, and valine. The amino acid group is (1)C. 1~6 Alkoxy, (2)C 1~6 alkylsulfinyl, (3) amino, as defined herein (e.g., unsubstituted amino (i.e., —NH) or substituted amino (i.e., —N(R N1 )2(wherein, R N1 is as defined for amino), (4) C 6~10 Aryl C 1~6 Alkoxy, (5) Azido, (6) Halo, (7) (C 2~9 heterocyclyl)oxy, (8) hydroxyl, (9) nitro, (10) oxo (e.g., carboxaldehyde or acyl), (11) C 1~7 Spirocyclyl, (12) thioalkoxy, (13) thiol, (14) -CO2R A’ (In the formula, R A’ is (a)C 1~20 Alkyl (e.g., C 1~6 alkyl), (b) C 2~20 Alkenyl (e.g., C 2~6 alkenyl), (c) C 6~10 Aryl, (d) hydrogen, (e) C 1~6 ALC-C 6~10 Aryl, (f) Amino-C 1~20 Alkyl, (g)-(CH2) s2 (OCH2CH2) s1 (CH2) s3OR' (wherein s1 is an integer of 1 to 10 (e.g., 1 to 6 or 1 to 4), s2 and s3 are each independently an integer of 0 to 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and R' is H or C 1~20 (h) -NR N1 (CH2) s2 (CH2CH2O) s1 (CH2) s3 NR N1 (wherein s1 is an integer of 1 to 10 (e.g., 1 to 6 or 1 to 4), s2 and s3 are each independently an integer of 0 to 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and each R N1 are independently hydrogen or optionally substituted C 1~6 (15) —C(O)NR B’ R C’ (In the formula, R B’ and R C’ each of which is independently (a) hydrogen, (b) C 1~6 Alkyl, (c) C 6~10 aryl, and (d) C 1~6 ALC-C 6~10 aryl), (16)-SO2R D’ (In the formula, R D’ is (a)C 1~6 Alkyl, (b) C 6~10 Aryl, (c) C 1~6 ALC-C 6~10 (17) -SO2NR E’ R F’ (In the formula, R E’ and R F’ each of which is independently (a) hydrogen, (b) C 1~6 Alkyl, (c) C 6~10 aryl, and (d) C 1~6 ALC-C 6~10 aryl), (18)—C(O)R G’ (In the formula, R G’ is (a)C1~20 Alkyl (e.g., C 1~6 alkyl), (b) C 2~20 Alkenyl (e.g., C 2~6 alkenyl), (c) C 6~10 Aryl, (d) hydrogen, (e) C 1~6 ALC-C 6~10 Aryl, (f) Amino-C 1~20 Alkyl, (g)-(CH2) s2 (OCH2CH2) s1 (CH2) s3 OR' (wherein s1 is an integer of 1 to 10 (e.g., 1 to 6 or 1 to 4), s2 and s3 are each independently an integer of 0 to 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and R' is H or C 1~20 (h) -NR N1 (CH2) s2 (CH2CH2O) s1 (CH2) s3 NR N1 (wherein s1 is an integer of 1 to 10 (e.g., 1 to 6 or 1 to 4), s2 and s3 are each independently an integer of 0 to 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and each R N1 are independently hydrogen or optionally substituted C 1~6 (19)-NR H’ C(O)R I’ (In the formula, R H’ is (a1) hydrogen and (b1) C 1~6 alkyl, and R I’ is (a2)C 1~20 Alkyl (e.g., C 1~6 alkyl), (b2) C 2~20 Alkenyl (e.g., C 2~6 alkenyl), (c2) C 6~10 Aryl, (d2) hydrogen, (e2) C 1~6 ALC-C 6~10 Aryl, (f2) Amino-C 1~20 Alkyl, (g2)-(CH2) s2(OCH2CH2) s1 (CH2) s3 OR' (wherein s1 is an integer of 1 to 10 (e.g., 1 to 6 or 1 to 4), s2 and s3 are each independently an integer of 0 to 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and R' is H or C 1~20 (h2)-NR N1 (CH2) s2 (CH2CH2O) s1 (CH2) s3 NR N1 (wherein s1 is an integer of 1 to 10 (e.g., 1 to 6 or 1 to 4), s2 and s3 are each independently an integer of 0 to 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and each R N1 are independently hydrogen or optionally substituted C 1~6 (20)-NR J’ C(O)OR K’ (In the formula, R J’ is (a1) hydrogen and (b1) C 1~6 alkyl, and R K’ is (a2)C 1~20 Alkyl (e.g., C 1~6 alkyl), (b2) C 2~20 Alkenyl (e.g., C 2~6 alkenyl), (c2) C 6~10 Aryl, (d2) hydrogen, (e2) C 1~6 ALC-C 6~10 Aryl, (f2) Amino-C 1~20 Alkyl, (g2)-(CH2) s2 (OCH2CH2) s1 (CH2) s3 OR' (wherein s1 is an integer of 1 to 10 (e.g., 1 to 6 or 1 to 4), s2 and s3 are each independently an integer of 0 to 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and R' is H or C 1~20 (h2)-NR N1(CH2) s2 (CH2CH2O) s1 (CH2) s3 NR N1 (wherein s1 is an integer of 1 to 10 (e.g., 1 to 6 or 1 to 4), s2 and s3 are each independently an integer of 0 to 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and each R N1 are independently hydrogen or optionally substituted C 1~6 (21) amidines), and (22) methyl-, ...

[0096] As used herein, the term "N-alkylated amino acid" refers to an amino acid containing a C1-C6 alkyl optionally substituted on the nitrogen of the amino acid that forms the peptide bond. N-alkylated amino acids include, but are not limited to, N-methyl amino acids such as N-methyl-alanine, N-methyl-threonine, N-methyl-phenylalanine, N-methyl-aspartic acid, N-methyl-valine, N-methyl-leucine, N-methyl-glycine, N-methyl-isoleucine, N(α)-methyl-lysine, N(α)-methyl-asparagine, and N(α)-methyl-glutamine.

[0097] The term "aryl" as used herein refers to a monocyclic, bicyclic, or polycyclic carbocyclic ring system having one or two aromatic rings, and is exemplified by phenyl, naphthyl, 1,2-dihydronaphthyl, 1,2,3,4-tetrahydronaphthyl, anthracenyl, phenanthrenyl, fluorenyl, indanyl, indenyl, and the like, and includes (1)C 1~7 Acyl (e.g., carboxaldehyde), (2) C 1~20 Alkyl (e.g., C 1~6 Alkyl, alkoxy-C 1~6 Alkyl, C1~6 Alkylsulfinyl-C 1~6 Alkyl, Amino-C 1~6 Alkyl, azido-C 1~6 Alkyl, (carboxaldehyde)-C 1~6 Alkyl, Halo-C 1~6 Alkyl (e.g., perfluoroalkyl), hydroxy-C 1~6 Alkyl, nitro-C 1~6 Alkyl or C 1~6 Thioalkoxy-C 1~6 alkyl), (3) C 1~20 Alkoxy (e.g., C 1~6 alkoxy, for example perfluoroalkoxy), (4) C 1~6 Alkylsulfinyl, (5)C 6~10 Aryl, (6) Amino, (7) C 1~6 ALC-C 6~10 Aryl, (8) Azide, (9) C 3~8 Cycloalkyl, (10)C 1~6 ALC-C 3~8 Cycloalkyl, (11) halo, (12) C 1~12 Heterocyclyl (e.g., C 1~12 Heteroaryl), (13)(C 1~12 (heterocyclyl)oxy, (14) hydroxyl, (15) nitro, (16) C 1~20 Thioalkoxy (e.g., thioalkoxy), (17)-(CH2) q CO2R A’ (wherein q is an integer of 0 to 4, and R A’ is (a) alkyl, (b) C 6~10 aryl, (c) hydrogen, and (d) C 1~6 ALC-C 6~10 aryl), (18)-(CH2) q CONR B’ R C’ (wherein q is an integer of 0 to 4, and R B’ and R C’ is (a) hydrogen, (b) C 1~6 Alkyl, (c) C 6~10 aryl, and (d) C 1~6 ALC-C 6~10aryl), (19)—(CH) q SO2R D’ (wherein q is an integer of 0 to 4, and R D’ is (a) alkyl, (b) C 6~10 aryl, and (c) alk-C 6~10 aryl), (20)-(CH2) q SO2NR E’ R F’ (wherein q is an integer of 0 to 4, and R E’ and R F’ are (a) hydrogen, (b) C 1~6 Alkyl, (c) C 6~10 aryl, and (d) C 1~6 ALC-C 6~10 (21) thiol; (22) C 6~10 Aryloxy, (23)C 3~8 Cycloalkoxy, (24)C 6~10 Aryl C 1~6 Alkoxy, (25)C 1~6 ALC-C 1~12 Heterocyclyl (e.g., C 1~6 ALC-C 1~12 Heteroaryl), (26)C 2~20 Alkenyl, and (27)C 2~20 alkynyl. In some embodiments, each of these groups can be further substituted as described herein. For example, the alkylene group of a C1-alkaryl or a C1-alkheterocyclyl can be further substituted with an oxo group to provide an aryloyl and (heterocyclyl)oyl substituent, respectively.

[0098] As used herein, an "arylalkyl" group refers to an aryl group, as defined herein, attached to the parent molecular group by an alkylene group, as defined herein. Exemplary unsubstituted arylalkyl groups include those having 7 to 30 carbons (e.g., 7 to 16 or 7 to 20 carbons, e.g., C 1~6 ALC-C6~10 Aryl, C 1~10 ALC-C 6~10 Aryl, or C 1~20 ALC-C 6~10 In some embodiments, alkylene and aryl can each be further substituted with 1, 2, 3, or 4 substituents as defined herein for that group. Other groups preceded by the prefix "alk-" are similarly defined, where "alk-" refers to any group selected from C 1~6 The alkylene means and the chemical structure to which it is attached is as defined herein.

[0099] The term "azido" refers to the group -N3, which may also be depicted as -N=N=N. As used herein, the terms "carbocyclic" and "carbocyclyl" refer to an optionally substituted C 3~12 It refers to monocyclic, bicyclic, or tricyclic non-aromatic ring structures. Carbocyclic structures include cycloalkyl, cycloalkenyl, and cycloalkynyl groups.

[0100] As used herein, a "carbocyclylalkyl" group represents a carbocyclic group, as defined herein, attached to the parent molecular group by an alkylene group, as defined herein. Exemplary unsubstituted carbocyclylalkyl groups include those having 7 to 30 carbons (e.g., 7 to 16 or 7 to 20 carbons, e.g., C 1~6 ALC-C 6~10 Carbocyclyl, C 1~10 ALC-C 6~10 Carbocyclyl, or C 1~20 ALC-C 6~10 In some embodiments, alkylene and carbocyclyl can each be further substituted with 1, 2, 3, or 4 substituents as defined herein for that group. Other groups preceded by the prefix "alk-" are similarly defined, where "alk-" refers to any group selected from C, C- ... 1~6 The alkylene means and the chemical structure to which it is attached is as defined herein.

[0101] The term "carbonyl" as used herein refers to a C(O) group, which may also be depicted as C=O. The term "carboxy" as used herein means -CO2H.

[0102] As used herein, the term "cyano" refers to a -CN group. The term "cycloalkyl," as used herein, unless otherwise specified, refers to a monovalent saturated or unsaturated non-aromatic cyclic hydrocarbon group of 3 to 8 carbons, and is exemplified by cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, bicycloheptyl, and the like. When a cycloalkyl group contains one carbon-carbon double bond, the cycloalkyl group can be referred to as a "cycloalkenyl" group. Exemplary cycloalkenyl groups include cyclopentenyl, cyclohexenyl, and the like. Cycloalkyl groups of the present invention include (1) C 1~7 acyl (e.g., carboxaldehyde), (2) C1-20 alkyl (e.g., alkyl, alkoxy-C1-6 alkyl, alkylsulfinyl-C1-6 alkyl, amino-C1-6 alkyl, azido-C1-6 alkyl, (carboxaldehyde)-C1-6 alkyl, halo-C1-6 alkyl (e.g., perfluoroalkyl), hydroxy-C1-6 alkyl, nitro-C1-6 alkyl, or C1-6 thioalkoxy-C1-6 alkyl), (3) C 1~20 Alkoxy (e.g., C 1~6 alkoxy, for example perfluoroalkoxy), (4) C 1~6 Alkylsulfinyl, (5)C 6~10 Aryl, (6) Amino, (7) C 1~6 ALC-C 6~10 Aryl, (8) Azide, (9) C 3~8 Cycloalkyl, (10)C 1~6 ALC-C 3~8 Cycloalkyl, (11) halo, (12) C 1~12 Heterocyclyl (e.g., C 1~12 Heteroaryl), (13)(C 1~12heterocyclyl)oxy, (14) hydroxyl, (15) nitro, (16) C1-20 thioalkoxy (e.g., C1-6 thioalkoxy), (17) —(CH2) q CO2R A’ (wherein q is an integer of 0 to 4, and R A’ is (a)C 1~6 Alkyl, (b) C 6~10 aryl, (c) hydrogen, and (d) C 1~6 ALC-C 6~10 aryl), (18)-(CH2) q CONR B’ R C’ (wherein q is an integer of 0 to 4, and R B’ and R C’ is (a) hydrogen, (b) C 6~10 Alkyl, (c) C 6~10 aryl, and (d) C 1~6 ALC-C 6~10 aryl), (19)—(CH) q SO2R D’ (wherein q is an integer of 0 to 4, and R D’ is (a)C 6~10 Alkyl, (b) C 6~10 aryl, and (c) C 1~6 ALC-C 6~10 aryl), (20)-(CH2) q SO2NR E’ R F’ (wherein q is an integer of 0 to 4, and R E’ and R F’ are (a) hydrogen, (b) C 6~10 Alkyl, (c) C 6~10 aryl, and (d) C 1~6 ALC-C 6~10 (21) thiol, (22) C 6~10 Aryloxy, (23)C 3~8 Cycloalkoxy, (24)C 6~10 Aryl C 1~6 Alkoxy, (25)C 1~6 ALC-C 1~12 Heterocyclyl (e.g., C1~6 ALC-C 1~12 Heteroaryl), (26) oxo, (27) C 2~20 Alkenyl, and (28)C 2~20 and (heterocyclyl)oyl. In some embodiments, each of these groups can be further substituted as described herein. For example, the alkylene group of a C1-alkaryl or a C1-alkheterocyclyl can be further substituted with an oxo group to provide an aryloyl and (heterocyclyl)oyl substituent, respectively.

[0103] As used herein, a "cycloalkylalkyl" group refers to a cycloalkyl group, as defined herein, attached to the parent molecular group by an alkylene group, as defined herein (e.g., an alkylene group of 1 to 4, 1 to 6, 1 to 10, or 1 to 20 carbons). In some embodiments, the alkylene and cycloalkyl can each be further substituted with 1, 2, 3, or 4 substituents, as defined herein for that group.

[0104] As used herein, the term "diastereomers" means stereoisomers that are not mirror images of one another and are not superimposable with respect to one another. As used herein, the term "enantiomer" refers to each individual optically active form of a compound of the present invention having an optical purity or enantiomeric excess (as determined by standard methods in the art) of at least 80% (i.e., at least 90% of one enantiomer and at most 10% of the other enantiomer), preferably at least 90%, more preferably at least 98%.

[0105] The term "halo" as used herein refers to a halogen selected from bromine, chlorine, iodine, or fluorine. As used herein, the term "heteroalkyl" refers to an alkyl group, as defined herein, in which one or two of the constituent carbon atoms are replaced by nitrogen, oxygen, or sulfur, respectively. In some embodiments, heteroalkyl groups can be further substituted with one, two, three, or four substituents as described herein for alkyl groups. As used herein, the terms "heteroalkenyl" and "heteroalkynyl" refer to alkenyl and alkynyl groups, as defined herein, respectively, in which one or two of the constituent carbon atoms are replaced by nitrogen, oxygen, or sulfur, respectively. In some embodiments, heteroalkenyl and heteroalkynyl groups can be further substituted with one, two, three, or four substituents as described herein for alkyl groups.

[0106] As used herein, the term "heteroaryl" refers to the subset of heterocyclyl, as defined herein, that is aromatic; i.e., they contain 4n+2 pi-electrons in a single ring or polycyclic ring system. Exemplary unsubstituted heteroaryl groups are 1 to 12 (e.g., 1 to 11, 1 to 10, 1 to 9, 2 to 12, 2 to 11, 2 to 10, or 2 to 9) carbons. In certain embodiments, heteroaryl is substituted with 1, 2, 3, or 4 substituents as defined for heterocyclyl groups.

[0107] The term "heteroarylalkyl" means a heteroaryl group, as defined herein, attached to the parent molecular group by an alkylene group, as defined herein. Exemplary unsubstituted heteroarylalkyl groups include those having 2 to 32 carbons (e.g., 2 to 22, 2 to 18, 2 to 17, 2 to 16, 3 to 15, 2 to 14, 2 to 13, or 2 to 12 carbons, e.g., C 1~6 ALC-C 1~12 Heteroaryl, C 1~10 ALC-C 1~12 Heteroaryl, or C 1~20 ALC-C 1~12In some embodiments, alkylene and heteroaryl can each be further substituted with 1, 2, 3, or 4 substituents, as defined herein for each group. Heteroarylalkyl groups are a subset of heterocyclylalkyl groups.

[0108] The term "heterocyclyl," as used herein, unless otherwise specified, refers to a 5-, 6-, or 7-membered ring containing 1, 2, 3, or 4 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur. Five-membered rings have two zero to double bonds, and 6- and 7-membered rings have three zero to double bonds. Exemplary unsubstituted heterocyclyl groups are 1 to 12 (e.g., 1 to 11, 1 to 10, 1 to 9, 2 to 12, 2 to 11, 2 to 10, or 2 to 9) carbons. The term "heterocyclyl" also refers to heterocyclic compounds having bridged polycyclic ring structures in which one or more carbons and / or heteroatoms bridge two non-adjacent members of a single ring, such as a quinuclidinyl group. The term "heterocyclyl" includes bicyclic, tricyclic, and tetracyclic groups in which any of the above heterocycles are fused to one, two, or three carbocyclic rings, such as an aryl ring, a cyclohexane ring, a cyclohexene ring, a cyclopentane ring, a cyclopentene ring, or other monocyclic heterocycles, such as indolyl, quinolyl, isoquinolyl, tetrahydroquinolyl, benzofuryl, benzothienyl, etc. Examples of fused heterocyclyls include tropane and 1,2,3,5,8,8a-hexahydroindolizine, including dihydro and tetrahydro forms in which one or more double bonds have been reduced and replaced with hydrogen. Heterocyclic compounds include pyrrolyl, pyrrolinyl, pyrrolidinyl, pyrazolyl, pyrazolinyl, pyrazolidinyl, imidazolyl, imidazolinyl, imidazolidinyl, pyridyl, piperidinyl, homopiperidinyl, pyrazinyl, piperazinyl, pyrimidinyl, pyridazinyl, oxazolyl, oxazolidinyl, isoxazolyl, isoxazolidinyl, morpholinyl, thiomorpholinyl, thiazolyl, thiazolidinyl, isothiazolyl, and isothiazolyl. zolidinyl, indolyl, indazolyl, quinolyl, isoquinolyl, quinoxalinyl, dihydroquinoxalinyl, quinazolinyl, cinnolinyl, phthalazinyl, benzimidazolyl, benzothiazolyl, benzoxazolyl, benzothiadiazolyl, furyl, thienyl, thiazolidinyl, isothiazolyl, triazolyl, tetrazolyl, oxadiazolyl (e.g., 1,2,3-oxadiazolyl), purinyl, thiadiazolyl (e.g., 1,2,3-thiadiazolyl), tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, dihydrothienyl, dihydroindolyl, dihydroquinolyl, tetrahydroquinolyl, tetrahydroisoquinolyl, dihydroisoquinolyl, pyranyl, dihydropyranyl, dithiazolyl, benzofuranyl, isobenzofuranyl, benzothienyl, etc. Still other exemplary heterocyclyls include 2,3,4,5-tetrahydro-2-oxooxazolyl, 2,3-dihydro-2-oxo-1H-imidazolyl, 2,3,4,5-tetrahydro-5-oxo-1H-pyrazolyl (e.g., 2,3,4,5-tetrahydro-2-phenyl-5-oxo-1H-pyrazolyl), 2,3,4,5-tetrahydro-2,4-dioxo-1H-imidazolyl (e.g., 2,3,4,5-tetrahydro-2,4-dioxo-1H-imidazolyl). 2,3-dihydro-2-thioxo-1,3,4-oxadiazolyl (e.g., 2,3-dihydro-2-thioxo-5-phenyl-1H-imidazolyl), 4,5-dihydro-5-oxo-1H-triazolyl (e.g., 4,5-dihydro-3-methyl-4-amino-5-oxo-1H-triazolyl), 1,2,3,4-tetrahydro-2,4-dioxopyridinyl (e.g., 1,2 ,3,4-tetrahydro-2,4-dioxo-3,3-diethylpyridinyl), 2,6-dioxo-piperidinyl (e.g., 2,6-dioxo-3-ethyl-3-phenylpiperidinyl), 1,6-dihydro-6-oxopyrimidinyl, 1,6-dihydro-4-oxopyrimidinyl (e.g., 2-(methylthio)-1,6-dihydro-4-oxo-5-methylpyrimidin-1-yl), 1,2,3,4-tetrahydro-2,4-dioxopyrimidinyl (e.g., For example, 1,2,3,4-tetrahydro-2,4-dioxo-3-ethylpyrimidinyl), 1,6-dihydro-6-oxo-pyridazinyl (for example, 1,6-dihydro-6-oxo-3-ethylpyridazinyl), 1,6-dihydro-6-oxo-1,2,4-triazinyl (for example, 1,6-dihydro-5-isopropyl-6-oxo-1,2,4-triazinyl), 2,3-dihydro-2-oxo-1H-indolyl (for example, 3,3-dimethyl-2,3-dihydro-2-oxo-1H-indolyl and 2,3-dihydro-2-oxo-3,3'-spiropropane-1H-indol-1-yl), 1,3-dihydro-1-oxo-2H-iso-indolyl, 1,3-dihydro-1,3-dioxo-2H-iso-indolyl, 1H-benzopyrazolyl (for example, 1-(ethoxycarbonyl)-1H-benzopyrazolyl), 2,3-dihydro-2-oxo-1H-benzimidazolyl 2,3-dihydro-2-oxo-benzoxazolyl (e.g., 3-ethyl-2,3-dihydro-2-oxo-1H-benzimidazolyl), 2,3-dihydro-2-oxo-benzoxazolyl (e.g., 5-chloro-2,3-dihydro-2-oxobenzoxazolyl), 2,3-dihydro-2-oxo-benzoxazolyl, 2-oxo-2H-benzopyranyl, 1,4-benzodioxanyl, 1,3-benzodioxanyl, 2,3-dihydro-3-oxo-4H-1, 3-benzothiazinyl, 3,4-dihydro-4-oxo-3H-quinazolinyl (e.g., 2-methyl-3,4-dihydro-4-oxo-3H-quinazolinyl), 1,2,3,4-tetrahydro-2,4-dioxo-3H-quinazolyl (e.g., 1-ethyl-1,2,3,4-tetrahydro-2,4-dioxo-3H-quinazolyl), 1,2,3,6-tetrahydro-2,6-dioxo-7H-purinyl (e.g., 1,2,3 ,6-tetrahydro-1,3-dimethyl-2,6-dioxo-1H-purinyl), 1,2,3,6-tetrahydro-2,6-dioxo-1H-purinyl (e.g., 1,2,3,6-tetrahydro-3,7-dimethyl-2,6-dioxo-1H-purinyl), 2-oxobenz[c,d]indolyl, 1,1-dioxo-2H-naphtho[1,8-c,d]isothiazolyl, and 1,8-naphthylenedicarboxamide. Additional heterocyclic compounds include 3,3a,4,5,6,6a-hexahydro-pyrrolo[3,4-b]pyrrol-(2H)-yl, and 2,5-diazabicyclo[2.2.1]heptan-2-yl, homopiperazinyl (or diazepanyl), tetrahydropyranyl, dithiazolyl, benzofuranyl, benzothienyl, oxepanyl, thiepanyl, azocanyl, oxecanyl, and thiocanyl. Heterocyclic groups also include those of the formula:

[0109] [ka]

[0110] (In the formula, E' is selected from the group consisting of -N- and -CH-; F' is selected from the group consisting of -N=CH-, -NH-CH2-, -NH-C(O)-, -NH-, -CH=N-, -CH2-NH-, -C(O)-NH-, -CH=CH-, -CH2-, -CH2CH2-, -CHO-, -OCH2-, -O-, and -S-; and G' is selected from the group consisting of -CH- and -N-. 1~7 Acyl (e.g., carboxaldehyde), (2) C 1~20 Alkyl (e.g., alkyl, alkoxy-C 1~6 Alkyl, alkylsulfinyl-C 1~6 Alkyl, Amino-C 1~6 Alkyl, azido-C 1~6 Alkyl, (carboxaldehyde)-C 1~6 Alkyl, Halo-C 1~6 Alkyl (e.g., perfluoroalkyl), hydroxy-C 1~6 Alkyl, nitro-C 1~6 Alkyl or C 1~6 Thioalkoxy-C 1~6 alkyl), (3) C 1~20 Alkoxy (e.g., C 1~6 alkoxy, for example perfluoroalkoxy), (4) C 1~6 Alkylsulfinyl, (5)C 6~10 Aryl, (6) Amino, (7) C 1~6 ALC-C 6~10 Aryl, (8) Azide, (9) C 3~8 Cycloalkyl, (10)C 1~6 ALC-C 3~8 Cycloalkyl, (11) halo, (12) C 1~12 Heterocyclyl (e.g., C2-12 heteroaryl), (13) (C 1~12(heterocyclyl)oxy, (14) hydroxyl, (15) nitro, (16) C 1~20 Thioalkoxy (e.g., C 1~6 Thioalkoxy), (17)-(CH2) q CO2R A’ (wherein q is an integer of 0 to 4, and R A’ is (a) alkyl, (b) C 6~10 aryl, (c) hydrogen, and (d) C 1~6 ALC-C 6~10 aryl), (18)-(CH2) q CONR B’ R C’ (wherein q is an integer of 0 to 4, and R B’ and R C’ is (a) hydrogen, (b) alkyl, (c) C 6~10 aryl, and (d) C 1~6 ALC-C 6~10 aryl), (19)—(CH) q SO2R D’ (wherein q is an integer of 0 to 4, and R D’ is (a)C 1~6 Alkyl, (b) C 6~10 aryl, and (c) C 1~6 ALC-C 6~10 aryl), (20)-(CH2) q SO2NR E’ R F’ (wherein q is an integer of 0 to 4, and R E’ and R F’ are (a) hydrogen, (b) C 1~6 Alkyl, (c) C 6~10 aryl, and (d) C 1~6 ALC-C 6~10 (21) thiol; (22) C 6~10 Aryloxy, (23)C 3~8 Cycloalkoxy, (24) arylalkoxy, (25) C 1~6 ALC-C 1~12 Heterocyclyl (e.g., C 1~6 ALC-C 1~12heteroaryl), (26) oxo, (27) (C 1~12 Heterocyclyl)imino, (28)C 2~20 Alkenyl, and (29)C 2~20 alkynyl. In some embodiments, each of these groups can be further substituted as described herein. For example, the alkylene group of a C1-alkaryl or a C1-alkheterocyclyl can be further substituted with an oxo group to provide an aryloyl and (heterocyclyl)oyl substituent, respectively.

[0111] As used herein, a "heterocyclylalkyl" group refers to a heterocyclyl group, as defined herein, attached to the parent molecular group by an alkylene group, as defined herein. Exemplary unsubstituted heterocyclylalkyl groups include those having 2 to 32 carbons (e.g., 2 to 22, 2 to 18, 2 to 17, 2 to 16, 3 to 15, 2 to 14, 2 to 13, or 2 to 12 carbons, e.g., C 1~6 ALC-C 1~12 Heterocyclyl, C 1~10 ALC-C 1~12 Heterocyclyl, or C 1~20 ALC-C 1~12 In some embodiments, the alkylene and heterocyclyl can each be further substituted with 1, 2, 3, or 4 substituents as defined herein for the respective group.

[0112] As used herein, the term "hydrocarbon" refers to a group consisting solely of carbon and hydrogen atoms. The term "hydroxyl," as used herein, refers to an -OH group. In some embodiments, the hydroxyl group can be substituted with 1, 2, 3, or 4 substituents (e.g., O-protecting groups) as defined herein for alkyl.

[0113] As used herein, the term "isomer" refers to any tautomer, stereoisomer, enantiomer, or diastereomer of any compound of the invention. It is recognized that the compounds of the invention may possess one or more chiral centers and / or double bonds and may therefore exist as stereoisomers, such as double bond isomers (i.e., E / Z geometric isomers) and diastereomers (e.g., enantiomers (i.e., (+) or (-)) or cis / trans isomers). In accordance with the present invention, the chemical structures, i.e., compounds of the invention, depicted herein encompass all corresponding stereoisomers, i.e., stereomerically pure forms (e.g., geometrically pure, enantiomerically pure, or diastereomerically pure forms), as well as mixtures of enantiomers or stereoisomers (e.g., racemates). Enantiomeric and stereoisomeric mixtures of the compounds of the present invention can typically be resolved into their component enantiomers or stereoisomers by well-known methods such as chiral-phase gas chromatography, chiral-phase high-performance liquid chromatography, crystallization of the compounds as chiral salt complexes, crystallization of the compounds in chiral solvents, etc. Enantiomers and stereoisomers can also be obtained from stereomerically or enantiomerically pure intermediates, reagents, and catalysts by well-known asymmetric synthetic methods.

[0114] The term "N-protected amino" as used herein means an amino group, as defined herein, bound to one or two N-protecting groups, as defined herein. As used herein, the term "N-protecting group" refers to a group intended to protect an amino group against undesired reactions during synthetic procedures. Commonly used N-protecting groups are disclosed in Greene, Protective Groups in Organic Synthesis, 3rd Edition (John Wiley & Sons, New York, 1999), which is incorporated herein by reference.N-protecting groups include acyl, aryloyl, or carbamyl groups, such as formyl, acetyl, propionyl, pivaloyl, t-butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl, phthalyl, o-nitrophenoxyacetyl, α-chlorobutyryl, benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl, and chiral auxiliaries, such as protected or deprotected D, L, or D,L-amino acids, such as α-chlorobenzoyl, ... alanine, leucine, phenylalanine, etc.; sulfonyl-containing groups, for example, benzenesulfonyl, p-toluenesulfonyl, etc.; carbamate-forming groups, for example, benzyloxycarbonyl, p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl, 3,5-dimethoxybenzyloxycarbonyl, 2, 4-Dimethoxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxycarbonyl, 3,4,5-trimethoxybenzyloxycarbonyl, 1-(p-biphenylyl)-1-methylethoxycarbonyl, α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzhydryloxycarbonyl, t-butyloxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl , methoxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxycarbonyl, fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl, adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl, etc., alkaryl groups such as benzyl, triphenylmethyl, benzyloxymethyl, etc., and silyl groups such as trimethylsilyl, etc. Suitable N-protecting groups are formyl, acetyl, benzoyl, pivaloyl, t-butylacetyl, alanyl, phenylsulfonyl, benzyl, t-butyloxycarbonyl (Boc), and benzyloxycarbonyl (Cbz).

[0115] The term "nitro" as used herein refers to the group --NO.sub.2. As used herein, the term "O-protecting group" refers to a group intended to protect an oxygen-containing (e.g., phenol, hydroxyl, or carbonyl) group from undesired reactions during synthetic procedures. Commonly used O-protecting groups are disclosed in Greene, Protective Groups in Organic Synthesis, 3rd Edition (John Wiley & Sons, New York, 1999), which is incorporated herein by reference. Exemplary O-protecting groups include acyl, aryloyl, or carbamyl groups, such as formyl, acetyl, propionyl, pivaloyl, t-butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl, phthalyl, o-nitrophenoxyacetyl, chlorobutyryl, benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl, t-butyldimethylsilyl, tri-isopropylsilyloxymethyl, 4,4'-dimethoxytrityl, isobutyryl, phenoxyacetyl, 4-isopropylphenoxyacetyl, dimethylformamidino, and 4-nitrobenzoyl; alkylcarbonyl groups such as acyl, acetyl, propionyl, and pivaloyl; optionally substituted arylcarbonyl groups such as benzoyl; silyl groups such as trimethylsilyl (TMS), tert-butyldimethylsilyl (TBDMS), tri-isopropylsilyloxymethyl (TOM), and triisopropylsilyl (TIPS); ether-forming groups with hydroxyl such as methyl, methoxymethyl, tetrahydropyranyl, benzyl, p-methoxybenzyl, and trityl; alkoxycarbonyl groups such as methoxycarbonyl, ethoxycarbonyl, isopropoxycarbonyl, n-isopropoxycarbonyl, n-butyloxycarbonyl, isobutyloxycarbonyl, sec-butyloxycarbonyl, and t-butyloxycarbonyl; alkoxyalkoxycarbonyl groups, such as methoxymethoxycarbonyl, ethoxymethoxycarbonyl, 2-methoxyethoxycarbonyl, 2-ethoxyethoxycarbonyl, 2-butoxyethoxycarbonyl, 2-methoxyethoxymethoxycarbonyl, allyloxycarbonyl, propargyloxycarbonyl, 2-butenoxycarbonyl, 3- methyl-2-butenoxycarbonyl, etc.; haloalkoxycarbonyl, for example, 2-chloroethoxycarbonyl, 2-chloroethoxycarbonyl, 2,2,2-trichloroethoxycarbonyl, etc.; optionally substituted arylalkoxycarbonyl groups, for example, benzyloxycarbonyl, p-methylbenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, 2,4-dinitrobenzyloxycarbonyl, 3,5-dimethylbenzyloxycarbonyl, p-chlorobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, fluorenylmethyloxycarbonyl, and the like, and optionally substituted aryloxycarbonyl groups, for example, phenoxycarbonyl, p-nitrophenoxycarbonyl, o-nitrophenoxycarbonyl, 2,4-dinitrophenoxycarbonyl, p-methylphenoxycarbonyl, m-methylphenoxycarbonyl, o-bromophenoxycarbonyl, 3,5-dimethylphenoxycarbonyl, carboxymethyl, p-chlorophenoxycarbonyl, 2-chloro-4-nitrophenoxycarbonyl, etc.), substituted alkyl, aryl, and alkaryl ethers (e.g., trityl, methylthiomethyl, methoxymethyl, benzyloxymethyl, siloxymethyl, 2,2,2-trichloroethoxymethyl, tetrahydropyranyl, tetrahydrofuranyl, ethoxyethyl, 1-[2-(trimethylsilyl)ethoxy]ethyl, 2-trimethylsilylethyl, t-butyl ether, p-chlorophenyl, p-methoxyphenyl, phenyl, p-nitrophenyl, benzyl, p-methoxybenzyl, and nitrobenzyl), silyl ethers (e.g., trimethylsilyl, triethylsilyl, triisopropylsilyl, dimethylisopropylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, tribenzylsilyl, triphenylsilyl, and diphenylmethylsilyl), carbonates (e.g., methyl, methoxymethyl, 9-fluorenylmethyl, ethyl, 2,2,2-trichloroethyl, 2-(trimethylsilyl)ethyl, biphenylsilyl, Protective groups include aryl, allyl, nitrophenyl, benzyl, methoxybenzyl, 3,4-dimethoxybenzyl, and nitrobenzyl), carbonyl protecting groups (e.g., acetal and ketal groups, such as dimethyl acetal and 1,3-dioxolane, acylal groups, and dithiane groups, such as 1,3-dithiane and 1,3-dithiolane), carboxylic acid protecting groups (e.g., ester groups, such as methyl ester, benzyl ester, t-butyl ester, orthoester, and the like), and oxazoline groups.

[0116] The term "oxo" as used herein refers to =O. As used herein, the prefix "perfluoro" refers to any group, as defined herein, in which each hydrogen radical bonded to an alkyl group has been replaced with a fluoride group. For example, perfluoroalkyl groups are exemplified by trifluoromethyl, pentafluoroethyl, and the like.

[0117] As used herein, the term "protected hydroxyl" means an oxygen atom bonded to an O-protecting group. As used herein, the term "spirocyclyl" refers to a C 1 -C 2 -C 3 -C 4 -C 5 -C 6 -C 7 -C 8 -C 9 -C 10 -C 11 -C 12 -C 13 -C 14 -C 15 -C 16 -C 17 -C 18 -C 19 -C 20 -C 21 -C 22 -C 23 -C 24 -C 2~7 It represents a divalent alkylene group, as well as a divalent heteroalkylene group having both termini bonded to the same atom. The heteroalkylene groups forming the spirocyclyl group can contain 1, 2, 3, or 4 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur. In some embodiments, the spirocyclyl group contains 1 to 7 carbon atoms, excluding the carbon atom to which the divalent group is bonded. The spirocyclyl groups of the present invention can be optionally substituted with 1, 2, 3, or 4 substituents provided herein as optional substituents for the cycloalkyl and / or heterocyclyl groups.

[0118] As used herein, the term "stereoisomer" refers to all different possible isomeric and conformational forms that a compound (e.g., a compound of any formula described herein) may exist in, particularly all possible stereochemical and conformational isomeric forms of the basic molecular structure, all diastereomers, enantiomers, and / or conformers. Some compounds of the present invention may exist in different tautomeric forms, and all of these tautomeric forms are included within the scope of the present invention.

[0119] The term "sulfonyl" as used herein refers to a -S(O)2- group. As used herein, the term "thiol" refers to a -SH group. definition In this application, unless otherwise clear from the context, (i) the term "a" may be understood to mean "at least one," (ii) the term "or" may be understood to mean "and / or," (iii) the terms "comprising" and "including" may be understood to include the itemized ingredient or step, whether presented by itself or with one or more additional ingredients or steps, (iv) the terms "about" and "approximately" may be understood to allow for standard deviation as understood by one of ordinary skill in the art, and (v) when ranges are provided, the endpoints are included.

[0120] As used herein, the term "π-effect interactions" refers to attractive non-covalent interactions between aromatic rings. As used herein, the term "active site" refers to the location on a protein (e.g., an enzyme) where a substrate molecule binds to initiate a chemical reaction. "Not bound to the active site" means that atoms of the compound or complex are not substantially involved in bonding with residues in the active site (e.g., residues involved in binding to the natural substrate molecule).

[0121] As used herein, the term "administration" refers to the administration of a composition (e.g., a compound, a conjugate, or a preparation comprising a compound or conjugate as described herein) to a subject or system. Administration to an animal subject (e.g., a human) can be by any suitable route. For example, in some embodiments, administration can be transbronchial (including by bronchial instillation), transbuccal, enteral, transcutaneous, intraarterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, transmucosal, intranasal, oral, rectal, subcutaneous, sublingual, topical, transtracheal (including by intratracheal instillation), transdermal, transvaginal, and intravitreal.

[0122] As known in the art, "affinity" is a measure of the tightness with which a particular ligand binds to its partner. Affinity can be measured in a variety of ways. In some embodiments, affinity is measured by a quantitative assay. In some such embodiments, the binding partner concentration may be fixed in excess of the ligand concentration to mimic physiological conditions. Alternatively, or additionally, in some embodiments, the binding partner concentration and / or the ligand concentration may be varied. In some such embodiments, affinity may be compared to a reference under equivalent conditions (e.g., concentrations).

[0123] As used herein, the term "analog" refers to a substance that shares one or more particular structural features, elements, components, or portions with a reference substance. Typically, an "analog" exhibits significant structural similarity to the reference substance, e.g., shares a core structure or consensus structure, but differs in certain individual respects. In some embodiments, an analog is a substance that can be produced from a reference substance by chemical manipulation of the reference substance. In some embodiments, an analog is a substance that can be produced by performing a synthetic process that is substantially similar (e.g., shares multiple steps) to the process that produces the reference substance. In some embodiments, an analog is produced or is producible by performing a synthetic process that is different from the process used to produce the reference substance.

[0124] As used herein, the term "animal" refers to any member of the animal kingdom. In some embodiments, "animal" refers to a human at any stage of development. In some embodiments, "animal" refers to a non-human animal at any stage of development. In some embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, a cow, a primate, and / or a pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, and / or worms. In some embodiments, animals may be transgenic (genetically engineered) and / or clones.

[0125] As used herein, the term "antagonist" refers to a compound that i) inhibits, reduces, or reduces the action of a target protein (e.g., a eukaryotic target protein, such as a mammalian target protein or a fungal target protein, or a prokaryotic target protein, such as a bacterial target protein), and / or ii) inhibits, reduces, decreases, or slows one or more biological events. Antagonists can be direct (where they affect their target directly) or indirect (where they affect something other than binding to its target, e.g., by interacting with a regulator of the target protein (e.g., a eukaryotic target protein, such as a mammalian target protein or a fungal target protein, or a prokaryotic target protein, such as a bacterial target protein), e.g., to alter the level or activity of the target protein).

[0126] As used herein, the terms "approximately" and "about," where appropriate in the relevant context, are intended to encompass normal statistical variations understood by one of ordinary skill in the art. In certain embodiments, the terms "approximately" or "about" refer to a range of values ​​that falls within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater or less than) the stated value, unless otherwise specified or otherwise apparent from the context (e.g., when such number exceeds 100% of the possible values).

[0127] Two events or entities, as that term is used herein, are "associated" with one another if the presence, level, and / or form of one correlates with that of the other. For example, a particular entity (e.g., a polypeptide) is considered to be associated with a particular disease, disorder, or condition if its presence, level, and / or form correlates with the incidence and / or susceptibility of the disease, disorder, or condition (e.g., across a relevant population). In some embodiments, two or more entities are physically "associated" with one another if they are in physical proximity to one another and interact, directly or indirectly, to maintain that proximity. In some embodiments, two or more entities that are physically associated with one another are covalently bonded to one another; in some embodiments, two or more entities that are physically associated with one another are not covalently bonded to one another, but are non-covalently associated, e.g., by hydrogen bonding, van der Waals interactions, hydrophobic interactions, magnetism, and combinations thereof.

[0128] It will be understood that the term "binding," as used herein, typically refers to an association (e.g., non-covalent or covalent) between two or more entities. "Direct" binding includes physical contact between the entities or moieties, while indirect binding includes physical interaction through physical contact with one or more intermediate entities. Binding between two or more entities can typically be assessed in any of a variety of contexts, including when the interacting entities or moieties are studied in isolation or in more complex systems (e.g., covalently or otherwise associated with a carrier entity and / or a biological system or cell).

[0129] The affinity between a molecule X and its partner Y is generally expressed as the dissociation constant (K D ) Affinity can be measured by conventional methods known in the art, including those described herein. Certain exemplary and representative embodiments for measuring binding affinity are described below. As used herein, "K DThe term "" is intended to mean the dissociation equilibrium constant of a particular compound-protein or complex-protein interaction. Typically, the compounds of the invention have a dissociation equilibrium constant of about 10, as determined, for example, by surface plasmon resonance (SPR) techniques using a presenter protein as the analyte and the compound as the ligand. -6 Less than M, for example, about 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M, or even lower dissociation equilibrium constant (K D The presenter protein / compound complex of the present invention binds to the presenter protein at about 10 s when determined, for example, by surface plasmon resonance (SPR) technology using the target protein as the analyte and the complex as the ligand. -6 Less than M, for example, about 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M, or even lower dissociation equilibrium constant (K D ) to bind to a target protein (e.g., a eukaryotic target protein, such as a mammalian target protein or a fungal target protein, or a prokaryotic target protein, such as a bacterial target protein).

[0130] As used herein, the term "binding free energy" refers to the energy difference between the bound and unbound states of a complex. Binding free energy may be determined by methods known in the art, including using the formula ΔG=-RTlnK using an experimentally derived dissociation constant, Kd. Binding free energies can be calculated using computational algorithms known in the art, such as molecular dynamics simulations, free energy perturbation, or Monte Carlo protocols, as implemented in commercially available software such as AMBER (Cornell et al., J. Am. Chem. Soc., 1995, 117, 5179), CHARMM (Brooks et al., J. Comp. Chem., 1983, 4, 187), or Desmond (Boowers et al., Proc. ACM / IEEE Conf. Supercomputing, 2006, SCO6).

[0131] As used herein, the term "buried surface area" refers to the surface area of ​​a protein or complex that is not exposed to solvent. Buried surface area may be determined by methods known in the art, including calculating solvent inaccessibility. Solvent inaccessibility may be calculated using a 1.4A rolling probe using a program such as PDBePISA release version 1.48 (http: / / www.ebi.ac.uk / pdbe / pisa / ).

[0132] As used herein, the term "cell-permeable" refers to a compound that is capable of entering the intracellular domain when added to a cellular environment without killing the cell. Whether a compound is cell-permeable may be determined using any method known in the art, for example, the biosensor methods described herein.

[0133] As used herein, the term "characteristic portion" is used in the broadest sense to mean a portion of a substance whose presence (or absence) correlates with the presence (or absence) of a particular characteristic, attribute, or activity of the substance. In some embodiments, a characteristic portion of a substance is a portion found in substances and related substances that share a particular characteristic, attribute, or activity, but not in substances that do not share the particular characteristic, attribute, or activity. In certain embodiments, a characteristic portion shares at least one functional property with the intact substance. For example, in some embodiments, a "characteristic portion" of a protein or polypeptide is a portion containing a contiguous stretch of amino acids or a collection of contiguous stretches of amino acids that are both specific to the protein or polypeptide. In some embodiments, each such contiguous stretch generally contains at least 2, 5, 10, 15, 20, 50, or more amino acids. Generally, a characteristic portion of a substance (e.g., a protein, antibody, etc.) is a portion that shares at least one functional property with the related intact substance, in addition to the sequence and / or structural identity specified above. In some embodiments, a characteristic portion may be biologically active.

[0134] As used herein, the phrase "characteristic sequence element" refers to a sequence element found in a polymer (e.g., a polypeptide or nucleic acid) that represents a characteristic portion of that polymer. In some embodiments, the presence of a characteristic sequence element correlates with the presence or level of a particular activity or property of the polymer. In some embodiments, the presence (or absence) of a characteristic sequence element defines a particular polymer as a member (or not) of a particular family or group of such polymers. A characteristic sequence element typically comprises at least two monomers (e.g., amino acids or nucleotides). In some embodiments, a characteristic sequence element comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, or more monomers (e.g., adjacently linked monomers). In some embodiments, a characteristic sequence element comprises at least first and second stretches of adjacent monomers separated by one or more spacer regions that may or may not be of variable length across the polymers sharing the sequence element. In certain embodiments, certain characteristic sequence elements may be referred to as "motifs."

[0135] As used herein, the term "clogP" refers to the calculated partition coefficient of a molecule or portion of a molecule. The partition coefficient is the ratio of the concentrations of a compound in a mixture of two immiscible phases (e.g., octanol and water) at equilibrium and is a measure of the hydrophobicity or hydrophilicity of the compound. Various methods are available in the art for determining clogP. For example, in some embodiments, clogP can be determined using quantitative structure-property relationship algorithms known in the art (e.g., using fragment-based prediction methods that predict the logP of a compound by determining the sum of non-overlapping molecular fragments). Several algorithms for calculating clogP are known in the art, including those used by molecular editing software such as CHEMDRAW® Pro, version 12.0.2.1092 (Cambridgesoft, Cambridge, MA) and MARVINSKETCH® (ChemAxon, Budapest, Hungary). A compound is considered to meet the threshold cLogP if it meets that threshold in at least one of the above methods.

[0136] As used herein, the term "collection" refers to a collection of 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 "Collection" refers to a group of individual or more distinct molecules. In some embodiments, at least 10% (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100%) of the compounds in the collection are compounds described herein (e.g., macrocyclic compounds).

[0137] As used herein, the term "combination therapy" refers to a situation in which a subject is exposed to two or more therapeutic regimens (e.g., two or more compounds, e.g., macrocyclic compounds) simultaneously. In some embodiments, the two or more compounds may be administered simultaneously, in some embodiments, such compounds may be administered sequentially, and in some embodiments, such compounds are administered in overlapping administration regimens.

[0138] As used herein, the term "equivalent" refers to two or more compounds, entities, circumstances, sets of conditions, etc. that are not necessarily identical to one another, but are sufficiently similar to allow a comparison to be made and conclusions to be reasonably drawn based on the observed differences or similarities. In some embodiments, equivalent sets of conditions, circumstances, individuals, or populations are characterized by multiple substantially identical characteristics and one or a few different characteristics. The level of identity required in any given situation to consider two or more such compounds, entities, circumstances, sets of conditions, etc. equivalent will be understood by one of ordinary skill in the art from the context. One of ordinary skill in the art will recognize that sets of circumstances, individuals, or populations are equivalent to one another when they are characterized by a sufficient number and type of substantially identical characteristics to provide a basis for a reasonable conclusion that differences in results obtained or phenomena observed under or by different sets of circumstances, individuals, or populations are caused by or indicative of variations in the various characteristics.

[0139] As used herein, the term "complex" refers to a group of two or more compounds and / or proteins bound together by binding interactions (e.g., non-covalent interactions such as hydrophobic interactions, electrostatic interactions, van der Waals interactions, π-effect interactions, etc.). An example of a complex is a "presenter protein / compound complex" comprising a compound of the invention bound to a presenter protein.

[0140] As used herein, the term "corresponding to" is often used to refer to a structural element or moiety in a subject compound that shares a position (e.g., in three-dimensional space or relative to other elements or moieties) with a position present in an appropriate reference compound. For example, in some embodiments, the term is used to refer to the position / identity of a residue in a polymer, such as an amino acid residue in a polypeptide or a nucleotide residue in a nucleic acid. For simplicity, a residue in a first polymer that "corresponds to" a residue at position 190 in a reference polymer need not actually be the 190th residue of the first polymer, although one of skill in the art will recognize that residues in such polymers are often represented using a canonical numbering system based on the reference related polymer so as to correspond to the residue found at position 190 in the reference polymer. One of skill in the art will readily recognize how to identify "corresponding" amino acids, including using one or more commercially available algorithms specifically designed for polymer sequence comparison.

[0141] As used herein, the term "designed" refers to (i) a compound whose structure is selected or chosen through the hand of man, (ii) a compound produced by a process requiring human intervention, and / or (iii) a compound that differs from natural materials and other known compounds.

[0142] Many of the methods described herein include a "determining" step. Those skilled in the art will recognize from reading this specification that such a "determining" step can utilize or be accomplished using any of a variety of techniques available to those skilled in the art, including, for example, certain techniques explicitly referenced herein. In some embodiments, the determining step involves manipulation of a physical sample. In some embodiments, the determining step involves reviewing and / or manipulating data or information, for example, using a computer or other processing unit adapted to perform relevant analyses. In some embodiments, the determining step involves receiving relevant information and / or materials from a source. In some embodiments, the determining step involves comparing one or more characteristics of the sample or entity to an equivalent reference.

[0143] As used herein, the term "Depsi bond" refers to the replacement of an ester bond with an amide bond. As used herein, the term "formulation" refers to a physically discrete unit of an active compound (e.g., a therapeutic or diagnostic agent) for administration to a subject. Each unit contains a predetermined amount of active agent. In some embodiments, such amount is a unit dose (or an integer fraction thereof) suitable for administration according to a dosing regimen determined to correlate with a desired or beneficial outcome when administered to a relevant population (i.e., using a therapeutic dosing regimen). Those skilled in the art will recognize that the total amount of a therapeutic composition or compound administered to a particular subject will be determined by one or more attending physicians and may require the administration of multiple formulations.

[0144] As used herein, the term "dosing regimen" refers to a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time. In some embodiments, a given therapeutic compound has a recommended dosing regimen that may include one or more doses. In some embodiments, a dosing regimen includes multiple doses, each separated from the other by periods of equal length. In some embodiments, a dosing regimen includes multiple doses separated into individual doses and at least two different periods of time. In some embodiments, all doses within a dosing regimen are the same unit dosage. In some embodiments, various doses within a dosing regimen are different amounts. In some embodiments, a dosing regimen includes a first dose at a first dosage amount, followed by one or more additional doses at a second dosage amount that is different from the first dosage amount. In some embodiments, a dosing regimen includes a first dose at a first dosage amount, followed by one or more additional doses at a second dosage amount that is the same as the first dosage amount. In some embodiments, the dosing regimen correlates with a desired or beneficial outcome when administered across a relevant population (ie, is a therapeutic dosing regimen).

[0145] As used herein, the term "engineered" is used to describe a compound whose design and / or production involves the action of the hand of man. For example, in some embodiments, an "engineered" compound is prepared by in vitro chemical synthesis. In some embodiments, an "engineered" compound is produced by cells that are genetically modified relative to a reference wild-type cell. In some embodiments, an "engineered" compound is produced by cells in culture. In some embodiments, an "engineered" compound is produced by cells in culture conditions that are specifically modified to enhance production of the compound. In some embodiments, an "engineered" compound has a structure that is designed or selected by in silico modeling.

[0146] As used herein, the term "flat surface site" as understood in the art refers to a site on the surface of a protein structure that has relatively flat characteristics (e.g., less than 500 Å). 2 Area of ​​over 400Å 3 "Flat" refers to a site that does not contain any well-defined pockets or cavities with a volume greater than 500 Å and a depth greater than 13 Å. In some embodiments, a site may be determined to be flat by utilizing commercially available algorithms known in the art. For example, a site may be flatter than 500 Å as determined by CAST (Liang et al., Prot. Sci. 1998, 7:1884) or Sitemap (Halgren, J. Chem. Inf. Model. 2009, 49:377). 2 Area of ​​over 400Å 3 A protein may be determined to be flat if it does not contain any clearly defined pockets or cavities with a volume greater than 100 Å and a depth greater than 13 Å. Those skilled in the art are familiar with the concept of flatness and are aware of its relationship to "druggability." In some embodiments, a protein is considered to have a flat surface site if it is undruggable as defined herein, and is determined to be undruggable, for example, using the program DOGSITESCORER®.

[0147] As used herein, the term "hydrophobic residue" refers to an amino acid having a hydropathic index value equal to or greater than that of proline. Examples of hydrophobic residues are valine, isoleucine, leucine, methionine, phenylalanine, tryptophan, alanine, glycine, and cysteine.

[0148] As used herein, the term "hydrophobic surface site" means a site on the surface of a protein structure that contains at least 30% hydrophobic residues. As used herein, the term "identity" refers to the overall relatedness between polymers, e.g., between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. For example, calculation of the percent identity of two nucleic acid sequences can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second nucleic acid sequences to ensure optimal alignment, and different sequences can be ignored for comparison purposes). In certain embodiments, the length of the sequences aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100% of the length of the reference sequence. The nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced to optimally align the two sequences. Comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two nucleotide sequences can be determined using the Meyers and Miller algorithm (Computer Applications in Biological Sciences (CABIOS), 1989, vol. 4, pp. 11-17) as incorporated into the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package using the NWSgapdna.CMP matrix.

[0149] As used herein, the term "isolated" refers to a substance and / or entity that is (1) separated from at least some of the components with which it was associated when originally created (whether in nature and / or experimental circumstances) and / or (2) designed, created, prepared, and / or manufactured by the hand of man. Isolated substances and / or entities may be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or greater than about 99% of the other components with which they were originally associated. In some embodiments, an isolated compound is about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or greater than about 99% pure. As used herein, a substance is "pure" if it is substantially free of other components. In some embodiments, as one of skill in the art will appreciate, a substance may still be considered "isolated" or even "pure" after combination with certain other components, such as one or more carriers or excipients (e.g., buffers, solvents, water, etc.), and in such embodiments, the percent isolation or purity of the substance is calculated without including such carriers or excipients. In some embodiments, isolation includes or requires disruption of covalent bonds (e.g., to isolate polypeptide domains from longer polypeptides and / or to isolate nucleotide sequence elements from longer oligonucleotides or nucleic acids).

[0150] As used herein, the term "macrocyclic compound" refers to a small molecule compound containing a ring with nine or more ring atoms. Macrocyclic compounds include macrolides, a group of small molecules containing macrocyclic lactones, such as erythromycin, rapamycin, and FK506. In some embodiments, a macrocyclic compound is a small molecule in which more than 25% (e.g., more than 30%, more than 35%, more than 40%, more than 45%) of the non-hydrogen atoms in the small molecule are contained in a single ring structure or a fused ring structure. In some embodiments, the macrocyclic compound is not a compound described in Benjamin et al., Nat. Rev. Drug. Discov., 2011, Vol. 10, No. 11, pp. 868-880, or Sweeney, ZK et al., J. Med. Chem., 2014, epub ahead of print, the structures of which are incorporated by reference.

[0151] As used herein, the term "target protein interacting moiety" refers to a group of ring atoms and their attached moieties (e.g., atoms within the 20 ring atoms, e.g., atoms within the 15 ring atoms, atoms within the 10 ring atoms, atoms within the 5 ring atoms) that specifically bind to a target protein (e.g., a eukaryotic target protein such as a mammalian target protein or a fungal target protein, or a prokaryotic target protein such as a bacterial target protein) when the compound is in a complex with a presenter protein.

[0152] The term "modulator" is used to mean an entity whose presence or level in a system in which an activity of interest is observed correlates with a change in the level and / or nature of that activity compared to that observed under otherwise equivalent conditions in the absence of the modulator. In some embodiments, a modulator is an activator, in the sense that the activity is increased in its presence compared to that observed under otherwise equivalent conditions in the absence of the modulator. In some embodiments, a modulator is an antagonist or inhibitor, in the sense that the activity is reduced in its presence compared to that under otherwise equivalent conditions in the absence of the modulator. In some embodiments, a modulator interacts directly with a target entity whose activity is of interest. In some embodiments, a modulator interacts indirectly with a target entity whose activity is of interest (i.e., interacts directly with an intermediate compound that interacts with the target entity). In some embodiments, a modulator affects the level of a target entity of interest; alternatively, or additionally, in some embodiments, a modulator affects the activity of a target entity of interest without affecting the level of the target entity. In some embodiments, a modulator affects both the level and activity of a target entity of interest, such that differences in observed activity are not entirely explained by or correspond to differences in observed level, hi some embodiments, the modulator is an allosteric modulator, such as an allosteric agonist.

[0153] As used herein, an atom "involved in a bond" is one that is within 4 Å of the bonding entity or is connected to an atom that is within 4 Å of the bonding entity. As used herein, the term "pharmaceutical composition" refers to an active compound formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, the active compound is present in a unit dosage suitable for administration of a therapeutic regimen that exhibits a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, the pharmaceutical compositions may be specially formulated for administration in solid or liquid form, including those adapted for oral administration, e.g., drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., buccal, sublingual, and those intended for systemic absorption, boluses, powders, granules, pastes for application to the tongue, those adapted for parenteral administration, e.g., subcutaneous, intramuscular, intravenous, or epidural injection, e.g., sterile solutions or suspensions, or sustained release formulations, those adapted for topical application, e.g., creams, ointments, or controlled-release patches or sprays applied to the skin, lungs, or oral cavity, those adapted vaginally or rectally, e.g., pessaries, creams, or foams, those adapted sublingually, those adapted ophthalmically, those adapted transdermally, or those adapted for pulmonary, nasal, and other mucosal surfaces.

[0154] As used herein, "pharmaceutically acceptable excipient" refers to any inactive ingredient (e.g., a medium in which an active compound can be suspended or dissolved) that is non-toxic and non-inflammatory in a subject. Typical excipients include, for example, antiadhesives, antioxidants, binders, coating agents, compression aids, disintegrants, dyes (colorants), emollients, emulsifiers, fillers (diluents), film-forming or coating agents, flavors, fragrances, glidants (glidants), lubricants, preservatives, printing inks, sorbents, suspending or dispersing agents, sweeteners, or water for hydration. Excipients include, but are not limited to, butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, cross-linked polyvinylpyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropylmethylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinylpyrrolidone, povidone, pregelatinized starch, propylparaben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol. Those of skill in the art are familiar with the wide variety of agents and materials useful as excipients.

[0155] As used herein, the term "pharmaceutically acceptable salt" refers to a salt of a compound described herein that is suitable for use in contact with the tissues of humans and animals without undue toxicity, irritation, allergic response, or the like, within the scope of sound medical judgment, and that is commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, pharmaceutically acceptable salts are described in Berge et al., J. Pharmaceutical Sciences, Vol. 66, pp. 1-19, 1977, and in Pharmaceutical Salts: Properties, Selection, and Use, by P.H. Stahl and C.G. Wermuth, Wiley-VCH, 2008. The salts can be prepared in situ during the final isolation and purification of the compounds described herein, or can be prepared separately by reaction of the free base group with a suitable organic acid.

[0156] The compounds of the present invention may have ionizable groups so that they can be prepared as pharmaceutically acceptable salts. Such salts may be acid addition salts with inorganic or organic acids, or, in the case of the acid form of the compounds of the present invention, salts may be prepared from inorganic or organic bases. In many cases, compounds are prepared or used as pharmaceutically acceptable salts prepared as addition products of pharmaceutically acceptable acids or bases. Suitable pharmaceutically acceptable acids and bases, such as hydrochloric acid, sulfuric acid, hydrobromic acid, acetic acid, lactic acid, citric acid, or tartaric acid to form acid addition salts, and potassium hydroxide, sodium hydroxide, ammonium hydroxide, caffeine, various amines, and the like to form base salts, are well known in the art. Methods for preparing suitable salts are well established in the art.

[0157] Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, and 2-hydroxyethanesulfonate. salts, such as ammonium salts, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like, as well as non-toxic ammonium, quaternary ammonium, and amine cations, such as, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like.

[0158] The term "polar surface area" refers to the sum of the surfaces covering all polar atoms of a molecule or part of a molecule, including bound hydrogens. Polar surface area can be determined computationally using a program such as CHEMDRAW® Pro, version 12.0.2.1092 (Cambridgesoft, Cambridge, MA).

[0159] The term "presenter protein" refers to a protein that binds to a small molecule to form a complex that binds to and modulates the activity of a target protein (e.g., a eukaryotic target protein, such as a mammalian target protein or a fungal target protein, or a prokaryotic target protein, such as a bacterial target protein). In some embodiments, the presenter protein is a relatively abundant protein (e.g., the presenter protein is sufficiently abundant that its participation in a tripartite complex does not substantially affect the biological role of the presenter protein in the cell and / or the viability or other attributes of the cell). In certain embodiments, the presenter protein is a protein that has chaperone activity in the cell. In some embodiments, the presenter protein is a protein that has multiple natural interaction partners in the cell. In certain embodiments, the presenter protein is one that is known to bind to a small molecule to form a binary complex that is known or predicted to bind to the target protein and modulate its biological activity.

[0160] The term "presenter protein binding moiety" refers to a compound that specifically binds to said presenter protein, e.g., with a KD of less than 10 μM (e.g., less than 5 μM, less than 1 μM, less than 500 nM, less than 200 nM, less than 100 nM, less than 75 nM, less than 50 nM, less than 25 nM, less than 10 nM), or with an IC of less than 1 μM (e.g., less than 0.5 μM, less than 0.1 μM, less than 0.05 μM, less than 0.01 μM). 50and refers to a group of ring atoms and moieties attached thereto (e.g., atoms within the 20 ring atoms, e.g., atoms within the 15 ring atoms, atoms within the 10 ring atoms, atoms within the 5 ring atoms) that are involved in binding to the presenter protein so as to inhibit the peptidyl-prolyl isomerase activity of the presenter protein. It will be understood that the presenter protein binding moiety does not necessarily encompass all of the atoms in a compound that interacts with the presenter protein. It will also be understood that one or more atoms of the presenter protein binding moiety can be within the target protein interacting moiety (e.g., a eukaryotic target protein interacting moiety such as a mammalian target protein interacting moiety or a fungal target protein interacting moiety, or a prokaryotic target protein interacting moiety such as a bacterial target protein interacting moiety).

[0161] The term "pure" means substantially pure or free from undesired components (e.g., other compounds and / or other components of a cell lysate), material contaminants, impurities, or defects.

[0162] The term "reference" is often used herein to describe a standard or control compound, individual, population, sample, sequence, or value with which a compound, individual, population, sample, sequence, or value of interest is compared. In some embodiments, the reference compound, individual, population, sample, sequence, or value is examined and / or determined substantially simultaneously with the examination or determination of the compound, individual, population, sample, sequence, or value of interest. In some embodiments, the reference compound, individual, population, sample, sequence, or value is a historical reference, optionally embodied in tangible media. Typically, as will be understood by one of skill in the art, the reference compound, individual, population, sample, sequence, or value is determined or characterized under conditions equivalent to those used to determine or characterize the compound, individual, population, sample, sequence, or value of interest.

[0163] The term "ring atom" refers to an atom of a cyclic compound, including the innermost portion of the ring. For example, using this method, FK506 has 21 ring atoms, and rapamycin has 29 ring atoms.

[0164] The term "naturally occurring protein-protein interaction site" refers to a location on the surface of a protein's structure that contains atoms involved in binding between the protein and other proteins in the protein's natural environment.

[0165] The term "small molecule" refers to organic and / or inorganic compounds with low molecular weights. Generally, a "small molecule" is a molecule less than about 5 kilodaltons (kD) in size. In some embodiments, a small molecule is less than about 4 kD, 3 kD, 2 kD, or 1 kD. In some embodiments, a small molecule is less than about 800 daltons (D), 600 D, 500 D, 400 D, 300 D, 200 D, or 100 D. In some embodiments, a small molecule is less than about 2000 g / mol, less than about 1500 g / mol, less than about 1000 g / mol, less than about 800 g / mol, or less than about 500 g / mol. In some embodiments, a small molecule is not a polymer. In some embodiments, a small molecule does not comprise a polymeric moiety. In some embodiments, a small molecule is not a protein or polypeptide (e.g., not an oligopeptide or peptide). In some embodiments, a small molecule is not a polynucleotide (e.g., not an oligonucleotide). In some embodiments, the small molecule is not a polysaccharide. In some embodiments, the small molecule does not comprise a polysaccharide (e.g., is not a glycoprotein, proteoglycan, glycolipid, etc.). In some embodiments, the small molecule is not a lipid. In some embodiments, the small molecule is a modulating compound. In some embodiments, the small molecule is biologically active. In some embodiments, the small molecule is detectable (e.g., comprises at least one detectable moiety). In some embodiments, the small molecule is a therapeutic agent.

[0166] Those skilled in the art will appreciate, upon reading this disclosure, that certain small molecule compounds described herein may be provided and / or utilized in any of a variety of forms, including, for example, salt forms, protected forms, prodrug forms, ester forms, isomeric forms (e.g., optical and / or structural isomers), and isotopic forms. In some embodiments, reference to a particular compound may refer to that particular form of the compound. In some embodiments, reference to a particular compound may refer to that compound in any form. In some embodiments, if a compound exists or is found in nature, the compound may be provided and / or utilized in accordance with the present invention in a form that differs from that in which it exists or is found in nature. Those skilled in the art will appreciate that a preparation of a compound that contains one or more individual forms in levels, amounts, or ratios that differ from a reference preparation or source (e.g., a natural source) of the compound may be considered a different form of the compound described herein. Thus, for example, in some embodiments, a preparation of a single stereoisomer of a compound may be considered a different form of the compound from a racemic mixture of the compound. A particular salt of a compound may be considered a different form of the compound from other salt forms of the compound. A preparation containing one conformational isomer of the double bond ((Z) or (E)) may be considered a different form from one containing the other conformational isomer of the double bond ((E) or (Z)), a preparation in which one or more atoms are isotopic differently than present in the reference preparation may be considered a different form, and so forth.

[0167] As used herein, the terms "specific binding" or "specific" refer to an interaction between a binder and a target entity. As will be understood by those skilled in the art, an interaction is considered "specific" if it favors binding with a KD of, for example, less than 10 μM (e.g., less than 5 μM, less than 1 μM, less than 500 nM, less than 200 nM, less than 100 nM, less than 75 nM, less than 50 nM, less than 25 nM, less than 10 nM) in the presence of alternative interactions. In many embodiments, the specific interaction depends on the presence of a particular structural feature of the target entity (e.g., an epitope, cleft, binding site). It should be understood that specificity need not be absolute. In some embodiments, specificity may be assessed relative to the specificity of a binder for one or more other possible target entities (e.g., competitors). In some embodiments, specificity is assessed relative to the specificity of a reference specific binder. In some embodiments, specificity is assessed relative to the specificity of a reference nonspecific binding agent.

[0168] The term "specific," when used in reference to a compound having activity, will be understood by those skilled in the art to mean that the compound distinguishes between potential target entities or states. For example, in some embodiments, a compound is said to bind "specifically" to a target if it preferentially binds to that target in the presence of one or more competing surrogate targets. In many embodiments, specific interaction depends on the presence of a particular structural feature (e.g., an epitope, cleft, binding site) of the target entity. It should be understood that specificity need not be absolute. In some embodiments, specificity may be assessed relative to the specificity of a binding agent for one or more other possible target entities (e.g., competitors). In some embodiments, specificity is assessed relative to the specificity of a reference specific binding agent. In some embodiments, specificity is assessed relative to the specificity of a reference nonspecific binding agent. In some embodiments, an agent or entity does not detectably bind to a competing surrogate target under conditions of binding to its target entity. In some embodiments, a binding agent binds to its target entity with a higher on-rate, a lower off-rate, increased affinity, decreased dissociation, and / or increased stability compared to a competing surrogate target.

[0169] The term "structural organization" refers to the average three-dimensional arrangement of atoms and bonds of a molecule. "Substantially unchanged structural organization" means that the root mean square deviation (RMSD) of two aligned structures is less than 1. RMSD can be calculated, for example, using the align command in PyMOL version 1.7rc1 (Schroedinger LLC). Alternatively, RMSD can be calculated using the Executive RMS parameter of the LigAlign algorithm (J. Mol. Graphics and Modeling, 2010, Vol. 29, pp. 93-101).

[0170] The term "substantially" refers to the qualitative condition of exhibiting the entire or nearly entire extent or degree of a property or characteristic of interest. Those skilled in the biological arts will understand that biological and chemical phenomena rarely, if ever, reach an end state and / or proceed perfectly or achieve or avoid an absolute result. Thus, the term "substantially" is used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0171] As used herein, the term "does not substantially bind" to a particular protein means, for example, that the target -4 M or more, alternatively 10 -5 M or more, alternatively 10 -6 M or more, alternatively 10 -7 M or more, alternatively 10 -8 M or more, alternatively 10 -9 M or more, alternatively 10 -10 M or more, alternatively 10 -11 M or more, alternatively 10 -12 K over M D or 10 -4 M~10 -12 M or 10 -6 M~10 -10 M or 10 -7 M~10 -9 K within M D The term "a" can be represented by a molecule or portion of a molecule having the formula:

[0172] The term "substantial structural similarity" refers to the presence of shared structural features, such as the presence and / or identity of particular amino acids at particular positions (see the definitions of "shared sequence homology" and "shared sequence identity"). In some embodiments, the term "substantial structural similarity" refers to the presence and / or identity of structural elements (e.g., loops, sheets, helices, H-bond donors, H-bond acceptors, glycosylation patterns, salt bridges, and disulfide bonds). In some embodiments, the term "substantial structural similarity" refers to the three-dimensional configuration and / or orientation of atoms or moieties relative to one another (e.g., distances and / or angles between a subject agent and a reference agent).

[0173] The term "target protein" refers to a protein other than mTOR or calcineurin that binds to a small molecule / presenter protein / compound complex described herein but does not substantially bind to a single small molecule or presenter protein. In some embodiments, the small molecule / presenter protein / compound complex does not substantially bind to mTOR or calcineurin. In some embodiments, the target protein is involved in a biological pathway associated with a disease, disorder, or condition. In some embodiments, the target protein is a naturally occurring protein. In some such embodiments, the target protein is naturally found in a particular mammalian cell (e.g., a mammalian target protein), fungal cell (e.g., a fungal target protein), bacterial cell (e.g., a bacterial target protein), or plant cell (e.g., a plant target protein). In some embodiments, the target protein is characterized by native interactions with one or more native presenter protein / native small molecule complexes. In some embodiments, the target protein is characterized by native interactions with multiple different native presenter protein / native small molecule complexes, and in some such embodiments, some or all of the complexes utilize the same presenter protein (and different small molecules). In some embodiments, the target protein does not substantially bind to a complex of cyclosporine, rapamycin, or FK506 and a presenter protein (e.g., FKBP). The target protein can be naturally occurring, e.g., wild-type. Alternatively, the target protein can differ from the wild-type protein but still maintain a biological function, e.g., an allelic variant, splice mutant, or biologically active fragment. Exemplary mammalian target proteins are GTPases, GTPase-activating proteins, guanine nucleotide exchange factors, heat shock proteins, ion channels, coiled-coil proteins, kinases, phosphatases, ubiquitin ligases, transcription factors, chromatin modifiers / remodelers, proteins with classical protein-protein interaction domains and motifs, or any other protein involved in a biological pathway associated with a disease, disorder, or condition.

[0174] The term "target protein interacting moiety" refers to a group of ring atoms and their attached moieties (e.g., atoms within the 20 ring atoms, e.g., atoms within the 15 ring atoms, atoms within the 10 ring atoms, atoms within the 5 ring atoms) that are involved in binding to a target protein (e.g., a eukaryotic target protein such as a mammalian target protein or a fungal target protein, or a prokaryotic target protein such as a bacterial target protein) when the compound is in complex with a presenter protein. It will be understood that the target protein interacting moiety does not necessarily encompass all of the atoms in a compound that interact with a target protein. It will also be understood that one or more atoms of the presenter protein binding moiety may also be present in the target protein interacting moiety.

[0175] By "therapeutic regimen" is meant a dosing regimen whose administration across a relevant population correlates with a desired or beneficial therapeutic outcome. The term "therapeutically effective amount" refers to an amount sufficient to treat a disease, disorder, and / or condition when administered to a disease population suffering from or susceptible to the disease, disorder, and / or condition according to a therapeutic dosing regimen. In some embodiments, a therapeutically effective amount is an amount that reduces the incidence and / or severity of a disease, disorder, and / or condition and / or delays the onset of one or more symptoms thereof. Those skilled in the art will appreciate that the term "therapeutically effective amount" does not require the achievement of effective treatment in a particular individual. More precisely, a therapeutically effective amount may be an amount that provides a specific desired pharmacological response in a significant number of subjects when administered to patients in need of such treatment. It is specifically understood that certain subjects may be "refractory" to a "therapeutically effective amount." By way of example, refractory subjects may have low bioavailability, resulting in a lack of clinical efficacy. In some embodiments, reference to a therapeutically effective amount may be a reference to an amount measured in one or more particular tissues (e.g., tissues affected by a disease, disorder, or condition) or fluids (e.g., blood, saliva, serum, sweat, tears, urine, etc.). One of skill in the art will appreciate that in some embodiments, a therapeutically effective amount may be formulated and / or administered in a single dose. In some embodiments, a therapeutically effective amount may be formulated and / or administered in multiple doses, e.g., as part of a dosing regimen.

[0176] The term "conventional binding pocket" refers to a cavity or pocket in a protein structure that has physiochemical and / or geometric properties comparable to those of a protein whose activity is modulated by one or more small molecules. In some embodiments, a conventional binding pocket is a protein having a 1000 A 3 A conventional binding pocket is a well-defined pocket with a volume greater than 1000 kJ / mol. Those skilled in the art are familiar with the concept of a conventional binding pocket and are aware of its relationship to "druggability." In certain embodiments, a protein is considered not to have a conventional binding pocket if it is undruggable, as defined herein.

[0177] The term "treatment," in its broadest sense, refers to any administration of a substance (e.g., a provided composition) that partially or completely alleviates, ameliorates, mitigates, inhibits, delays the onset of, reduces the severity of, and / or reduces the incidence of one or more symptoms, characteristics, and / or causes of a particular disease, disorder, and / or condition. In some embodiments, such treatment may be administered to subjects who do not exhibit signs of the relevant disease, disorder, and / or condition and / or who exhibit only early signs of the disease, disorder, and / or condition. Alternatively or additionally, in some embodiments, treatment may be administered to subjects who exhibit one or more established signs of the relevant disease, disorder, and / or condition. In some embodiments, treatment may be administered to subjects who have been diagnosed with the relevant disease, disorder, and / or condition. In some embodiments, treatment may be administered to subjects known to have one or more susceptibility factors statistically correlated with an increased risk of developing the relevant disease, disorder, and / or condition.

[0178] The term "undruggable target" refers to a protein that is not a member of a protein family known to be a drug target and / or does not have a binding site suitable for high-affinity binding to a small molecule. Methods for determining whether a target protein is undruggable are known in the art. For example, whether a target protein is undruggable can be determined using a structure-based algorithm such as that used by the program DOGSITESCORER® (Universität Hamburg, Hamburg, Germany), which evaluates druggability based on calculated parameters for the binding pocket on a protein, including volume, surface area, lipophilic surface area, depth, and / or hydrophobicity ratio.

[0179] As used herein, the term "van der Waals interactions" refers to attractive or repulsive forces between atoms that are not due to covalent bonds, electrostatic interactions, or hydrogen bonds. The term "variant" refers to an entity that exhibits significant structural identity with a reference entity but differs structurally from the reference entity in the presence or level of one or more chemical moieties relative to the reference entity. In many embodiments, variants also differ functionally from the reference entity. Generally, whether a particular entity is properly considered a "variant" of a reference entity is based on the degree of structural identity with the reference entity. As one of skill in the art will appreciate, any biological or chemical reference entity possesses certain characteristic structural elements. A variant, by definition, is an identifiable chemical entity that shares one or more such characteristic structural elements. To name a few examples, small molecules can have a characteristic core structural element (e.g., a macrocyclic core) and / or one or more characteristic pendant moieties; variants of small molecules share a core structural element and characteristic pendant moieties but differ in other pendant moieties and / or in the type of bond (single vs. double bond, E vs. Z, etc.) present in the core; polypeptides can have characteristic sequence elements comprising multiple amino acids with specified relative positions in linear or three-dimensional space and / or that contribute to a particular biological function; and nucleic acids can have characteristic sequence elements comprising multiple nucleotide residues with specified relative positions in linear or three-dimensional space. For example, variant polypeptides can differ from a reference polypeptide as a result of one or more differences in amino acid sequence and / or one or more differences in chemical moieties (e.g., carbohydrates, lipids, etc.) covalently attached to the polypeptide backbone. In some embodiments, the variant polypeptide exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% overall sequence identity with the reference polypeptide. Alternatively or additionally, in some embodiments, the variant polypeptide does not share at least one characteristic sequence element with the reference polypeptide. In some embodiments, the reference polypeptide has one or more biological activities. In some embodiments, the variant polypeptide shares one or more of the biological activities with the reference polypeptide.In some embodiments, a variant polypeptide lacks one or more biological activities of a reference polypeptide. In some embodiments, a variant polypeptide exhibits one or more biological activities at a reduced level compared to the reference polypeptide. In many embodiments, a subject polypeptide is considered a "variant" of a parent or reference polypeptide if the subject polypeptide has an amino acid sequence identical to that of the parent, except for minor sequence changes at specific positions. Typically, less than 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, or 2% of the variant's residues are substituted compared to the parent. In some embodiments, a variant has 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substituted residue compared to the parent. Often, a variant has only a small number (e.g., less than 5, 4, 3, 2, or 1) of substituted functional residues (i.e., residues responsible for a particular biological activity). Furthermore, a variant typically has no more than 5, 4, 3, 2, or 1 additions or deletions compared to the parent, and often no additions or deletions. Furthermore, any additions or deletions will typically be less than about 25, 20, 19, 18, 17, 16, 15, 14, 13, 10, 9, 8, 7, 6, and usually less than about 5, 4, 3, or 2 residues. In some embodiments, the parent or reference polypeptide is one found in nature. As will be appreciated by those of skill in the art, multiple variants of a particular subject polypeptide will typically be found in nature.

[0180] The term "wild-type" refers to an entity having a structure and / or activity that is found in nature in a "normal" (as opposed to mutant, diseased, modified, etc.) state or situation. Those skilled in the art will recognize that wild-type genes and polypeptides often exist in multiple alternative forms (e.g., alleles). [Brief explanation of the drawings]

[0181] [Figure 1] Table of exemplary compounds of the present invention. [Figure 2] Table of exemplary compounds of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0182] Small molecules have limited targeting capabilities because their interactions with targets are driven by adhesive forces, the strength of which is roughly proportional to the contact surface area. Due to their small size, the only way for small molecules to create sufficient intermolecular contact surface area to effectively interact with a target protein is by directly wrapping around the protein. In fact, a large body of both experimental and computational data supports the view that only proteins with hydrophobic "pockets" on their surface can bind small molecules. In this case, binding is enabled by wrapping.

[0183] Nature has evolved strategies to allow small molecules to interact with target proteins at sites other than hydrophobic pockets. This strategy is exemplified by the naturally occurring immunosuppressants cyclosporin A, rapamycin, and FK506. The biological activity of these drugs requires the formation of high-affinity complexes between the small molecule and a small, displayed protein. The complex surface of the small molecule and the displayed protein engages the target. Thus, for example, the binary complex formed between cyclosporin A and cyclophilin A targets calcineurin with high affinity and specificity, whereas neither cyclosporin A nor cyclophilin A alone binds to calcineurin with measurable affinity.

[0184] Many important therapeutic targets exert their function through complexation with other proteins. In many of these systems, protein / protein interaction surfaces contain an inner core of hydrophobic side chains surrounded by a broad ring of polar residues. Because hydrophobic residues contribute nearly all of the energetically favorable contacts, this cluster has been represented as a "hot spot" for protein-protein interaction engagement. Importantly, in the above-listed complexes of naturally occurring small molecules with small, displayed proteins, the small molecule provides a cluster of hydrophobic features similar to the hot spot, while the protein provides a ring of primarily polar residues. In other words, the displayed small molecule systems mimic the surface architecture commonly utilized in natural protein / protein interaction systems.

[0185] Nature has demonstrated the ability to reprogram the target specificity of presented small-molecule-portable hotspots through evolutionary diversification. In the best-characterized example, the complex formed between FK506-binding protein (FKBP) and FK506 targets calcineurin. However, FKBP can also form a complex with the related molecule rapamycin, and this complex interacts with an entirely different target, TorC1. So far, no method has been developed to reprogram the binding and modulation capabilities of presenter protein / ligand interfaces to interact with and modulate other target proteins previously considered undruggable.

[0186] Additionally, it is widely recognized that some drug candidates fail because they modulate the activity of both their intended target and other unintended proteins in the same way. This problem is particularly challenging when the drug-binding site of the target protein is similar to that of a non-target protein. The insulin-like growth factor receptor (IGF-1R), whose ATP-binding pocket is structurally similar to that of the non-target insulin receptor (IR), is one such example. Small molecule development candidates designed to target IGF-1R also typically have unacceptable side effects that also modulate the insulin receptor. However, structural dissimilarity exists between these two proteins in the region surrounding the ATP-binding pocket. Despite this knowledge, there is currently no method to take advantage of these differences to develop drugs that are more specific to IGF-1R than IR.

[0187] The invention features compounds (e.g., macrocyclic compounds) that can modulate biological processes, e.g., by binding to a presenter protein (e.g., a member of the FKBP family, a member of the cyclophilin family, or PIN1) and a target protein. In some embodiments, the target protein and / or presenter protein are intracellular proteins. In some embodiments, the target protein and / or presenter protein are mammalian proteins. In some embodiments, provided compounds participate in a tripartite presenter protein / compound / target protein complex within a cell, e.g., a mammalian cell. In some embodiments, provided compounds may be useful in the treatment of diseases and disorders, such as cancer, inflammation, and infectious diseases.

[0188] compound The present invention features compounds (e.g., macrocyclic compounds) that can modulate biological processes, for example, by binding to a presenter protein (e.g., a member of the FKBP family, a member of the cyclophilin family, or PIN1) and a target protein (e.g., a eukaryotic target protein, such as a mammalian target protein or a fungal target protein, or a prokaryotic target protein, such as a bacterial target protein). Briefly, such compounds bind to an endogenous intracellular presenter protein, such as FKBP, and the resulting binary complex selectively binds to and modulates the activity of the intracellular target protein. Without wishing to be bound by any particular theory, we propose that the formation of a tripartite complex between the presenter protein, compound, and target protein is driven by both protein-compound and protein-protein interactions, and both are required for modulation (e.g., positive or negative modulation) of the target protein's activity. In some embodiments, the compounds of the invention provide the ability to modulate (e.g., positively or negatively modulate) the activity of these new targets by "reprogramming" the binding of presenter proteins to protein targets that do not normally bind to the presenter protein and whose binding is not significantly enhanced in the presence of the compound.

[0189] As described herein, the compounds of the present invention comprise a presenter protein-binding moiety and a target protein-interacting moiety. In some embodiments, the presenter protein-binding moiety and the target protein-interacting moiety are separate parts of a ring structure, e.g., they do not overlap. In some embodiments, the presenter protein-binding moiety and the target protein-interacting moiety are connected to each other on one or both sides by a linker.

[0190] In some embodiments, the compounds of the present invention do not substantially bind to the target protein in the absence of complex formation, as described herein. In some embodiments, the complex of the compound of the present invention and a presenter protein, as described herein, binds to the target protein with an affinity that is at least 5 times (at least 10 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 100 times) greater than the affinity of the compound for the target protein in the absence of complex formation. In certain embodiments, the compounds of the present invention do not substantially modulate the activity of the target protein in the absence of complex formation with a presenter protein. For example, in some embodiments, the compounds of the present invention have an IC of greater than 10 μM (e.g., greater than 20 μM, greater than 50 μM, greater than 100 μM, greater than 500 μM). 50 Alternatively, compounds of the invention inhibit the activity of a target protein at AC values ​​greater than 10 μM (e.g., greater than 20 μM, greater than 50 μM, greater than 100 μM, greater than 500 μM). 50 In certain embodiments, the complex of the compound and the presenter protein is at least 5-fold more active than the compound alone (i.e., 1 / 5th the IC 50 or AC 50 (having).

[0191] The compounds (e.g., macrocyclic compounds) of the invention generally bind strongly to presenter proteins. For example, in some embodiments, the compounds (e.g., macrocyclic compounds) of the invention have a K of less than 10 μM (e.g., less than 5 μM, less than 1 μM, less than 500 nM, less than 200 nM, less than 100 nM, less than 75 nM, less than 50 nM, less than 25 nM, less than 10 nM). D or, for example, an IC of less than 1 μM (e.g., less than 0.5 μM, less than 0.1 μM, less than 0.05 μM, less than 0.01 μM). 50 inhibits the peptidyl-prolyl isomerase activity of presenter proteins.

[0192] In some embodiments, the present invention provides compounds of formulas XIV-XVIII described herein:

[0193] [ka]

[0194] This includes compounds having the structure shown in In certain embodiments, the compound has the structure of any of the compounds in FIG. 1 or FIG.

[0195] In some embodiments, the compound is a naturally occurring compound (e.g., synthesized by a genetically unmodified bacterial strain). In some embodiments, the compound is a variant of a naturally occurring compound (e.g., a semi-synthetic compound). In some embodiments, the variant shares a ring size with the reference naturally occurring compound. In some embodiments, the variant differs from the reference naturally occurring compound only in the identity of one or more substituents (e.g., for at least one position, the variant has a different substituent or set of substituents than found at the corresponding position in the appropriate reference compound).

[0196] In some embodiments, compounds of the invention (e.g., macrocyclic compounds of the invention) contain 12 to 40 ring atoms (e.g., 12 to 20 ring atoms, 14 to 20 ring atoms, 17 to 25 ring atoms, 21 to 26 ring atoms, 20 to 30 ring atoms, 25 to 35 ring atoms, 30 to 40 ring atoms). In some embodiments, such compounds contain 19 ring atoms. In some embodiments, such compounds have an even number of ring atoms. In certain embodiments, at least 25% (e.g., at least 30%, at least 35%, at least 40%, at least 45%) of the atoms in the compound are included in a single ring or fused ring system.

[0197] In some embodiments, the compounds of the present invention comprise rings (e.g., macrocycles) whose ring atoms are selected from the group consisting of carbon atoms, hydrogen atoms, nitrogen atoms, oxygen atoms, sulfur atoms, phosphorus atoms, silicon atoms, and combinations thereof, and in some embodiments, all of the ring atoms in the compounds are selected from this group. In some embodiments, the compounds of the present invention comprise rings (e.g., macrocycles) whose ring atoms are selected from the group consisting exclusively of carbon atoms, hydrogen atoms, nitrogen atoms, oxygen atoms, and combinations thereof, and in some embodiments, all of the ring atoms in the compounds are selected from the group consisting exclusively of carbon atoms, hydrogen atoms, nitrogen atoms, oxygen atoms, and combinations thereof.

[0198] In certain embodiments, the ring bond in the provided compounds (eg, macrocyclic compounds) of the present invention comprises a ketone, ester, amide, ether, thioester, urea, amidine, or hydrocarbon.

[0199] In some embodiments, provided compounds are non-peptides. In certain embodiments, provided compounds comprise one or more amino acid residues. In some embodiments, provided compounds comprise only amino acid residues.

[0200] In some embodiments, the compounds of the invention have a molecular weight of 400-2000 daltons (e.g., 400-600 daltons, 500-700 daltons, 600-800 daltons, 700-900 daltons, 800-1000 daltons, 900-1100 daltons, 1000-1200 daltons, 1100-1300 daltons, 1200-1400 daltons, 1300-1500 daltons, 1400-1600 daltons, 1500-1700 daltons, 1600-1800 daltons, 1700-1900 daltons, 1800-2000 daltons, 400-1000 daltons, 1000-2000 daltons). In some embodiments, the molecular weight of the compounds of the invention is less than 2000 daltons (e.g., less than 500 daltons, less than 600 daltons, less than 700 daltons, less than 800 daltons, less than 900 daltons, less than 1000 daltons, less than 1100 daltons, less than 1200 daltons, less than 1300 daltons, less than 1400 daltons, less than 1500 daltons, less than 1600 daltons, less than 1700 daltons, less than 1800 daltons, less than 1900 daltons).

[0201] In certain embodiments, provided molecular compounds are hydrophobic. For example, in some embodiments, the compounds have a cLogP of 2 or greater (e.g., 2.5 or greater, 3.0 or greater, 3.5 or greater, 4 or greater, 4.5 or greater, 5 or greater, 5.5 or greater, 6 or greater, 6.5 or greater, 7 or greater). Alternatively, in some embodiments, the compounds have a cLogP of 2 to 7 (e.g., 2 to 4, 3.5 to 4.5, 4 to 5, 4.5 to 5.5, 5 to 6, 5.5 to 6.5, 6 to 7, 4 to 7, 4 to 6, 4 to 5.5). Provided compounds can also be characterized as hydrophobic by having low aqueous solubility. For example, in some embodiments, the compounds have an aqueous solubility of greater than 1 μM (e.g., greater than 1 μM, greater than 2 μM, greater than 5 μM, greater than 10 μM, greater than 20 μM, greater than 30 μM, greater than 40 μM, greater than 50 μM, greater than 75 μM, or greater than 100 μM). Alternatively, in some embodiments, the compound has an aqueous solubility of 1-100 μM (eg, 1-10 μM, 5-10 μM, 5-20 μM, 10-50 μM, 5-50 μM, 20-100 μM).

[0202] In some embodiments, the compounds of the invention are cell-permeable (e.g., capable of entering the intracellular domain of a cell without killing the cell and / or capable of entering the extracellular domain upon contact with the extracellular surroundings).

[0203] The compounds of the present invention may be naturally occurring or non-naturally occurring. In some embodiments, the compounds of the present invention are not naturally occurring. In certain embodiments, the compounds of the present invention are engineered. An engineered compound is a compound whose design and / or production requires the intervention of man (e.g., a compound prepared by chemical synthesis, a compound prepared by cells genetically engineered relative to a reference wild-type cell, a compound produced by cells under modified culture conditions to enhance production of the compound).

[0204] In certain embodiments, a provided compound (e.g., a macrocyclic compound) is not a compound described in Benjamin et al., Nat. Rev. Drug. Discov., 2011, Vol. 10, No. 11, pp. 868-880, or Sweeney, ZK et al., J. Med. Chem., 2014, epub ahead of print, the structures of which are incorporated by reference, and / or a compound having the structure:

[0205] Presenter protein binding site The compounds of the invention comprise a presenter protein binding moiety such that the provided compounds specifically bind to said presenter protein, e.g., with a KD of less than 10 μM (e.g., less than 5 μM, less than 1 μM, less than 500 nM, less than 200 nM, less than 100 nM, less than 75 nM, less than 50 nM, less than 25 nM, less than 10 nM), or with an IC of less than 1 μM (e.g., less than 0.5 μM, less than 0.1 μM, less than 0.05 μM, less than 0.01 μM). 50and includes a group of ring atoms (e.g., 5-20 ring atoms, 5-10 ring atoms, 10-20 ring atoms) and their attached moieties (e.g., atoms within 20 ring atoms, e.g., atoms within 15 ring atoms, atoms within 10 ring atoms, atoms within 5 ring atoms) involved in binding to the presenter protein so as to inhibit the peptidyl-prolyl isomerase activity of the presenter protein. In some embodiments, the presenter protein binding moiety does not encompass all of the atoms in the provided compound that interact with the presenter protein. In some embodiments, one or more atoms of the presenter protein binding moiety can be within the target protein interacting moiety (e.g., a eukaryotic target protein interacting moiety such as a mammalian target protein interacting moiety or a fungal target protein interacting moiety, or a prokaryotic target protein interacting moiety such as a bacterial target protein interacting moiety). In certain embodiments, one or more atoms of the presenter protein binding moiety do not interact with the presenter protein.

[0206] In some embodiments, the presenter protein binding moiety comprises an N-acylproline moiety, an N-acyl-pipecolic acid moiety, an N-acyl3-morpholinocarboxylic acid moiety, and / or an N-acylpiperazine acid moiety (e.g., either nitrogen atom is acylated). In certain embodiments, the presenter protein binding moiety comprises an N-acyl-pipecolic acid moiety. In some embodiments, the presenter protein binding moiety comprises an N-acylproline moiety. In certain embodiments, the presenter protein binding moiety comprises an N-acyl3-morpholinocarboxylic acid moiety. In some embodiments, the presenter protein binding moiety comprises an N-acylpiperazine acid moiety.

[0207] In some embodiments, at least one atom of the presenter protein binding moiety is involved in binding to one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) of Tyr27, Phe37, Asp38, Arg41, Phe47, Gln54, Glu55, Val56, Ile57, Trp60, Ala82, Try83, His88, Ile92, and / or Phe100 of FKBP12. In some embodiments, at least one atom of the presenter protein binding moiety is involved in binding to at least one (e.g., 2, 3, or 4) of Arg41, Gln54, Glu55, and / or Ala82 of FKBP12.

[0208] In some embodiments, the presenter protein binding moiety has Formulas I-VIII:

[0209] [ka]

[0210] It has the structure shown below. In some embodiments, the presenter protein binding moiety has the formula Ia-IVa:

[0211] [ka]

[0212] It has the structure shown below. In some embodiments, the presenter protein binding moiety has the structure:

[0213] [ka]

[0214] JPEG2026015411000030.jpg199170

[0215] JPEG2026015411000031.jpg172170

[0216] or comprises or consists of a stereoisomer thereof. In certain embodiments, the presenter protein binding moiety has the structure:

[0217] [ka]

[0218] or includes the structure thereof. Target protein interaction moiety The compounds of the invention comprise a target protein interacting moiety (e.g., a eukaryotic target protein interacting moiety, such as a mammalian target protein interacting moiety or a fungal target protein interacting moiety, or a prokaryotic target protein interacting moiety, such as a bacterial target protein interacting moiety). This moiety comprises a group of ring atoms (e.g., 5-20 ring atoms, 5-10 ring atoms, 10-20 ring atoms) and their attached moieties (e.g., atoms within 20 ring atoms, e.g., atoms within 15 ring atoms, atoms within 10 ring atoms, atoms within 5 ring atoms) that specifically bind to the target protein when the compound is complexed with the presenter protein. In some embodiments, the target protein interacting moiety comprises multiple atoms in the compound that interact with the target protein. In some embodiments, one or more atoms of the target protein interacting moiety can be within the presenter protein binding moiety. In certain embodiments, one or more atoms of the target protein interacting moiety do not interact with the target protein.

[0219] The target protein can be bound to a ring atom in the target protein interaction moiety. Alternatively, the target protein can be bound to two or more ring atoms in the target protein interaction moiety. Alternatively, the target protein can be bound to a substituent bound to one or more ring atoms in the target protein interaction moiety. Alternatively, the target protein can be bound to a ring atom in the target protein interaction moiety and a substituent bound to one or more ring atoms in the target protein interaction moiety. Alternatively, the target protein is bound to a group that mimics the target protein's natural ligand, and the group that mimics the target protein's natural ligand is bound to the target protein interaction moiety. Alternatively, the target protein is bound to a presenter protein, and the affinity of the target protein for the presenter protein in the binary complex is increased compared to the affinity of the target protein for the presenter protein in the absence of the complex. In these examples, the binding is typically, but not exclusively, via a non-covalent interaction of the target protein with the target protein interaction moiety.

[0220] In some embodiments, the target protein interacting moiety is hydrophobic. For example, in some embodiments, the target protein interacting moiety has a cLogP of 2 or greater (e.g., 2.5 or greater, 3 or greater, 3.5 or greater, 4 or greater, 4.5 or greater, 5 or greater, 5.5 or greater, 6 or greater, 6.5 or greater, 7 or greater). Alternatively, in some embodiments, the target protein interacting moiety has a cLogP of 2-7 (e.g., 2-4, 2.5-4.5, 3-5, 3.5-5.5, 4-6, 4.5-6.5, 5-7, 3-6, 3-5, 3-5.5). The target protein interacting moiety may also be characterized as hydrophobic because of its low polar surface area. For example, in some embodiments, the target protein interacting moiety has a cLogP of 350 Å or greater. 2 Less than (e.g., 300 Å 2 Less than 250Å 2 Less than 200Å 2 Less than 150Å 2 Less than 125Å 2 It has a polar surface area of ​​less than 1000 nm.

[0221] In some embodiments, the target protein interacting moiety comprises one or more hydrophobic pendant groups (e.g., one or more methyl, ethyl, isopropyl, phenyl, benzyl, and / or phenethyl groups). In some embodiments, the pendant group comprises fewer than 30 total atoms (e.g., fewer than 25 total atoms, fewer than 20 total atoms, fewer than 15 total atoms, fewer than 10 total atoms). Alternatively, in some embodiments, the pendant group comprises 10-30 total atoms (e.g., 10-20 total atoms, 15-25 total atoms, 20-30 total atoms). In certain embodiments, the pendant group has a molecular weight of less than 200 daltons (e.g., less than 150 daltons, less than 100 daltons, less than 75 daltons, less than 50 daltons). Alternatively, in some embodiments, the pendant group has a molecular weight of 50-200 daltons (e.g., 50-100 daltons, 75-150 daltons, 100-200 daltons).

[0222] In some embodiments, the target protein interacting moiety is hydrocarbon-based (e.g., the moiety comprises primarily carbon-carbon bonds). In some embodiments, the target protein interacting moiety is hydrocarbon-based and comprises a linear divalent C4-C6 moiety composed primarily of carbon and hydrogen, optionally containing one or more double bonds. 30 (For example, C6~C 20 , C6~C 15 ) aliphatic groups. In some embodiments, the divalent aliphatic groups can also be replaced with groups that mimic natural ligands that bind to the target protein. Examples include phosphotyrosine mimetics and ATP mimetics.

[0223] In some embodiments, the target protein interacting moiety is peptide-based (e.g., the moiety comprises a peptide bond). In some embodiments, the target protein interacting moiety is peptide-based and comprises one or more (e.g., 2, 3, 4, 5, 6, 7, or 8) alanine residues, one or more (e.g., 2, 3, 4, 5, 6, 7, or 8) valine residues, one or more (e.g., 2, 3, 4, 5, 6, 7, or 8) isoleucine residues, one or more (e.g., 2, 3, 4, 5, 6, 7, or 8) leucine residues, one or more (e.g., 2, 3, 4, 5, 6, 7, or 8) cyclohexyl 1,2,3,4,5,6,7, or 8 cyclohexyl 1 ... In some embodiments, the target protein interacting moiety comprises one or more (e.g., 2, 3, 4, 5, 6, 7, or 8) methionine residues, one or more (e.g., 2, 3, 4, 5, 6, 7, or 8) phenylalanine residues, one or more (e.g., 2, 3, 4, 5, 6, 7, or 8) tyrosine residues, one or more (e.g., 2, 3, 4, 5, 6, 7, or 8) tryptophan residues, one or more (e.g., 2, 3, 4, 5, 6, 7, or 8) glycine residues, and / or one or more (e.g., 2, 3, 4, 5, 6, 7, or 8) proline residues. In some embodiments, the target protein interacting moiety is peptide-based and comprises one or more (e.g., 2, 3, 4, 5, 6, 7, or 8) arginine residues or one or more (e.g., 2, 3, 4, 5, 6, 7, or 8) lysine residues. In some embodiments, the target protein interacting portion is peptide-based and comprises one or more (e.g., 2, 3, 4, 5, 6, 7, or 8) unnatural amino acids, one or more (e.g., 2, 3, 4, 5, 6, 7, or 8) D-amino acids, and / or one or more (e.g., 2, 3, 4, 5, 6, 7, or 8) N-alkylated amino acids. In some embodiments, the target protein interacting portion is peptide-based and comprises predominantly D-amino acids (e.g., at least 50% of the amino acids are D-amino acids, at least 75% of the amino acids are D-amino acids, or 100% of the amino acids are D-amino acids).In certain embodiments, the target protein interacting portion is peptide-based and comprises predominantly N-alkylated amino acids (e.g., at least 50% of the amino acids are N-alkylated amino acids, at least 75% of the amino acids are N-alkylated amino acids, or 100% of the amino acids are N-alkylated amino acids). In certain embodiments, the target protein interacting portion is peptide-based and comprises one or more (e.g., 2, 3, 4, 5, 6, 7, or 8) Depsi bonds.

[0224] In some embodiments, the target protein interacting moiety (e.g., a eukaryotic target protein interacting moiety, such as a mammalian target protein interacting moiety or a fungal target protein interacting moiety, or a prokaryotic target protein interacting moiety, such as a bacterial target protein interacting moiety) has Formula IX:

[0225] [ka]

[0226] (wherein u is an integer of 1 to 20, and Each Y is independently any amino acid, O, NR20, S, S(O), SO2, or a group represented by Formulas X-XIII:

[0227] [ka]

[0228] (where each R 20 are independently selected from hydrogen, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted aryl, C3-C7 carbocyclyl, optionally substituted C6-C 10 arylC1-C6 alkyl, and optionally substituted C3-C7 carbocyclylC1-C6 alkyl, or R 19 is any R 20 , R 21 , R22 , R 23 , R 24 , R 25 , R 26 , R 27 , R 28 , R 29 , or R 30 in combination with an optionally substituted C3-C 10 Carbocyclyl, optionally substituted C-C 10 forming an aryl or an optionally substituted C2-C9 heteroaryl; Each R 21 and R 22 are independently hydrogen, halogen, optionally substituted hydroxyl, optionally substituted amino, or R 20 and R 21 are, in combination, ═O, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C3-C 10 Carbocyclyl, optionally substituted C-C 10 Aryl, optionally substituted C-C 10 arylC1-C6 alkyl, optionally substituted C2-C9 heteroaryl, optionally substituted C2-C9 heteroarylC1-C6 alkyl, optionally substituted C2-C9 heterocyclyl, or optionally substituted C2-C9 heterocyclylC1-C6 alkyl, or R 21 Or R 22 is any R 20 , R 21 , R 22 , R 23 , R 24 , R 25 , R 26 , R 27 , R 28 , R 29 , or R 30 in combination with an optionally substituted C3-C 10 Carbocyclyl, optionally substituted C-C 10 forming an aryl or an optionally substituted C2-C9 heteroaryl; Each R 23 , R 24 , R 25 , and R 26 are independently hydrogen and hydroxyl, or R 23 and R 24 combine to form =O or R 23 , R 24 , R 25 , or R 26 is any R 20 , R 21 , R 22 , R 23 , R 24 , R 25 , R 26 , R 27 , R 28 , R 29 , or R 30 in combination with an optionally substituted C3-C 10 Carbocyclyl, optionally substituted C-C 10 aryl or optionally substituted C2-C9 heteroaryl; and Each R 27 , R 28 , R 29 , and R 30 are independently hydrogen, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C3-C 10 Carbocyclyl, optionally substituted C-C 10 Aryl, optionally substituted C-C 10 arylC1-C6 alkyl, optionally substituted C2-C9 heteroaryl, optionally substituted C2-C9 heteroarylC1-C6 alkyl, optionally substituted C2-C9 heterocyclyl, or optionally substituted C2-C9 heterocyclylC1-C6 alkyl, or R 27 , R 28 , R 29 , or R 30 is any R 20 , R 21 , R 22 , R 23, R 24 , R 25 , R 26 , R 27 , R 28 , R 29 , or R 30 in combination with an optionally substituted C3-C 10 Carbocyclyl, optionally substituted C-C 10 aryl, or optionally substituted C2-C9 heteroaryl) (having a structure represented by one of the following): It has the structure shown below.

[0229] In some embodiments, the target protein interacting moiety has formula IXa:

[0230] [ka]

[0231] It has the structure shown below. In certain embodiments, the target protein interacting moiety (e.g., a eukaryotic target protein interacting moiety such as a mammalian target protein interacting moiety or a fungal target protein interacting moiety or a prokaryotic target protein interacting moiety such as a bacterial target protein interacting moiety) has the structure:

[0232] [ka]

[0233] Does not include. Linker The compounds of the present invention comprise a linker (e.g., two linkers connecting a presenter protein binding moiety and a target protein interacting moiety (e.g., a eukaryotic target protein interacting moiety such as a mammalian target protein interacting moiety or a fungal target protein interacting moiety or a prokaryotic target protein interacting moiety such as a bacterial target protein interacting moiety). The linker component of the present invention is, in the simplest case, a bond, but may also provide a linear, cyclic, or branched molecular scaffold with pendant groups covalently linking the two moieties.

[0234] In some embodiments, at least one atom of the linker is involved in binding to the presenter protein and / or the target protein, while in certain embodiments, at least one atom of the linker is not involved in binding to the presenter protein and / or the target protein.

[0235] Thus, attachment of the two moieties is achieved by covalent means involving bond formation through one or more functional groups located on both moieties. Examples of chemically reactive functional groups that can be utilized for this purpose include, but are not limited to, carbonyl, carbohydrate groups, vicinal diols, thioethers, 2-amino alcohols, 2-aminothiols, guanidinyl, imidazolyl, and phenol groups.

[0236] Covalent bonding of two moieties can be achieved using a linker that contains a reactive moiety capable of reacting with such functional groups present on both moieties. For example, an amine group on a moiety can react with a carboxyl group or an activated derivative thereof on the linker, thus forming an amide linking the two.

[0237] Examples of moieties capable of reacting with sulfhydryl groups include α-haloacetyl compounds of the XCH2CO- type (where X = Br, Cl, or I). These compounds not only exhibit specific reactivity toward sulfhydryl groups, as described by Gurd, Methods Enzymol. 11, 532 (1967), but can also be used to modify imidazolyl, thioether, phenolic, and amino groups. N-Maleimide derivatives are also considered selective for sulfhydryl groups, but may also be useful for coupling with amino groups under certain conditions. Reagents such as 2-iminothiolane, which introduces a thiol group by conversion of an amino group (Traut et al., Biochemistry 12, 3266 (1973)), can be considered sulfhydryl reagents when coupling is achieved by disulfide bridge formation.

[0238] Examples of reactive moieties capable of reacting with amino groups include, for example, alkylating and acylating agents. Representative alkylating agents include: (i) α-haloacetyl compounds, which are specific for amino groups in the absence of reactive thiol groups and are of the XCHCO- type (where X = Br, Cl, or I), as described, for example, by Wong, Biochemistry, 24, 5337, 1979; (ii) N-maleimide derivatives, which can react with amino groups via a Michael-type reaction or via acylation by addition to a ring carbonyl group, as described, for example, in Smyth et al., J. Am. Chem. Soc., 82, 4600 (1960) and Biochem. J., 91, 589 (1964); (iii) aryl halides, e.g., reactive nitrohaloaromatic compounds; (iv) alkyl halides, for example, McKenzie et al., J. Protein Chem., Vol. 7, p. 581, 1988; (v) Aldehydes and ketones capable of forming Schiff bases with amino groups, the adducts formed usually affording stable amines upon reduction. (vi) epoxide derivatives, such as epichlorohydrin and bisoxiranes, which can react with amino, sulfhydryl, or phenolic hydroxyl groups; (vii) chlorine-containing derivatives of s-triazines, which are highly reactive towards nucleophiles such as amino, sulfhydryl and hydroxyl groups; (viii) Aziridines based on the s-triazine compounds detailed above, such as those described by Ross in J. Adv. Cancer Res., Vol. 2, p. 1, 1954, which react with nucleophiles such as amino groups by ring-opening. (ix) squaric acid diethyl ester, as described in Tietze, Chem. Ber., 124, 1215, 1991, and (x) α-haloalkyl ethers, which are more reactive alkylating agents than conventional alkyl halides due to the activation caused by the ether oxygen atom, as described by Benneche et al., Eur. J. Med. Chem., Vol. 28, p. 463, 1993; Examples include:

[0239] Representative amino-reactive acylating agents include: (i) isocyanates and isothiocyanates, especially aromatic derivatives, which form stable urea and thiourea derivatives, respectively; (ii) sulfonyl chlorides, as described by Herzig et al., Biopolymers, Vol. 2, p. 349, 1964; (iii) acid halides, (iv) active esters, such as nitrophenyl esters or N-hydroxysuccinimidyl esters; (v) acid anhydrides, such as mixed, symmetric, or N-carboxy anhydrides; (vi) Other useful reagents for amide bond formation, e.g., M. Bodansky, Principles of Peptide Synthesis Synthesis, Springer-Verlag, 1984, (vii) acyl azides, the azide group of which is generated from a preformed hydrazide derivative using sodium nitrite as described by Wetz et al., Anal. Biochem., 58, 347, 1974; (viii) imidoesters, which react with amino groups to form stable amidines, as described, for example, by Hunter and Ludwig, J. Am. Chem. Soc., 84, 3491 (1962), and (ix) a haloheteroaryl group, such as a halopyridine or halopyrimidine; Examples include:

[0240] Aldehydes and ketones can react with amines to form Schiff bases, which can be advantageously stabilized by reductive amination. Alkoxylamino moieties can be synthesized, for example, as described by Webb et al., Bioconjugate Chemistry, Vol. 1, No. 1, pp. 111-114, 1999. Chem., Vol. 1, p. 96, 1990, it readily reacts with ketones and aldehydes to produce stable alkoxamines.

[0241] Examples of reactive moieties that can react with carboxyl groups include diazo compounds, such as diazoacetate esters and diazoacetamides, which react with high specificity to produce ester groups, as described, for example, in Herriot, Adv. Protein Chem., 3, 169, 1947. Carboxyl-modifying reagents such as carbodiimides are also available, which react via O-acylurea formation and subsequent amide bond formation.

[0242] It will be appreciated that, if desired, functional groups on either moiety may be converted to other functional groups prior to reaction, for example to impart additional reactivity or selectivity. Examples of methods useful for this purpose include conversion of amines to carboxyls using reagents such as dicarboxylic acid anhydrides, conversion of amines to thiols using reagents such as N-acetylhomocysteine ​​thiolactone, S-acetylmercaptosuccinic anhydride, 2-iminothiolane, and thiol-containing succinimidyl derivatives, conversion of thiols to carboxyls using reagents such as α-haloacetates, conversion of thiols to amines using reagents such as ethyleneimine and 2-bromoethylamine, conversion of carboxyls to amines using reagents such as carbodiimides followed by diamines, and conversion of alcohols to thiols using reagents such as tosyl chloride followed by transesterification with thioacetate and hydrolysis to thiols using sodium acetate.

[0243] If desired, so-called zero-length linkers may be used in accordance with the present invention, which involve a direct covalent bond between a reactive chemical group on one moiety and a reactive chemical group on another moiety without the introduction of additional linking material.

[0244] More commonly, however, the linker will contain two or more reactive moieties connected by a spacer element, as described above. The presence of such a spacer allows the bifunctional linker to react with a specific functional group in either moiety to form a covalent bond between the two. The reactive moieties in the linker can be the same (homo-bifunctional linker) or different (heterobifunctional linker, or, if several dissimilar reactive moieties are present, a multifunctional linker), providing a variety of possible reagents that can form a covalent bond between the two moieties.

[0245] The spacer element in the linker typically consists of a straight or branched chain, 1~10 Alkyl, C 2~10 Alkenyl, C 2~10 Alkynyl, C 2~ 6Heterocyclyl, C 6~12 Aryl, C 7~14 Alkaril, C 3~10 Alkheterocyclyl, C2-C 100 Polyethylene glycol, or C 1~10 It may include heteroalkyl.

[0246] In some cases, the linker is described by Formula V: Examples of homobifunctional linkers useful in preparing the conjugates of the present invention include, but are not limited to, diamines and diols selected from ethylenediamine, propylenediamine and hexamethylenediamine, ethylene glycol, diethylene glycol, propylene glycol, 1,4-butanediol, 1,6-hexanediol, cyclohexanediol, and polycaprolactone diol.

[0247] In some embodiments, a linker is a bond or a linear chain of up to 10 atoms independently selected from carbon, nitrogen, oxygen, sulfur, or phosphorus atoms, each atom in the chain optionally substituted with one or more substituents independently selected from alkyl, alkenyl, alkynyl, aryl, heteroaryl, chloro, iodo, bromo, fluoro, hydroxyl, alkoxy, aryloxy, carboxy, amino, alkylamino, dialkylamino, acylamino, carboxamido, cyano, oxo, thio, alkylthio, arylthio, acylthio, alkylsulfonate, arylsulfonate, phosphoryl, and sulfonyl, and any two atoms in the chain together with the substituents attached thereto can form a ring, which can be further substituted and / or fused to one or more optionally substituted carbocyclic, heterocyclic, aryl, or heteroaryl rings.

[0248] In some embodiments, the linker has Formula XIX: A 1 -(B 1 ) a -(C 1 ) b -(B 2 ) c -(D)-(B 3 ) d -(C 2 )e-(B 4 ) f -A 2 Formula XIX (In the formula, A 1 is the bond between the linker and the presenter protein binding moiety, and A 2 is the bond between the mammalian target interacting moiety and the linker, and B 1 , B 2 , B 3 , and B 4 are each independently an optionally substituted C1-C2 alkyl, an optionally substituted C1-C3 heteroalkyl, O, S, and NR N Selected from R N is hydrogen, optionally substituted C 1~4Alkyl, optionally substituted C 3~4 Alkenyl, optionally substituted C 2~4 Alkynyl, optionally substituted C 2~6 Heterocyclyl, optionally substituted C 6~12 aryl, or optionally substituted C 1~7 Heteroalkyl, C 1 and C 2 are each independently selected from carbonyl, thiocarbonyl, sulfonyl, or phosphoryl; a, b, c, d, e, and f are each independently 0 or 1; and D is optionally substituted C 1~10 Alkyl, optionally substituted C 2~10 Alkenyl, optionally substituted C 2~10 Alkynyl, optionally substituted C2-6 heterocyclyl, optionally substituted C 6~12 Aryl, optionally substituted C-C 10 Polyethylene glycol or optionally substituted C 1~10 Heteroalkyl, or A 1 -(B 1 )a-(C 1 )b-(B 2 )c- and -(B 3 )d-(C 2 )e-(B 4 )fA 2 (This is a chemical bond that connects It has the following structure.

[0249] Compound properties Pharmacokinetic parameters To demonstrate bioavailability, for example, an in vivo rat early phase pharmacokinetic (EPK) study design can be used to assess the preliminary exposure characteristics of a compound. For example, a particular formulation can be administered to male Sprague-Dawley rats via oral (PO) gavage. Blood samples can then be collected from the animals at six time points up to four hours post-dose. Pharmacokinetic analysis is then performed based on LC-MS / MS-measured concentrations of each compound at each time point. Cell permeability In some embodiments, the compounds are cell-permeable. Any method known in the art may be used to determine the permeability of a compound, such as the biosensor assays described herein.

[0250] protein Presenter Protein A presenter protein can bind to a small molecule to form a complex, and this complex can bind to and modulate the activity of a target protein (e.g., a eukaryotic target protein, such as a mammalian target protein or a fungal target protein, or a prokaryotic target protein, such as a bacterial target protein). In some embodiments, the presenter protein is a mammalian presenter protein (e.g., a human presenter protein). In some embodiments, the presenter protein is a fungal presenter protein. In certain embodiments, the presenter protein is a bacterial presenter protein. In some embodiments, the presenter protein is a plant presenter protein. In some embodiments, the presenter protein is a relatively abundant protein (e.g., the presenter protein is sufficiently abundant that its association with a tripartite complex does not substantially adversely affect the biological role of the presenter protein in the cell and / or the viability or other attributes of the cell). In some embodiments, the presenter protein is a more abundant target protein. In certain embodiments, the presenter protein is a protein that has chaperone activity within the cell. In some embodiments, the presenter protein has multiple natural interaction partners within the cell. In certain embodiments, the presenter protein is known to bind to a small molecule to form a binary complex that is known or predicted to bind to a target protein and modulate its biological activity. Immunophilins are a class of presenter proteins known to have this function, including FKBPs and cyclophilins. In some embodiments, the reference presenter protein exhibits peptidyl prolyl isomerase activity. In some embodiments, the presenter protein exhibits activity comparable to that of the reference presenter protein.In certain embodiments, the presenter protein is a member of the FKBP family (e.g., FKBP12, FKBP12.6, FKBP13, FKBP19, FKBP22, FKBP23, FKBP25, FKBP36, FKBP38, FKBP51, FKBP52, FKBP60, FKBP65, and FKBP133), a member of the cyclophilin family (e.g., PP1A, CYPB, CYPC, CYP40, CYPE, CYPD, NKTR, SRCyp, CYPH, CWC27, CYPL1, CYP60, CYPJ, PPIL4, PPIL6, RANBP2, PPWD1, PPIAL4A, PPIAL4B, PPIAL4C, PPIAL4D, or PPIAL4G), or PIN1. The "FKBP family" is a family of proteins with prolyl isomerase activity that function as protein folding chaperones for proteins containing proline residues. Genes encoding proteins of this family include AIP, AIPL1, FKBP1A, FKBP1B, FKBP2, FKBP3, FKBP4, FKBP5, FKBP6, FKBP7, FKBP8, FKBP9, FKBP9L, FKBP10, FKBP11, FKBP14, FKBP15, and LOC541473.

[0251] The "cyclophilin family" is a family of proteins that bind to cyclosporine. Genes encoding proteins in this family include PPIA, PPIB, PPIC, PPID, PPIE, PPIF, PPIG, PPIH, SDCCAG-10, PPIL1, PPIL2, PPIL3, PPIL4, P270, PPWD1, and COAS-2. Exemplary cyclophilins include PP1A, CYPB, CYPC, CYP40, CYPE, CYPD, NKTR, SRCyp, CYPH, CWC27, CYPL1, CYP60, CYPJ, PPIL4, PPIL6, RANBP2, PPWD1, PPIAL4A, PPIAL4B, PPIAL4C, PPIAL4D, and PPIAL4G.

[0252] In some embodiments, the presenter protein is a chaperone protein such as GRP78 / BiP, GRP94, GRP170, calnexin, calreticulin, HSP47, ERp29, protein disulfide isomerase (PDI), or ERp57.

[0253] In some embodiments, the presenter protein is an allelic variant or splice variant of an FKBP or cyclophilin disclosed herein. In some embodiments, a presenter protein is a polypeptide whose amino acid sequence i) shows significant identity to that of a reference presenter protein, ii) contains a portion that shows significant identity to a corresponding portion of the reference presenter protein, and / or iii) contains at least one characteristic sequence found in a presenter protein. In many embodiments, identity of 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more is considered "significant" for the purposes of defining a presenter protein. In some embodiments, the portion showing significant identity is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 20, 21, 22, 23, 24 6, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 300, 350, 450, 500, 550, 600 or more amino acids in length.

[0254] Representative presenter proteins are encoded by the genes listed in Table 1 or homologs thereof. In some embodiments, a reference presenter protein is encoded by the set of genes shown in Table 1. Those skilled in the art can also refer to Table 3 to readily identify sequences characteristic of presenter proteins in general and / or specific subsets of presenter proteins.

[0255] [Table 1]

[0256] Table 1. Genes encoding selected presenter proteins Target protein Target proteins (e.g., eukaryotic target proteins, such as mammalian target proteins or fungal target proteins, or prokaryotic target proteins, such as bacterial target proteins) are proteins that mediate disease pathology or symptoms of disease pathology. Therefore, by modulating (inhibiting or increasing) their activity, a desired therapeutic effect can be achieved. Target proteins useful in the complexes and methods of the invention include those that are not naturally associated with a presenter protein, for example, those that have an affinity for the presenter protein of greater than 1 μM, preferably greater than 5 μM, and more preferably greater than 10 μM, in the absence of a binary complex with a compound of the invention. Alternatively, target proteins that are not naturally associated with a presenter protein are those that have an affinity for the compound of the invention of greater than 1 μM, preferably greater than 5 μM, and more preferably greater than 10 μM, in the absence of a binary complex. Alternatively, the target protein not naturally associated with the presenter protein has an affinity of greater than 1 μM, preferably greater than 5 μM, and more preferably greater than 10 μM for a binary complex of the presenter protein with cyclosporine, rapamycin, or FK506 (e.g., FKBP). Alternatively, the target protein not naturally associated with the presenter protein is other than calcineurin or mTOR. The choice of a suitable target protein for the complexes and methods of the invention may depend on the presenter protein. For example, a target protein with low affinity for cyclophilin may have high affinity for FKBP, in which case it would not be used with the latter.

[0257] A target protein can be naturally occurring, e.g., wild-type. Alternatively, a target protein can differ from the wild-type protein but still maintain a biological function, e.g., an allelic variant, splice mutant, or biologically active fragment.

[0258] In some embodiments, the target protein is a transmembrane protein. In some embodiments, the target protein has a coiled-coil structure. In certain embodiments, the target protein is one protein of a dimeric complex.

[0259] In some embodiments, target proteins of the invention comprise one or more surface sites (e.g., flat surface sites) that are characterized by low or undetectable binding of small molecules to the site in the absence of presenter protein / compound complex formation. In some embodiments, target proteins comprise one or more surface sites (e.g., flat surface sites) that exhibit low or undetectable binding of a particular small molecule (e.g., a compound) in the absence of presenter protein / compound complex formation (e.g., at most 2 / 3, 4 / 5, 6 / 7, 8 / 9, 10 / 10, 20 / 30, 40 / 50, 100 / 100, or less, of the binding observed for a presenter protein / compound complex containing the same compound). In some embodiments, the target protein has a surface (in some embodiments, the entire surface) characterized by one or more sites that lack any conventional binding pocket, e.g., lacks protein structural cavities or pockets with physiochemical and / or geometric properties comparable to proteins whose activity is modulated by one or more small molecules. In certain embodiments, the target protein has conventional binding pockets and sites suitable for protein-protein interactions. In some embodiments, the target protein is an undruggable target, e.g., the target protein is not a member of a protein family known to be a drug target and / or does not have a binding site that is expected to be suitable for binding to a small molecule (e.g., according to art-accepted understanding as discussed herein).

[0260] The most common type is the GTP interfaceば、DIRAS1、DIRAS2、DIRAS3、ERAS、GEM HRAS, KRAS, MRAS, NKIRAS1, NKIRAS2, NRAS, RALA, RALB, RAP1A, RAP1B, RAP 2A, RAP2B, RAP2C, RASD1, RASD2, RASL10A, RASL10B, RASL11A, RASL11B, RAS L12, REM1, REM2, RERG, RERGL, RRAD, RRAS, RRAS2, RHOA, RHOB, RHOBTB1, RHO BTB2、RHOBTB3、RHOC、RHOD、RHOF、RHOG、RHOH、RHOJ、RHOQ、RHOU、RHOV、RND1 RND2, RND3, RAC1, RAC2, RAC3, CDC42, RAB1A, RAB1B, RAB2, RAB3A, RAB3B AB3C, RAB3D, RAB4A, RAB4B, RAB5A, RAB5B, RAB5C, RAB6A, RAB6B, RAB6C, RAB 7A、RAB7B、RAB7L1、RAB8A、RAB8B、RAB9、RAB9B、RABL2A、RABL2B、RABL4、RAB1 0、RAB11A、RAB11B、RAB12、RAB13、RAB14、RAB15、RAB17、RAB18、RAB19、RAB2 0、RAB21、RAB22A、RAB23、RAB24、RAB25、RAB26、RAB27A、RAB27B、RAB28、RAB 2B, RAB30, RAB31, RAB32, RAB33A, RAB33B, RAB34, RAB35, RAB36, RAB37, RAB 38. RAB39, RAB39B, RAB40A, RAB40AL, RAB40B, RAB40C, RAB41, RAB42, RAB43 RAP1A, RAP1B, RAP2A, RAP2B, RAP2C, ARF1, ARF3, ARF4, ARF5, ARF6, ARL1, A RL2, ARL3, ARL4, ARL5, ARL5C, ARL6, ARL7, ARL8, ARL9, ARL10A, ARL10B, ARL 10C、ARL11、ARL13A、ARL13B、ARL14、ARL15、ARL16、ARL17、TRIM23、ARL4D、A RFRP1, ARL13B, RAN, RHEB, RHEBL1, RRAD, GEM, REM, REM2, RIT1, RIT2, RHOT1In some embodiments, the target protein is a GTPas-activating protein, such as NF1, IQGAP1, PLEXIN-B1, RASAL1, RASAL2, ARHGAP5, ARHGAP8, ARHGAP12, ARHGAP22, ARHGAP25, BCR, DLC1, DLC2, DLC3, GRAF, RALBP1, RAP1GAP, SIPA1, TSC2, AGAP2, ASAP1, or ASAP3. In some embodiments, the target protein is a guanine nucleotide exchange factor, e.g., CNRASGEF, RASGEFlA, RASGRF2, RASGRPl, RASGRP4, SOSl, RALGDS, RGLl, RGL2, RGR, ARHGEFlO, ASEF / ARHGEF4, ASEF2, DBS, ECT2, GEF-Hl, LARG, NETl, OBSCURIN, P-REXl, P-REX2, PDZ-RHOGEF, TEM4, TIAMl, TRIO, VAVl, VAV2, VAV3, DOCKl, DOCK2, DOCK3, DOCK4, DOCK8, DOCKlO, C3G, BIG2 / ARFGEF2, EFA6, FBX8, or GEP100. In certain embodiments, the target protein is a protein having a protein-protein interaction domain, such as ARM, BAR, BEACH, BH, BIR, BRCT, BROMO, BTB, C1, C2, CARD, CC, CALM, CH, CHROMO, CUE, DEATH, DED, DEP, DH, EF-hand, EH, ENTH, EVH1, F-box, FERM, FF, FH2, FHA, FYVE, GAT, GEL, GLUE, GRAM, GRIP, GY In some embodiments, the target protein is a heat shock protein, e.g., Hsp20, Hsp27, Hsp70, Hsp84, Hsp85, Hsp86, Hsp87, Hsp88, Hsp89, Hsp80, Hsp81, Hsp82, Hsp83, Hsp84, Hsp84, Hsp85, Hsp86, Hsp87, Hsp88, Hsp89, Hsp81, Hsp82, Hsp83, Hsp84, Hsp85, Hsp86, Hsp87, Hsp88, Hsp89 ...In certain embodiments, the target protein is an ion channel, e.g., Cav2.2, Cav3.2, IKACh, Kv1.5, TRPA1, NAv1.7, Nav1.8, Nav1.9, P2X3, or P2X4. In some embodiments, the target protein is a coiled-coil protein, e.g., geminin, SPAG4, VAV1, MAD1, ROCK1, RNF31, NEDP1, HCCM, EEA1, vimentin, ATF4, Nemo, SNAP25, syntaxin1a, FYCO1, or CEP250. In certain embodiments, the target protein is a kinase, e.g., ABL, ALK, AXL, BTK, EGFR, FMS, FAK, FGFR1, 2, 3, 4, FLT3, HER2 / ErbB2, HER3 / ErbB3, HER4 / ErbB4, IGF1R, INSR, JAK1, JAK2, JAK3, KIT, MET, PDGFRA, PDGFRB, RETRON, ROR1, ROR2, ROS, SRC, SYK, TIE1, TIE2, TRKA, TRKB, KDR, AKT1, AKT In some embodiments, the target protein is a phosphatase, such as WIP1, SHP2, SHP1, PRL-3, PTP1B, or STEP. In certain embodiments, the target protein is a ubiquitin ligase, e.g., BMI-1, MDM2, NEDD4-1, Beta-TRCP, SKP2, E6AP, or APC / C. In some embodiments, the target protein is a chromatin modifier / remodeler, e.g., the genes BRG1, BRM, ATRX, PRDM3, ASH1L, CBP, KAT6A, KAT6B, MLL, NSD1, SETD2, EP300,In some embodiments, the target protein is a chromatin modifier / remodeler encoded by KAT2A or CREBBP. In some embodiments, the target protein is a transcription factor, e.g., a transcription factor encoded by the genes EHF, ELF1, ELF3, ELF4, ELF5, ELK1, ELK3, ELK4, ERF, ERG, ETS1, ETV1, ETV2, ETV3, ETV4, ETV5, ETV6, FEV, FLI1, GAVPA, SPDEF, SPI1, SPIC, SPIB, E2F1, E2F2, E2F3, E2F4, E2F7, E2F8, ARNTL, BHLHA15, BHLHB2, BHLBHB3, BHLHE22, BHLHE23, BHLHE41, CLO CK, FIGLA, HAS5, HES7, HEY1, HEY2, ID4, MAX, MESP1, MLX, MLXIPL, MNT, MSC, MYF6, NEUROD2, NEUROG2, NHLH1, OLIG1, OLIG2, OLIG3, SREBF2, TCF3, TCF4, TFAP4, TFE3, TFEB, TFEC, USF1, ARF4, ATF7, BATF3, CEBPB, CEBPD, CEBPG, CREB3L1, DBP, HLF, JDP2, MAFF, MAFG, MAFK, NRL, NFE2, NFIL3, TEF, XBP1, PROX1, TEAD1, TEAD3, TEAD4, ONECUT3, ALX3, ALX4, ARX, BARHL2, BARX, BSX, CART1, CDX1, CDX2, DLX1, DLX2, DLX3, DLX4, DLX5, DLX6, DMBX1, DPRX, DRGX, DUXA, EMX1, EMX2, EN1, EN2, ESX1, EVX1, EVX2, GBX1, GBX2, GSC, GSC2, GSX1, GSX2, HESX1, HMX1, HMX2, HMX3, HNF1A, HNF1B, HOMEZ, HOXA 1, HOXA1, HOXA13, HOXA2, HOXAB13, HOXB2, HOXB3, HOXB5, HOXC10, HOXC11, HOXC12, HOXC13, HOXD11, HOXD12, HOXD13, HOXD8, IRX2, IRX5, ISL2, IS X, LBX2, LHX2, LHX6, LHX9, LMX1A, LMX1B, MEIS1, MEIS2, MEIS3, MEOX1, MEOX2, MIXL1, MNX1, MSX1, MSX2, NKX2-3, NKX2-8, NKX3-1, NKX3-2, NKX6-1,NKX6-2、NOTO、ONECUT1、ONECUT2、OTX1、OTX2、PDX1、PHOX2A、PHOX2B、PITX1、PITX3、PKNOX1、PROP1、PRRX1、PRRX2、RAX、RAXL1、RHOXF1、SHOX、SHOX2、TGIF1、TGIF2、TGIF2LX、UNCX、VAX1、VAX2、VENTX、VSX1、VSX2、CUX1、CUX2、POU1F1、POU2F1、POU2F2、POU2F3、POU3F1、POU3F2、POU3F3、POU3F4、POU4F1、POU4F2、POU4F3、POU5F1P1、POU6F2、RFX2、RFX3、RFX4、RFX5、TFAP2A、TFAP2B、TFAP2C、GRHL1、TFCP2、NFIA、NFIB、NFIX、GCM1、GCM2、HSF1、HSF2、HSF4、HSFY2、EBF1、IRF3、IRF4、IRF5、IRF7、IRF8、IRF9、MEF2A、MEF2B、MEF2D、SRF、NRF1、CPEB1、GMEB2、MYBL1、MYBL2、SMAD3、CENPB、PAX1、PAX2、PAX9、PAX3、PAX4、PAX5、PAX6、PAX7、BCL6B、EGR1、EGR2、EGR3、EGR4、GLIS1、GLIS2、GLI2、GLIS3、HIC2、HINFP1、KLF13、KLF14、KLF16、MTF1、PRDM1、PRDM4、SCRT1、SCRT2、SNAI2、SP1、SP3、SP4、SP8、YY1、YY2、ZBED1、ZBTB7A、ZBTB7B、ZBTB7C、ZIC1、ZIC3、ZIC4、ZNF143、ZNF232、ZNF238、ZNF282、ZNF306、ZNF410、ZNF435、ZBTB49、ZNF524、ZNF713、ZNF740、ZNF75A、ZNF784、ZSCAN4、CTCF、LEF1、SOX10、SOX14、SOX15、SOX18、SOX2、SOX21、SOX4、SOX7、SOX8、SOX9、SRY、TCF7L1、FOX03、FOXB1、FOXC1、FOXC2、FOXD2、FOXD3、FOXG1、FOXI1、FOXJ2、FOXJ3、FOXK1、FOXL1、FOXO1、FOXO4、FOXO6、FOXP3、EOMES、MGA、NFAT5、NFATC1、NFKB1、NFKB2、TΡ63, RUNX2, RUNX3, T, TBR1, TΒΧ1, TΒΧ15, TΒΧ19, TΒΧ2, TΒΧ20, TΒΧ21, TΒΧ4, TΒΧ5, AR, ESR1 , ESRRA, ESRRB, HNF4A, NR2C2, NR2E1, NR2F1, NR2F6, NR3C1, NR3C2, NR4A2, RARA, RARB, RARG, The transcription factor is RORA, RXRA, RXRB, RXRG, THRA, THRB, VDR, GATA3, GATA4, or GATA5, or C-myc, Max, Stat3, androgen receptor, C-Jun, C-Fox, N-Myc, L-Myc, MITF, Hif-1α, Hif-2α, Bcl6, E2F1, NF-κB, Stat5, or ER(coact). In certain embodiments, the target protein is TrkA, P2Y14, and mPEGS, ASK1, ALK, Bcl-2, BCL-XL, mSIN1, RORγt, IL17RA, eIF4E, TLR7R, PCSK9, IgER, CD40, CD40L, Shn-3, TNFR1, TNFR2, IL31RA, OSMR, IL12beta1, 2, Tau, FASN, KCTD6, KCTD9, Raptor, Rictor, RALGAPA, RAL GAPB, annexin family members, BCOR, NCOR, β-catenin, AAC, PLD1, PLD2, Frizzled7, RaLP11, MLL-1, Myb, Ezh2, RhoGD12, EGFR, CTLA4R, GCGC (coact), adiponectin R2, GPR81, IMPDH2, IL-4R, IL-13R, IL-1R, IL2-R, IL-6R, IL-22R, TNF-R, TLR4, Nrlp3, or OTR.

[0261] Complex Presenter protein / compound complex In naturally occurring protein-protein interactions, binding events are primarily driven by hydrophobic residues on the flat surface regions of the two proteins, in contrast to many small molecule protein interactions that are driven by interactions between small molecules in cavities or pockets on the proteins. Hydrophobic residues on the flat surface regions form hydrophobic hot spots on the two interacting proteins, and most of the binding interactions between the two proteins are van der Waals interactions. Small molecules can serve as portable hot spots for proteins lacking one another (e.g., presenter proteins), which then participate in pseudoprotein-protein interactions (e.g., form tripartite complexes with target proteins) by forming complexes (e.g., presenter protein / compound complexes).

[0262] Many mammalian proteins can bind to any of several different partners, and in some cases, these alternative binding interactions contribute to the protein's biological activity. Many of these proteins display identical residues in different structural contexts, adapting to the inherent variability of hotspot protein regions. More specifically, protein-protein interactions can be mediated by a class of natural products produced by a select group of fungal and bacterial species. These molecules exhibit a common structural organization and consequently function that provides the ability to modulate protein-protein interactions. These molecules contain a highly conserved presenter protein-binding portion and a target protein-interacting portion that exhibits a high degree of variability among different natural products. The presenter protein-binding portion confers specificity for the presenter protein, allowing the molecule to bind to the presenter protein and form a binary complex. The mammalian target protein-interacting portion confers specificity for the target protein and allows the binary complex to bind to the target protein, typically modulating its activity (e.g., positively or negatively).

[0263] These natural products are presented by presenter proteins, such as FKBP and cyclophilin, and act as diffusible, cell-permeable, orally bioavailable adaptors for protein-protein interactions. Examples include well-known, clinically relevant molecules such as rapamycin (sirolimus), FK506 (tacrolimus), and cyclosporine. Briefly, these molecules bind to endogenous intracellular presenter proteins, such as FKBP (e.g., rapamycin and FK506), or cyclophilin (e.g., diluent), and the resulting binary complex of presenter protein-binding molecules selectively binds to and inhibits the activity of intracellular target proteins. The formation of a tripartite complex between the presenter protein, molecule, and target protein is driven by both protein-molecule and protein-protein interactions, both of which are required for target protein inhibition. In the example of the FKBP-rapamycin complex, the intracellular target is the serine-threonine kinase mTOR, while in the FKBP-FK506 complex, the intracellular target is the phosphatase calcineurin. What is particularly interesting about these two examples is that FKBP12 is utilized as a partner presentation protein by both rapamycin and FK506-presented ligands. Furthermore, while the substructural components of rapamycin and FK506 involved in binding to FKBP12 are structurally closely related, i.e., the so-called "conserved regions," there are significant structural differences between rapamycin and FK506 in the non-FKBP12-binding regions, i.e., the "variable regions," resulting in the specific targeting of two distinct intracellular proteins, mTOR and calcineurin, respectively. Thus, the variable regions of rapamycin and FK506 function as contributors to the binding energy required to enable presenter protein-target protein interactions.

[0264] In some embodiments, presenter protein / compound complexes of the invention bind to target proteins with an affinity that is at least 5-fold (e.g., at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 100-fold) greater than the affinity with which the complex binds to mTOR and / or calcineurin, respectively.

[0265] In some embodiments, the presenter protein / compound complexes of the invention bind to a target protein with an affinity that is at least 5-fold (e.g., at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 100-fold) greater than the affinity of the compound for the target protein when the compound is not bound in complex with the presenter protein.

[0266] In certain embodiments, the presenter protein / compound complexes of the invention bind to a target protein with an affinity that is at least 5-fold (e.g., at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 100-fold) greater than the affinity of the presenter protein for the target protein when the presenter protein is not bound in a complex with a compound.

[0267] In some embodiments, the presenter protein / compound complexes of the invention inhibit a naturally occurring interaction between a target protein and a ligand, e.g., a protein or small molecule that specifically binds to the target protein.

[0268] In certain embodiments, when the presenter protein is a prolyl isomerase, prolyl isomerase activity is inhibited by the formation of a presenter protein / compound complex. In some embodiments of the presenter protein / compound complexes of the invention, the compound has a K of less than 10 μM (e.g., less than 5 μM, less than 1 μM, less than 500 nM, less than 200 nM, less than 100 nM, less than 75 nM, less than 50 nM, less than 25 nM, less than 10 nM). Dor specifically binds to the presenter protein with an IC of less than 1 μM (e.g., less than 0.5 μM, less than 0.1 μM, less than 0.05 μM, less than 0.01 μM). 50 inhibits peptidyl-prolyl isomerase activity against presenter proteins. Tripartite Complex The vast majority of small molecule drugs act by binding to functionally important sites on target proteins, thereby modulating (e.g., positively or negatively) the activity of those proteins. For example, cholesterol-lowering drugs, statins, bind to the enzyme active site of HMG-CoA reductase, thereby preventing the enzyme from engaging its substrate. The fact that many such drug / target interaction pairs are known may lead one to mistakenly believe that, with the appropriate amount of time, effort, and resources, it is possible to discover small molecule modulators for most, if not all, proteins. This is far from the truth. Current estimates suggest that only about 10% of all human proteins are potential small molecule targets. The remaining 90% are currently considered too cumbersome or difficult for small molecule drug discovery. Such targets are commonly referred to as "undruggable." These undruggable targets encompass a vast and largely untapped reservoir of medically important human proteins. Therefore, there is great interest in discovering new molecular modalities that can modulate the function of such undruggable targets.

[0269] The present invention encompasses the recognition that small molecules typically have limited targeting capabilities because their interactions with targets are driven by adhesive forces, the strength of which is roughly proportional to the surface area of ​​contact. Due to their small size, the only way for small molecules to create sufficient intermolecular surface area of ​​contact to effectively interact with a target protein is by directly enveloping the protein. In fact, many sets of experimental and computational data support the view that only proteins with hydrophobic "pockets" on their surfaces can bind small molecules. In such cases, binding is enabled by envelopment. There are no examples of small molecules that bind with high affinity to proteins outside of the hydrophobic pocket.

[0270] Nature has evolved strategies to allow small molecules to interact with target proteins at sites other than hydrophobic pockets. This strategy is exemplified by the naturally occurring immunosuppressants cyclosporin A, rapamycin, and FK506. The activity of these drugs requires the formation of a high-affinity complex between the small molecule and a small display protein. The complex surface of the small molecule and the display protein then engages the target. Thus, for example, the binary complex formed between cyclosporin A and cyclophilin A targets calcineurin with high affinity and specificity, whereas neither cyclosporin A nor cyclophilin A alone binds to calcineurin with measurable affinity.

[0271] Many important therapeutic targets exert their function through complexation with other proteins. In many of these systems, protein / protein interaction surfaces contain an inner core of hydrophobic side chains surrounded by a broad ring of polar residues. Because hydrophobic residues contribute nearly all of the energetically favorable contacts, this cluster has been represented as a "hot spot" for protein-protein interaction engagement. Importantly, in the above-listed complexes of naturally occurring small molecules with small, displayed proteins, the small molecule provides a cluster of hydrophobic features similar to the hot spot, while the protein provides a ring of primarily polar residues. In other words, the displayed small molecule systems mimic the surface architecture commonly utilized in natural protein / protein interaction systems.

[0272] The compounds (e.g., macrocyclic compounds) of the present invention can modulate biological processes, for example, by binding to a presenter protein (e.g., a member of the FKBP family, a member of the cyclophilin family, or PIN1) to form the presenter protein / compound complex described above, which then binds to a target protein to form a tripartite complex. The formation of such a tripartite complex allows for the modulation of proteins that do not have a conventional binding pocket and / or are considered undruggable. The presenter protein / compound complex can modulate biological processes through cooperative binding between the compound and the presenter protein. Both the compound and the presenter protein alone have low affinity for the target protein, but the presenter protein / compound complex has high affinity for the target protein. Cooperative binding can be determined by measuring the buried surface area of ​​the target protein, including atoms from the compound and / or the presenter protein, and / or by measuring the free binding energy contribution of the compound and / or the presenter protein. Binding is considered cooperative if at least one atom from each of the compound and the presenter protein is involved in binding to the target protein.

[0273] Binding of the presenter protein / compound complex to the target protein is achieved by forming a combinatorial binding site that includes residues of both the presenter protein and the compound, allowing for increased affinity not possible with either the presenter protein or the compound alone, for example, at least 20% (e.g., at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%) of the total buried surface area of ​​the target protein in the tripartite complex includes one or more atoms involved in binding to the compound, and / or at least 20% (e.g., at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%) of the total buried surface area of ​​the target protein in the tripartite complex includes one or more atoms involved in binding to the presenter protein. Alternatively, the compound contributes at least 10% (e.g., at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%) of the total binding free energy of the tripartite complex, and / or the presenter protein contributes at least 10% (e.g., at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%) of the total binding free energy of the tripartite complex.

[0274] In some embodiments, the presenter protein / compound complex binds to a flat surface site on the target protein. In some embodiments, the compound (e.g., a macrocyclic compound) in the presenter protein / compound complex binds to a hydrophobic surface site on the target protein, e.g., a site comprising at least 50% hydrophobic residues. In some embodiments, at least 70% of the binding interactions between one or more atoms of the compound and one or more atoms of the target protein are van der Waals interactions and / or π-effect interactions. In certain embodiments, the presenter protein / compound complex binds to the target protein at the site of a naturally occurring protein-protein interaction between the target protein and a protein that specifically binds to the target protein. In some embodiments, the presenter protein / compound complex does not bind to the active site of the target protein. In some embodiments, the presenter protein / compound complex binds to the active site of the target protein.

[0275] A feature of the compounds of the present invention that form tripartite complexes with presenter proteins and target proteins is the lack of major structural reorganization in the presenter protein / compound complex compared to the tripartite complex. This lack of major structural reorganization reduces the entropic cost of reorganizing the presenter protein / compound complex into a configuration favorable for tripartite complex formation after its formation. For example, threshold quantification of RMSD can be measured using the align command in PyMOL version 1.7rc1 (Schroedinger LLC). Alternatively, RMSD can be calculated using the Executive RMS parameter of the LigAlign algorithm (J. Mol. Graphics and Modeling, 2010, Vol. 29, pp. 93-101). In some embodiments, the structural organization of the compound (i.e., the average three-dimensional arrangement of the atoms and bonds of the molecule) is substantially unchanged in the tripartite complex compared to the compound in the presenter protein / compound complex prior to binding to the target protein, e.g., the root mean square deviation (RMSD) between the two aligned structures is less than 1.

[0276] Usefulness and Administration The compounds and presenter protein / compound complexes described herein are useful in the methods of the present invention and, without being bound by theory, are believed to exert their desired effects through their ability to modulate (e.g., positively or negatively modulate) the activity of the target protein (e.g., a eukaryotic target protein, such as a mammalian target protein or a fungal target protein, or a prokaryotic target protein, such as a bacterial target protein) through interaction between the presenter protein and the target protein.

[0277] kit In some embodiments, the present invention relates to kits for conveniently and effectively practicing the methods of the present invention. Typically, pharmaceutical packs or kits include one or more containers filled with one or more components of the pharmaceutical compositions of the present invention. Such kits are particularly suitable for the delivery of solid oral formulations, such as tablets or capsules. Such kits may preferably include a card containing several unit doses and indicating the dosages in the order of their intended use. Optionally, for example, if the subject is suffering from Alzheimer's disease, a memory aid can be provided, e.g., in the form of numbers, letters, or other designations, or by designating a treatment schedule with days on a calendar by which administration may occur. Alternatively, a kit can be provided that includes placebo doses (or calcium dietary supplements) similar to or different from the dosages of the pharmaceutical composition, so that a dosage is taken daily. Optionally, associated with such containers can be a notice in the form specified by a government agency regulating the manufacture, use, or sale of pharmaceuticals. The notice reflects the regulatory agency's approval for manufacture, use, or sale for human administration.

[0278] Pharmaceutical Composition For use in treating human and animal subjects, the compounds of the present invention can be formulated as pharmaceutical or veterinary compositions. Depending on the subject to be treated, the mode of administration, and the type of treatment desired (e.g., prevention, prophylaxis, or therapy), the compounds are formulated in a manner consistent with these parameters. Overviews of such techniques can be found in Remington: The Science and Practice of Pharmacy, 21st ed., Lippincott Williams & Wilkins, 2005; and Encyclopedia of Pharmaceutical Technology, edited by J. Swarbrick and JC Boylan, 1988-1999, Marcel Dekker, New York (each of which is incorporated herein by reference).

[0279] The compounds described herein may be present in a total amount of 1 to 95% by weight of the total weight of the composition. The compositions may be provided in a dosage form suitable for intraarticular, oral, parenteral (e.g., intravenous, intramuscular), rectal, cutaneous, subcutaneous, topical, transdermal, sublingual, nasal, vaginal, intravesicular, intraurethral, ​​intrathecal, epidural, otic, or ocular administration, or for injection, inhalation, or direct contact with nasal, urogenital, genital, or oral mucosa. Thus, pharmaceutical compositions may take the form of, for example, tablets, capsules, pills, powders, granules, suspensions, emulsions, solutions, gels, including hydrogels, pastes, ointments, creams, plasters, drenches, osmotic delivery devices, suppositories, enemas, injectables, implants, sprays, preparations suitable for iontophoretic delivery, or aerosols. The compositions may be formulated according to conventional pharmaceutical practice.

[0280] Generally, for therapeutic use, the compounds described herein may be used alone or in combination with one or more other active agents. Examples of other pharmaceuticals that may be combined with the compounds described herein would include pharmaceuticals for the treatment of the same indication. Other examples of pharmaceuticals that may be combined with the compounds described herein would include pharmaceuticals for the treatment of different, but related, conditions or indications. Depending on the mode of administration, the compounds will be formulated into a suitable composition to allow for easy delivery. Each compound of the combination therapy may be formulated in various ways known in the art. For example, the first and second active agents of the combination therapy may be formulated together or separately. Desirably, the first and second active agents are formulated together for simultaneous or near-simultaneous administration of the agents.

[0281] The compounds of the present invention may be prepared and used as pharmaceutical compositions comprising an effective amount of a compound described herein and a pharmaceutically acceptable carrier or excipient, as is well known in the art. In some embodiments, the composition comprises at least two different pharmaceutically acceptable excipients or carriers.

[0282] Formulations may be prepared in a manner suitable for systemic or local administration. Systemic formulations include those designed for injection (e.g., intramuscular, intravenous, subcutaneous injection), or may be prepared for transdermal, transmucosal, or oral administration. Formulations will generally include a diluent and, in some cases, adjuvants, buffers, preservatives, etc. The compounds may also be administered as liposomal compositions or microemulsions.

[0283] For injection, the formulations can be prepared in conventional forms, either as liquid solutions or suspensions, or as solid preparations suitable for dissolution or suspension in liquid prior to injection, or as emulsions. Suitable excipients include, for example, water, saline, dextrose, glycerol, etc. Such compositions may also contain various amounts of nontoxic auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, and the like, for example, sodium acetate, sorbitan monolaurate, and the like.

[0284] Various sustained release systems for drugs have also been devised, see, for example, U.S. Patent No. 5,624,677 (incorporated herein by reference). Systemic administration can also include relatively non-invasive methods, such as suppositories, transdermal patches, transmucosal delivery, and intranasal administration. Oral administration is also suitable for the compounds of the present invention. Suitable formulations include syrups, capsules, and tablets, as is understood in the art.

[0285] Each compound of the combination therapy described herein may be formulated in a variety of ways known in the art, for example, the first and second agents of the combination therapy may be formulated together or separately.

[0286] Individually or separately formulated agents can be packaged together as a kit. Examples include, but are not limited to, a kit containing two pills, a pill plus a powder, a suppository plus a liquid in a vial, two topical creams, etc. The kit may include optional components to aid in administering the unit dose to a subject, such as a vial for reconstituting a powder form, a syringe for injection, a customized IV delivery system, an inhaler, etc. Additionally, a unit dose kit may contain instructions for preparing and administering the composition. The kit may be manufactured as a single-use unit dose for one subject, as multiple uses for a particular subject (either at a fixed dose or with varying potencies of individual compounds over the course of treatment), or the kit may contain multiple doses suitable for administration to multiple subjects ("bulk packaging"). The kit components may be assembled into cartons, blister packs, bottles, tubes, etc.

[0287] Formulations for oral use include tablets containing the active ingredient(s) in a mixture with non-toxic pharmaceutically acceptable excipients. Such excipients may be, for example, inert diluents or fillers (e.g., sucrose, sorbitol, sugars, mannitol, microcrystalline cellulose, starches such as potato starch, calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate, or sodium phosphate), granulating and disintegrating agents (e.g., cellulose derivatives such as microcrystalline cellulose, starches such as potato starch, croscarmellose sodium, alginates, or alginic acid), binders (e.g., sucrose, glucose, sorbitol, acacia, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, magnesium aluminum silicate, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, ethylcellulose, polyvinylpyrrolidone, or polyethylene glycol), and lubricants, glidants, and antiadherents (e.g., magnesium stearate, zinc stearate, stearic acid, silica, hydrogenated vegetable oils, or talc). Other pharmaceutically acceptable excipients can be colorants, flavoring agents, plasticizers, humectants, buffering agents, and the like.

[0288] The two or more compounds may be mixed together in a tablet, capsule, or other vehicle, or may be separated. For example, a first compound is contained on the inside of a tablet and a second compound on the outside, such that a substantial portion of the second compound is released before the release of the first compound.

[0289] Formulations for oral use may also be provided as chewable tablets, or as hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent (e.g., potato starch, lactose, microcrystalline cellulose, calcium carbonate, calcium phosphate, or kaolin), or as soft gelatin capsules in which the active ingredient is mixed with water or an oil medium, such as peanut oil, liquid paraffin, or olive oil. Powders, granules, and pellets may be prepared using the above-listed ingredients in tablets and capsules in a conventional manner, for example, in a mixer, fluid bed apparatus, or spray drying apparatus.

[0290] Dissolution or diffusion controlled release can be achieved by applying a suitable coating to tablets, capsules, pellets, or granules of the compound or by incorporating the compound in a suitable matrix, such as one or more of the coating materials listed above and / or, for example, shellac, beeswax, glycowax, castor wax, carnauba wax, stearyl alcohol, glyceryl monostearate, glyceryl distearate, glycerol palmitostearate, ethyl cellulose, acrylic resin, dl-polylactic acid, cellulose acetate butyrate, polyvinyl chloride, polyvinyl acetate, vinylpyrrolidone, polyethylene, polymethacrylate, methyl methacrylate, two hydroxymethacrylates, methacrylate hydrogels, 1,3-butylene glycol, ethylene glycol methacrylate, and / or polyethylene glycol. Additionally, in controlled release matrix formulations, the matrix material may include, for example, hydrated methylcellulose, carnauba wax and stearyl alcohol, Carbopol 934, silicone, glyceryl tristearate, methyl acrylate-methyl methacrylate, polyvinyl chloride, polyethylene, and / or halogenated fluorocarbons.

[0291] Liquid preparations into which the compounds and compositions of the present invention may be incorporated for oral administration include aqueous solutions, suitably flavored syrups, aqueous or oily suspensions, and flavored emulsions, including edible oils such as Mentha oil, sesame oil, coconut oil, peanut oil, and the like, as well as elixirs and similar pharmaceutical vehicles.

[0292] Generally, when administered to humans, the oral dosage of any of the compounds of the combination of the present invention will depend on the properties of the compound, but can be readily determined by one skilled in the art. Typically, such dosages will usually be about 0.001 mg to 2000 mg per day, preferably about 1 mg to 1000 mg per day, and more preferably about 5 mg to 500 mg per day. Doses of up to 200 mg per day may be necessary.

[0293] Administration of each agent of the combination therapy described herein can independently be four times daily for one day to one year, or even for the life of the subject. Chronic long-term administration may be required.

[0294] The following examples are intended to illustrate the synthesis of a representative number of compounds and the use of these compounds to elicit chemotactic and antifungal activity. Accordingly, the examples are intended to illustrate, but not limit, the invention. Additional compounds not specifically exemplified may be synthesized using conventional methods in combination with the methods described herein.

[0295] (Example) Example 1. General Fermentation and Isolation Protocol Compounds synthesized by bacterial strains can be fermented and isolated using the following general protocol. General Fermentation Protocol Strains: Bacterial strains producing FKBP ligands (e.g., F1, F2, F3, or their structurally related compounds and analogs), such as Streptomyces malaysiensis DSM 41697, other producing species, or genetically modified derivatives, were grown axenically on solid medium (e.g., ISP4).

[0296] Working cell bank: Spores or mycelium from cultures grown on solid medium plates at 30°C for 3-14 days were used to inoculate liquid cultures (e.g., 40 ml ATCC172 liquid medium in a 250 ml Erlenmeyer flask). The cultures were incubated with shaking at 30°C for 2-3 days. The resulting cell suspension was mixed with sterile 50% glycerol to give a mixture containing a final concentration of 15-25% glycerol. Aliquots (approximately 1 ml) of the glycerol-mycelium mixture were stored in sterile cryovials at -80°C until further use.

[0297] Primary seed culture: Primary seed culture (e.g., 40 mL in a 250 mL Erlenmeyer flask) ATCC 172 medium) was inoculated with 1 mL of the working cell bank suspension. The culture was incubated on a 50.8 mm (2 inch) throw shaker at 200-220 rpm at 30°C for 2-3 days.

[0298] Secondary seed culture: Secondary seed cultures (e.g., 100-200 mL ATCC172 in a 500 mL Erlenmeyer flask) were inoculated with the primary seed culture (5% v / v) and incubated as described above for various incubation times (e.g., 18-48 h).

[0299] Production fermentations in flasks: Production fermentations were carried out in 1.8 L Fernbach or Erlenmeyer flasks containing 0.5 L production medium (e.g., medium 8430 or its derivatives) that supports the biosynthesis of these compounds. Cultures were inoculated at 2-5% (v / v) with the seed culture prepared as described above and incubated for 3-7 days under the conditions described above.

[0300] Production fermentation in bioreactors: Production fermentations were performed in bioreactors (7.5 L capacity, New Brunswick Scientific, NJ, USA) controlled by a BioFlo 300 module. The bioreactors containing 5 L of sterile medium (e.g., 8430 and its derivatives) were inoculated with a seed culture (2–5%, v / v) and incubated for 3–7 days with or without control of parameters such as dissolved oxygen (e.g., 10–50%), propeller speed (e.g., 200–500 rpm), pH (e.g., pH 4.5–7.0), temperature (e.g., 25–35°C), and nutrient supply, if appropriate.

[0301] ISP4 (per liter) Soluble starch 10.0g Dipotassium phosphate 1.0g Magnesium Sulfate USP 1.0g Sodium chloride 1.0g Ammonium sulfate 2.0g Calcium carbonate 2.0g Ferrous sulfate 1.0mg Manganese chloride 1.0mg Zinc sulfate 1.0mg Agar 20.0g

[0302] [Table 2]

[0303] Table 2. ATCC#172 Media (per liter)

[0304] [Table 3]

[0305] Table 3.8430 Medium

[0306] [Table 4]

[0307] Table 4.*R2 trace element solution General isolation protocol The fermentation broth of a strain producing a specific compound was separated from the supernatant and centrifuged to pellet the microorganisms. The target compound in the supernatant could be extracted by partition extraction using a water-immiscible solvent such as dichloromethane (DCM) or ethyl acetate (EtOAc), or by solid-phase extraction using a nonpolar resin such as HP20 or HP20ss. The target compound in the pellet could be extracted repeatedly (4x) using ethyl EtOAc-methanol (9:1, v / v). The microbial extracts were pooled and concentrated under vacuum. To this extract, HP20 beads (using organic solvents such as methanol (MeOH), DCM, acetonitrile, or isopropanol (IPA)) and / or material eluted from the organic phase of a liquid / liquid extraction of the original supernatant could be added.

[0308] The combined extracts are filtered through Celite and dried under vacuum to produce the primary crude product, which is then weighed. The primary crude product is dissolved in a minimum of 100% MeOH or DCM and tetrahydrofuran (THF). A binding medium such as silica gel powder is added to the mixture in a flask, and the mixture is re-dried under vacuum for normal-phase silica gel column chromatography. The crude to silica gel ratio in the column bed is preferably approximately 1:5 (wt / wt). The crude material can be fractionated using a RediSep® normal-phase silica flash column using step gradient, linear gradient, or isocratic elution conditions. Elution solvents can include hexane, heptane, ethyl acetate, ethanol, acetone, isopropanol, or other organic solvents or combinations. Fractions containing the enriched target compound are pooled, dried, and subjected to further purification after LC / MS and / or thin-layer chromatography (TLC) analysis.

[0309] Further purification can be achieved via normal-phase preparative or specific preparative HPLC columns such as Waters Spherisorb CN, Waters Prep Silica, or Kromacil 60-5DIOL. Elution solvents can include hexane, heptane, ethyl acetate, ethanol, acetone, isopropanol, or other organic solvents, or combinations. Fractions containing the enriched or pure target compound are pooled, dried, and subjected to further workup after LC / MS and / or thin-layer chromatography (TLC) analysis.

[0310] Further purification can be achieved with a variety of reversed-phase preparative HPLC columns, depending on the complexities of the concentrated material and the properties of the target compound, such as polarity and solubility. The reversed-phase preparative HPLC columns used for the separations included Waters Sunfire Prep C18 OBD, Waters Xbridge Prep C18 OBD, Kromacil C4, Thermo Acclaim Polar Advantage 2, and Phenomenex Luna C18. The common solvent system was a mixture of water and acetonitrile or methanol, with or without 0.1% formic acid or 0.01% trifluoroacetic acid modifier or 25 mM ammonium formate buffer. The elution mode could be either linear gradient or isocratic. Fractions with pure target compound are pooled, dried and subjected to further work-up after LC / MS and / or thin layer chromatography (TLC) analysis.

[0311] Fractions containing the pure compounds are subjected to workup and drying processes to obtain pure solid materials. Certain target compounds can be purified from aqueous matrices using ethyl acetate or dichloromethane after reverse-phase column chromatography. Solvent removal and drying techniques include rotavap, speedvac, and lyophilization. The purity and chemical structure of the purified target compounds are determined by LC-MS( / MS) and NMR techniques.

[0312] Example 2. Isolation of F2 and F3 A 10 L fermentation broth of Streptomyces malaysiensis (NRRL B-24313, ATCC BAA-13, DSM41697, JCM10672, KCTC9934, NBRC16446, CGMCC, 4.1900, IFO16448) producing F1 (target mass 595), F2 (target mass 609), and compound 3 (target mass 623) was separated by centrifugation. F1 and F2 were present in both the clarified broth and the microbial pellet. The target compounds in the supernatant were extracted once with EtOAc at a volumetric ratio (1:1, v / v). The pellet was extracted three times with 1.5 L of EtOAc-MeOH (9:1, v / v) with stirring using an overhead stirrer for 1 to 1.5 hours for each extraction. The organic extract was filtered through Celite. The combined filtrate was evaporated to dryness at 35 °C to give approximately 30 g of crude extract. The residue was then dissolved in 90 mL of DCM-THF (80:20, v / v), to which 60 g of silica gel was added at 35 °C and dried in vacuo. The dried residue / silica mixture was loaded onto a 120 g RediSep silica gold cartridge. The compounds were eluted with a linear gradient of 100% heptane to heptane-EtOAc (6:4, v / v) at 85 mL / min for 30 min and analyzed by a Teledyne ISCO Combiflash Rf (Teledyne ISCO Combiflash Rf). Fractions of 50 mL were collected using a Rf apparatus.

[0313] Fractions enriched by TLC for F2 were eluted with 20%–30% EtOAc in heptane. The pooled fractions were then concentrated at 35 °C to provide 900 mg of enriched F2 material, which was further purified on a silica gel cartridge. Approximately 1 mL of DCM was used to dissolve the fraction, and 1.8 g of silica gel was added. The dried mixture was loaded onto an 80 g RediSep silica gold cartridge. The compound was eluted with a linear gradient of 100% heptane to heptane-EtOAc (6:4, v / v) at 60 mL / min for 30 min, collecting 50 mL fractions. TLC-pure fractions 25–28 were combined and subjected to solvent removal in vacuo at 35 °C to yield 300 mg of pure F2 (β-form) for structural elucidation and animal experiments.

[0314] F2: 1 H NMR (500MHz, benzene d6) δ7.20-7.13(m,4H),7.0-7.05(m,1H),5.82(s,1H),5. 79-5.69(m,2H),5.51(m,1H),5.46-5.35(m,3H),4.60(d,J=12Hz,1H),3.98- 3.90(m,1H),3.63(dqd,J=13,6.5,3.0Hz,1H),3.22(d,J=3.6Hz,1H),3.07(t d,J=12,2.8Hz,1H),3.00(t,J=9.9Hz,1H),2.93(dd,J=13,4.4Hz,1H),2.63- 2.54(m,3H),2.20(d,J=13Hz,1H),2.11-2.03(m,1H),1.99-1.86(m,2H),1. 79-1.71(m,1H),1.68-1.60(m,1H),1.51-1.47(m,1H),1.45(d,J=6.6Hz,3H) ,1.37(m,4H),1.31(m,1H),1.30(d,J=6.6Hz,3H),1.29-1.22(m,2H),1.16-1 .08(m,1H),1.04-0.94(m,1H),0.82(t,J=7.4Hz,3H),0.69(d,J=6.7Hz,3H). 13 C NMR (125MHz, benzene d6) δ 209.9, 169.7, 167.5, 141.3, 132.2, 129.6, 129.4, 128.7, 128.0, 127.7, 126.4, 98.2, 79.7, 75.5, 71.1, 51.9, 46.9, 44.2, 44.0, 40.4, 36.2, 35.3, 35.3, 35.2, 34.0, 33.3, 25.4, 25.3, 22.5, 21.1, 17.4, 17.1, 11.6, 9.7. HR-MS [M+Na] + : Calculated value [C36H51NO7+Na] + 632.3563, observed value 632.3569.

[0315] Compound 3-enriched fractions were eluted with 30% to 40% EtOAc in heptane, as determined by TLC and LC-MS analysis. The pooled fractions were then concentrated at 35 °C to provide 500 mg of enriched compound 3, which was then repurified by reverse-phase preparative HPLC on a Thermo Polar Advantage II column (5 μm, 250 × 21.2 mm). Preparative HPLC conditions included 70% acetonitrile + 0.1% formic acid in water, isocratic elution mode, 15 mL / min, 254 nm. The enriched compound 3 sample was dissolved in 10 mL of methanol and subjected to 10 repeated injections. The target compound 3 peak was collected at 23.5 min. After extraction with EtOAc from the pooled preparative HPLC fractions, the organic solvent was removed in vacuo to yield 250 mg of pure compound 3. Its chemical structure was subsequently determined by various LC-MS and NMR techniques.

[0316] F3: 1¹H NMR (500MHz, d6, 1:1 mixture of cyclohexane and cyclohexane) δ 7.30 (m, 1H), 7.20–7.10 (m, 6H), 7.10–7.06 (m, 3H), 7.00 (m, 2H), 5.65–5.55 (m, 2H), 5.45 (m, 1H), 5.25–5.15 (m, 2H), 4.98 (dd, J=15, 7.3Hz, 1H), 4.89 (dd, J=8.9, 5.0Hz, 1H), 4.67 (dd, J=15, 8.8Hz, 1H), 4.45 (m, 2H), 4.20 (m, 1H), 4.13 (m, 1H), 3.87 (m, 1H), 3.57 (m, 2H) ,3.35-3.05(m,3H),2.72(m,2H),2.65-2.50(m,2H),2.50-2.30(m,6H),2.0 8(m,1H),1.93(m,1H),1.80-0.90(m,50H)[1.71(d,J=6.8Hz,3H),1.54(d,J =6.8Hz,3H)],1.24(d,J=6.5Hz,3H),1.18(d,J=6.6Hz,3H),1.08(d,J=6.8H z,3H),1.01(m,J=6.7Hz,3H)],0.73(t,J=7.5Hz,3H),0.69(t,J=7.5Hz,3H). 13 CNMR(125MHz,ベンゼンd6)δ201.5,199.9,197.8,191.6,170.4,169.5,166.8,166.6,145.4,144.8,140.6,140.5,133. 9,131.3,129.7,129.4,129.3,128.8,128.8,128.4,128.3,126.6,126.5,126.0,100.0,99.3,80.6,78.2,73.1,72 .4,71.7,70.6,57.1,52.6,51.9,51.1,45.8,45.4,44.2,42.5,42.2,39.8,35.8,35.7,35.6,34.1,33.6,33.4,29. 9,29.9,29.3,28.2,27.4,27.1,25.1,25.1,22.3,22.2,21.3,21.2,16.6,16.2,14.6,13.7,11.2,11.1,10.6,9.5. HR-MS[M+H] + : Calculation value [C 36 H 49 NO8+H] + 624.3536, viewing value 624.3547.

[0317] Example 3. Isolation of F22 A 10 L fermentation broth produced from the recombinant strain S1806 was centrifuged to obtain a pellet and supernatant. The pellet was extracted three times with 1.5 L of EtOAc / MeOH (9:1, v / v). The combined organic solvent was concentrated in vacuo to obtain 1.8 g of crude extract. 2 mL of heptane-THF (4:1, v / v) was added to dissolve the extract, followed by the addition of 2 g of Celite. The solvent was removed by rotary evaporation at 30 °C to obtain a dry mixture. The dry residue / Celite mixture was loaded onto a 40 g RediSep silica gold cartridge for column chromatography. Compounds were fractionated using a linear gradient elution from 100% n-heptane to 40% EtOAc in heptane (v / v) at 20 mL / min over 25 min, collecting 50 mL fractions. F22 (target mass 607) was primarily enriched in fraction 14, as identified by LC-MS analysis. Fraction 14 was then dried in vacuo at 30 °C to give 17.8 mg of solid material, which was further purified by preparative HPLC on a Thermo Polar Advantage II column (5 μm, 250 × 21.2 mm). The preparative HPLC conditions included 90% acetonitrile + 0.1% formic acid in water, isocratic elution mode, 15 mL / min, and 254 nm. The sample was dissolved in 1.78 mL of methanol and subjected to five repeated injections. The target F22 peak was collected at 11.5 min. After solvent removal in vacuo, 3.64 mg of pure F22 was obtained. Its chemical structure was subsequently determined by various LC-MS / MS and NMR techniques.

[0318] Example 4. Synthesis of selected compounds Device: Purification was performed by preparative HPLC using an Agilent SD-1 system. Electrospray LC / MS analysis was performed using an Agilent 1260 Infinity system equipped with an Agilent 1260 series LC pump. The method used was as follows:

[0319] Analytical HPLC method 1: Agilent Zorbax Extend C-18 reversed-phase column (2.1 x 50 mm), 1.8 μm: Solvent A: Water + 0.1% formic acid Solvent B: Acetonitrile + 0.1% formic acid Flow rate: 0.5mL / min Injection volume: 5μl Column temperature: 40℃ Gradient:

[0320] [Table 5]

[0321] Analytical HPLC method 2: ThermoScientific Acclaim, Polar Advantage II, 4.6 x 150 mm, 5 μm Solvent A: Water + 0.1% formic acid Solvent B: Acetonitrile + 0.1% formic acid Flow rate: 0.8mL / min Injection volume: 5μl Column temperature: 40°C: Isocratic:

[0322] [Table 6]

[0323] Electrospray UHPLC / MS was performed using an Agilent 1290 Infinity system equipped with an Agilent 1290 series LC pump. The columns used were identical.

[0324] Analytical UHPLC method 1: Agilent Zorbax Extend C-18 reversed-phase column (2.1 x 50 mm), 1.8 μm: Solvent A: Water + 0.1% formic acid Solvent B: Acetonitrile + 0.1% formic acid Flow rate: 0.5mL / min Injection volume: 5μl Column temperature: 40℃ Gradient:

[0325] [Table 7]

[0326] Purification Method A: ACCLAIM Polar Advantage II The analysis was performed using an Advantage II (21.2 x 250 mm) column. Flow rate was 17 mL / min, isocratic 70% B. Solvent A was 0.1% aqueous formic acid, and solvent B was 100% acetonitrile containing 0.1% formic acid.

[0327] Synthesis of F11 Synthesis of (2S)-1-((4R,7S)-7-((2R,3S,4R,11S,12R)-12-benzyl-3,11-dihydroxy-4-methyltetradecan-2-yl-2-hydroxy-4-methyl-3-oxooxepane-2-carbonyl)piperidine-2-carboxylic acid C-11 lactone.F11

[0328] [ka]

[0329] Ethyl acetate (1 mL) was added to a mixture of (2S)-1-((4R,7S)-7-((2R,3S,4R,6E,9E,11R,12R)-12-benzyl-3,11-dihydroxy-4-methyltetradeca-6,9-dien-2-yl)-2-hydroxy-4-methyl-3-oxooxepane-2-carbonyl)piperidine-2-carboxylic acid C-11 lactone (5 mg, 8.2 μmol), 10% palladium on carbon (2 mg), and stirrer beads under nitrogen. The flask was charged with hydrogen and stirred vigorously for 1.5 hours. The hydrogen atmosphere was replaced with nitrogen, and the reaction was filtered through Celite. The Celite pad was washed with more ethyl acetate, and the solvent was evaporated in vacuo. The residue was purified by silica gel chromatography with gradient elution ethyl acetate:hexane 40:60 to 100:0 to give the title compound.

[0330] 1 H NMR (CDCl3,500MHz): δ7.28(m,2H),7.19(m,1H),7.13(d,J=6.98Hz,2H),5.65(s,1H),5.26(d,J=4.9 2Hz,1H),5.11(m,1H),4.67(d,J=13.02Hz,1H),4.02(dd,J=10.67,1.13Hz,1H),3.35(m,1H),3.23-3. 10(m,2H),2.72(dd,J=13.85,5.50Hz,1H),2.50(dd,J=13.93,9.25Hz,1H),2.39(m,1H),1.95-1.73(m ,5H),1.71-1.15(m,25H),1.03(d,J=6.71Hz,3H),0.85(t,J=7.42Hz,3H),0.79(d,J=6.82Hz,3H)ppm. 13 C NMR(CDCl3,500MHz):δ210.5,170.3,167.4,140.5,129.0,128.3,126.0,97.8,79.1,76.9,71.1,52.0,45.9,43.9,43. 5,39.9,36.3,35.1,33.1,32.3,31.9,29.1,27.9,27.1,25.8,25.1,23.4,22.1,21.1,20.0,17.0,16.6,11.5,8.9ppm. MS (ESI): calculated for (C36H55NO7+H)+ 614.4057, found 614.4066.

[0331] Synthesis of F24 Synthesis of (S)-1-(2-((2R,3R,6S)-6-((2R,3R,4S,6E,9E,11R,12R)-12-benzyl-3,11-dihydroxy-4-methyl-5-oxotetradeca-6,9-dien-2-yl-2-hydroxy-3-methyltetrahydro-2-H-pyran-2-yl-2-oxoacetyl)piperidine-2-carboxylic acid C-11 lactone

[0332] [ka]

[0333] To a solution of F3 (24.2 mg, 36.7 μmol) in ethyl acetate (1 mL) under nitrogen was added 10% Pd / C (12 mg, 50% w / w). The flask was charged with hydrogen and the suspension was stirred at room temperature for 30 minutes. The hydrogen was replaced with nitrogen, and the reaction mixture was then filtered through Celite. The filtrate was concentrated under vacuum to give 24 mg of crude product, a portion of which was purified by Method A to give tetrahydro WDB-003 as a white solid (11 mg, 47.8%). TLC: (50 / 50 heptane / ethyl acetate) Rf=0.45.

[0334] 1 H NMR (400 MHz, C6D6, 1:0.3 mixture of rotamers, asterisk ( * ) indicates peaks related to minor isomers) δ 7.25-7.0 (m, 5H), 6.18 * (s,1H),5.33-5.28(m,2H),5.11 * (d, J = 12 Hz, 1 H), 4.92 * (m,1H),4.45 * (d,J=12Hz,1H), 4.24(m,1H), 4.06 *(td,J=8Hz,1H),3.40(dd,J=4Hz,1H),3.88(t,J=8Hz,1H),3.65(d,J=12Hz,1H),3.30(td,J=12Hz,1H),3.02 * (td,J=12,4Hz,1H),2.84 * (m,1H),2.74(dd,16,8Hz,1H),2.65(q,8Hz,1H),2.61-2.48(m,2H),2.38-2.09(m, 5H),1.73-1.54(m,6H),1.47-1.02(m,28H),0.90(m,4H),0.80(t,J=8Hz,3H),0.73 * (t, J = 8 Hz, 3 H) ppm. 13 C NMR(400MHz,C6D6)δ:212.59,197.51,170.64,166.70,140.93,129.39,1 28.77,128.17,127.94,126.43,99.30,76.54,72.64,71.61,52.46,51.24 ,46.46,45.30,41.62,40.82,36.20,35.31,32.09,29.96,29.47,27.67,2 6.17,25.71,24.92,22.57,21.78,21.59,16.59,13.68,11.49,10.37ppm. MS(ESI):(C 36 H 53 NO8+Na) + The calculated value was 650.37 and the measured value was 650.3.

[0335] Synthesis of F25 Synthesis of (2S)-1-((4R,7S)-7-((2R,3R,4S,11S,12R)-12-benzyl-3,11-dihydroxy-4-methyl-5-oxotetradecan-2-yl)-2-hydroxy-4-methyl-3-oxooxepane-2-carbonyl)piperidine-2-carboxylic acid C-11 lactone.

[0336] [ka]

[0337] (S)-1-(2-((2R,3R,6S)-6-((2R,3R,4S,6E,9E,11R,12R)-12-benzyl-3,11-dihydroxy-4-methyl-5-oxotetradeca-6,9-dien-2-yl)-2-hydroxy-3-methyltetrahydro-2H-pyran-2-yl-2-oxoacetyl)piperidine-2-carboxylic acid C-11 lactate in dichloromethane (2 mL) under nitrogen An ice-cold solution of acetone-F24 (18.8 mg, 30.1 μL) and triethylamine (4.0 μL, 30.1 μL) was prepared. The resulting solution was stirred at 0 °C for 15 min and then warmed to room temperature for 2 h. The reaction was cooled to 0 °C, and a second portion of triethylamine (4.0 μL, 30.1 μL) and tert-butyldimethylsilyl trifluoromethanesulfonate (6.9 μL, 30.1 μmol) was added. The reaction was warmed to room temperature again and stirred under nitrogen for 16 h. Dichloromethane (10 mL) and 0.5 M aqueous sodium bicarbonate solution (10 mL) were added, and the organic layer was separated, washed with 5% brine solution (10 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated in vacuo. The crude product was purified using Method A to give the starting material as a white solid (2.52 mg) and the title compound (4.51 mg) as a white solid.

[0338] 1 H NMR(400MHz,C6D6)δ7.26-7.16(m,4H),7.07(tt,J=6.4,2Hz,1H),5.58(s,1H),5.39(d,J=4.8Hz,1H),5.17 (m,1H),4.67(d,J=12.4Hz,1H),4.29(d,J=10.8Hz,1H),3.42(m,1H),3.06(td,J=11.2,2.8Hz,1H),2.92(t ,J=10Hz,1H),2.85(m,1H),2.75(s,1H),2.66(dd,J=14,5.6Hz,1H),2.52(dd,J=14,9.2Hz,1H),2.35(m,1H) ),2.28(m,1H),1.84(m,2H),1.68-1.59(m,2H),1.46-1.06(m,23H),0.88(m,4H),0.82(t,3H,J=7.2Hz)ppm. 13C NMR(400MHz,C6D6)δ:226.15,210.53,209.8,179.03,167.59,140.84,12 9.40,128.77,128.18,127.9,126.45,98.16,79.21,76.85,70.48,52.20 ,46.07,44.46,43.88,42.76,36.67,35.30,35.14,32.88,30.53,27.94, 25.49,25.32,24.57,22.39,21.36,20.72,16.98,15.16,11.68,9.08ppm. MS(ESI):(C 36 H 53 NO8+Na) + The calculated value was +650.37, and the actual value was 650.3.

[0339] Example 5. Synthesis of cyclosporine analogues General Protocol Using solution-phase peptide synthesis, over 2,000 analogs of cyclosporine have been prepared, for example, according to Li et al., J. Org. Chem. 2000, 65, 2951. The amino acid sequence of cyclosporine is: cyclo-(D-Ala)-(C1H-C1H-C2H-C3H-O-C4H-O-C5H-O-C6H-O-C7H-O-C8H-O-C9H-O-C9H-O-C1H-O-C1H-O-C1H-O-C1H-O-C1H-O-C2H-O-C1H-O-C2H-O-C3H-O-C4H-O-C5H-O-C6H-O-C7H-O-C8H-O-C9H-O-C9H-O-C1H-O-C1H-O-C1H- 8 -MeLeu 9 -MeLeu 10 -MeVal 11 -MeLeu 1 -Nva 2 -Sar 3 -MeLeu 4 -Val 5 -MeLeu 6 -Ala7) D-Ala 8 ~Sar 3 This polypeptide stretch can be considered the "constant" region that is largely responsible for binding to cyclophilin A. Therefore, cyclosporin analogs that preserve cyclophilin binding are those that contain MeLeu. 4 -Val 5 -MeLeu 6 -Ala 7 They can be made by synthesis of tetrapeptide surrogates of the fragments, followed by elongation and cyclization.

[0340] Cyclo-(D-Ala 8 -MeLeu 9 -MeLeu 10 -MeVal 11 -MeLeu 1 -Nva 2 -Sar 3 -Gly 4 -Gly 5 -Gly 6 -Gly 7 In a specific example of the synthesis of cyclosporine analogue cyclo-(D-Ala), Fmoc-Gly-OH is coupled with Ala-OBzl in the presence of 2,6-lutidine and BDMP (5-(1H-benzotriazol-1-yloxy)-3,4-dihydro-1-methyl 2H-pyrrolium hexachloroantimonate) to produce Fmoc-Gly-Gly-OBzl. Removal of the Fmoc group with diethylamine and subsequent coupling with Fmoc-Gly-OH (promoted by BDMP) produces Fmoc-Gly-Gly-Gly-OBzl. Repeated Fmoc removal and Fmoc-Gly-OH coupling produces Fmoc-Gly-Gly-Gly-Gly-OBz. This suitably protected tetrapeptide can be converted to the cyclosporine analogue cyclo-(D-Ala) according to the method provided by Li et al. 8 -MeLeu 9 -MeLeu 10 -MeVal 11 -MeLeu 1 -Nva 2 -Sar 3 -Gly 4 -Gly 5 -Gly 6 -Gly 7 ) can be changed to

[0341] Example 6. Synthesis of cyclic peptide compounds of the present invention General Protocol The general method described by Ishizawa et al., J. Am. Chem. Soc., 2013, vol. 135, p. 5433, can be used. Synthetic constant regions terminated in carboxylic acids and (2-chloroacetamido)-acylated amines (both directions) are prepared. Subsequently, peptide variable regions are prepared using standard Fmoc solid-phase peptide synthesis (SPPS) starting from Fmoc-Gly-Wang resin. Cysteine ​​residues are incorporated into internal positions for subsequent macrocyclization. A linear polypeptide is coupled to the synthetic constant region and then cleaved from the resin using trifluoroacetic acid. To facilitate macrocyclization, the peptide is treated with triethylamine in DMSO.

[0342] Example 7. Binding of compounds to cyclophilin A The binding of the compounds of the invention to cyclophilin A can be determined using the following protocol.

[0343] General Protocol This protocol utilizes the Perkin Elmer AlphaLISA technology platform to detect cyclosporine analogs by measuring the inhibition of binding of biotinylated cyclosporine A to FLAG-tagged cyclophilin A.

[0344] Reagents: 10x TBST buffer (Boston BioProducts IBB-181), biotinylated cyclosporine A (in-house), FLAG-tagged cyclophilin A (in-house), anti-FLAG donor beads (PerkinElmer AS103) and streptavidin acceptor beads (PerkinElmer AL125), compound cyclosporine A (LC Labs Cat# C-6000) in DMSO (in-house).

[0345] Equipment: Biotek Synergy2, Janus MTD Head pipettor, Eppendorf Repeat Pipettor.

[0346] Supplies: White 96-well Corning 1 / 2 area plate (Cat. #3642), 96-well polypropylene full-skirt (180 μl) PCR plate, 96-well Viaflow P20 tips for Janus MTD Head pipettor.

[0347] Experimental Protocol / Assay Details: Add 20 μL of 6 nM biotinylated CsA working stock to each well of a 96-well plate. Add 1 μL of test compound (100% DMSO) to each well of the plate using a Janus MTD Head and P20 tip (except control wells). Add 1 μL of DMSO to negative control wells and 1 μL of 500 μM cyclosporine A solution to positive control wells. Add 20 μL of combined donor / acceptor beads to each well in the dark. Incubate in the dark at room temperature for 30 minutes. Add 10 μL of 25 nM Flag-tagged CypA working stock to each well in the dark. Incubate in the dark at room temperature for 60 minutes. Protect plates from light until reading on a Biotek Synergy2 plate reader (Alphalisa 96-well protocol (680 excitation / 615 emission)).

[0348] Results: The binding affinities of 104 cyclosporine analogs to cyclophilin A were determined as shown in Table 5.

[0349] [Table 8]

[0350] JPEG2026015411000048.jpg118170

[0351] Table 5. Cyclophilin A binding of cyclosporine analogs Example 8. Binding of compounds to FKBP12 The binding of the compounds of the invention to FKBP12 can be determined using the following protocol.

[0352] General Protocol This protocol utilizes the Perkin-Elmer AlphaLISA technology platform to detect FKBP binders by measuring the inhibition of binding of biotinylated FK506 to FLAG-tagged FKBP12.

[0353] Reagents: 10x TBST buffer (Boston BioProducts IBB-181), biotinylated FK506 (in-house), FLAG-tagged FKBP (in-house), anti-FLAG donor beads (PerkinElmer AS103) and streptavidin acceptor beads (PerkinElmer AL125), compound FK506 in DMSO (in-house).

[0354] Equipment: Biotek Synergy2, Janus MTD Head pipettor, Eppendorf Repeat Pipettor.

[0355] Supplies: White 96-well Corning 1 / 2 area plate (Cat. #3642), 96-well polypropylene full-skirt (180 μl) PCR plate, 96-well Viaflow P20 tips for Janus MTD Head pipettor.

[0356] Experimental Protocol / Assay Details: Add 20 μL of 12.5 nM FKBP-FLAG working stock to each well of a 96-well plate. Add 1 μL of test compound (100% DMSO) to each well of the plate using a Janus MTD Head and P20 tip (except control wells). Add 1 μL of DMSO to negative control wells and 1 μL of 500 μM FK506 solution to positive control wells. Add 20 μL of combined donor / acceptor beads to each well in the dark. Incubate in the dark at room temperature for 30 minutes. Add 10 μL of 5 nM biotinylated FK506 working stock to each well in the dark. Incubate in the dark at room temperature for 60 minutes. Protect plates from light until reading on a Biotek Synergy2 plate reader (Alphalisa 96-well protocol (680 excitation / 615 emission)).

[0357] Results: FKBP12 binding of selected compounds was determined as shown in Table 6.

[0358] [Table 9]

[0359] Table 6.FKBP12 binding Example 9. SPR protocol for measuring binding of compounds to FKBP12 In this protocol, we utilize surface plasmon resonance (SPR) as a method to determine the binding kinetics (KD, Ka, Kd) of a compound (analyte) to immobilized FKBP12 (ligand).

[0360] Reagents: Compounds in 100% DMSO (in-house), 10x HBS-P+ buffer (GE Healthcare BR-1006-71), assay buffer (1x HBS-P+ buffer, 1% DMSO), 12x HIS-tagged FKBP12 (in-house).

[0361] Equipment: Biacore™ X100 (GE Healthcare). Supplies: NTA sensor chip (GE Healthcare BR-1000~34).

[0362] Experimental protocol: Experiments are performed at 25°C. A stock solution of 12xHIS-tagged FKBP12 is diluted to 100 nM in assay buffer (final 1% DMSO). Approximately 500-600 RU of FKBP12 is immobilized on one of two flow cells of an activated NTA chip. The second flow cell is unactivated as a reference for nonspecific interactions between the analyte and the sensor chip. Various concentrations of compound (ranging from 1 nM to 1 μM) serially diluted in the same assay buffer (final 1% DMSO) are injected over the FKBP12 and reference surfaces at a flow rate of 10 μl / min. The surfaces are regenerated with 350 mM EDTA between analyte injections.

[0363] Data fitting: The BiaEvaluation software program is used for data fitting. All data are reference subtracted for both the reference flow cell and buffer injections. For kinetic analysis, data are locally fitted to a 1:1 interaction model.

[0364] [Table 10]

[0365] Table 7. FKBP12 binding data Example 10. Determination of binding of F2 and F11 to FKBP12 by SPR In this protocol, surface plasmon resonance (SPR) is used as a method to determine the binding kinetics (KD, Ka, Kd) of F2 and F11 (analytes) to immobilized FKBP12 (ligand).

[0366] Reagents: F2 and F11 in 100% DMSO (in-house), 10x HBS-P+ buffer (GE Healthcare BR-1006-71), assay buffer (1x HBS-P+ buffer, 1% DMSO), 12x HIS-tagged FKBP12 (in-house).

[0367] Equipment: Biacore™ X100 (GE Healthcare). Supplies: NTA sensor chip (GE Healthcare BR-1000~34).

[0368] Experimental protocol: Experiments are performed at 25°C. A stock solution of 12xHIS-tagged FKBP12 is diluted to 100 nM in assay buffer (final 1% DMSO). Approximately 500-600 RU of FKBP12 is immobilized on one of two flow cells of an activated NTA chip. The second flow cell is unactivated as a reference for nonspecific interactions between the analyte and the sensor chip. Various concentrations of F2 or F11 (ranging from 1 nM to 1 μM), serially diluted in the same assay buffer (final 1% DMSO), are injected over the FKBP12 and reference surfaces at a flow rate of 10 μl / min. The surfaces are regenerated with 350 mM EDTA between analyte injections.

[0369] Data fitting: The BiaEvaluation software program is used for data fitting. All data are reference subtracted for both the reference flow cell and buffer injections. For kinetic analysis, data are locally fitted to a 1:1 interaction model.

[0370] Results: The binding value of F2 to FKBP12 was Ka(1 / Ms): 4.50 × 10 4 , Kd(1 / s): 5.94 × 10 -4 , and KD: 13.2 nM. The binding value of F11 with FKBP12 is Ka(1 / Ms): 5.67 × 10 5 , Kd(1 / s): 8.8 × 10 -3, and KD: 15.6 nM.

[0371] Example 11. Determination of cell permeability of compounds The cell permeability of the compounds is determined using the following protocol. General Protocol This protocol utilizes modified FKBP or cyclophilin destabilizing mutants to determine the bioactivity of FKBP- or cyclophilin-binding compounds in whole cell assays.

[0372] Reagents: DMEM, DMEM without phenol red, 10% FBS, 1× sodium pyruvate, 1× Glutamax. Add 125 μl of medium with compound to each well.

[0373] Equipment: Biotek Synergy2, Janus MTD Head pipettor, Eppendorf Repeat Pipettor.

[0374] Supplies: White 96-well Corning 1 / 2 area plate (Cat. #3642), 96-well polypropylene full-skirt (180 μl) PCR plate, 96-well Viaflow P20 tips for Janus MTD Head pipettor.

[0375] Experimental Protocol / Assay: HeLa-FKBP12 cells (for FKBP-binding compounds) or HeLa-Cyclophilin A cells (for cyclophilin-binding compounds) are plated and seeded overnight (approximately 18 hours) at 5k / well. Using a multichannel pipette, remove old medium and add approximately 125ul of new medium with compound. Dilute compounds using phenol red-free DMEM, 10% FBS, 1x sodium pyruvate, and 1x Glutamax. Add 125µl of medium with compound to each well. Treat cells with compound at concentrations of 30, 10, 3.33, 1.11, 0.37, 0.12, 0.04, and 0.013µM. The time point is 72 hours, and the plate is read using a plate reader at excitation / emission of 575 / 620.

[0376] Calculation: Cell binding / permeability is calculated as fold change (total RFU of treated samples / total RFU of DMSO-treated samples or total RFU above background (total RFU minus total RFU of DMSO-treated samples)).

[0377] Results: Cell permeability data for selected compounds was collected as shown in Table 8.

[0378] [Table 11]

[0379] Table 8. Biosensor permeability Example 12. Binding of presenter protein / compound complex to target protein The binding of the presenter protein / compound complex of the present invention to a target protein can be determined using the following protocol.

[0380] General protocol for cyclophilin A complex This protocol utilizes the Perkin Elmer AlphaLISA technology platform to detect cyclosporine analogs by measuring the binding of cyclosporine compounds to a 6xHIS-tagged target protein plus FLAG-tagged cyclophilin A.

[0381] Reagents: 10x TBST buffer (Boston BioProducts IBB-181), MgCl2 (Sigman), 6xHIS-tagged target protein (in-house), FLAG-tagged cyclophilin A (in-house), anti-FLAG donor beads (PerkinElmer AS103) and streptavidin acceptor beads (PerkinElmer AL125), compound in DMSO (in-house), cyclosporine A (LC Labs Cat# C-6000).

[0382] Equipment: Biotek Synergy2, Janus MTD Head pipettor, Eppendorf Repeat Pipettor.

[0383] Supplies: White 96-well Corning 1 / 2 area plate (Cat. #3642), 96-well polypropylene full-skirt (180 μl) PCR plate, 96-well Viaflow P20 tips for Janus MTD Head pipettor.

[0384] Experimental Protocol / Assay: Add 20 μL of 250 nM 6xHIS-tagged target protein working stock to each well of a 96-well plate. Add 1 μL of test compound (100% DMSO) to each well of the plate using a Janus MTD Head and P20 tip (except for control wells). Add 1 μL of DMSO to the reference well. Add 20 μL of combined donor / acceptor beads to each well in the dark. Incubate in the dark for 30 minutes at room temperature. Add 10 μL of 10 nM Flag-tagged CypA working stock to each well in the dark. Incubate in the dark for 60 minutes at room temperature. Biotek Synergy 2 Protect plates from light until reading on a Synergy2 plate reader (Alphalisa 96-well protocol (680 excitation / 615 emission)). General protocol for FKBP12 complexes This protocol utilizes the Perkin-Elmer AlphaLISA technology platform to detect compounds by measuring the binding of a 6xHIS-tagged target protein plus FLAG-tagged FKBP12 to an FKBP-binding compound.

[0385] Reagents: 10x TBST buffer (Boston BioProducts IBB-181), MgCl2 (Sigman), 6xHIS-tagged target protein (in-house), FLAG-tagged FKBP12 (in-house), anti-FLAG donor beads (PerkinElmer AS103) and streptavidin acceptor beads (PerkinElmer AL125), compound FK506 in DMSO (in-house).

[0386] Equipment: Biotek Synergy2, Janus MTD Head pipettor, Eppendorf Repeat Pipettor.

[0387] Supplies: White 96-well Corning 1 / 2 area plate (Cat. #3642), 96-well polypropylene full-skirt (180 μl) PCR plate, 96-well Viaflow P20 tips for Janus MTD Head pipettor.

[0388] Experimental Protocol / Assay: Add 20μL of 250nM 6xHIS-tagged target protein working stock to each well of a 96-well plate. Add 1μL of test compound (100% DMSO) to each well of the plate using the Janus MTD Head and P20 tip (except control wells). Add 1μL of DMSO to the reference well. Add 20μL of combined donor / acceptor beads to each well. Incubate in the dark for 30 minutes at room temperature. Add 10μL of 10nM Flag-tagged FKBP12 working stock to each well in the dark. Incubate in the dark for 60 minutes at room temperature. Protect the plate from light until reading on a Biotek Synergy2 plate reader (Alphalisa 96-well protocol (680 excitation / 615 emission)).

[0389] Example 13. Determining the binding between a presenter protein / compound complex and a target protein by SPR In this protocol, we utilize surface plasmon resonance (SPR) as a method to determine the binding kinetics (KD, Ka, Kd) of a mammalian target protein (analyte) to an immobilized FKBP12-compound binary complex (ligand).

[0390] Reagents: Compounds in 100% DMSO (in-house), 10x HBS-P+ buffer (GE Healthcare BR-1006-71), assay buffer (1x HBS-P+ buffer, 1% DMSO, 1 µM F2), 12x HIS-tagged FKBP12 (in-house), mammalian target protein (in-house).

[0391] Equipment: Biacore™ X100 (GE Healthcare). Supplies: NTA sensor chip (GE Healthcare BR-1000~34).

[0392] Experimental Protocol: Experiments are performed at 25°C. A stock solution of 12x HIS-tagged FKBP12 is diluted to 100 nM in assay buffer containing 1 μM compound (final 1% DMSO). Approximately 200-400 RU of FKBP12 is immobilized on one of two flow cells of an activated NTA chip. The second flow cell is unactivated to serve as a reference for nonspecific interactions between the analyte and the sensor chip. Various concentrations of target protein (ranging from 1 nM to 1 μM) serially diluted in the same assay buffer containing 1 μM compound (final 1% DMSO) are injected over the FKBP12 and reference surfaces at a flow rate of 10 μl / min. The surfaces are regenerated with 350 mM EDTA between analyte injections.

[0393] Data fitting: The BiaEvaluation software program is used for data fitting. All data are reference subtracted for both the reference flow cell and buffer injections. For kinetic analysis, data are locally fitted to a 1:1 interaction model.

[0394] Example 14. Determination of binding between FKBP12 / F2 complex and CEP250 by SPR In this protocol, we utilize surface plasmon resonance (SPR) as a method to determine the kinetics (KD, Ka, Kd) of binding between CEP250 (analyte) and immobilized FKBP12-F2 binary complex (ligand).

[0395] Reagents: F2 in 100% DMSO (in-house), 10x HBS-P+ buffer (GE Healthcare BR-1006-71), assay buffer (1x HBS-P+ buffer, 1% DMSO, 1 µM F2), 12x HIS-tagged FKBP12 (in-house), CEP25029.2 (residues 1982-2231), and CEP250 11.4 (residues 2134–2231) (in-house production).

[0396] Equipment: Biacore™ X100 (GE Healthcare). Supplies: NTA sensor chip (GE Healthcare BR-1000~34).

[0397] Experimental Protocol: Experiments are performed at 25°C. A stock solution of 12x HIS-tagged FKBP12 is diluted to 100 nM in assay buffer containing 1 μM F2 (final 1% DMSO). Approximately 200-400 RU of FKBP12 is immobilized on one of two flow cells of an activated NTA chip. The second flow cell is unactivated as a reference for nonspecific interactions between the analyte and the sensor chip. Various concentrations of CEP250 (ranging from 1 nM to 1 μM) serially diluted in the same assay buffer containing 1 μM F2 (final 1% DMSO) are injected over the FKBP12 and reference surfaces at a flow rate of 10 μl / min. The surfaces are regenerated with 350 mM EDTA between analyte injections.

[0398] Data fitting: The BiaEvaluation software program is used for data fitting. All data are reference subtracted for both the reference flow cell and buffer injections. For kinetic analysis, data are locally fitted to a 1:1 interaction model.

[0399] Results: FKBP12 / F2 complex and CEP250 11.4 and CEP250 29.2The k values ​​for binding to and are K, respectively. a (1 / Ms):5.71×10 5 , K. d (1 / s): 3.09 x 10 -3 , and K D : 5.4 nM, and Ka (1 / Ms): 3.11 × 10 5 , Kd(1 / s): 9.25 × 10 -5 , and KD: 0.29 nM.

[0400] Example 15. Determining binding between presenter protein / compound complexes and target proteins by ITC General Protocol In this protocol, isothermal titration calorimetry (ITC) is utilized to directly measure the heat change associated with the binding of a presenter protein (e.g., FKBP, cyclophilin)-compound binary complex to a target protein. The measurement of the heat change determines the association constant (K a ), reaction stoichiometry (N), and change in bond enthalpy (ΔH).

[0401] Reagents: Compounds in 100% DMSO (in-house), protein buffer (10 mM HEPES, pH 7.5, 75 mM NaCl, 0.5 mM TCEP), assay buffer (protein buffer + 1% DMSO), presenter protein (e.g., FKBP, cyclophilin) ​​(in-house), target protein (in-house).

[0402] Device: MicroCal™ ITC 200 (MicroCal TM ITC 200 )(GE Healthcare). Experimental protocol: Presenter protein (e.g., FKBP, cyclophilin) ​​stock solution is diluted to 10 μM in assay buffer (1% DMSO final). Compounds are added to the presenter protein to 20 μM (1% DMSO final). After a 5-10 min preincubation period, the binary complex is loaded into the reaction cell of the ITC instrument. Target protein stock is diluted to 50 μM in assay buffer, and 20 μM compound is added (1% DMSO final). The target protein is then loaded into the injection syringe. A control experiment is also performed in the absence of compound to determine operational artifacts and heat associated with dilution of the titrant during injection from the syringe into the reaction cell. Data collection and analysis are as described for the binding of FKBP12-F2 and FKBP12-F11 binary complexes to CEP250.

[0403] Example 16. Determination of binding of FKBP12 / F2 and FKBP12 / F2 complex to CEP250 by ITC In this protocol, isothermal titration calorimetry (ITC) is used to directly measure the heat change associated with the binding of FKBP12-F2 and FKBP12-F11 binary complexes to CEP250. Measurement of the heat change allows for the accurate determination of the association constant (Ka), reaction stoichiometry (N), and change in binding enthalpy (ΔH).

[0404] Reagents: F2 and F11 in 100% DMSO (in-house), protein buffer (10 mM HEPES, pH 7.5, 75 mM NaCl, 0.5 mM TCEP), assay buffer (protein buffer + 1% DMSO), FKBP12 (in-house), CEP250 29.4 (residues 1982–2231), and CEP250 11.4 (residues 2134–2231) (in-house production).

[0405] Device: MicroCal™ ITC200 (GE Healthcare). Experimental protocol: FKBP12 stock solution was diluted to 10 μM in assay buffer (1% DMSO final). Compounds were added to FKBP12 to a concentration of 20 μM (1% DMSO final). After a 5-10 minute preincubation period, the binary complex was loaded into the reaction cell of the ITC instrument. CEP250 protein stock was diluted to 50 μM in assay buffer, and 20 μM compound was added (1% DMSO final), then loaded into the injection syringe. A control experiment was also performed in the absence of compound to determine operational artifacts and heat associated with dilution of the titrant during injection from the syringe into the reaction cell. More detailed experimental parameters are shown in Tables 11 and 12 below. Table 9. ITC experimental parameters Test equipment: MicroCal TM iT 200 (GE Healthcare)

[0406] [Table 12]

[0407] Experimental parameters

[0408] [Table 13]

[0409] Injection parameters

[0410] [Table 14]

[0411] Table 10. Protein and ligand concentrations for ITC Final protein and ligand concentrations

[0412] [Table 15]

[0413] Data fitting: Data fitting was performed using Origin ITC200 software according to the following procedure. 1) Reading raw data.

[0414] 2) "mRawlTC": Integrates all peaks by adjusting the integrated peaks and baseline. 3) "ΔH" - Data management: Remove bad data (injection #1 and other artifacts) and subtract a line (background subtraction).

[0415] 4) "ΔH" - Model fitting: Select one set of site models and perform fitting using the Levenberg-Marquardt algorithm until χ2 no longer decreases, then click "done" to terminate (the parameters N, Ka, and ΔH are calculated based on the fitting). The ITC measurements of the binding of FKBP12-F2 and FKBP12-F11 binary complexes with CEP250 are summarized in Table 11 below.

[0416] [Table 16]

[0417] Table 11.ITC measurements Results: Overall, the data for the FKBP12-F2 and FKBP12-F11 binary complexes binding to CEP25011.4 and CEP25029.4 show similar interaction parameters. Kd values ​​were similar for all combinations. All interactions showed nearly identical thermodynamic profiles, and binding was characterized by a purely enthalpic binding mode (-T * The AS term is positive and does not contribute to the Gibbs free energy. The binding stoichiometry for all interactions is N = 0.5–0.6, and CEP250 11.4 This supports a 1:2 binding ratio for one CEP250 homodimer bound to two FKBP12 molecules as demonstrated by the crystal structure of / F2 / FKBP12.

[0418] Example 17. Crystal structure determination of the ternary complex General Protocol In this protocol, we describe crystallization and structure determination methods for the structure of a specific FKBP12-compound-target protein ternary complex.

[0419] Reagents: compounds in 100% DMSO (in-house), FKBP12 (in-house), and mammalian target protein (in-house). Device: Superdex 200 (GE Healthcare).

[0420] Experimental protocol: A 3:1 molar excess of compound is added to FKBP12 in 12.5 mM HEPES pH 7.4, 75 mM NaCl buffer and incubated overnight at 4 °C. Ternary complex formation is terminated by adding a 3:1 molar excess of the FKBP12-compound binary complex to the target protein and incubating overnight at 4 °C. The pure ternary complex is isolated by gel filtration purification using a Superdex 200 column in 12.5 mM HEPES pH 7.4, 75 mM NaCl. The purified complex (10–20 mg / ml) is subjected to crystallization at 22 °C by sitting-drop vapor diffusion using various buffers, detergents, and salt solutions. For data collection, the crystals are transferred to a solution containing mother liquor supplemented with 20–25% glycerol and frozen in liquid nitrogen. Diffraction datasets are collected at the Advanced Photon Source (APS) and processed with the HKL program. A molecular exchange solution is obtained with the program PHASER from the CCP4 suite using the published structure of FKBP12 (PDB-ID 1FKD) as a search model. Subsequent model building and refinement are carried out according to standard protocols using the software packages CCP4 and COOT.

[0421] Example 18. Crystal structure determination of tertiary complexes of FKBP12 / F2 and FKBP12 / F11 complexes with CEP250 In this protocol, we describe the crystallization and structure determination methods for the structures of the FKBP12-Compound 2-CEP250 and FKBP12-F11-CEP250 ternary complexes.

[0422] Reagents: F2 and F11, FKBP12 (in-house), and CEP250 in 100% DMSO (in-house). 11.4 (residues 2134–2231) (in-house production). Device: Superdex 200 (GE Healthcare).

[0423] Experimental protocol: A 3:1 molar excess of F2 or F11 was added to FKBP12 in 12.5 mM HEPES pH 7.4, 75 mM NaCl buffer and incubated overnight at 4 °C. Ternary complex formation was completed by adding a 3:1 molar excess of FKBP12-F2 or FKBP12-F11 binary complex to CEP250.4 and incubating overnight at 4 °C. The pure ternary complex was isolated by gel filtration purification using a Superdex 200 column in 12.5 mM HEPES pH 7.4, 75 mM NaCl. The purified complex (10–20 mg / ml) was subjected to crystallization at 22 °C using sitting-drop vapor diffusion. FKBP12-F2-CEP250 crystals were grown in a well solution containing 0.2 M sodium malonate, 0.1 M HEPES 7.0, and 21% PEG3350. FKBP12-F11-CEP250 crystals were grown in a well solution containing 0.1 M Tris pH 8.5, 0.2 M trimethylamine N-oxide, and 22-24% PEG2000 MME. For data collection, the crystals were transferred to a solution containing mother liquor supplemented with 20-25% glycerol and frozen in liquid nitrogen. Diffraction datasets were collected at the Advanced Photon Source (APS) and processed with the HKL program. Molecular exchange solutions were obtained using the PHASER program from the CCP4 suite using the published structure of FKBP12 (PDB-ID 1FKD) as a search model. Subsequent model building and refinement were performed according to standard protocols using the software packages CCP4 and COOT.

[0424] Results: Overall structure of FKBP12-F2-CEP250: In the structure of FKBP12 with CEP250 in complex with F2, two FKBP12 monomers are bound to a CEP250 homodimer. Two CEP250 monomers form a coiled-coil structure. Four heterodimers exist in the asymmetric unit with essentially identical overall conformations. The model includes residues Met1 to Glu108 of FKBP12 and Asp2142 to His2228 of CEP250. The electron density reveals a clear binding mode for the ligand F2, including the ligand's orientation and conformation.

[0425] The CEP250 residues involved in binding to F2 are L2190, Q2191, V2193, A2194, M2195, F2196, L2197, and Q2198. The CEP250 residues involved in binding to FKBP12 are A2185, S2186, S2189, Q2191, M2195, Q2198, V2201, L2202, R2204, D2205, S2206, Q2208, Q2209, and Q2212.

[0426] The total buried surface area of ​​the ternary complex is 1759 Å. The total buried surface area of ​​CEP250 is 865 Å, of which 663 Å is contributed by FKBP12 and 232 Å is contributed by F2.

[0427] 100% of the binding interactions in the ternary complex between F2 and CEP250 are van der Waals or π-π interactions. In comparison, 100% of the binding interactions between rapamycin and mTOR are van der Waals or π-π interactions, and 89% of the binding interactions between FK506 and calcineurin are van der Waals or π-π interactions, but 11% are hydrogen bonds (two H-bonds from OMe at C13 and C15 to the NH of Trp352).

[0428] Overall structure of FKBP12-F11-CEP250: In the structure of FKBP12 with CEP250 in complex with F11, one FKBP12 monomer is bound to a CEP250 homodimer. Two CEP250 monomers form a coiled-coil structure. The crystal contains a heterotrimer (one FKBP12 and two CEP250) in the asymmetric unit. The model includes residues Met1 to Glu108 of FKBP12 and Ser2143 to His2228 of CEP250. One short loop region of FKBP12 (18-19) is not fully defined by the electron density and is not included in the model. The electron density reveals a clear binding mode for the ligand F11, including the ligand's orientation and conformation.

[0429] The CEP250 residues involved in binding to F2 are L2190, Q2191, V2193, A2194, M2195, F2196, L2197, and Q2198. The CEP250 residues involved in binding to FKBP12 are Q2182, A2185, S2186, S2189, Q2191, M2195, Q2198, V2201, L2202, R2204, D2205, S2206, Q2208, Q2209, and Q2212.

[0430] The total buried surface area of ​​the ternary complex is 1648 Å. The total buried surface area of ​​CEP250 is 831 Å, of which 590 Å is contributed by FKBP12 and 241 Å is contributed by F2.

[0431] The statistics of the final structure are listed in Tables 12 and 13 below.

[0432] [Table 17]

[0433] Table 12.FKBP12-F2-CEP250

[0434] [Table 18]

[0435] Table 13.FKBP12-F11-CEP250 While the invention has been described in connection with particular embodiments thereof, it will be understood that further modifications are possible, and this application is intended to cover any such variations, uses, or adaptations of the invention in accordance with its principles generally, and to cover such departures from the present disclosure as come within known or customary practice within the art to which this invention pertains and are applicable to the essential features hereinabove set forth.

[0436] All publications, patents, and patent applications are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety. [Appendix 1] A macrocyclic compound or a pharmaceutically acceptable salt thereof comprising 14 to 40 ring atoms, the compound comprising (a) a mammalian target protein interacting portion and (b) a presenter protein binding portion, wherein the compound and the presenter protein form a complex that specifically binds to the target protein, and wherein each of the compound and the presenter protein does not substantially bind to the target protein in the absence of formation of the complex, or the compound and the presenter protein form a complex that binds to the target protein with an affinity that is at least 5 times greater than the affinity of each of the compound and the presenter protein for the target protein in the absence of formation of the complex. [Supplementary Note 2] The presenter protein binding moiety has Formula I: [ka] (wherein n is 0 or 1; X1 and X3 are each independently O, S, CR3R4, or NR5; X2 is O, S, or NR5; and R1, R2, R3, and R4 are each independently hydrogen, hydroxyl, optionally substituted amino, halogen, thiol, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C1-C6 heteroalkyl, optionally substituted C2-C6 heteroalkenyl, optionally substituted C1 ... optionally substituted C2-C6 heteroalkynyl, optionally substituted C3-C10 carbocyclyl, optionally substituted C6-C10 aryl, optionally substituted C6-C10 arylC1-C6 alkyl, optionally substituted C2-C9 heteroaryl, optionally substituted C2-C9 heteroarylC1-C6 alkyl, optionally substituted C2-C9 heterocyclyl, or optionally substituted C2-C9 heterocyclylC1-C6 alkyl; or any two of R1, R2, R3, or R4 are together with the atom or atoms to which they are bonded form an optionally substituted carbocyclyl, an optionally substituted heterocyclyl, an optionally substituted aryl, or an optionally substituted heteroaryl, and each R5 is independently hydrogen, hydroxyl, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C1-C6 heteroalkyl, optionally substituted C2-C6 heteroalkenyl, optionally substituted C1-C6 alkynyl, optionally substituted C1-C6 heteroalkyl, optionally substituted C2-C6 heteroalkenyl, optionally substituted C1-C6 alkynyl, optionally substituted C1-C6 heteroalkyl, optionally substituted C2-C6 heteroalkenyl, optionally substituted C1-C6 heteroaryl, optionally substituted C2-C6 alkynyl, optionally substituted C1-C6 heteroalkyl, optionally substituted C2-C6 heteroaryl, optionally substituted C2-C6 alkynyl, optionally substituted C1-C6 heteroalkyl, optionally substituted C2-C6 heteroaryl, optionally substituted C2-C6 alkynyl, optionally substituted C2-C6 heteroaryl, optionally substituted C2-C6 heteroaryl, optionally substituted C2-C6 alkynyl, optionally substituted C2 ... heteroaryl, optionally substituted C2-C6 alkynyl, optionally substituted C2- optionally substituted C2-C6 heteroalkynyl, optionally substituted C3-C10 carbocyclyl, optionally substituted C6-C10 aryl, optionally substituted C6-C10 arylC1-C6 alkyl, optionally substituted C2-C9 heteroaryl, optionally substituted C2-C9 heteroarylC1-C6 alkyl, optionally substituted C2-C9 heterocyclyl, or optionally substituted C2-C9 heterocyclylC1-C6 alkyl; or R5 and one of R1, R2, R3, or R4 isor a pharmaceutically acceptable salt thereof. [Supplementary Note 3] The presenter protein binding moiety is represented by Formulae II to IV: [ka] (wherein o and p are independently 0, 1, or 2; q is an integer from 0 to 7; r is an integer from 0 to 4; X4 and X5 are each independently absent, CH2, O, S, SO, SO2, or NR11; and each R6 and R7 is independently hydrogen, hydroxyl, optionally substituted amino, halogen, thiol, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C1-C6 heteroalkyl ... C6 heteroalkenyl, optionally substituted C2-C6 heteroalkynyl, optionally substituted C3-C10 carbocyclyl, optionally substituted C6-C10 aryl, optionally substituted C6-C10 arylC1-C6 alkyl, optionally substituted C2-C9 heteroaryl, optionally substituted C2-C9 heteroarylC1-C6 alkyl, optionally substituted C2-C9 heterocyclyl, optionally substituted C2-C9 heterocyclylC1-C6 alkyl, or R6 and R7 are the groups to which they are attached. and R8 is independently selected from the group consisting of hydroxyl, optionally substituted amino, halogen, thiol, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C1-C6 heteroalkyl, optionally substituted C2-C6 heteroalkenyl, optionally substituted C2-C6 heteroalkynyl, optionally substituted C3-C10 carbocyclyl, optionally substituted C6-C10 aryl, and any optionally substituted C6-C10 arylC1-C6 alkyl, optionally substituted C2-C9 heteroaryl, optionally substituted C2-C9 heteroarylC1-C6 alkyl, optionally substituted C2-C9 heterocyclyl, or optionally substituted C2-C9 heterocyclylC1-C6 alkyl; or two R8 combine to form an optionally substituted C3-C10 carbocyclyl, optionally substituted C6-C10 aryl, or optionally substituted C2-C9 heteroaryl, and R9 isOptionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C1-C6 heteroalkyl, optionally substituted C2-C6 heteroalkenyl, optionally substituted C2-C6 heteroalkynyl, optionally substituted C3-C10 carbocyclyl, optionally substituted C6-C10 aryl, optionally substituted C6-C10 arylC1-C6 alkyl, optionally substituted C2-C9 heteroaryl, optionally optionally substituted C2-C9 heteroarylC1-C6 alkyl, optionally substituted C2-C9 heterocyclyl, or optionally substituted C2-C9 heterocyclylC1-C6 alkyl, R10 is optionally substituted C1-C6 alkyl, and each R11 is independently hydroxyl, cyano, optionally substituted amino, halogen, thiol, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C1 1 to C6 heteroalkyl, optionally substituted C2 to C6 heteroalkenyl, optionally substituted C2 to C6 heteroalkynyl, optionally substituted C3 to C10 carbocyclyl, optionally substituted C6 to C10 aryl, optionally substituted C6 to C10 arylC1 to C6 alkyl, optionally substituted C2 to C9 heteroaryl, optionally substituted C2 to C9 heteroarylC1 to C6 alkyl, optionally substituted C2 to C9 heterocyclyl, or optionally substituted C2 to C9 heterocyclyl. and R12 and R13 are each independently hydrogen, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted aryl, C3-C7 carbocyclyl, optionally substituted C6-C10 arylC1-C6 alkyl, and optionally substituted C3-C7 carbocyclylC1-C6 alkyl, or a pharmaceutically acceptable salt thereof. [Supplementary Note 4] The presenter protein binding moiety has formula V: [ka] or a pharmaceutically acceptable salt thereof. 100. The compound of Appendix 3, comprising the structure represented by the formula: wherein R14 is hydrogen, hydroxyl, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C1-C6 heteroalkyl, optionally substituted C2-C6 heteroalkenyl, optionally substituted C2-C6 heteroalkynyl, optionally substituted C3-C10 carbocyclyl, optionally substituted C6-C10 aryl, optionally substituted C6-C10 arylC1-C6 alkyl, optionally substituted C2-C9 heteroaryl, optionally substituted C2-C9 heteroarylC1-C6 alkyl, optionally substituted C2-C9 heterocyclyl, or optionally substituted C2-C9 heterocyclylC1-C6 alkyl. [Supplementary Note 5] The presenter protein binding moiety has formula VI or VII: [ka] (wherein s and t are each independently an integer of 0 to 7; X6 and X7 are each independently O, S, SO, SO2, or NR19; R15 and R17 are each independently hydrogen, hydroxyl, or optionally substituted C1-C6 alkyl; R16 and R18 are each independently hydroxyl, optionally substituted amino, halogen, thiol, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C1-C6 heteroalkyl, optionally substituted C2-C6 heteroalkenyl, optionally substituted C2-C6 heteroalkynyl, optionally substituted C3-C10 carbocyclyl, optionally substituted C6-C10 aryl, optionally substituted C6-C10 arylC1-C6 alkyl, or any or a pharmaceutically acceptable salt thereof. [Additional Note 6] The target interaction moiety has the formula IX: [ka] wherein u is an integer from 1 to 20, and each Y is independently any amino acid, O, NR20, S, S(O), SO2, or a group represented by Formulas X-XIII: [ka] wherein each R20 is independently hydrogen, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted aryl, C3-C7 carbocyclyl, optionally substituted C6-C10 arylC1-C6 alkyl, and optionally substituted C3-C7 carbocyclylC1-C6 alkyl; or R19 in combination with any of R20, R21, R22, R23, R24, R25, R26, R27, R28, R29, or R30 to form any optionally substituted C3-C10 carbocyclyl, optionally substituted C6-C10 aryl, or optionally substituted C2-C9 heteroaryl; each R21 and R22 is independently hydrogen, halogen, optionally substituted hydroxyl, or optionally substituted amino; or R20 and R21 taken in combination form =O, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, or optionally substituted C3-C10 carbocyclyl. , optionally substituted C6-C10 aryl, optionally substituted C6-C10 arylC1-C6 alkyl, optionally substituted C2-C9 heteroaryl, optionally substituted C2-C9 heteroarylC1-C6 alkyl, optionally substituted C2-C9 heterocyclyl, or optionally substituted C2-C9 heterocyclylC1-C6 alkyl; or R21 or R22 may be combined with any of R20, R21, R22, R23, R24, R25, R26, R27, R28, R29, or R30 to form any optionally substituted C3-C10 carbocyclyl, optionally substituted C6-C10 aryl, or optionally substituted C2-C9 heteroaryl; each R23, R24, R25, or R26 is independently hydrogen, hydroxyl, or R22 and R23 combine to form =0, or R23, R24, R25, or R26 combines with any of R20, R21, R22, R23, R24, R25, R26, R27, R28, R29, or R30 to form an optionally substituted C3-C10 carbocyclyl;optionally substituted C6-C10 aryl, or optionally substituted C2-C9 heteroaryl, and each R27, R28, R29, and R30 independently represent hydrogen, halogen, optionally substituted hydroxyl, optionally substituted amino, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C3-C10 carbocyclyl, optionally substituted C6-C10 aryl, optionally substituted C6-C10 arylC1-C6 alkyl, optionally substituted C2-C9 heteroaryl, optionally substituted C2-C9 heteroaryl, optionally substituted C2-C9 alkyl, optionally substituted C3-C10 aryl, optionally substituted C6-C10 arylC1-C6 alkyl, optionally substituted C2-C9 heteroaryl ... or a pharmaceutically acceptable salt thereof). [Appendix 7] The compound is represented by formulas XIV to XVIII: [ka] or a pharmaceutically acceptable salt thereof. [Supplementary Note 8] The portion of the molecule containing each ring atom involved in binding to the target protein has the structure: [ka] 8. The compound according to any one of Appendices 1 to 7, or a pharmaceutically acceptable salt thereof, which does not have [Appendix 9] The compound has the structure: [ka] 9. The compound according to any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof, which does not contain: [Appendix 10] The compound according to any one of Appendices 1 to 9, or a pharmaceutically acceptable salt thereof, wherein the presenter protein is prolyl isomerase. [Appendix 11] The compound according to any one of Appendices 1 to 10, or a pharmaceutically acceptable salt thereof, wherein the presenter protein is a member of the FKBP family, a member of the cyclophilin family, or PIN1. [Appendix 12] The compound according to any one of Appendices 1 to 11, or a pharmaceutically acceptable salt thereof, wherein the mammalian target protein is a GTPase, a GTPase-activating protein, a guanine nucleotide exchange factor, a heat shock protein, an ion channel, a coiled-coil protein, a kinase, a phosphatase, a ubiquitin ligase, a transcription factor, a chromatin modifier / remodeler, or a protein having a classical protein-protein interaction domain and motif. [Appendix 13] A presenter protein / compound complex comprising the compound according to any one of Appendices 1 to 12 and a presenter protein. [Appendix 14] A pharmaceutical composition comprising the compound according to any one of Appendices 1 to 12 and a pharmaceutically acceptable excipient, or a pharmaceutically acceptable salt thereof. [Appendix 15] A method for preparing the compound according to any one of Appendices 1 to 12, or a pharmaceutically acceptable salt thereof, comprising culturing a bacterial strain of the genus Streptomyces modified to produce the compound, and isolating the compound from the fermentation broth. [Appendix 16] A method for preparing the compound according to any one of Appendices 1 to 12, or a pharmaceutically acceptable salt thereof, comprising culturing a bacterial strain of the genus Streptomyces under conditions in which the strain produces the compound, and isolating the compound from the fermentation broth. [Appendix 17] A tripartite complex comprising (i) a mammalian target protein and (ii) a presenter protein / compound complex, wherein the presenter protein / compound complex comprises a presenter protein and a macrocyclic compound according to any one of 1 to 12, or a pharmaceutically acceptable salt thereof.

Claims

1. The following structure 【Chemistry 1】 A compound having the formula: During the ceremony, A is a compound of formula IX 【Chemistry 2】 having the structure u is an integer from 1 to 20; Each Y is independently any amino acid, O, NR ２０ ,S,S(O),SO ２ or of formula X-XIII 【Transformation 3】 and Each R ２１ and R ２２ are independently hydrogen, halogen, optionally substituted hydroxyl, optionally substituted amino, optionally substituted C １ ~C ６ alkyl, or R ２１ and R ２２ and combine to form =O, Each R ２７ , R ２８ , R ２９ , and R ３０ are independently hydrogen, halogen, optionally substituted hydroxyl, optionally substituted amino, or optionally substituted C １ ~C ６ is alkyl, Each R ２０ are independently hydrogen, optionally substituted C １ ~C ６ Alkyl, optionally substituted C ２ ~C ６ Alkenyl, optionally substituted C ２ ~C ６ alkynyl, optionally substituted aryl, C ３ ~C ７ Carbocyclyl, optionally substituted C ６ ~C １０ Aryl C １ ~C ６ Alkyl, and optionally substituted C ３ ~C ７ Carbocyclyl C １ ~C ６ alkyl, or R ２０ is any R ２０ , R ２１ , R ２２ , R ２３ , R ２４ , R ２５ , R ２６ , R ２７ , R ２８ , R ２９ , or R ３０ in combination with optionally substituted C ３ ~C １０ Carbocyclyl, optionally substituted C ６ ~C １０ aryl or optionally substituted C ２ ~C ９ forming a heteroaryl, L １ and L ２ each is independently a bond; B has the following structure: 【Chemistry 4】 and X ６ and X ７ are each independently O, S, SO, SO ２ , or NR １９ and R ６ and R ７ Each of the is independently hydrogen, optionally substituted C １ ~C ６ alkyl, or R ６ and R ７ are bonded to the carbon atom to which they are attached to form C=O, R １４ is an optionally substituted —(C １ ~C ６ alkylene)-C ６ ~C １０ is aryl, R １５ and R １７ are each independently hydrogen, hydroxyl, or an optionally substituted C １ ~C ６ is alkyl, R １６ and R １８ are each independently selected from hydroxyl, optionally substituted amino, halogen, thiol, optionally substituted C １ ~C ６ Alkyl, optionally substituted C ２ ~C ６ Alkenyl, optionally substituted C ２ ~C ６ Alkynyl, optionally substituted C １ ~C ６ Heteroalkyl, optionally substituted C ２ ~C ６ Heteroalkenyl, optionally substituted C ２ ~C ６ Heteroalkynyl, optionally substituted C ３ ~C １０ Carbocyclyl, optionally substituted C ６ ~C １０ Aryl, optionally substituted C ６ ~C １０ Aryl C １ ~C ６ Alkyl, optionally substituted C ２ ~C ９ Heteroaryl, optionally substituted C ２ ~C ９ Heteroaryl C １ ~C ６ Alkyl, optionally substituted C ２ ~C ９ heterocyclyl or optionally substituted C ２ ~C ９ Heterocyclyl C １ ~C ６ is alkyl, R １９ is an optionally substituted C １ ~C ６ Alkyl, optionally substituted C ２ ~C ６ Alkenyl, optionally substituted C ２ ~C ６ alkynyl, optionally substituted aryl, C ３ ~C ７ Carbocyclyl, optionally substituted C ６ ~C １０ Aryl C １ ~C ６ Alkyl and optionally substituted C ３ ~C ７ Carbocyclyl C １ ~C ６ is alkyl, s and t are each independently an integer from 0 to 7; o is 1 or 2; A has the following structure: 【Transformation 5】 or a pharmaceutically acceptable salt thereof, wherein

2. R １５ and R １７ 10. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein each is hydroxyl.

3. X ６ and X ７ and each is O, or a pharmaceutically acceptable salt thereof.

4. R １４ is optionally substituted -(C １ ~C ２ alkylene)-C ６ The compound according to any one of claims 1 to 3, which is aryl, or a pharmaceutically acceptable salt thereof.

5. R １４ 5. The compound of claim 4, or a pharmaceutically acceptable salt thereof, wherein is optionally substituted phenethyl.

6. B has the following structure 【Transformation 6】 2. The compound of claim 1, comprising:

7. A pharmaceutical composition comprising the compound according to any one of claims 1 to 6 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

8. A presenter protein-compound complex comprising the compound according to any one of claims 1 to 6 or a pharmaceutically acceptable salt thereof, and a presenter protein.

9. The complex of claim 8 , wherein the presenter protein is a prolyl isomerase.

10. The complex according to claim 8 or 9, wherein the presenter protein is FKBP12, FKBP12.6, FKBP25, or FKBP4.

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