WO2020076738A2

WO2020076738A2 - Protein-binding compounds

Info

Publication number: WO2020076738A2
Application number: PCT/US2019/055067
Authority: WO
Inventors: Joseph Henri Bayle; Slawomir Szymanski; Matthew Robert Collinson-Pautz; Steven Mark TOLER; David Michael Spencer
Original assignee: Bellicum Pharmaceuticals, Inc
Priority date: 2018-10-12
Filing date: 2019-10-07
Publication date: 2020-04-16
Also published as: WO2020076738A3

Abstract

The technology relates in part to compounds that bind to proteins. In certain examples, the compounds can bind to proteins that bind to rapamycin. In some examples, the compounds can bind to cellular proteins, and/or variant forms of cellular proteins, that bind to rapamycin and/or analogs of rapamycin. In certain examples, the compounds bind to and multimerize proteins that bind to rapamycin and/or analogs of rapamycin, such as for example, an FKBP12 protein (and/or variants threreof) and an mTOR polypeptide and/or region such as FRB, and/or variants thereof. Compounds provided include those having a structure of Formula A Formula A, where R²⁰, R²¹ and R²² moieties are described herein.

Description

PROTEIN-BINDING COMPOUNDS

Reference to Related Patent Application

[0001] This application claims the benefit of the filing date of United States Provisional Application 62/744,813, filed October 12, 2018, the contents of which are incorporated by reference in their entirety for all purposes.

Field

[0002] The technology relates in part to compounds that bind to proteins. In certain examples, the compounds can bind to proteins that bind to rapamycin. In some examples, the compounds can bind to cellular proteins, and/or variant forms of cellular proteins, that bind to rapamycin and/or analogs of rapamycin. In certain examples, the compounds bind to and multimerize proteins that bind to rapamycin and/or analogs of rapamycin, such as for example, an FKBP protein (and/or variants threreof) and an mTOR protein and/or region thereof such as FRB, and/or variants thereof.

Background

[0003] Chemical induction of protein dimerization with small molecules can be used to switch protein function and alter cell physiology. An example of a high specificity, efficient dimerizer is rapamycin (also referred to as sirolimus). Rapamycin is a chemical compound produced by the bacterium Streptomyces hygroscopicus that can inhibit growth of some eukaryotic cells.

Rapamycin binds to the immunophilin FKBP12 and forms a complex that binds with mTOR, which is a protein kinase contained in the protein complex TORC1 involved in activation of protein translation and inhibition of autophagy. The portion of mTOR bound by FKBP12- rapamycin is termed FRB (FKBP-rapamycin binding domain). The heterodimerization that occurs upon the binding of the FKBP12-rapamycin complex to FRB can inhibit mTOR function. Rapamycin has generally been used as an immunosuppressive and/or antiproliferative drug or antibiotic. However, because rapamycin has limited solubility and stability in aqueous solution, there is a need for compounds that have similar binding characteristics of rapamycin but with more suitable pharmacologic properties.

[0004] Rapamycin-directed, as well as rapamycin analog (rapalog)-directed, protein dimerization has also been used to approximate proteins fused to FKBP12 and proteins fused to FRB. Such systems can provide for rapamycin (or rapalog)-controlled cell signaling to regulate, for example, transcription and apoptosis in cells expressing the fusion proteins. However, use of rapamycin (or a rapalog capable of binding to mTOR) as an agent to induce dimerization of fusion proteins in target cells can result in mTOR inhibition as an undesired side effect.

Accordingly, to increase the applicability of FKBP12-FRB multimerization-based cell-signaling systems for in vivo use, there is a need for FKBP12/rapalog/variant FRB binding partners that have a higher binding affinity than that of the FKBP12-rapalog complex for wild type mTOR.

[0005] Provided herein are compounds that bind to proteins. In certain embodiments, the compound binds to one or more proteins to which rapamycin and/or a rapamycin analog binds, such as, for example, cellular proteins, including, but not limited to, proteins of animal (e.g., human) cells. Such peptides and polypeptides include, for example, a variant or wild type FRB (FKBP-rapamycin binding domain) of the mTOR domain of TORCI and an FK506-binding protein (FKBP) protein such as FKBP12 or variant thereof.

[0006] In some embodiments, a compound provided herein selectively binds to a variant FRB polypeptide. In certain embodiments, a compound provided herein binds to a variant FRB polypeptide with greater affinity than it has for binding to a wild type FRB polypeptide. In such embodiments, the compound may also bind to wild type FKBP12 with similar affinity as rapamycin. In some embodiments, a compound provided herein possesses properties (e.g., solubility and/or stability) that are more favorable for pharmaceutical or in vivo use than rapamycin. In certain embodiments, compounds provided herein have greater solubility and/or stability than rapamycin and/or a particular rapamycin analog in water and/or in other pharmaceutically acceptable aqueous solutions.

[0007] In certain aspects, provided herein are compounds having a structure of the following Formula B:

or a pharmaceutically acceptable salt thereof, wherein:

R^20A is hydrogen and R^20B is -R²³ or -R^F-R²³, or R^20B is hydrogen and R^20A is -R²³ or -R^F-R²³; R^21A is hydrogen and R^{21 B} is hydroxy, -R^G-R²⁴ -R^G-R²⁴-R²⁵ or -R^G-R²⁴-R²⁶-R²⁵, or R^{21 B} is hydrogen and R^21A is hydroxy, -R^G-R²⁴ -R^G-R²⁴-R²⁵ or -R^G-R²⁴-R²⁶-R²⁵;

R^22A is hydrogen and R^22B is halogen, -NR²⁷R²⁸, -R^H-R²⁹-R³⁰ or -R^H-R²⁹-R³¹-R³⁰, or R^22B is hydrogen and R^22A is halogen, -NR²⁷R²⁸, -R^H-R²⁹-R³⁰ or -R^H-R²⁹-R³¹-R³⁰;

R^F, R^G and R^H each independently is -0-, -C(O)-, -O-C(O)-, -C(0)-0-, -S-, -S(0)_n-, -O- S(0)_n-, -S(0)_n-0-, -NH-S(0)_n-, -S(O) _n-NH-, -NH-C(O)-, -C(0)-NH-, -NH-C(S)-, -C(S)- NH-, -S-C(S)-, -C(S)-S-, or -C(S)-;

n is 1 or 2;

R²³ independently is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, aryl, heterocycloalkyl or heteroaryl, which independently is optionally substituted by one or more substituents chosen from halogen, hydroxy, alkyl, alkenyl, alkynyl, heteroalkyl, haloalkyl, perhaloalkyl, perhaloalkoxy, alkoxy, haloalkoxy, alkoxyalkyl, acyl, oxo, acyloxy, carboxyl, amido, cyano, amino, alkylamino, alkylaminoalkyl, thiol, alkylthio, alkylthioalkyl, haloalkylthio, perhaloalkylthio, nitro, aryl, arylalkyl, cycloalkylalkyl, heterocycle-alkyl, cycloalkyl, heterocycloalkyl, heteroaryl, heteroarylalkyl, alkylsulfonyl, sulfonamide, alkylsulfonamido, alkylsilyloxy, alkylsulfinamido, arylsulfinamido and arylsulfonamido;

R²⁴ is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl, which independently is optionally substituted by one or more substituents chosen from halogen, hydroxy, alkyl, alkenyl, alkynyl, heteroalkyl, haloalkyl, perhaloalkyl, perhaloalkoxy, alkoxy, haloalkoxy, alkoxyalkyl, acyl, oxo, acyloxy, carboxyl, amido, cyano, amino, alkylamino, alkylaminoalkyl, thiol, alkylthio, alkylthioalkyl, haloalkylthio, perhaloalkylthio, nitro, aryl, arylalkyl, cycloalkylalkyl, heterocycle-alkyl, cycloalkyl, heterocycloalkyl, heteroaryl, heteroarylalkyl, alkylsulfonyl, sulfonamide, alkylsulfonamido and alkylsilyloxy;

R²⁹ is cycloalkyl, heterocycloalkyl, aryl or heteroaryl, which independently is optionally substituted by one or more substituents chosen from halogen, hydroxy, alkyl, alkenyl, alkynyl, heteroalkyl, hydroxyalkyl, haloalkyl, perhaloalkyl, perhaloalkoxy, alkoxy, haloalkoxy, alkoxyalkyl, acyl, oxo, acyloxy, carboxyl, amido, cyano, amino, alkylamino, alkylaminoalkyl, thiol, alkylthio, alkylthioalkyl, haloalkylthio, perhaloalkylthio, nitro, aryl, arylalkyl, cycloalkylalkyl, heterocycle- alkyl, cycloalkyl, heterocycloalkyl, heteroaryl, heteroarylalkyl, alkylsulfonyl, sulfonamide, alkylsulfonamido and alkylsilyloxy;

R²⁵, R³⁰ and R³³ each independently is halogen, cycloalkyl, heterocycloalkyl, aryl or heteroaryl, which independently is optionally substituted by one or more substituents chosen from halogen, hydroxy, alkyl, alkenyl, alkynyl, heteroalkyl, haloalkyl, perhaloalkyl, perhaloalkoxy, alkoxy, haloalkoxy, alkoxyalkyl, acyl, oxo, acyloxy, carboxyl, amido, cyano, amino, alkylamino, alkylaminoalkyl, thiol, alkylthio, alkylthioalkyl, haloalkylthio, perhaloalkylthio, nitro, aryl, arylalkyl, cycloalkylalkyl, heterocycle-alkyl, cycloalkyl, heterocycloalkyl, heteroaryl, heteroarylalkyl, alkylsulfonyl, sulfonamide, alkylsulfonamido and alkylsilyloxy;

R²⁶, R³¹ and R³² each independently is an alkyl, alkenyl or alkynyl linker or heteroalkyl linker, which independently is optionally substituted with one or more substituents chosen from hydroxy, halogen, acyl, oxo, acyloxy, carboxyl, amido, cyano, amino, alkylamino,

alkylaminoalkyl, thiol, alkylthio, alkylthioalkyl, haloalkylthio, perhaloalkylthio and nitro; and

R²⁷ and R²⁸ each independently is -R³²-R³³, or hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, perhaloalkyl, alkoxy, cycloalkyl, aryl, heterocycloalkyl, heterocycle-alkyl or heteroaryl, which independently is optionally substituted with one or more substituents chosen from halogen, hydroxy, alkyl, alkenyl, alkynyl, heteroalkyl, haloalkyl, perhaloalkyl, perhaloalkoxy, alkoxy, haloalkoxy, alkoxyalkyl, acyl, oxo, acyloxy, carboxyl, amido, cyano, amino, alkylamino, alkylaminoalkyl, thiol, alkylthio, alkylthioalkyl, haloalkylthio, perhaloalkylthio, nitro, aryl, arylalkyl, cycloalkylalkyl, heterocycle-alkyl, cycloalkyl, heterocycloalkyl, heteroaryl, heteroarylalkyl, alkylsulfonyl, sulfonamide, alkylsulfonamido, alkylsilyloxy, alkylsulfinamido, arylsulfinamido, and arylsulfonamido with the proviso that R²⁷ and R²⁸ are not both hydrogen when -R^F is -O- and R²³ is alkyl.

[0008] In certain embodiments, a compound provided herein has the structure of Formula B, and R^20A, R^20B, R^21A, R^{21 B}, R^F, R^G, R^H, R²³, R²⁴, R²⁵, R²⁶, R²⁷, R²⁸, R²⁹, R³⁰, R³¹ , R³², R³³ and n as set forth above, except that R^22A is hydrogen and R^22B is halogen, -NR²⁷R²⁸ or -R^H-R²⁹-R³¹- R³⁰, or R^22B is hydrogen and R^22A is halogen, -NR²⁷R²⁸ or -R^H-R²⁹-R³¹-R³⁰. In certain aspects, compounds provided herein have a structure of Formula B, or a

pharmaceutically acceptable salt thereof, wherein:

R^20A is hydrogen and R^20B is -R²³ or -R^F-R²³, or R^20B is hydrogen and R^20A is -R²³ or -R^F-R²³; R^21A is hydrogen and R^{21 B} is hydrogen, hydroxy or -R^G-R³⁴, or R^{21 B} is hydrogen and R^21A is hydrogen, hydroxy or -R^G-R³⁴;

R^22A is hydrogen and R^22B is -R^H-R³⁵, or R^22B is hydrogen and R^22A is -R^H-R³⁵;

R^F, R^G and R^H each independently is -0-, -C(O)-, -O-C(O)-, -C(0)-0-, -S-, -S(0)_n-, -O- S(0)_n— ,— S(0)_n— O— , -NH-S(0)_n-, -S(O) _n-NH-, -NH-C(O)-, -C(0)-NH-, -NH-C(S)-, -C(S)- NH-, -S-C(S)-, -C(S)-S-, or -C(S)-;

n is 1 or 2;

R²³ independently is:

(1) C3-C10 alkyl, C3-C10 heteroalkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl, which independently is optionally substituted by one or more substituents chosen from halogen, hydroxy, alkyl, alkenyl, alkynyl, heteroalkyl, haloalkyl, perhaloalkyl, perhaloalkoxy, alkoxy, haloalkoxy, alkoxyalkyl, acyl, oxo, acyloxy, carboxyl, amido, cyano, amino, alkylamino, alkylaminoalkyl, thiol, alkylthio, alkylthioalkyl, haloalkylthio, perhaloalkylthio, nitro, aryl, arylalkyl, cycloalkylalkyl, heterocycle-alkyl, cycloalkyl, heterocycloalkyl, heteroaryl, heteroarylalkyl, alkylsulfonyl, sulfonamide, alkylsulfonamido and alkylsilyloxy, with the proviso that if R²³ is aryl, R^F is not -O- or

(2) C3-C10 alkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl, which independently is optionally substituted by one or more substituents chosen from halogen, hydroxy, alkyl, alkenyl, alkynyl, heteroalkyl, haloalkyl, perhaloalkyl, perhaloalkoxy, alkoxy, haloalkoxy, alkoxyalkyl, acyl, oxo, acyloxy, carboxyl, amido, cyano, amino, alkylamino, alkylaminoalkyl, thiol, alkylthio, alkylthioalkyl, haloalkylthio, perhaloalkylthio, nitro, aryl, arylalkyl, cycloalkylalkyl, heterocycle- alkyl, cycloalkyl, heterocycloalkyl, heteroaryl, heteroarylalkyl, alkylsulfonyl, sulfonamide, alkylsulfonamido and alkylsilyloxy, with the proviso that if R²³ is aryl, R^F is not -0-; and

R³⁴ and R³⁵ each independently is a C2-C6 alkyl substituted with one or more hydroxy substituents.

[0009] In certain aspects, compounds provided herein have a structure of Formula B, or a pharmaceutically acceptable salt thereof, wherein:

R^20A is hydrogen and R^20B is -R²³, -R^F-R²³ or -R'-R³⁴, or R^20B is hydrogen and R^20A is -R²³,

- R^F-R²³ or -R'-R³⁴;

R^21A is hydrogen and R^{21 B} is hydrogen, hydroxy, -R^G-R²⁴ _, -R^G-R²⁴-R²⁵ or -R^G-R²⁴-R²⁶-R²⁵, or R^21B is hydrogen and R^21A is hydrogen, hydroxy, -R^G-R²⁴ -R^G-R²⁴-R²⁵ or -R^G-R²⁴-R²⁶-R²⁵; R^22A is hydrogen and R^22B is hydrogen, hydroxy, halogen, -N³, -NR²⁷R²⁸ -R^H-R²⁹, -R^H-R²⁹-R³⁰ or -R^H-R²⁹-R³¹-R³⁰, or R^22B is hydrogen and R^22A is hydrogen, hydroxy, halogen, -N³, -NR²⁷R²⁸ -

n is 1 or 2;

R' is -0-S(0)_n-, -S(0)_n-, -S(0)_n-0-, -S(O) _n-NH-, -NH-C(O)-, or -NH-S(O)-;

n is 1 or 2;

R²³ independently is a C5-C7 cycloalkyl or 5-7 membered heteroaryl containing 2 or more heteroatoms, which independently is optionally substituted by one or more substituents chosen from halogen, hydroxy, alkyl, alkenyl, alkynyl, heteroalkyl, haloalkyl, perhaloalkyl, perhaloalkoxy, alkoxy, haloalkoxy, alkoxyalkyl, acyl, oxo, acyloxy, carboxyl, amido, cyano, amino, alkylamino, alkylaminoalkyl, thiol, alkylthio, alkylthioalkyl, haloalkylthio, perhaloalkylthio, nitro, aryl, arylalkyl, cycloalkylalkyl, heterocycle-alkyl, cycloalkyl, heterocycloalkyl, heteroaryl, heteroarylalkyl, alkylsulfonyl, sulfonamide, alkylsulfonamido and alkylsilyloxy;

R²⁴ and R²⁹ each independently is:

(1) alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl, which independently is optionally substituted by one or more substituents chosen from halogen, hydroxy, alkyl, alkenyl, alkynyl, heteroalkyl, haloalkyl, perhaloalkyl, perhaloalkoxy, alkoxy, haloalkoxy, alkoxyalkyl, acyl, oxo, acyloxy, carboxyl, amido, cyano, amino, alkylamino, alkylaminoalkyl, thiol, alkylthio, alkylthioalkyl, haloalkylthio, perhaloalkylthio, nitro, aryl, arylalkyl, cycloalkylalkyl, heterocycle-alkyl, cycloalkyl, heterocycloalkyl, heteroaryl, heteroarylalkyl, alkylsulfonyl, sulfonamide, alkylsulfonamido and alkylsilyloxy

or

(2) cycloalkyl, heterocycloalkyl, aryl or heteroaryl, which independently is optionally substituted by one or more substituents chosen from halogen, hydroxy, alkyl, alkenyl, alkynyl, heteroalkyl, haloalkyl, perhaloalkyl, perhaloalkoxy, alkoxy, haloalkoxy, alkoxyalkyl, acyl, oxo, acyloxy, carboxyl, amido, cyano, amino, alkylamino, alkylaminoalkyl, thiol, alkylthio, alkylthioalkyl, haloalkylthio, perhaloalkylthio, nitro, aryl, arylalkyl, cycloalkylalkyl, heterocycle-alkyl, cycloalkyl, heterocycloalkyl, heteroaryl, heteroarylalkyl, alkylsulfonyl, sulfonamide, alkylsulfonamido and alkylsilyloxy;

alkylaminoalkyl, thiol, alkylthio, alkylthioalkyl, haloalkylthio, perhaloalkylthio and nitro;

R²⁷ and R²⁸ each independently is -R³²-R³³, or hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, perhaloalkyl, alkoxy, cycloalkyl, aryl, heterocycloalkyl, heterocycle-alkyl or heteroaryl, which independently is optionally substituted with one or more substituents chosen from halogen, hydroxy, alkyl, alkenyl, alkynyl, heteroalkyl, haloalkyl, perhaloalkyl, perhaloalkoxy, alkoxy, haloalkoxy, alkoxyalkyl, acyl, oxo, acyloxy, carboxyl, amido, cyano, amino, alkylamino, alkylaminoalkyl, thiol, alkylthio, alkylthioalkyl, haloalkylthio, perhaloalkylthio, nitro, aryl, arylalkyl, cycloalkylalkyl, heterocycle-alkyl, cycloalkyl, heterocycloalkyl, heteroaryl, heteroarylalkyl, alkylsulfonyl, sulfonamide, alkylsulfonamido and alkylsilyloxy; and

R³⁴ independently is alkyl, alkenyl, alkynyl, heteroalkyl or amino which independently is optionally substituted by one or more substituents chosen from halogen, hydroxy, alkyl, alkenyl, alkynyl, heteroalkyl, haloalkyl, perhaloalkyl, perhaloalkoxy, alkoxy, haloalkoxy, alkoxyalkyl, acyl, oxo, acyloxy, carboxyl, amido, cyano, amino, alkylamino, alkylaminoalkyl, thiol, alkylthio, alkylthioalkyl, haloalkylthio, perhaloalkylthio, nitro, aryl, arylalkyl, cycloalkylalkyl, heterocycle- alkyl, cycloalkyl, heterocycloalkyl, heteroaryl, heteroarylalkyl, alkylsulfonyl, sulfonamide, alkylsulfonamido and alkylsilyloxy, with the proviso that when R' is -NH-S(O)-, R³⁴ is not straight-chain alkyl.

[0010] In certain embodiments, a compound provided herein has the structure of Formula B as defined directly above, and R^20A, R^20B, R^21A, R^{21 B}, R^F, R^G, R^H, R', R²³, R²⁴, R²⁵, R²⁶, R²⁷, R²⁸, R²⁹, R³⁰, R³¹ , R³², R³³ and n as set forth above, except that R^22A is hydrogen and R^22B is hydrogen, hydroxy, halogen, N₃, -NR²⁷R²⁸ or -R^H-R²⁹-R³¹-R³⁰, or R^22B is hydrogen and R^22A is hydrogen, hydroxy, halogen, N₃, -NR²⁷R²⁸ or -R^H-R²⁹-R³¹-R³⁰.

[0011] In certain aspects, compounds provided herein have a structure of Formula B, or a pharmaceutically acceptable salt thereof, wherein:

R^20A is hydrogen and R^20B is -R²³ or -R^F-R²³, or R^20B is hydrogen and R^20A is -R²³ or -R^F-R²³;

R^21A is hydrogen and R^{21 B} is hydroxy, -R^G-R²⁴ -R^G-R²⁴-R²⁵ or -R^G-R²⁴-R²⁶-R²⁵, or R^{21 B} is hydrogen and R^21A is hydroxy, -R^G-R²⁴ -R^G-R²⁴-R²⁵ or -R^G-R²⁴-R²⁶-R²⁵; R^22A is hydrogen and R^22B is halogen, -N₃, -NR²⁷R²⁸, -R^H-R²⁹, -R^H-R²⁹-R³⁰ or -R^H-R²⁹-R³¹-R³⁰, or R^22B is hydrogen and R^22A is halogen, -N₃, -NR²⁷R²⁸, -R^H-R²⁹, -R^H-R²⁹-R³⁰ or -R^H-R²⁹-R³¹-

R³⁰;

n is 1 or 2;

R²³ independently is cycloalkyl, aryl, heterocycloalkyl or heteroaryl, which independently is optionally substituted by one or more substituents chosen from halogen, hydroxy, alkyl, alkenyl, alkynyl, heteroalkyl, haloalkyl, perhaloalkyl, perhaloalkoxy, alkoxy, haloalkoxy, alkoxyalkyl, acyl, oxo, acyloxy, carboxyl, amido, cyano, amino, alkylamino, alkylaminoalkyl, thiol, alkylthio, alkylthioalkyl, haloalkylthio, perhaloalkylthio, nitro, aryl, arylalkyl, cycloalkylalkyl, heterocycle- alkyl, cycloalkyl, heterocycloalkyl, heteroaryl, heteroarylalkyl, alkylsulfonyl, sulfonamide, alkylsulfonamido, alkylsilyloxy, alkylsulfinamido, arylsulfinamido and arylsulfonamido;

R²⁴ and R²⁹ each independently is cycloalkyl, heterocycloalkyl, aryl or heteroaryl, which independently is optionally substituted by one or more substituents chosen from halogen, hydroxy, alkyl, alkenyl, alkynyl, heteroalkyl, haloalkyl, perhaloalkyl, perhaloalkoxy, alkoxy, haloalkoxy, alkoxyalkyl, acyl, oxo, acyloxy, carboxyl, amido, cyano, amino, alkylamino, alkylaminoalkyl, thiol, alkylthio, alkylthioalkyl, haloalkylthio, perhaloalkylthio, nitro, aryl, arylalkyl, cycloalkylalkyl, heterocycle-alkyl, cycloalkyl, heterocycloalkyl, heteroaryl, heteroarylalkyl, alkylsulfonyl, sulfonamide, alkylsulfonamido and alkylsilyloxy;

R²⁶, R³¹ and R³² each independently is an alkyl linker or heteroalkyl linker, which independently is optionally substituted with one or more substituents chosen from hydroxy, halogen, acyl, oxo, acyloxy, carboxyl, amido, cyano, amino, alkylamino, alkylaminoalkyl, thiol, alkylthio,

alkylthioalkyl, haloalkylthio, perhaloalkylthio and nitro; and

R²⁷ and R²⁸ each independently is -R³²-R³³, or hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, perhaloalkyl, alkoxy, cycloalkyl, aryl, cycloalkyl, heterocycloalkyl, heterocycle-alkyl or heteroaryl, which independently is optionally substituted with one or more substituents chosen from halogen, hydroxy, alkyl, alkenyl, alkynyl, heteroalkyl, haloalkyl, perhaloalkyl, perhaloalkoxy, alkoxy, haloalkoxy, alkoxyalkyl, acyl, oxo, acyloxy, carboxyl, amido, cyano, amino, alkylamino, alkylaminoalkyl, thiol, alkylthio, alkylthioalkyl, haloalkylthio, perhaloalkylthio, nitro, aryl, arylalkyl, cycloalkylalkyl, heterocycle-alkyl, cycloalkyl, heterocycloalkyl, heteroaryl, heteroarylalkyl, alkylsulfonyl, sulfonamide, alkylsulfonamido, alkylsilyloxy, alkylsulfinamido, arylsulfinamido, and arylsulfonamido.

[0012] Also provided, in some aspects, are compositions containing a compound having a structure of a formula provided herein, which optionally include a pharmaceutically acceptable carrier or diluent. Pharmaceutical compositions containing a compound provided herein are also provided.

[0013] Also provided, in certain aspects, are methods for using compounds having a structure of a formula provided herein, or compositions containing such compounds. In some aspects, provided are methods for administering a compound having a structure of a formula provided herein, or a pharmaceutically acceptable salt thereof, or a composition comprising such a compound, to an in vitro, in vivo or ex vivo system. In certain aspects, provided are methods for administering a compound having a structure of a formula provided herein, or a

pharmaceutically acceptable salt thereof, or a composition comprising such a compound, to a cell, tissue and/or subject. Methods provided herein include, but are not limited to, methods of approximating or multimerizing two or more peptides or polypeptides within a cell and methods of activating or inhibiting the growth of a cell containing an FKBP (or portion thereof) protein fusion and an FRB (or portion thereof) fusion protein by contacting the cell with a compound provided herein.

[0014] Also provided, in certain aspects, are methods of treating, preventing and/or delaying the onset of a disease, disorder or condition using a compound provided herein. Also provided herein are methods of regulating a treatment (e.g., a cell-based treatment) administered for therapy, prevention, and/or delaying the onset of a disease, disorder or condition using a compound provided herein alone or in combination with one or more other compounds.

[0015] Certain embodiments are described further in the following description, examples, claims and drawings.

Incorporation by Reference [0016] All publications, patents and patent applications, GENBANK sequences (e.g., available at the World Wide Web Uniform Resource Locator (URL) ncbi.nlm.nih.gov of the National Center for Biotechnology Information (NCBI)), sequences available through other databases, and websites and other published materials referred to throughout the entire disclosure herein, unless noted otherwise, are incorporated by reference in their entirety. Citation of any publications, patents and patent applications, GENBANK (and other database) sequences, websites and other published materials herein is not an admission that any of the foregoing is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents. Their citation is not an indication of a search for relevant disclosures. All statements regarding the date(s) or contents of the documents is based on available information and is not an admission as to their accuracy or correctness.

Brief Description of the Drawings

[0017] The drawings illustrate certain embodiments of the technology and are not limiting. For clarity and ease of illustration, the drawings are not made to scale and, in some instances, various aspects may be shown exaggerated or enlarged to facilitate an understanding of particular embodiments.

[0018] FIG. 1 depicts the chemical structure of rapamycin.

[0019] FIG. 2 illustrates one version of the possible combinations of multiple components that can be used to create a dual switch activation/elimination system that can be implemented in a CAR T cell. In the exemplary system depicted in FIG. 2, a co-stimulatory T cell activation component is shown in the diagram of a cell on the left side of the figure. The cell membrane is depicted as two parallel dotted lines in the figure. The first activation signal is provided through the chimeric antigen receptor, which includes an extracellular, antibody-derived single chain variable fragment (scFv), that specifically recognizes a target tumor cell antigen and which is fused to a Q-bend 10 (Q) epitope derived from CD34 (for use in assessing transduction efficiency) which is fused, through a transmembrane domain, to Oϋ3z. The second activation signal, which is in the form of an“on” switch,” can be induced by administration of a rapalog.

This switch is formed from fusion proteins containing the KLW mutant of FRB (i.e.,

FRB(leu2098) or FRB_L), FKBP12, a portion of MyD88 (designated“M”) and a portion of CD40 (designated“C”) and is referred to as iRMC. The rapalog contains an FKBP12-binding domain (depicted as a circle in the figure) and an FRB_L-binding domain (depicted as an arrowhead) and binds to the FKBP12 portion of one fusion protein and the FRBL portion of another fusion protein forming heteromultimers of the fusion proteins and activating the MyD88 and CD40 proteins in the process. The right side of FIG. 2 shows an inducible cell death component of an exemplary cell activation/elimination system. This component includes a fusion protein (designated iC9) of the FKBP12 variant FKBP12v36 (designated as FKBPv) and a portion of caspase 9 (designated AC9). This fusion protein serves as an“off’ safety switch which can be induced by

administration of, for example, rimiducid, analog thereof, or other compound that binds to and multimerizes FKBP12v36. Rimiducid contains two FKBP12v36-binding domains (depicted as circles on opposite sides of a dumbbell) that bind to two different iC9 fusion proteins forming a homodimer and activating the caspase9 protein in the process. When both the activation and elimination components (dual switches), which can be induced by distinct inducing agents, are included in a single CAR T cell, the cell is referred to as DS-CAR-T.

[0020] FIG. 3 is a graphic representation of the results of transcriptional switch assays of rapamycin and 7(S)-dimethoxyphenol-rapamycin (CMP001) using transfected cells expressing a mutant or wild type FRB-HSV VP16 fusion protein, a GAL4 DNA binding domain-FKPB3) fusion protein and a GAL4 DNA recognition site-SeAP fusion protein. The graph is a plot of secreted alkaline phosphatase (SeAP) activity (in arbitrary units) vs. concentration (nM) of either rapamycin or CM P001. The plots of circle symbols and triangle symbols are those of the results of assays using cells expressing a wild type (KTW) FRB-HSV VP16 fusion protein treated with increasing concentrations of rapamycin and CMP001 , respectively. The plots of square symbols and inverted triangle symbols are those of the results of assays using cells expressing a mutant (TLW) FRB-HSV VP16 fusion protein treated with increasing concentrations of rapamycin and CMP001 , respectively.

[0021] FIG. 4 is a graphic representation of the results of transcriptional switch assays of compounds using transfected cells expressing a wild type FRB-HSV VP16 fusion protein, a GAL4 DNA binding domain-FKPB12(3) fusion protein and a GAL4 DNA recognition site-SeAP fusion protein. The graphs are plots of SeAP activity (in arbitrary units) vs. concentration (nM) of rapamycin, CMP001 or a compound as provided herein. The structure of each compound is shown next to the graph for the results of assays in which the compound was tested. The compounds shown on the left side of the figure (from top to bottom of the figure) are CMP001 , CMP013 and CMP015. The compounds shown on the right side of the figure (from top to bottom of the figure) are CMP01 1 , CMP014 and CMP012.

[0022] FIG. 5 shows a western blot of human PBMC lysates using antibodies against human S6 protein (pS6) phosphorylated at serine residues 240 and 244, unphosphorylated human S6 protein (S6), human 4E-BP1 protein and human vinculin. The lanes contain (from the left): molecular weight markers (lane 1) and lysates of PMBCs that had been activated with anti-CD3 and anti-CD28 antibodies and treated with no compound (lane 2), 1 nM and 10 nM rapamycin (RAP; lanes 3 and 4, respectively), and 1 nM, 5 nM, 10 nM, 50 nM, 100 nM, 500 nM and 1000 nM 7(S)-dimethoxyphenol-rapamycin (CMP001 ; lanes 5-1 1 , respectively).

[0023] FIG. 6 is a map of retroviral vector pM006 which can be used to generate retrovirus for transducing cells to develop CAR cells expressing a dual switch system. pM006 contains nucleic acid encoding the following elements in a dual switch system in the 5’ to 3’ direction:

(1) a fusion protein containing a KLW mutant of human FRB having a Thr2098Leu substitution (FRB_L) fused, through a 2-amino acid linker peptide, to a wild type human FKBP12 polypeptide fused to a truncated human MyD88 polypeptide (containing a death domain and intermediary domain but lacking a TIR domain) fused, through a 2-amino acid linker peptide, to a portion of a human CD40 polypeptide labeled as CD40IC (the entire fusion protein is termed MC-Rap or iRMC),

(2) a 3-amino acid linker peptide,

(3) a P2A self-cleaving peptide,

(4) a 5-amino acid linker peptide,

(5) a mutant human FKBP12 protein (FKBP12(F36V) also known as FKBP12v36, Fv36, FKBPv, or F_v) in which the phenylalanine at amino acid position 36 (or 37 if the initial methionine of the protein is counted) is substituted by a valine which is fused, through an 8-amino acid linker to a portion of human caspase 9 polypeptide (Acaspase9) (the entire fusion protein is termed iC9),

(6) a 5-amimo acid linker,

(7) a T2A self-cleaving peptide,

(8) a 4-amino acid linker,

(9) a membrane signal peptide fused to light (4d5 vl) and heavy (4d5 vh) chain variable regions of anti-HER2 monoclonal antibody 4D5 (with an intervening 15-amino acid flexible glycine-serine linker, i.e., flex peptide, between the chains) fused, through a 2-amino acid linker, to a human CD34 epitope peptide which is fused to an alpha stalk region of human CD8 (CD8 stalk) which is fused to the transmembrane domain of human CD8 (CD8 atm) which is fused to a portion of human Oϋ3z.

[0024] FIG. 7 is a map of retroviral vector pM007 which can be used to generate retrovirus for transducing cells to develop CAR cells expressing an inducible cell activation system or for developing CAR cells expressing a dual switch cell activation/cell elimination system when the cells are also transduced with retrovirus generated using a vector such as, for example, pM006 shown in FIG. 6 and/or pM008 shown in FIG. 10. pM007 contains nucleic acid encoding the following elements in a cell activation switch system in the 5’ to 3’ direction: (1) a fusion protein containing a KLW mutant of human FRB having a Thr2098Leu substitution (FRB_L) fused, through an 8-amino acid linker peptide, to a wild type human FKBP12 polypeptide fused, through a 5-amino acid linker peptide, to a truncated human MyD88 polypeptide fused, through a 2-amino acid linker peptide, to a portion of a human CD40 polypeptide (the entire fusion protein is termed MC-Rap or iRMC and is labeled as“MC” in the figure),

(2) a 6-amino acid linker peptide,

(3) a P2A self-cleaving peptide,

(4) a 2-amino acid linker peptide,

(5) a membrane signal peptide fused to light (4d5 vl) and heavy (4d5 vh) chain variable regions of anti-HER2 monoclonal antibody 4D5 (with an intervening 15-amino acid flexible glycine-serine linker, i.e., flex peptide between the chains) fused, through a 2-amino acid linker, to a human CD34 epitope peptide which is fused to an alpha stalk region of human CD8 (CD8 stalk) which is fused to the transmembrane domain of human CD8 (CD8 atm) which is fused to a portion of human Oϋ3z.

[0025] FIG. 8 is a map of retroviral vector pM009 which can be used to generate retrovirus for transducing cells to develop CAR cells expressing a dual switch system. pM009 contains nucleic acid encoding the following elements in a dual switch system in the 5’ to 3’ direction:

(1) a fusion protein containing a KLW mutant of human FRB having a Thr2098Leu substitution (FRB_L) fused, through a 2-amino acid linker peptide, to a wild type human FKBP12 polypeptide fused to a portion of the human MyD88 polypeptide (containing a death domain and intermediary domain but lacking a TIR domain) fused, through a 2-amino acid linker peptide, to a portion of the human CD40 polypeptide, referred to as CD40IC, fused to a wild type human FKBP12 polypeptide fused to FRB_L (the entire fusion protein is termed MC-Rap or iRMC),

(2) a 3-amino acid linker peptide,

(3) a P2A self-cleaving peptide,

(4) a 5-amino acid linker peptide,

(5) a mutant human FKBP12 protein (FKBP12(F36V) also known as FKBP12v36, Fv36, FKBPv, or F_v) in which the phenylalanine at amino acid position 36 (or 37 if the initial methionine of the protein is counted) is substituted by a valine which is fused, through an 8-amino acid linker, to a portion of human caspase 9 polypeptide (Acaspase9) (the entire fusion protein is termed iC9),

(6) a 5-amimo acid linker,

(7) a T2A self-cleaving peptide,

(8) a 4-amino acid linker, (9) a membrane signal peptide fused to a heavy variable region (FRP5 VH) and light chain variable region (FRP5 VL) of anti-HER2 monoclonal antibody FRP5 (with an intervening 15-amino acid flexible glycine-serine linker, i.e., flex peptide, between the chains) fused, through a 2-amino acid linker, to a human CD34 epitope peptide which is fused to an alpha stalk region of human CD8 (CD8 stalk) which is fused to the transmembrane domain of human CD8 (CD8 atm) which is fused to a portion of human Oϋ3z polypeptide.

[0026] FIG. 9 (A) and (B) show the results of fluorescence assays of cocultured transduced PMBCs (i.e., T cells) expressing a red fluorescent protein and OE19 tumor cells expressing a green fluorescent protein over time using an IncuCyte cell imaging system (coculture effectortarget cell ratios of 1 :15). Results shown in (A) and (B) are from assays of cocultures of OE19 cells and one of the following T cell lines: control T cells (circles), CAR T cells, which were transduced with nucleic acid encoding a chimeric antigen receptor fusion of an anti-HER2 scFv with Oϋ3z (triangles), CAR T cells in the presence of CMP001 (blue diamonds), CAR T cells (designated DS-CAR-T) that were also transduced with nucleic acid encoding an FRBP12- FRBL-MyD88-CD40 fusion protein (designated iMC) and nucleic acid encoding an FKBP12V- Acaspase 9 fusion (designated iC9) (green diamonds) and DS-CAR-T cells in the presence of CMP001 (inverted triangles). Results shown in (C) and (D) are cytokine levels (IFN-g in (C) and IL-2 in (D)) of coculture (effectortarget cell ratios of 1 :5) supernatants as measured by ELISA assay. The results are shown for cultures in the absence (“no drug”) or presence of 5nM CMP001.

[0027] FIG. 10 is a map of retroviral vector pM008 which can be used to generate retrovirus for transducing cells to develop CAR cells expressing an inducible cell elimination system or for developing CAR cells expressing a dual switch cell activation/cell elimination system when the cells are also transduced with retrovirus generated using a vector such as, for example, pM006 shown in FIG. 6 or pM007 shown in FIG. 7. pM008 contains nucleic acid encoding the following elements in a cell elimination switch system in the 5’ to 3’ direction:

(1) a fusion protein containing an FKBP12v36 mutant of human FKBP12 having an F36 to V substitution fused, through a 6-amino acid linker peptide, to a portion of human caspase 9 polypeptide (Acaspase9) (the entire fusion protein is termed iC9),

(2) a T2A self-cleaving peptide to provide for separation of the fusion protein from the protein encoded by the DNA 3’ of the T2A-encoding DNA in the plasmid, and

(3) a non-signaling portion of human CD19 that serves as a surface marker.

[0028] FIG. 1 1 (left side) shows a schematic depiction of an example of a timeline for the development a mouse xenograft model. This animal model can be used to evaluate the efficacy of multimerizing compounds in affecting in vivo proliferation of immune cells transduced with nucleic acid encoding an FRBP12-FRBL-MyD88-CD40 fusion protein (iRMC) and an FKBP12V- Acaspase 9 fusion protein (iC9). These cells are designated as“iRMC-GoCAR-T +iC9” in the figure. The animal model can also be used to evaluate the efficacy of multimerizing compounds in affecting tumor cell inhibition in vivo. In this exemplary protocol, tumor cells (e.g., OE19 cells transduced with nucleic acid encoding firefly luciferase), designated OE19.GFP/uc, were implanted subcutaneously into immunodeficient (e.g., NSG) mice. Four days later, the mice received 5 x10⁶ transduced T cells (designated CAR T in FIG. 1 1) via intravenous infusion. Beginning the day after infusion with transduced T cells, compound, e.g., CMP001 (i.e.,“Go” drug), was administered to the mice intraperitoneally and again weekly thereafter at 1 mg/kg body weight. The mice were monitored for at least 55 days post T cell infusion for transduced T cell and OE19 cell proliferation and serum cytokine levels.

[0029] The right side of FIG. 11 shows a photograph of Renilla luciferase-derived

bioluminescence imaging of mice that had been engrafted with OE19 tumor cells (transduced with nucleic acid encoding GFP FFluc) and infused with CAR T cells (transduced with retrovirus containing plasmids encoding iRMC and iC9 proteins and ONL Rluc Renilla luciferase), which are designated as“iRMC-GoCAR-T + iC9” in the figure. One group of these mice had been administered CMP001 and another group of these mice had been administered a vehicle lacking CMP001. Luciferase-derived bioluminescence Imaging of control mice is also shown in the figure. The control mice (designated“iC9: in the figure) had been engrafted with OE19 tumor cells (transduced with nucleic acid encoding GFP FFluc) and then infused with T cells transduced with retrovirus containing nucleic acid encoding the iC9 protein and ONL Rluc, but lacking nucleic acid encoding iRMC and a chimeric antigen receptor. Control mice had also been administered a vehicle instead of CMP001 on the day after T cell infusion and weekly thereafter. All of the mice imaged were injected with coelenterazine (Renilla luciferase substrate) by an intraperitoneal (i.p.) route in the lower abdomen prior to imaging. The symbol† in place of a photo of an imaged mouse indicates that the mouse expired by the specified time point after T cell infusion.

[0030] FIG. 12 (left side) shows a photograph of firefly luciferase-derived bioluminescence imaging of mice that had been engrafted with OE19 tumor cells (transduced with nucleic acid encoding GFP FFluc) and infused with CAR T cells (transduced with retrovirus containing plasmids encoding iRMC and iC9 proteins and ONL Rluc Renilla luciferase), which are designated as“iRMC-GoCAR-T + iC9” in the figure. One group of these mice had been administered CMP001 and another group of these mice had been administered a vehicle lacking CMP001. Firefly luciferase-derived bioluminescence imaging of control mice is also shown in the figure. The control mice (designated“iC9: in the figure) had been engrafted with OE19 tumor cells (transduced with nucleic acid encoding GFP FFluc) and then infused with T cells transduced with retrovirus containing nucleic acid encoding the iC9 protein and ONL Rluc, but lacking nucleic acid encoding iRMC and a chimeric antigen receptor. Control mice had also been administered a vehicle instead of CMP001 on the day after T cell infusion and weekly thereafter. All of the mice imaged were injected with luciferin (firefly luciferase substrate) by an intraperitoneal (i.p.) route in the lower abdomen prior to imaging. The symbol† in place of a photo of an imaged mouse indicates that the mouse expired by the specified time point after T cell infusion.

[0031] The right side of FIG. 12 shows the results of Kaplan-Meier analysis from the in vivo assay of the control and iRMC-GoCAR-T + iC9 mice that were imaged as shown in FIG. 1 1 and FIG. 12.

[0032] FIG. 13 provides a graphic depiction of the relative levels of 29 cytokines in serum collected from the xenograft mice that were studied by imaging as presented in FIGs. 11 and 12. Serum was collected from control mice and mice infused with T cells coexpressing anti- HER2/003z CAR, iRMC and iC9 fusion proteins (“iRMC-GoCAR-T + iC9” administered CMP001 or vehicle) on the ninth day following cell infusion. Cytokine levels were assessed in the serum samples using a multiplex assay system (LUMINEX). The color shading bar to the right of the results correlates with a relative level of cytokine ranging from -1/0 (dark blue/dark violet) to +2/+3 (deep pink/lighter pink).

[0033] FIG. 14 is a map of plasmid pM010, which can be utilized to stably label cells (e.g., PBMCs) with nuclear-localized red fluorescent protein (RFP) protein, and which can be utilized to evaluate cell proliferation over time. Plasmid pM010 contains the following polynucleotides in the 5’ to 3’ direction: polynucleotide encoding an SP163 translation enhancer, a polynucleotide encoding a linker polypeptide, a polynucleotide encoding RFP, a polynucleotide encoding a linker peptide, and a polynucleotide encoding three nuclear localization sequences fused in succession. Detailed

Rapamycin and rapalogs

Structural, functional and binding properties of rapamycin

[0034] Rapamycin, also known as sirolimus, is a macrolide lactone natural product having the following structure:

[0035] Rapamycin was initially identified as a chemical compound produced by the bacterium Streptomyces hygroscopicus. It is a chiral compound and has 15 defined stereocenters in its molecular structure. There are several different schemes for numbering of the carbon atoms in the structure of rapamycin. For example, the carbon atoms designated as numbers 7, 29 and 40 in the structure depicted above (and in FIG. 1), are also referred to in the art as carbon numbers 16, 28 and 43, respectively. All references herein to a carbon atom number in rapamycin, or analogs or derivatives thereof, are based on the numbering shown in the structure of rapamycin provided in FIG. 1 , unless specifically stated otherwise. In solutions of differing solvents, rapamycin exists as a mixture of conformational isomers which can be distinguished by NMR spectroscopy. Typically, the B (or b) isomer is the main component of a rapamycin solution with about 3-10% being the C (or y) isomer and less than 0.5% being the A (or a) isomer (see, e.g., Sobhani et al. (2013) Iran J Pharm Res 12 (Suppl):77-81). Rapamycin has very low solubility in water.

[0036] Rapamycin binds with high affinity (K_D < 1 nM) to FKBP12. The FKBP12-binding surface of rapamycin (also referred to as the binding domain of rapamycin) is localized to the portion of the molecule extending from about carbon atoms 9 to 21 that includes the piperidinyl moiety (see, e.g., Branazynski et al. (2005) J Am Chem Soc 127:4715-4721). Human FKBP12 is a 108-amino acid (SEQ ID NO: 85 encoded by SEQ ID NO: 3) 12-kDa intracellular enzyme that is a cytoplasmic receptor for the immunosuppressive drug FK506 (also known as tacrolimus). Binding of FK506 to FKBP12 and calcineurin inhibits the phosphatase activity of calcineurin, which is involved in T cell activation (see, e.g., Bierer et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87:9231-9235 and Liu et al. (1991) Cell 66:807-815). FKBP12 also binds to calcium release channels (see, e.g., Brilliantes et al. (1994) Cell 77:513-523) and interacts with the TGF- b type I receptor to inhibit receptor-mediated signal transduction (see, e.g., Wang et al. (1996) Cell 86:435-444). FKBP12 is not only expressed in immune tissues, but also in other cells, including abundant expression in nervous tissue and the brain.

[0037] The binding of rapamycin to FKBP12 forms a complex that initiates a high-affinity, inhibitory interaction with the FKBP-Rapamycin-Binding (FRB) domain of mTOR (mammalian target of rapamycin; see, e.g., Sabatini et al. (1994) Cell 78:35-43). Thus, rapamycin can function as a protein dimerizer that binds with affinity to FKBP12 and then, as a rapamycin- FKBP12 complex, binds with nanomolar affinity to mTOR (see, e.g., Brown et al. (1994) Nature 369:756-758; Kunz et al. (1993) Cell 73:585-596). The FRB-binding surface of rapamycin (also referred to as the effector domain of rapamycin) is localized to the portion of the molecule around carbon atoms 29-33 and 1-7 (see, e.g., Branazynski et al. (2005) J Am Chem Soc 127:4715-4721). Rapamycin binds in a cleft between two helices of FRB, which is an ~89- amino acid 4-helix bundle (see, e.g., Choi et al. (1996) Science 273:239). The minimal FRB domain (SEQ ID NO: 77 encoded by SEQ ID NO: 1) is an ~1 1-kDa protein that includes amino acids 2025-21 14 of the human mTOR protein (amino acid SEQ ID NO: 76; nucleotide SEQ ID NO: 15) and has a rapamycin dissociation constant (Kd) of about 4 nM.

[0038] mTOR (also known as FRAP, RAFT, RAPT1 and SEP) is a 289-kDa cytoplasmic phosphatidylinositol-3-kinase (PI3K)-related kinase (EC 2.7.1 1.1) and is the catalytic component of two multicomponent complexes (mTORCI and mTORC2). The portion of mTOR bound by FKBP12-rapamycin is a four-helical bundle centered between amino acids 2025 and 21 14 of the FRB parent molecule (see, e.g., Chen et al. (1995) Proc Natl Acad Sci U.S.A. 92:4947-4951). mTOR is a part of an intracellular signaling pathway involved in cell cycle regulation including cell metabolism, growth, proliferation, autophagy and survival (see, e.g., Saxton and Sabatini (2017) Cell 168:960-976 and Saxton and Sabatini (2017) Cell 169:361-371). mTOR has a controlling role in the pathway, which it exerts through a phosphotransfer process using ATP as a phosphate donor (see, e.g., Guertin and Sabatini (2007) Cancer Cell 12:9-22). Substrates for mTOR include S6KI and 4E-BP1 which are regulators of mRNA translation. The elF4E complex serves as a scaffold that facilitates mTORCI-dependent phosphorylation of S6KI and 4E-BP1. The binding of the rapamycin-FKBP12 complex to the FRB domain of mTOR allosterically inhibits the mTOR catalytic site and thus inhibits S6KI activation. [0039] As used herein, the term“rapalog” refers to an analog of the macrolide rapamycin. Rapalogs are molecules that generally are structurally similar to rapamycin but are variants of rapamycin in that they are not identical to rapamycin. For example, a rapalog may differ from rapamycin in one or more atoms and/or functional groups. Rapalogs may have functional, physical, binding, pharmacological, pharmacokinetic and/or other properties that differ from those of rapamycin. In some instances, a rapalog can be a prodrug. In some instances, a rapalog can be metabolized in vivo to yield rapamycin or another rapalog. In some instances, a rapalog is not a prodrug. In some instances, a rapalog does not yield rapamycin or another rapalog, including, but not limited to, demethoxy-o,p-dimethoxyphenylrapamycin, under in vivo conditions or after being administered to a subject.

[0040] Compounds provided herein include rapalogs. Some embodiments of rapalogs provided herein have certain properties such as, for example, enhanced solubility, stability in serum and/or modified (e.g., decreased or increased) affinity for binding to wild type or variant FRB and/or FKBP12 as compared to rapamycin. For commercial purposes, in some embodiments, compounds provided herein have useful scaling and production properties.

Uses of rapamycin and rapalogs

Rapamycin and rapalog use as therapeutic agents

[0041] The cellular effects of rapamycin make it useful in treating a number of conditions. Rapamycin has antifungal properties (see, e.g., Bastidas et al. (2012) Eukaryot Cell 1 1 (3):270- 281) and has been investigated as a treatment for microbial infections. Rapamycin also inhibits cytokine- and mitogen-induced T-cell and B-cell proliferation and reduces immunoglobulin synthesis (see, e.g., Sehgal (1998) Clin Biochem 31 :335-340; Kay et al. (1991) Immunol 72:544-549; Kim et al. (1994) Clin Exp Immunol 96:508-512) and has been studied for use as an immunosuppressive agent in organ transplantation to reduce rejection and renal failure (see, e.g., Shitrit et al (2004) Respiratory Med 98:892-897). Antiproliferative and antiangiogenesis effects of rapamycin (see, e.g., Aissat et al. (2008) Cancer Chemother Pharmacol 62:305-313; Guba et al. (2002) Nat Med 8(2): 128-135) are properties that indicate its use in cancer therapies (see, e.g., Dai et al. (2013) Int J Mol Sci 14(1):273-285) and proliferative dysregulation disorders, such as, for example, lymphangiomyomatosis, angiolipomas, neurofibromatosis, Cowden’s syndrome and tuberous sclerosis (see, e.g., Bissler et al. (2008) N Engl J Med 358:140-151 ; McCormack et al. (201 1) A/ Eng/ J ec/ 364: 1595- 1606). Additionally, improper regulation and increased activation of the mTORCI pathway has been observed in cancers associated with oncogene and tumor suppressor mutations (see, e.g., Li et al. (2014) Cell Metab 19(3):373-379), and rapamycin and analogs thereof (rapalogs) have been investigated in the treatment of such cancers (see, e.g., Cloughesy et al. (2008) PLOS Med 5(1):

e8.doi: 10.1371/journal. pmed.0050008 and Meric-Bernstam and Gonzalez-Angulo (2009) J Clin Oncol 27(13):2278-2287). Therapeutic use of rapamycin, for example as anti-growth immunosuppressants and anti-tumor agents, is facilitated by its cell permeability, in vivo stability and high target affinity and specificity.

[0042] The term“cancer” as used herein is defined as a hyperproliferation of cells whose unique trait— loss of normal controls— can result in unregulated growth, lack of differentiation, local tissue invasion, and metastasis. Examples include but are not limited to, melanoma, nonsmall cell lung, small-cell lung, lung, hepatocarcinoma, leukemia, retinoblastoma, astrocytoma, glioblastoma, gum, tongue, neuroblastoma, head, neck, breast, pancreatic, prostate, renal, bone, testicular, ovarian, mesothelioma, cervical, gastrointestinal, lymphoma, brain, colon, sarcoma or bladder.

[0043] Rapamycin, which can act in cells to induce heterodimerization of FKBP12 and the FRB domain of mTOR, can also be used as an agent in the chemical induction of dimerization (CID). CID can be employed as a biological tool to spatially manipulate specific molecules, e.g., peptides and polypeptides, within cells at precise times to control a particular activity. Uses of CID include experimental investigations to elucidate cellular systems and therapeutic uses to regulate cell-based therapies. U.S. Patent Application number no. 15/377,776 (publication no. US 2017/0166877 entitled“Dual Controls for Therapeutic Cell Activation or Elimination”) describes methods for orthogonal control of the activation and elimination of therapeutic cells using molecular switches that employ distinct multimeric ligands, in conjunction with binding partner fusion proteins that can affect intracellular signaling pathways. Exemplary uses of the technology include activation or elimination of cells used to promote engraftment, to treat diseases or conditions, or to control or modulate the activity of therapeutic cells that express chimeric antigen receptors or recombinant T cell receptors.

[0044] A CID system generally involves aggregation of surface receptors and other cell surface proteins, or non-surface cytosolic proteins, to effectively activate downstream signaling cascades. A CID system typically makes use of a synthetic bivalent ligand to rapidly crosslink signaling molecules that are fused to ligand binding domains. This system has been used, for example, to trigger the oligomerization and activation of cell surface proteins (see, e.g., Spencer et al. (1993) Science 262:1019-1024; Spencer et al. (1996) Curr Biol 6:839-847 ; Blau et al. (1997) Proc Natl Acad Sci USA 94:3076-3081), or cytosolic proteins (see, e.g., Luo et al. (1996) Nature 383:181-185; MacCorkle et al. (1998) Proc Natl Acad Sci USA 95:3655-3660), the recruitment of transcription factors to DNA elements to modulate transcription (see, e.g., Ho et al. (1996) Nature 382:822-826; Rivera et al. (1996) Nat ecf 2: 1028-1032) and the recruitment of signaling molecules to the plasma membrane to stimulate signaling (see, e.g., Spencer et al. (1995) Proc Natl Acad Sci USA 92:9805-9809; Holsinger et al. (1995) Proc Natl Acad Sci USA 92:9810-9814). In addition, the synthetic ligands used in CID are resistant to protease degradation, making them more efficient at activating receptors in vivo than most delivered protein agents.

Use ofrapamycin and rapalogs in inducible FKBP12/FRB-based multimerization systems

FKBP12/FRB multimerization-based transcription induction, protein localization and protein stabilization

[0045] Coexpression of a fusion protein of FRB and a target protein of interest in cells with FKBP12, or a fusion protein of FKBP12 and another target protein, provides the elements for rapamycin- or rapalog-controlled approximation of the FRB fusion protein and FKBP12, or an FKBP12 fusion protein, with high affinity and specificity (see, e.g., Bayle et al. (2006) Chem Biol 13:99-107; Ho et al. (1996) Nature 382:822-826). Rapamycin- or rapalog-directed protein dimerization can thus be used in the development of inducible systems, or molecular switches, to control cell signaling. Strategies that can employ rapamycin- or rapalog-directed protein dimerization include, for example, transcription induction through recruitment of activating or repressing moieties to DNA-binding proteins in a drug-sensitive fashion (see, e.g., Bayle et al. (2006) Chem Biol 13:99-107), directing the localization or mislocalization of signaling proteins to or from their normal site of action (see, e.g., Klemm et al. (1997) Curr Biol 7:638-644; Liberies et al. (1997) Proc Natl Acad Sci U.S.A. 94:7825-7830), stabilization/destabilization of proteins (see e.g., Stankunas et al. (2003) Mol Cell 12:1615-1624), induction of apoptosis or programmed cell death and activation of growth-promoting signaling intermediates (see, e.g., U.S. Patent Application publication no. US 2017/0166877).

[0046] Rapamycin-controlled protein switches such as these are designed to effect specific outcomes based on dimerization of the target proteins. However, if rapamycin is used as the dimerizing agent in these systems, mTOR inhibition, and thus reduction in cell growth and proliferation and possible immunosuppression, may occur as a side-effect. One approach to reducing or eliminating mTOR inhibition in FKBP12/FRB-based CID systems is to use a multimerizing agent having reduced or no ability to bind endogenous (e.g., wild type) mTOR (i.e., FRB domain) in combination with a variant (or mutant) FRB protein to which the multimerizing agent specifically and sufficiently binds. Such a multimerizing agent would also retain the ability to bind FKBP12 for use in this exemplary system. Rapalogs are examples of multimerizing agents for potential use in this system.

[0047] The C7 position of rapamycin (also referred to as C16 in alternative numbering schemes), which is bound to a methoxy group, is located in the FRB-binding region of the compound. Some rapalogs that differ from rapamycin at the C7 position retain FKBP12-binding ability and have a reduced immunosuppressant activity relative to rapamycin (see, e.g., Luengo et al. (1995) Chem Biol 2:471-481) and have reduced or no ability to bind wild type FRB (see, e.g., Liberies et al. (1997) Proc Natl Acad Sci U.S.A. 94:7825-7830). Another rapalog (C20- methylallylrapamycin), which differs from rapamycin at the C20 position (also referred to as C3 in alternative numbering schemes), also retains FKBP12-binding ability with reduced or no ability to bind wild type FRB (see e.g., Stankunas et al. (2003) Mol Cell 12:1615-1624).

[0048] Certain variant FRB proteins are bound by some rapalogs. For example, a mutant human FRB referred to as“PLF” containing three amino acid substitutions, K2095P, T2098L and W2101 F (amino acid position numbering is based on the full human mTOR protein), is bound by some rapalogs. Such rapalogs include C16-(R)-OiPR (an analog in which the methyl group of the methoxy moiety bound to C16 (which is referred to as C7 in the rapamycin atom numbering system used herein) is substituted with an isopropyl group), C16-(R)- methylallylrapamycin (an analog in which the methoxy moiety bound to C16 (referred to as C7 herein) is substituted with a methallyl group) and C20-methylallylrapamycin (an analog in which the unsaturation of the C19-C20 bond (C20 is also referred to as C3 elsewhere) is removed and a methallyl group is added to C20).

[0049] The three amino acid substitutions in the PLF mutant of human FRB destabilize the protein which confers instability to proteins to which it may be fused. The PLF mutant is more susceptible to thermal denaturation in vitro than the wild type FRB (which is referred to as KTW relative to the PLF mutant) and is more readily degraded in vivo in cells. Dimerization of a PLF mutant, or PLF mutant fusion protein, through binding of FKBP-rapamycin (or FKBP-rapalog) stabilizes the protein. Similarly, a mutant human FRB that contains a single amino acid substitution (T2098L), referred to as KLW, reportedly is also unstable, although mutant human FRB proteins that have only one amino acid substitution at position 2095 (K to P) or 2101 (W to F) are generally as stable as wild type FRB. In the PLF mutant of human FRB, the T2098L substitution is primarily responsible for the instability of the mutant protein (see, e.g.,

Stankounas et al. (2007) ChemBioChem 8:1 162-1 169). Chimeric antigen receptor (CAR) T cells incorporating FKBP12/FRB- based multimerization systems to control cell activation and/or elimination

[0050] T cells (also referred to as T lymphocytes) belong to a group of white blood cells referred to as lymphocytes. Lymphocytes generally are involved in cell-mediated immunity.

The“T” in“T cells” refers to cells derived from or whose maturation is influenced by the thymus. T cells can be distinguished from other lymphocytes types such as B cells and Natural Killer (NK) cells by the presence of cell surface proteins known as T cell receptors. The term “activated T cells” as used herein, refers to T cells that have been stimulated to produce an immune response (e.g., clonal expansion of activated T cells) by recognition of an antigenic determinant, such as, for example, presented in the context of a Class II major histocompatibility (MHC) marker. T-cells are activated by the presence of an antigenic determinant, cytokines and/or lymphokines and cluster of differentiation cell surface proteins (e.g., CD3, CD4, CD8, the like and combinations thereof). Cells that express a cluster of differential protein often are said to be“positive” for expression of that protein on the surface of T-cells (e.g., cells positive for CD3 or CD 4 expression are referred to as CD3⁺ or CD4⁺). CD3 and CD4 proteins are cell surface receptors or co-receptors that may be directly and/or indirectly involved in signal transduction in T cells.

[0051] T cells express receptors on their surfaces (i.e., T cell receptors) that recognize antigens presented on the surface of cells. During a normal immune response, binding of these antigens to the T cell receptor, in the context of MHC antigen presentation, initiates intracellular changes leading to T cell activation. Chimeric antigen receptors (CARs) are artificial receptors designed to convey antigen specificity to T cells without the requirement for MHC antigen presentation. They include an antigen-specific component, a transmembrane component, and an intracellular component selected to activate the T cell and provide specific immunity.

Chimeric antigen receptor-expressing T cells may be used in various therapies, including cancer therapies. Co-stimulating polypeptides may be used to enhance the activation of CAR- expressing T cells against target antigens, and therefore increase the potency of adoptive immunotherapy. Inducible FKBP12/FRB-based multimerization systems can also be incorporated into chimeric antigen receptor (CAR) T cells which can be used, for example, in immunotherapy applications. Such CAR T cells incorporating an FKBP12/FRB-based multimerization system have a built-in control mechanism that can be regulated by

administration one or more compounds, e.g., a rapalog, that binds to FKBP12 and wild type or mutant FRB. [0052] By“chimeric antigen receptor” or“CAR” is meant, for example, a chimeric polypeptide which comprises a polypeptide sequence that recognizes a target antigen (an antigen- recognition domain) linked to a transmembrane polypeptide and intracellular domain polypeptide selected to activate the T cell and provide specific immunity. The antigen- recognition domain may be a single-chain variable fragment (scFv), or may, for example, be derived from other molecules such as, for example, a T cell receptor or Pattern Recognition Receptor. The intracellular domain comprises at least one polypeptide which causes activation of the T cell, such as, for example, but not limited to, CD3 zeta, and, for example, co-stimulatory molecules, for example, but not limited to, CD28, 0X40 and 4-1 BB. The term“chimeric antigen receptor” may also refer to chimeric receptors that are not derived from antibodies, but are chimeric T cell receptors. These chimeric T cell receptors may comprise a polypeptide sequence that recognizes a target antigen, where the recognition sequence may be, for example, but not limited to, the recognition sequence derived from a T cell receptor or an scFv. The intracellular domain polypeptides are those that act to activate the T cell. Chimeric T cell receptors are discussed in, for example, Gross, G., and Eshar, Z., FASEB Journal 6:3370-3378 (1992), and Zhang, Y„ et al., PLOS Pathogens 6:1- 13 (2010).

Multimerization-based control of CAR T cell activation

[0053] Immunotherapy strategies for treating cancer involve enlisting a patient’s immune system to attack and kill tumor cells. One type of immunotherapy is adoptive cell transfer in which a subject’s immune cells are collected and modified ex vivo to provide for specific and targeted tumor cell killing when the modified cells are returned to the body. A particular adoptive cell transfer method uses CAR-modified T cells and holds great promise for the treatment of a variety of malignancies. In this therapy, T cells are extracted from a patient’s blood and genetically engineered to express chimeric antigen receptors (CARs) on the cell surface. The components of a CAR typically include an extracellular, antibody-derived single chain variable fragment (scFv), which specifically recognizes a target tumor cell antigen, and one or multiple intracellular T cell-derived signaling sequences (e.g., ΰϋ3z; see SEQ ID NO:

133 for full-length amino acid sequence and SEQ ID NO: 62 for a nucleotide sequence encoding it) fused to the scFv. Binding of the scFv region to an antigen results in activation of the T cell through the signaling domains of the CAR. Cytolytic effector function in effector T cells is mediated by the release of pre-formed granzymes and perforin following tumor recognition, and activation through ΰϋ3z is sufficient to induce this process without the need for co-stimulation. [0054] While CARs were first designed with a single signaling domain, for example, Oϋ3z (see, e.g., Becker et al. (1989) Cell 58:91 1-921 ; Goverman et al. (1990) Cell 60:929-939; Gross et al. (1989) Proc Natl Acad Sci U.S.A. 86:10024-10028; Kuwana et al. (1987) Biochem Biophys Res Commun 149:960-968), clinical trials evaluating the feasibility of CAR immunotherapy showed limited clinical benefit (see, e.g., Till et al. (2012) Blood 1 19:3040-3050; Pule et al.

(2008) Nat Med 14:1264-1270; Jensen et al. (2010) Biol Blood Marrow Transplant 16:1245- 1256; Park et al. (2007) Mol Ther 15:825-833). This has been primarily attributed to the incomplete activation of T cells following tumor recognition, which leads to limited persistence and expansion of the cells in vivo (see, e.g., Ramos et al. (201 1) Expert Opin Biol Ther 1 1 :855- 873).

[0055] To address this deficiency, CARs have been engineered to include another stimulating domain, often derived from the cytoplasmic portion of T cell co-stimulating molecules, including CD28, 4-1 BB, 0X40, ICOS and DAP10 (see, e.g., Carpenito et al. (2009) Proc Natl Acad Sci U.S.A. 106:3360-3365; Finney et al. (1998) J Immunol 161 :2791-2797; Hombach et al. J Immunol 167:6123-6131 ; Maher et al. (2002) Nat Biotechnol 20:70-75; Imai et al. (2004) Leukemia 18:676-684; Wang et al. (2007) Hum Gene Ther 18:712-725; Zhao et al. (2009) J Immunol 183:5563-5574; Milone et al. (2009) Mol Ther 17:1453-1464; Yvon et al. (2009) Clin Cancer Res 15:5852-5860), which allow CAR T cells to receive appropriate co-stimulation upon engagement of the target antigen. Clinical trials conducted with anti-CD19 CARs having CD28 or 4-1 BB signaling domains for the treatment of refractory acute lymphoblastic leukemia (ALL) have demonstrated significant T cell persistence, expansion and serial tumor killing following adoptive transfer (Kalos et al. (201 1) Sci Trans I Med 3:95ra73; Porter et al. (201 1) N Engl J Med 365:725-733; Brentjens et al. (2013) Sci Trans! Med 5:177ra38).

[0056] CD28 co-stimulation provides a clear clinical advantage for the treatment of CD19⁺ lymphomas. In a CAR T cell clinical trial comparing first (CD19.Q and second generation CARs (CD19.28.Q, CD28-enhanced T cell persistence and expansion was reported following adoptive transfer (Savoldo et al. (201 1) J Clin Invest 121 :1822-1826). First generation CAR T cells (e.g., CARs constructed with only the ΰϋ3z cytoplasmic region) can lyse tumor cells; however, survival and proliferation are impaired due to lack of co-stimulation. Hence, the addition of CD28 or 4-1 BB co-stimulating domain constructs has significantly improved the survival and proliferative capacity of CAR T cells.

[0057] One of the principal functions of second generation CARs is the ability to produce IL-2 that supports T cell survival and growth through activation of the nuclear factor of activated T cells (NFAT) transcription factor by ΰϋ3z (signal 1) and NF-kB (signal 2) by CD28 or 4-1 BB.32. Other molecules that similarly activate NF-kB may also be paired with the Oϋ3z chain within a CAR molecule. One approach employs a T cell co-stimulating molecule that was originally developed as an adjuvant for a dendritic cell (DC) vaccine (Narayanan et al. (201 1) J Clin Invest 121 :1524-1534; Kemnade et al. (2012) Mol Ther 20(7): 1462-1471). For full activation or licensing of DCs, Toll-like receptor (TLR) signaling is usually involved. In TLR signaling, the cytoplasmic TLR/IL-1 domains (referred to as TIR domains) of TLRs dimerize which leads to recruitment and association of cytosolic adaptor proteins such as, for example, the myeloid differentiation primary response protein (MyD88; see SEQ ID NO: 101 for full length amino acid sequence and SEQ ID NO: 29 for a nucleotide sequence encoding it). MyD88 also contains a TIR domain through which it is able to heterodimerize with TLRs and homodimerize with other MyD88 proteins. This in turn results in recruitment and activation of IRAK family kinases through interaction of the death domains (DD) at the amino terminus of MyD88 and IRAK kinases which thereby initiates a signaling pathway that leads to activation of JNK, p38 MAPK (mitogen-activated protein kinase) and NF-kB, a transcription factor that induces expression of cytokine- and chemokine-encoding genes (as well as other genes). TLR signaling also upregulates expression of CD40 (see SEQ ID NO: 104 for full length amino acid sequence and SEQ ID NO: 32 for a nucleotide sequence encoding it), a member of the tumor necrosis factor receptor (TNFR) family, which interacts with CD40 ligand (CD154 or CD40L) on antigen-primed CD4⁺ T cells. The CD40/CD154 signaling system is an important component in T cell function and B cell— T cell interactions. CD40 signaling proceeds through formation of CD40

homodimers and interactions with TNFR-associated factors (TRAFs), carried out by recruitment of TRAFs to the cytoplasmic domain of CD40, which leads to T cell activation involving several secondary signals such as the NF-kB, JNK and AKT pathways.

[0058] One approach to co-stimulation of CAR T cells is to express a fusion protein (referred to as MC) of the signaling elements of MyD88 and CD40 (see, e.g., U.S. patent application publication no. 2016/0058857 entitled Costimulation of Chimeric Antigen Receptors by MyD88 and CD40 Proteins). Survival and growth of such cells can be enhanced through activation of the NFAT transcription factor by Oϋ3z, which is part of the chimeric antigen receptor (signal 1), and NF-kB (signal 2) by MyD88 and CD40. The activation of CAR T cells expressing MC is observed with a cytoplasmic MyD88/CD40 chimeric fusion protein, lacking a membrane targeting region, and with a chimeric fusion protein comprising MyD88/CD40 and a membrane targeting region, such as, for example, a myristoylation region.

[0059] An inducible MyD88/CD40 (iMC) switch has been used to synergistically activate dendritic cells for enhanced antitumor efficacy (see, e.g., Narayanan et al. (201 1) J Clin Invest 121 :1524-1534). These cells expressed a fusion protein (referred to as iMC) of the signaling elements of MyD88 and CD40 and one or more proteins that bind to a chemical inducer of dimerization. iMC is a potent, dimerizing drug-inducible, molecule that provides for

simultaneous activation of MyD88- and CD40-dependent signaling pathways in cells expressing the fusion protein. In generating iMC fusion proteins, the cytoplasmic domain of CD40 and the DD and intermediary domains of MyD88 are included in order to achieve optimal NF-KB activation; however, the C-terminal TIR domain of MyD88 is not required to be present. The fusion proteins further include elements that bind a chemical inducer of dimerization in the fusion protein, thereby making it possible to exercise temporal control over NF-kB activation through the administration of a CID to cells expressing iMC in a manner designed to minimize potential adverse effects of enhanced immune cell activation. One example of a protein that binds a chemical inducer of dimerization is the FKBP12 protein and variants (e.g., FKBP12v36). FKBP12 and/or variants thereof bind to homomultimer-inducing agents such as, e.g., FK506 dimer (or a dimeric FK506 analog ligand), AP1903 (rimiducid) or AP20187. Another example of a protein that binds a chemical inducer of dimerization is the FRB domain of mTOR and variants thereof (e.g., the KLW mutant of FRB) that bind to heteromultimer-inducing agents, such as rapamycin or a rapalog, which will also bind to FKBP12.

[0060] Apart from survival and growth advantages, iMC-induced co-stimulation may also provide additional functions to CAR-modified T cells. MyD88 signaling is critical for both Th1 and Th17 responses and acts via IL-1 to render CD4⁺ T cells refractory to regulatory T cell (Treg)-driven inhibition (see, e.g., Schenten et al. (2014) Immunity 40:78-90). In addition, CD40 signaling in CD8⁺ T cells via Ras, PI3K and protein kinase C, results in NF-KB-dependent induction of cytotoxic mediators granzyme and perforin that lyse CD4⁺CD25⁺ Treg cells (Martin et al. (2010) J Immunol 184:5510-5518). Thus, MyD88 and CD40 co-activation may render CAR-T cells resistant to the immunosuppressive effects of Treg cells, a function that could be critically important in the treatment of solid tumors and other types of cancers.

Multimerization-based control of CAR T cell elimination

[0061] CAR-expressing T cells may be used in various therapies, including cancer therapies.

In some instances, there is a need to increase the activity of the therapeutic cell. For example, co-stimulating polypeptides may be used to enhance the activation of T cells, and of CAR- expressing T cells against target antigens, which would increase the potency of adoptive immunotherapy. These treatments are used, for example, to target tumors for elimination, and to treat cancer and blood disorders, but these therapies may have negative side effects.

Overzealous on-target effects, such as those directed against large tumor masses, can lead to cytokine storms associated with tumor lysis syndrome (TLS), cytokine release syndrome (CRS) or macrophage activation syndrome (MAS). In some instances of therapeutic cell-induced adverse events, there is a need for rapid and near complete elimination of the therapeutic cells. In cases in which therapeutic cells trigger serious adverse events (SAEs), or become obsolete following treatment, it is useful to include a stable, reliable“suicide gene” in the cells that can eliminate the transferred T cells or stem cells. Yet, the need for therapy may remain. To safely provide continued therapy, methods have been developed for controlled activation or elimination of therapeutic cells. Such methods reduce or eliminate the possible negative effects of donor cells used in cellular therapy, while retaining part or all of the beneficial effects of the therapy.

[0062] In one approach to balanced therapeutic cell treatment, the modified cell, e.g., a CAR T cell, is engineered to also express a regulatable cell elimination product such as, for example, an inducible pro-apoptotic molecule. A pro-apoptotic molecule is one that is involved in apoptosis, or programmed cell death, which is tightly regulated and naturally uses scaffolds, such as Apaf-1 , CRADD/RAIDD, or FADD/Mort1 , to oligomerize and activate caspases (intracellular enzymes which are a family of cysteine-aspartic acid proteases) that can ultimately kill the cell. Apoptosis generally is triggered by stress conditions (e.g., DNA damage) that cause release of pro-apoptotic factors, such as cytochrome c, from mitochondria which interact with apoptotic protease-activating factor-1 (Apaf-1) to induce its oligomerization and formation of a complex called an apoptosome. The apoptosome provides a scaffold for initiation of a cascade of enzyme-mediated reactions that result in cell death. Apoptosis occurs through sequential activation of caspases which exist in cells as inactive zymogens. Most caspases that are involved in the downstream steps of the apoptotic cascade, referred to as“effector” caspases (e.g., caspase 3 and 7), are activated by proteolytic cleavage through the action of upstream or “initiator” caspases such as caspase 9. Caspase 9 (see, e.g., amino acid SEQ ID NO: 146) activation occurs in connection with its binding to an apoptosome and caspase 9

homodimerization through CARD (caspase recruitment domain; approximately amino acids 17- 90 of SEQ ID NO: 146) regions contained in the protein (see, e.g., Pop et al. (2006) Mol Cell 22:269-275; Wu and Bratton (2017) Mol Cell Oncol 4(2), e1281865, DOI:

10.1080/23723556.2017.1281865). Activated caspase 9 can, in turn, activate effector caspases leading to a chain of events that ultimately results in cell death.

[0063] If there is a need, for example, to reduce the number of transferred chimeric antigen receptor modified T cells, an inducing ligand may be administered to the subject being treated, thereby inducing apoptosis specifically of the modified T cells (see, e.g., U.S. Patent Application number no. 15/377,776 (publication no. US 2017/0166877) which is incorporated by reference herein in its entirety). For example, multimeric versions of the ligand binding domains FRB and/or FKBP12 fused to caspase proteins and expressed in a modified therapeutic cell can serve as scaffolds that permit the spontaneous dimerization and activation of the caspase units upon recruitment through the FRB and/or FKBP12 with a chemical inducing agent including for example, but not limited to, rapamycin, a rapalog, rimiducid, analog of rimiducid or other compound that binds to and dimerizes FKBP12 or a variant thereof. Examples of compounds that bind to and multimerize FKBP12 and/or a variant (e.g., FKBP12(F36V)) thereof are described in U.S. patent application no. 62/608,552 (attorney docket no. BEL-2027-PV) filed December 20, 2017, and entitled“Multimeric Compounds” which is incorporated by reference herein in its entirety.

[0064] As an example of this type of cell activation/elimination system, homodimerization with rimiducid, analog thereof or other dimerizing compound, can be used in the context of an inducible caspase safety switch, and as an inducible activation switch for cellular therapy, where co-stimulatory polypeptides including MyD88 and CD40 polypeptides are used to stimulate immune activity. Rimiducid (AP1903) is a high specificity, efficient dimerizer which has two identical, protein-binding surfaces arranged tail-to-tail, each with high affinity and specificity for a mutant or variant of FKBP12 referred to as FKBP12(F36V) (also known as FKBP12v36, F_V36 or F_v; see, e.g., amino acid SEQ ID NO: 93 and nucleotide SEQ ID NO: 12). Attachment of one or more F_v domains onto one or more cell signaling molecules that normally rely on

homodimerization can convert that protein to rimiducid control. However, because, in this example, a rimiducid-inducible caspase safety switch and a rimiducid-inducible MyD88/CD40 activation switch rely on the same ligand inducer, it is difficult to control both functions using these switches within the same cell. In order to separately regulate therapeutic cell activation and cell elimination, one of the molecular switches can be controlled by a distinct dimerizer ligand, such rapamycin or a rapamycin analog (rapalog). Thus, for example, a rapamycin or rapalog-inducible co-stimulatory polypeptide (e.g., MyD88/CD40 (iMC)) can be used in combination with a rimiducid-inducible pro-apoptotic polypeptide (e.g., caspase-9), or, conversely, a rimiducid-inducible chimeric fusion stimulating polypeptide, such as, for example, iMC, can be used in combination with a rapamycin- or rapalog-inducible pro-apoptotic polypeptide (e.g., caspase-9) to produce dual-switches in a modified therapeutic cell. These dual- switches can be used to control both cell proliferation and apoptosis selectively by administration of either of two distinct ligand inducers.

[0065] Figure 2 illustrates one version of the possible combinations of multiple components that can be used to create a dual switch activation/elimination system that can be implemented in a CAR T cell. In the exemplary system depicted in FIG. 2, a co-stimulatory T cell activation component is shown in the diagram of a cell on the left side of the figure. The first activation signal is provided through the chimeric antigen receptor, which includes an extracellular, antibody-derived single chain variable fragment (scFv), that specifically recognizes a target tumor cell antigen, fused to Q-bend 10 (Q) epitope derived from CD34 (for use in assessing transduction efficiency) which is fused, through a transmembrane linker, to a portion of Oϋ3z. Binding of the tumor cell antigen by scFv activates Oϋ3z which leads to NFAT activation. The second activation signal, which is in the form of a“go” switch,” can be induced by administration of a rapalog, such as, for example, an allele-specific rapalog, e.g., 7-demethoxy-7(S)-o,p- dimethoxyphenylrapamycin (referred to as CMP001 herein). This signal is initiated through the heteromultimerization of the KLW mutant of FRB (i.e., FRB(leu2098) or FRB_L) and FKBP12 in fusion proteins expressed in the cell. Heteromultimerization brings the co-stimulatory MyD88 (designated“M”) and CD40 (designated“C”) domains of the MC fusion protein (which is fused to the FRB_L-FKBP12 fusion at the amino terminus of MyD88) into proximity and results in further activation and proliferation of the CAR T cell. This inducible activation system is referred to as iRMC (inducible rapalog MyD88/CD40).

[0066] The right side of FIG. 2 shows an inducible cell death component of an exemplary cell activation/elimination system. This component includes fusion proteins (designated iC9) of the FKBP12 variant FKBP12v36 (designated as FKBPv) and caspase 9 lacking the CARD domain (designated AC9). These fusion proteins serve as a“stop” or“off” safety switch which can be induced by administration of rimiducid (or analog thereof or other compound that binds to and multimerizes FKBP12v36) which homodimerizes the FKBP12v36 proteins and approximates and activates the caspase 9 proteins to initiate apoptosis in the cell. When both the activation and elimination components (dual switches), which can be induced by distinct inducing agents, are included in a single CAR T cell, the cell is referred to as DS-CAR-T. Despite the fact that both iRMC and iC9 incorporated FKBP12 domains, because rimiducid is highly specific for the F36V variant of FKBP12, the co-stimulatory and safety switches are orthogonally regulated in this exemplary system. In another exemplary dual switch system, the co-stimulatory component can be generated through fusion of one or more of the F36V variant of FKBP12 proteins with MyD88 and CD40 to generate iMC while the elimination component (safety switch) can be formed from a fusion of FRB (or variant thereof) and FKBP12 and Acaspase 9 to generate iC9.

Compounds

Binding characteristics

[0067] Provided herein are compounds that bind to proteins, e.g., cellular proteins. In certain embodiments, the compound binds to one or more, or at least two, proteins to which rapamycin binds. Such proteins include those that participate in multimer, e.g., homodimer and/or heterodimer, formation. Examples include a variant or wild type FRB (FKBP-rapamycin binding domain) of the mTOR domain of TORC1 or TORC2 and an FKBP12 protein.

[0068] The term "protein" as used herein refers to a molecule having a sequence of amino acids linked by peptide bonds. As used herein the term protein is interchangeable with the terms“polypeptide” and“peptide”. This term includes fusion proteins, oligopeptides, peptides, cyclic peptides, polypeptides and polypeptide derivatives, whether native or recombinant, and also includes fragments, derivatives, homologs, and variants thereof. Proteins include, for example, proteins of intracellular origin (e.g., located in the nucleus, cytosol, organelle (e.g., mitochondria or peroxisome) or interstitial space of cells in vivo) and cell membrane proteins in vivo.

[0069] The term“variant” when used herein in connection with a protein refers to any modified form of a particular reference or standard protein, such as, for example, a wild type protein. Variants of a protein include, for example, analogs, mutants and isoforms of the protein. A variant protein may differ from a reference protein in a number of ways. For example, a variant protein may have amino acid sequence variations, e.g., substitutions, deletions, insertions, relative to the reference protein. Modifications in a variant protein relative to a reference protein may, in some instances, result in one or more differences in the properties of the variant protein relative to the those of the reference protein. For example, a variant protein may have different binding characteristics, e.g. affinities, for other molecules (e.g., proteins and compounds) than the reference protein.

[0070] A“region” or“domain” of a polypeptide refers to a portion or portions of a polypeptide that maintains a particular aspect or function (e.g., ligand-binding, catalytic ability) of the polypeptide. For example, a ligand-binding domain or region of a polypeptide refers to any portion or portions of the polypeptide that are able to bind the ligand. In another example, a pro- apoptotic domain or region of a polypeptide, e.g., a caspase-9 polypeptide or truncated caspase-9 polypeptide, refers to a portion(s) thereof that, upon dimerization or multimerization, can participate in the caspase cascade, allowing for, or causing, apoptosis.

[0071] The term“FRB” (FKBP12-Rapamycin-Binding) refers to a domain within the mTOR region of MTORC1 or MTORC2 to which FKBP12-rapamycin binds (for example, approximately residues 2025-21 14 within mTOR; see SEQ ID NO: 76 for the amino acid sequence of a human mTOR protein and SEQ ID NO: 77 for the amino acid sequence of a human FRB domain), and variants thereof. Reference herein to an amino acid position in an FRB protein is based on the amino acid position numbering of the human mTOR sequence (SEQ ID NO: 76), unless specifically noted otherwise. Based on the crystal structure of FRB conjugated to rapamycin, there are 3 key rapamycin-interacting residues that have been most analyzed, K2095, T2098, and W2101. In this context, a wild type FRB protein is often referred to as“KTW.” Examples of FRB mutants are discussed in Bayle et al. ((2006) Chem & Bio 13: 99-107), Stankunas et al. ((2007) Chembiochem 8:1 162-1 169) and Liberies et al. ((1997) Proc Natl Acad Sci 94:7825- 7830). One or more properties of an FRB variant, e.g., stability (some variants are more labile than others) and ability to bind to various rapalogs may differ from those of wild type FRB. For example, an FRB variant polypeptide may bind to a rapalog, and may bind, or may not bind, to rapamycin. Mutation of all three of the“key” rapamycin-interacting residues (i.e., K2095, T2098, and W2101) of FRB results in an unstable protein (e.g., K2095P, T2098L and W2101 F or“PLF”) that can be stabilized in the presence of rapamycin or some rapalogs (referred to as chemically induced stabilization). Additional unstable FRB mutants include KLF (T2098L, W2101 F), TLF (K2095T, T2098L, W2101 F), and RLF (K2095R, T2098L, W2101 F). This feature can be used to further increase the signal oise ratio in some applications.

[0072] In certain embodiments of the compounds provided herein, the compound, or a pharmaceutically acceptable salt thereof, bind to a wild type, e.g., human, FRB protein or to one or more variant FRB proteins with a K_D less than about 200 nM, or less than about 150 nM, or less than about 100 nM, or less than about 50 nM, or less than about 25 nM, or less than about 20 nM, or less than about 10 nM, or less than about 5 nM or less than about 1 nM.

[0073] In certain embodiments of the compounds provided herein, the compound, or a pharmaceutically acceptable salt thereof, is able to bind to one or more variant FRB proteins. In some embodiments, a compound provided herein is able to selectively bind to a variant FRB protein relative to a wild type FRB protein. Methods of identifying variant FRB proteins that bind to a compound provided herein include, for example, structure-based methods using a mammalian three-hybrid transcription assay of mutant FRB-encoding cDNAs (see, e.g., Liberies et al. (1997) Proc Natl Acad Sci 94:7825-7830). Examples of FRB variant polypeptides include, but are not limited to, KLW (T2098L), PLW (K2095P, T2098L), TLW (K2095T, T2098L), KTF (W2101 F), ATF (K2095A, W2101 F), PTF (K2095P, W2101 F), KLF (T2098L, W2101 F), TLF (K2095T, T2098L, W2101 F) and RLF (K2095R, T2098L, W2101 F). FRB variant KLW is also referred to as FRBL polypeptide (SEQ ID NO: 79). By comparing the KLW variant of SEQ ID NO: 79 with the wild type FRB polypeptide (SEQ ID NO: 77), one can determine the sequence of other FRB variants, including those listed herein.

[0074] In some embodiments, a compound, or a pharmaceutically acceptable salt thereof, provided herein binds to an FRB polypeptide variant that has an amino acid substitution in place of a threonine at residue 2098 of human FRB or a homolog thereof. In certain embodiments, a compound provided herein, or a pharmaceutically acceptable salt thereof, binds to an FRB polypeptide variant that has a leucine at residue 2098 of human FRB (i.e., FRB(T2098L)) or homolog thereof. In some embodiments, a compound provided herein, or a pharmaceutically acceptable salt thereof, binds to an FRB polypeptide variant that has an amino acid substitution in place of a lysine at residue 2095 of human FRB or a homolog thereof. In certain

embodiments, a compound provided herein, or a pharmaceutically acceptable salt thereof, binds to an FRB polypeptide variant that has a threonine (i.e., FRB(K2095T)) or proline (i.e., FRB(K2095P)) at residue 2095 of human FRB or homolog thereof. In some embodiments, a compound provided herein, or a pharmaceutically acceptable salt thereof, binds to an FRB polypeptide variant that has an amino acid substitution in place of a tryptophan at residue 2101 of human FRB or homolog thereof. In certain embodiments, a compound provided herein, or a pharmaceutically acceptable salt thereof, binds to an FRB polypeptide variant that has a phenylalanine at residue 2101 of human FRB (i.e., FRB(W2101 F)) or homolog thereof. In some embodiments, a compound provided herein, or a pharmaceutically acceptable salt thereof, binds to an FRB polypeptide variant that has a threonine at residue 2095 and a leucine at residue 2098 of human FRB (i.e., FRB(K2095T, T2098L)) or homolog thereof. In some embodiments, a compound provided herein, or a pharmaceutically acceptable salt thereof, binds to an FRB polypeptide variant that has a proline at residue 2095, a leucine at residue 2098 and a phenylalanine at residue 2101 of human FRB (i.e., FRB(K2095P, T2098L,

W2101 F)) or homolog thereof. In some embodiments, a compound provided herein, or a pharmaceutically acceptable salt thereof, binds with greater affinity to one or more of the following variant FRB polypeptides than to wild type FRB: KLW (T2098L), PLW (K2095P, T2098L), TLW (K2095T, T2098L), KTF (W2101 F), ATF (K2095A, W2101 F), PTF (K2095P, W2101 F), KLF (T2098L, W2101 F), TLF (K2095T, T2098L, W2101 F) and RLF (K2095R,

T2098L, W2101 F). In some embodiments, a compound provided herein, or a pharmaceutically acceptable salt thereof, is able to bind a human FRB variant protein and bind a wild type human FKBP12 protein. In certain embodiments, a compound provided herein, or a pharmaceutically acceptable salt thereof, is able to bind a human FRB variant protein and bind a wild type human FKBP12 protein with greater affinity than it binds to a variant human FKBP12 protein.

[0075] The term“FKBP” refers to the cellular FK506-binding proteins. There are multiple FKBPs and together they represent a subset of immunophilins. Most FKBPs exhibit peptidylprolyl cis/trans isomerase (PPIase) activity and bind to the immunosuppressive compound FK506 (also called tacrolimus). Different FKBPs have different molecular weights. Smaller FKBPs, such as FKBP12, basically contain only an FK506-binding domain, whereas larger FKBPs may contain additional domains. The term“FKBP12” refers to a specific member of the FKBP family (see SEQ ID NO: 85 for the amino acid sequence of a full-length human FKBP12), and variants thereof. In some FKBP12 protein variants, the phenylalanine at amino acid position 36 (or 37 if the initial methionine of the protein is counted) in the wild type FKBP12 polypeptide is substituted with a different amino acid. For example, the amino acid substitution can be to a valine, leucine, isoleucine, alanine or other amino acid. In a particular FKBP12 variant, the amino acid substitution is to valine and is referred to as FKBP12v36 (also referred to as FKBP12(F36V), Fvse, FKBP_V, or F_v).

[0076] In certain embodiments of the compounds provided herein, the compound, or a pharmaceutically acceptable salt thereof, is able to bind to one or more variant human FKBP12 proteins or homologs thereof. In some embodiments, a compound provided herein binds to a human FKBP12 polypeptide variant that has an amino acid substitution in place of

phenylalanine at residue 36 of human FKBP12, or a homolog thereof. In certain embodiments, a compound provided herein, or a pharmaceutically acceptable salt thereof, binds to a human FKBP12 polypeptide variant that has a valine at residue 36 of human FKBP12 (i.e.,

FKBP12v36) or homolog thereof. In some embodiments, a compound provided herein, or a pharmaceutically acceptable salt thereof, is able to selectively bind to a variant human FKBP12 protein relative to a wild type human FKBP12 protein. In some embodiments, a compound provided herein, or a pharmaceutically acceptable salt thereof, selectively binds to wild type human FKBP12 protein relative to a variant human FKBP12 protein.

[0077] In certain embodiments of the compounds provided herein, the compound, or a pharmaceutically acceptable salt thereof, bind to a wild type, e.g., human, FKBP12 protein or to one or more variant FKBP12 proteins with a K_D less than about 200 nM, or less than about 150 nM, or less than about 100 nM, or less than about 50 nM, or less than about 25 nM, or less than about 20 nM, or less than about 10 nM , or less than about 5 nM or less than about 1 nM.

[0078] In some embodiments, a compound described herein, or a pharmaceutically acceptable salt thereof, binds to a variant FRB polypeptide with an IC50, EC50 and/or KD at least 10 times lower than the IC50, EC50 and/or K_D of the compound binding to a wild type FRB polypeptide. In some embodiments, a compound described herein, or a pharmaceutically acceptable salt thereof, binds to a variant FRB polypeptide with an IC50, EC50 and/or KD at least 100 times lower than the IC50, EC50 and/or K_D of the compound binding to the wild type FRB polypeptide. In some embodiments, a compound described herein, or a pharmaceutically acceptable salt thereof, binds to a variant FRB polypeptide with an IC50, EC50 and/or K_D at least 1000 times lower than the IC50, EC50 and/or KD of the compound binding to the wild type FRB polypeptide. In some embodiments a compound described herein, or a pharmaceutically acceptable salt thereof, has a binding affinity (IC50, EC50 and/or KD) for binding to FRBL (or KLW) of 100 nM or less. In some embodiments, a compound provided herein, or a pharmaceutically acceptable salt thereof, binds to a variant FRB polypeptide with an IC50, EC50 and/or KD at least 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3500, 4000, 4500, or 5000, times lower than the IC50, EC50 and/or K_D of the binding of the compound to the wild type FRB polypeptide. In some embodiments, the FRB polypeptide variant used to measure binding affinity has an amino acid substitution at amino acid residue or position 2098. In some embodiments, the FRB polypeptide variant used to measure binding affinity has a substitution at position 2098 to leucine. In some embodiments, the FRB polypeptide variant used to measure binding affinity is FRB_L (or KLW).

[0079] In some embodiments, a compound described herein, or a pharmaceutically acceptable salt thereof, binds to an FKBP12 polypeptide variant with an IC50, EC50 and/or K_D at least 10 times lower than the IC50, EC50 and/or K_D of the binding of the compound to the wild type FKBP12 polypeptide. In some embodiments, a compound described herein, or a pharmaceutically acceptable salt thereof, binds to anFKBP12 polypeptide variant with an IC50, EC50 and/or K_D at least 100 times lower than the IC50, EC50 and/or K_D of the binding of the compound to the wild type FKBP12 polypeptide. In some embodiments, a compound described herein, or a pharmaceutically acceptable salt thereof, binds to an FKBP12 polypeptide variant with an IC50, EC50 and/or K_D at least 1000 times lower than the IC50, EC50 and/or K_D of the binding of the compound to the wild type FKBP12 polypeptide. In some embodiments a compound described herein, or a pharmaceutically acceptable salt thereof, has a binding affinity (IC50, EC50 and/or K_D) for binding to FKBP12v36 of 100 nM or less. In some embodiments, a compound provided herein, or a pharmaceutically acceptable salt thereof, binds to an FKBP12 polypeptide variant with an IC50, EC50 and/or K_D at least 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3500, 4000, 4500, or 5000, times lower than the IC50,

EC50 and/or KD of the binding of the compound to the wild type FKBP12 polypeptide. In some embodiments, the FKBP12 polypeptide variant used to measure binding affinity has an amino acid substitution at amino acid residue or position 36. In some embodiments, the FKBP12 polypeptide variant used to measure binding affinity has a substitution at position 36 to an amino acid chosen from valine, leucine, isoleucine and alanine. In some embodiments, the FKBP12 polypeptide variant used to measure binding affinity has an amino acid substitution at position 36 to valine. In some embodiments, the FKBP12 polypeptide variant used to measure binding affinity is FKBP12v36. [0080] In some embodiments, a compound described herein, or a pharmaceutically acceptable salt thereof, binds to a wild type FKBP12 polypeptide with an IC₅o, EC₅o and/or K_D at least 10 times lower than the IC50, EC50 and/or KD of the binding of the compound to a variant FKBP12 polypeptide. In some embodiments, a compound described herein, or a

pharmaceutically acceptable salt thereof, binds to a wild type FKBP12 polypeptide with an IC50, EC50 and/or K_D at least 100 times lower than the IC50, EC50 and/or K_D of the binding of the compound to a variant FKBP12 polypeptide. In some embodiments, a compound described herein, or a pharmaceutically acceptable salt thereof, binds to a wild type FKBP12 polypeptide with an IC50, EC50 and/or K_D at least 1000 times lower than the IC50, EC50 and/or K_D of the binding of the compound to a variant FKBP12 polypeptide. In some embodiments, a compound provided herein, or a pharmaceutically acceptable salt thereof, binds to a wild type FKBP12 polypeptide with an IC50, EC50 and/or K_D at least 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3500, 4000, 4500, or 5000, times lower than the IC50, EC50 and/or K_D of the binding of the compound to a variant FKBP12 polypeptide. In some embodiments, the FKBP12 polypeptide variant used to measure binding affinity has an amino acid substitution at amino acid residue or position 36. In some embodiments, the FKBP12 polypeptide variant used to measure binding affinity has a substitution at position 36 to an amino acid chosen from valine, leucine, isoleucine and alanine. In some embodiments, the FKBP12 polypeptide variant used to measure binding affinity has an amino acid substitution at position 36 to valine. In some embodiments, the FKBP12 polypeptide variant used to measure binding affinity is FKBP12v36.

Multimeric compounds

[0081] In some embodiments, compounds provided herein are able to crosslink proteins that include a region that binds to the compound, such as, for example, a multimeric ligand binding region or a multimerizing region. Such proteins can be endogenous cellular proteins including, for example, wildtype FRB, or mTOR, and FKBP12. Binding of endogenous cellular proteins by a compound provided herein can be used, for example, in therapeutic applications such as treating diseases, disorders or conditions. For example, therapeutic applications include, but are not limited to, treatment of conditions which benefit from inhibition of immune cells or immunosuppression. In some embodiments, a compound provided herein can be used to crosslink proteins in modified cells, e.g., in vitro, ex vivo or in vivo. For example, by attaching one or more multimerizing regions to one or more cell signaling proteins that are able to dimerize, the protein, or proteins, can be multimerized by contact with a compound provided herein. In one example, contacting modified cells that express chimeric fusion polypeptides that include one or more multimerizing regions and an apoptosis-inducing polypeptide with a compound provided herein, activates a cellular safety switch, resulting in apoptosis. Contacting modified cells that express chimeric fusion polypeptides that include one or more multimerizing regions and one or more co-stimulatory polypeptides with a compound provided herein, activates co-stimulatory activity.

[0082] Certain embodiments of compounds provided herein are referred to herein as “multimeric compounds.” A multimeric compound may also be referred to herein as a multimerizing agent, a multimerizing compound, a multimerizing ligand, a multimeric agent, a multimeric compound, or a multimeric ligand. The term“multimerize” or multimerization refers to the dimerization of two peptides or polypeptides, or the multimerization of more than two peptides or polypeptides. Polypeptides that are dimerized or multimerized can be referred to as multimeric ligand binding polypeptides. A portion of a multimeric ligand-binding polypeptide that is capable of binding a multimeric compound may be referred to as a“ligand binding” region or domain,“dimerizing” region or domain,“dimerization” region or domain,“multimerizing” polypeptide, region or domain,“dimeric ligand binding” polypeptide, region or domain, “multimerization” polypeptide, region or domain, and“multimeric ligand binding” polypeptide, region or domain. In some embodiments, multimeric compounds provided herein are capable of multimerizing, or heteromerizing, peptides or polypeptides that are different from each other.

[0083] Included in compounds provided herein are compounds referred to generally as multimeric compounds which can exhibit favorable specificity for, and efficient dimerization of, FKBP polypeptides and FRB polypeptides (including, for example, variant forms of FRB such as the KLW, TLW, PLW and PLF variants of FRB), and can be considered favorable multimeric ligands relative to rapamycin. Multimeric compounds described herein, or pharmaceutically acceptable salts thereof, in some embodiments bind with relatively high affinity to FKBP and/or FRB polypeptides, and sometimes with high binding affinity to FKBP12 and/or FRB polypeptide variants to which rapamycin binds with high affinity. Sometimes compounds described herein, or pharmaceutically acceptable salts thereof, exhibit about the same or better binding to a FKBP12 and/or FRB polypeptide variant as compared to rapamycin (e.g., as determined by similar or greater binding affinity (such as a lower IC50, EC50 and/or K_D)..

[0084] In some embodiments, methods are provided for multimerizing polypeptides expressed in a cell, comprising contacting the cell with a compound, including, for example, a compound or composition provided herein, wherein the polypeptides comprise at least one FRB polypeptide, or variant thereof, and/or at least one FKBP12 polypeptide, or variant thereof. In some embodiments, the at least one FRB polypeptide can be a variant FRB polypeptide that has an amino acid substitution at a position corresponding to position 2098 in the wild type FRB polypeptide. In some embodiments, the amino acid substitution is to a leucine. In some embodiments, the FRB polypeptide variant is FRBL (or KLW).

Rapalogs

[0085] Included in compounds provided herein are analogs of rapamycin (rapalogs). A rapalog can differ from rapamycin in one or more ways. For example, a rapalog may be modified at one or more of the atoms (such as, for example, a carbon atom or oxygen atom) within the compound relative to rapamycin. In some embodiments, a rapalog may have a substituent at one or more of the atoms (such as, for example, a carbon atom or oxygen atom) within the compound that differs from the substituent at the same position in the rapamycin natural product. Because rapamycin is a chiral compound containing 15 defined stereocenters, a rapalog can also differ from rapamycin in the absolute stereochemical configuration at one or more chiral centers. When a rapalog has a different absolute configuration from rapamycin at only one of the chiral centers, it is referred to as an epimer of rapamycin. In some

embodiments, a compound provided herein can differ from rapamycin in the absolute configuration of a chiral center at C40, C7 and/or C29. The configurations at these positions in rapamycin are R for C40, S for C7 and S for C29. For example, in some embodiments, the compound is a C40 epimer (S configuration), a C7 (R configuration) epimer or a C29 epimer (R configuration) of the chiral carbons at the corresponding positions in rapamycin.

[0086] The dimerizer rapamycin has a low solubility of approximately 5-10 mg/ml in water at 25°C. A rapalog, 7-demethoxy-7(S)-o,p-dimethoxyphenylrapamycin (also referred to as CMP001 , 7(S)-DMOP-rapamycin, (S)-DMOP-rapamycin and DMOP-rapamycin herein), also has low solubility in water (~5-10 mg/ml) at 25°C. Compounds provided herein, or

pharmaceutically acceptable salts thereof, can exhibit greater solubility in water and/or other pharmaceutically acceptable aqueous solutions relative to rapamycin, CMP001 and other rapalogs. For example, some embodiments of compounds provided herein, or pharmaceutically acceptable salts thereof, have a solubility in water at 25°C of at least about 20 mg/ml, at least about 25 mg/ml, at least about 30 mg/ml, at least about 35 mg/ml, at least about 40 mg/ml, at least about 45 mg/ml, at least about 50 mg/ml, at least about 55 mg/ml, at least about 60 mg/ml, at least about 65 mg/ml, at least about 70 mg/ml, at least about 75 mg/ml, at least about 80 mg/ml, at least about 85 mg/ml, at least about 90 mg/ml, at least about 95 mg/ml, at least about 100 mg/I, at least about 120 mg/ml, at least about 140 mg/ml, at least about 150 mg/ml, at least about 160 mg/ml, at least about 180 mg/ml, at least about 200 mg/ml, at least about 250 mg/ml, at least about 300 mg/ml, at least about 350 mg/ml, at least about 400 mg/ml, at least about 450 mg/ml, at least about 500 mg/ml, at least about 550 mg/ml, at least about 600 mg/ml, at least about 650 mg/ml, at least about 700 mg/ml, at least about 750 mg/ml, at least about 800 mg/ml or more. In some embodiments, compounds provided herein, or pharmaceutically acceptable salts thereof, are at least about 2-fold or more, at least about 3-fold or more, at least about 4- fold or more, at least about 5-fold or more, at least about 6-fold or more, at least about 7-fold or more, at least about 8-fold or more, at least about 9-fold or more, at least about 10-fold or more, at least about 20-fold or more, at least about 30-fold or more, at least about 40-fold or more or at least about 50-fold or more soluble in water at 25°C than rapamycin or CMP001.

[0087] In some embodiments, compounds described herein, or pharmaceutically acceptable salts thereof, are soluble in water. In some embodiments, compounds described herein, or pharmaceutically acceptable salts thereof, are soluble in an acetate buffer having a pH of 6 or less. In some embodiments, compounds described herein, or pharmaceutically acceptable salts thereof, are soluble in an acetate buffer having a pH of 4 or less. In some embodiments, compounds described herein, or pharmaceutically acceptable salts thereof, are soluble in an acetate buffer having a pH of 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less.

[0088] In some embodiments, compounds described herein, or pharmaceutically acceptable salts thereof, have a solubility in an acetate buffer having a pH of 6 or less that is greater than the solubility of rapamycin or CMP001 in an acetate buffer having a pH of 6 or less. In some embodiments, compounds described herein, or pharmaceutically acceptable salts thereof, have a solubility in an acetate buffer having a pH of 5 or less that is greater than the solubility of rapamycin or CMP001 in an acetate buffer having a pH of 5 or less. In some embodiments, compounds described herein, or pharmaceutically acceptable salts thereof, have a solubility in an acetate buffer having a pH of 4 or less that is greater than the solubility of rapamycin or CMP001 in an acetate buffer having a pH of 4 or less. In some embodiments, compounds described herein, or pharmaceutically acceptable salts thereof, are soluble at a concentration greater than 4 mg.mL^-1 in an acetate buffer having a pH of 6 or less. In some embodiments, compounds described herein, or pharmaceutically acceptable salts thereof, are soluble at a concentration greater than 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5. 7, or 8 mg.mL^-1 in an acetate buffer having a pH of 6 or less.

[0089] In some embodiments, compounds described herein, or pharmaceutically acceptable salts thereof, are soluble at a concentration greater than 4 mg.mL^-1 in an acetate buffer having a pH of 5 or less. In some embodiments, compounds described herein, or pharmaceutically acceptable salts thereof, are soluble at a concentration greater than 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5. 7, or 8 mg.mL^-1 in an acetate buffer having a pH of 5 or less. [0090] In some embodiments, compounds described herein, or pharmaceutically acceptable salts thereof, are soluble at a concentration greater than 0.2 mg.mL^-1 in an acetate buffer having a pH of 4 or less. In some embodiments, compounds described herein, or pharmaceutically acceptable salts thereof, are soluble at a concentration greater than 4 mg.mL^-1 in an acetate buffer having a pH of 4 or less. In some embodiments, compounds described herein, or pharmaceutically acceptable salts thereof, are soluble at a concentration greater than 0.2, 0.5, 0.75, 1 , 1.25, 1.5, 1.75, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5. 7, or 8 mg.mL^-1 in an acetate buffer having a pH of 4 or less.

[0091] In some embodiments, compounds described herein, or pharmaceutically acceptable salts thereof, are soluble in phosphate buffer. In some embodiments, compounds described herein, or pharmaceutically acceptable salts thereof, are soluble in a phosphate buffer having a pH of 6 or less. In some embodiments, compounds described herein, or pharmaceutically acceptable salts thereof, are soluble in a phosphate buffer having a pH of 4 or less. In some embodiments, compounds described herein, or pharmaceutically acceptable salts thereof, are soluble in a phosphate buffer having a pH of 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less.

[0092] In some embodiments, compounds described herein, or pharmaceutically acceptable salts thereof, have a solubility in a phosphate buffer having a pH of 6 or less that is greater than the solubility of rapamycin or CMP001 in a phosphate buffer having a pH of 6 or less. In some embodiments, compounds described herein, or pharmaceutically acceptable salts thereof, have a solubility in a phosphate buffer having a pH of 5 or less that is greater than the solubility of rapamycin or CMP001 in a phosphate buffer having a pH of 5 or less. In some embodiments, compounds described herein, or pharmaceutically acceptable salts thereof, have a solubility in a phosphate buffer having a pH of 4 or less that is greater than the solubility of rapamycin or CMP001 in a phosphate buffer having a pH of 4 or less. In some embodiments, compounds described herein, or pharmaceutically acceptable salts thereof, are soluble at a concentration greater than 4 mg.mL^-1 in a phosphate buffer having a pH of 6 or less. In some embodiments, compounds described herein, or pharmaceutically acceptable salts thereof, are soluble at a concentration greater than 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5. 7, or 8 mg.mL^-1 in a phosphate buffer having a pH of 6 or less.

[0093] In some embodiments, compounds described herein, or pharmaceutically acceptable salts thereof, are soluble at a concentration greater than 4 mg.mL^-1 in a phosphate buffer having a pH of 5 or less. In some embodiments, compounds described herein, or pharmaceutically acceptable salts thereof, are soluble at a concentration greater than 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5. 7, or 8 mg.mL^-1 in a phosphate buffer having a pH of 5 or less.

[0094] In some embodiments, compounds described herein, or pharmaceutically acceptable salts thereof, are soluble at a concentration greater than 0.2 mg.mL^-1 in a phosphate buffer having a pH of 4 or less. In some embodiments, compounds described herein, or

pharmaceutically acceptable salts thereof, are soluble at a concentration greater than 4 mg.mL^-1 in a phosphate buffer having a pH of 4 or less. In some embodiments, compounds described herein, or pharmaceutically acceptable salts thereof, are soluble at a concentration greater than 0.2, 0.5, 0.75, 1 , 1.25, 1.5, 1.75, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5. 7, or 8 mg.mL^-1 in a phosphate buffer having a pH of 4 or less.

[0095] Embodiments of some of the compounds provided herein exhibit increased metabolic stability relative to rapamycin and/or certain rapamycin analogs, including, for example, but not limited to, 7-demethoxy-7(S)-o,p-dimethoxyphenylrapamycin (CMP001). For example, in some embodiments, compounds provided herein have a half-life (e.g., as measured in minutes using animal, e.g., human, liver microsome assays or animal model assays) that is at least 1 .2 times,

1.5 times, 1.75 times, 2.0 times, 2.2 times, 2.5 times, 2.75 times, 3.0 times, 3.2 times, 3.5 times, 3.75 times, 4 times, 4.2 times, 4.5 times, 4.75 times or 5.0 times longer in duration than the half- life of rapamycin or a rapalog such as, e.g., 7-demethoxy-7(S)-o,p-dimethoxyphenylrapamycin (CMP001). In some embodiments, compounds provided herein have an intrinsic clearance (e.g., as measured in ml/min/mg protein using animal, e.g., human, liver microsome assays or animal model assays) that is at least 1.2-fold less, 1.5-fold less, 1.75-fold less, 2.0-fold less, 2.2- fold less, 2.5-fold less, 2.75-fold less, 3.0-fold less, 3.2-fold less, 3.5-fold less, 3.75-fold less, 4- fold less, 4.2-fold less, 4.5-fold less, 4.75-fold less or 5.0-fold less than the intrinsic clearance of rapamycin or a rapalog such as, e.g., 7-demethoxy-7(S)-o,p-dimethoxyphenylrapamycin (CMP001).

Stereochemistry

[0096] Asymmetric centers exist in embodiments of the compounds disclosed herein. These centers are designated by the symbols“R” or“S,” depending on the configuration of substituents around the chiral carbon atom. Included for compounds herein are all

stereochemical isomeric forms, including diastereomeric, enantiomeric, and epimeric forms, as well as d-isomers and 1 -isomers, and mixtures thereof, unless otherwise specified. Individual stereoisomers of compounds can be prepared synthetically from commercially available starting materials that contain chiral centers. Individual stereoisomers of compounds can be generated by preparing mixtures of enantiomeric products followed by separation, non-limiting examples of which include conversion to a mixture of diastereomers followed by separation or

recrystallization, chromatographic techniques, direct separation of enantiomers on chiral chromatographic columns, or any other appropriate method known in the art. Starting compounds of particular stereochemistry are commercially available or can be made and resolved by techniques known in the art. Additionally, compounds disclosed herein may exist as geometric isomers. Included for compounds herein are all cis, trans, syn, anti, entgegen (E), and zusammen (Z) isomers as well as the appropriate mixtures thereof. Additionally, compounds may exist as tautomers; all tautomeric isomers are included herein unless otherwise specified. Additionally, the compounds disclosed herein can exist in unsolvated as well as solvated forms with pharmaceutically acceptable solvents, non-limiting examples of which include water, ethanol, and the like. In general, the solvated forms are considered equivalent to the unsolvated forms.

[0097] In some embodiments, compounds provided herein are rapalogs that are epimers of rapamycin. In some embodiments, compositions containing a compound provided herein that is an epimer of rapamycin are substantially free of the homologous epimer that has the stereochemistry as it naturally occurs in rapamycin. In some embodiments, compositions containing a compound provided herein that is an epimer of rapamycin contain less than about 45% of the homologous naturally occurring epimer, less than about 40% of the homologous naturally occurring epimer, less than about 35% of the homologous naturally occurring epimer, less than about 30% of the homologous naturally occurring epimer, less than about 25% of the homologous naturally occurring epimer, less than about 20% of the homologous naturally occurring epimer, less than about 15% of the homologous naturally occurring epimer, less than about 10% of the homologous naturally occurring epimer, less than about 5% of the

homologous naturally occurring epimer, or less than about 1 % of the homologous naturally occurring epimer on either a weight or molar basis.

Examples of compounds

[0098] Compounds described herein often have a structure of Formula B, or a pharmaceutical salt thereof

Formula B

[0099] In some embodiments, compounds provided herein have a structure of Formula B, or a pharmaceutically acceptable salt thereof, wherein:

R^20A is hydrogen and R^20B is -R²³ or -R^F-R²³, or R^20B is hydrogen and R^20A is -R²³ or -R^F-R²³; R^21A is hydrogen and R^{21 B} is hydroxy, -R^G-R²⁴ _, -R^G-R²⁴-R²⁵ or -R^G-R²⁴-R²⁶-R²⁵, or R^{21 B} is hydrogen and R^21A is hydroxy, -R^G-R²⁴ _, -R^G-R²⁴-R²⁵ or -R^G-R²⁴-R²⁶-R²⁵;

n is 1 or 2;

R²⁷ and R²⁸ each independently is -R³²-R³³, or hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, perhaloalkyl, alkoxy, cycloalkyl, aryl, heterocycloalkyl, heterocycle-alkyl or heteroaryl, which independently is optionally substituted with one or more substituents chosen from halogen, hydroxy, alkyl, alkenyl, alkynyl, heteroalkyl, haloalkyl, perhaloalkyl, perhaloalkoxy, alkoxy, haloalkoxy, alkoxyalkyl, acyl, oxo, acyloxy, carboxyl, amido, cyano, amino, alkylamino, alkylaminoalkyl, thiol, alkylthio, alkylthioalkyl, haloalkylthio, perhaloalkylthio, nitro, aryl, arylalkyl, cycloalkylalkyl, heterocycle-alkyl, cycloalkyl, heterocycloalkyl, heteroaryl, heteroarylalkyl, alkylsulfonyl, sulfonamide, alkylsulfonamido, alkylsilyloxy, alkylsulfinamido, arylsulfinamido, and arylsulfonamido, with the proviso that R²⁷ and R²⁸ are not both hydrogen when -R^F is -O- and R²³ is alkyl; and

R²⁹ has a structure of Formula C-1 :

Formula C-1

wherein:

X⁴⁰, X⁴¹, X⁴², X⁴³, X⁴⁴ and X⁴⁵ together form an aryl or heteroaryl ring; zero, one, two or three of X⁴⁰, X⁴¹, X⁴², X⁴³, X⁴⁴ and X⁴⁵ are oxygen, nitrogen or sulfur heteroatoms, and the remaining X⁴⁰, X⁴¹ , X⁴², X⁴³, X⁴⁴ and X⁴⁵ are carbon; and

one, two or three of R⁴¹ , R⁴², R⁴³, R⁴⁴ and R⁴⁵ independently is R³¹-R³⁰, and each of the remaining R⁴¹, R⁴², R⁴³, R⁴⁴ and R⁴⁵ independently is not present or independently is hydrogen, halogen, hydroxy, C1 -C3 alkyl, C1-C3 heteroalkyl, C1 -C3 haloalkyl, C1 -C3 perhaloalkyl, C1-C3 perhaloalkoxy, C1-C3 alkoxy, C1 -C3 haloalkoxy, C1 -C3 alkoxy C1-C3 alkyl, C1 -C4 acyl, oxo, C1 -C4 acyloxy, carboxyl, amido, cyano, amino, C1-C3 alkylamino, C1-C3 alkylamino C1 -C3 alkyl, thiol, C1 -C3 alkylthio, C1 -C3 alkylthio C1 -C3 alkyl, C1 -C3 haloalkylthio, C1 -C3

perhaloalkylthio, nitro, C5-C7 aryl, C5-C7 aryl C1 -C3 alkyl, C5-C7 cycloalkyl, C5-C7 cycloalkyl C1 -C3 alkyl, 5-7 membered heterocycloalkyl, 5-7 membered heterocycle C1 -C3 alkyl, 5-7 membered heteroaryl, 5-7 membered heteroaryl C1 -C3 alkyl, C1 -C3 alkylsulfonyl, sulfonamide, C1 -C3 alkylsulfonamido or C1 -C3 alkylsilyloxy.

[0100] In certain embodiments of compounds having a structure of the Formula B as defined directly above, wherein R²⁹ has a structure of Formula C-1 , R^22A is hydrogen and R^22B is halogen, -NR²⁷R²⁸ or -R^H-R²⁹-R³¹-R³⁰, or R^22B is hydrogen and R^22A is halogen, -NR²⁷R²⁸ or - R^H_R²⁹_R³¹_R³⁰ |_n certain embodiments of compounds having a structure of the above- defined Formula B, wherein R²⁹ has a structure of Formula C-1 , R^22A is hydrogen and R^22B is - R^H— R²⁹— R³¹— R³⁰ or R^22B is hydrogen and R^22A is -R^H-R²⁹-R³¹-R³⁰. In certain embodiments of the above-defined Formula B, wherein R²⁹ has a structure of Formula C-1 , R^H is -O-C(O)-.

[0101] In certain embodiments of compounds having a structure of Formula B as defined above, wherein R²⁹ has a structure of Formula C-1 , when X⁴¹ , X⁴², X⁴³, X⁴⁴ and X⁴⁵

independently is carbon, R⁴¹, R⁴², R⁴³, R⁴⁴ and R⁴⁵, respectively, independently is a Group A moiety chosen from R³¹-R³⁰, hydrogen, halogen, hydroxy, C1 -C3 alkyl, C1 -C3 heteroalkyl, C1 - C3 haloalkyl, C1 -C3 perhaloalkyl, C1 -C3 perhaloalkoxy, C1 -C3 alkoxy, C1 -C3 haloalkoxy, C1 - C3 alkoxy C1 -C3 alkyl, acyl, oxo, acyloxy, carboxyl, amido, cyano, amino, C1 -C3 alkylamino, C1 -C3 alkylaminoalkyl, thiol, C1 -C3 alkylthio, C1 -C3 alkylthio C1 -C3 alkyl, C1 -C3 haloalkylthio, C1 -C3 perhaloalkylthio, nitro, C5-C7 aryl, C5-C7 aryl C1 -C3 alkyl, C5-C7 cycloalkyl C1 -C3 alkyl, 5-7 membered heterocycle-C1 -C3 alkyl, C5-C7 cycloalkyl, 5-7 membered heterocycloalkyl, 5-7 membered heteroaryl, 5-7 membered heteroaryl C1 -C3 alkyl, C1 -C3 alkylsulfonyl, sulfonamide, C1 -C3 alkylsulfonamido and C1 -C3 alkylsilyloxy. Stated another way, when X⁴¹ is carbon, R⁴¹ is a Group A moiety; when X⁴² is carbon, R⁴² is a Group A moiety; when X⁴³ is carbon, R⁴³ is a Group A moiety; when X⁴⁴ is carbon, R⁴⁴ is a Group A moiety; and when X⁴⁵ is carbon, R⁴⁵ is a Group A moiety. In certain of these embodiments, R^22A or R^22B is -R^H-R²⁹-R³¹-R³⁰. In certain of these embodiments, R^H is -O-C(O)-. [0102] In certain embodiments of the above-defined Formula B, wherein R²⁹ has a structure of Formula C-1 , when X⁴¹ , X⁴², X⁴³, X⁴⁴ and X⁴⁵ independently is nitrogen, R⁴¹ , R⁴², R⁴³, R⁴⁴ and R⁴⁵, respectively, independently is a Group B moiety chosen from hydrogen, C1 -C3 alkyl or C1 - C3 alkyl substituted with hydroxyl, halogen or NR²⁷R²⁸. Stated another way, when X⁴¹ is nitrogen, R⁴¹ is a Group B moiety; when X⁴² is nitrogen, R⁴² is a Group B moiety; when X⁴³ is nitrogen, R⁴³ is a Group B moiety; when X⁴⁴ is nitrogen, R⁴⁴ is a Group B moiety; and when X⁴⁵ is nitrogen, R⁴⁵ is a Group B moiety. In certain of these embodiments, R^22A or R^22B is -R^H-R²⁹- R3I_R3O I _n certain of these embodiments, R^H is -O-C(O)-.

[0103] In certain embodiments of compounds having a structure of the above-defined Formula B, wherein R²⁹ has a structure of Formula C-1 , when X⁴¹ , X⁴², X⁴³, X⁴⁴ and X⁴⁵ independently is oxygen or sulfur, R⁴¹, R⁴², R⁴³, R⁴⁴ and R⁴⁵, respectively, independently is not present. Stated another way, when X⁴¹ is oxygen or sulfur, R⁴¹ is not present; when X⁴² is oxygen or sulfur, R⁴² is not present; when X⁴³ is oxygen or sulfur, R⁴³ is not present; when X⁴⁴ is oxygen or sulfur, R⁴⁴ is not present; and when X⁴⁵ is oxygen or sulfur, R⁴⁵ is not present. In certain of these embodiments, R^22A or R^22B is -R^H-R²⁹-R³¹-R³⁰. In certain of these embodiments, R^H is -O- C(O)-.

[0104] In some embodiments, compounds provided herein have a structure of Formula B, or a pharmaceutically acceptable salt thereof, wherein:

R^20A is hydrogen and R^20B is-R²³ or -R^F-R²³, or R^20B is hydrogen and R^20A is -R²³ or -R^F-R²³; R^21A is hydrogen and R^{21 B} is hydroxy, -R^G-R²⁴ -R^G-R²⁴-R²⁵ or -R^G-R²⁴-R²⁶-R²⁵, or R^{21 B} is hydrogen and R^21A is hydroxy, -R^G-R²⁴ _, -R^G-R²⁴-R²⁵ or -R^G-R²⁴-R²⁶-R²⁵;

R^F, R^G and R^H each independently is -0-, -C(O)-, -O-C(O)-, -C(0)-0-, -S-, -S(0)_n-, -O- S(0)n-, -S(0)n-0-, -NH-S(0)n-, -S(O) n-NH-, -NH-C(O)-, -C(0)-NH-, -NH-C(S)-, -C(S)- NH-, -S-C(S)-, -C(S)-S-, or -C(S)-;

n is 1 or 2;

R²³ independently is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, aryl, heterocycloalkyl or heteroaryl, which independently is optionally substituted by one or more substituents chosen from halogen, hydroxy, alkyl, alkenyl, alkynyl, heteroalkyl, haloalkyl, perhaloalkyl, perhaloalkoxy, alkoxy, haloalkoxy, alkoxyalkyl, acyl, oxo, acyloxy, carboxyl, amido, cyano, amino, alkylamino, alkylaminoalkyl, thiol, alkylthio, alkylthioalkyl, haloalkylthio, perhaloalkylthio, nitro, aryl, arylalkyl, cycloalkylalkyl, heterocycle-alkyl, cycloalkyl, heterocycloalkyl, heteroaryl, heteroarylalkyl, alkylsulfonyl, sulfonamide, alkylsulfonamido, alkylsilyloxy, alkylsulfinamido, arylsulfinamido and arylsulfonamido; R²⁴ is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl, which independently is optionally substituted by one or more substituents chosen from halogen, hydroxy, alkyl, alkenyl, alkynyl, heteroalkyl, haloalkyl, perhaloalkyl, perhaloalkoxy, alkoxy, haloalkoxy, alkoxyalkyl, acyl, oxo, acyloxy, carboxyl, amido, cyano, amino, alkylamino, alkylaminoalkyl, thiol, alkylthio, alkylthioalkyl, haloalkylthio, perhaloalkylthio, nitro, aryl, arylalkyl, cycloalkylalkyl, heterocycle-alkyl, cycloalkyl, heterocycloalkyl, heteroaryl, heteroarylalkyl, alkylsulfonyl, sulfonamide, alkylsulfonamido and alkylsilyloxy;

R²⁵ and R³³ each independently is halogen, cycloalkyl, heterocycloalkyl, aryl or heteroaryl, which independently is optionally substituted by one or more substituents chosen from halogen, hydroxy, alkyl, alkenyl, alkynyl, heteroalkyl, haloalkyl, perhaloalkyl, perhaloalkoxy, alkoxy, haloalkoxy, alkoxyalkyl, acyl, oxo, acyloxy, carboxyl, amido, cyano, amino, alkylamino, alkylaminoalkyl, thiol, alkylthio, alkylthioalkyl, haloalkylthio, perhaloalkylthio, nitro, aryl, arylalkyl, cycloalkylalkyl, heterocycle-alkyl, cycloalkyl, heterocycloalkyl, heteroaryl, heteroarylalkyl, alkylsulfonyl, sulfonamide, alkylsulfonamido and alkylsilyloxy;

R³⁰ has a structure of Formula F-1 :

Formula F-1

wherein:

X⁶⁰, X⁶¹ , X⁶², X⁶³, X⁶⁴ and X⁶⁵ together form a heterocycloalkyl ring;

one, two or three of X⁶⁰, X⁶¹ , X⁶², X⁶³, X⁶⁴ and X⁶⁵ are oxygen, nitrogen or sulfur heteroatoms, and the remaining X⁶⁰, X⁶¹ , X⁶², X⁶³, X⁶⁴ and X⁶⁵ are carbon; and

R⁶¹ , R⁶², R⁶³, R⁶⁴ and R⁶⁵ each represent zero, one or two substituents and each of which substituents independently is hydrogen, halogen, hydroxy, C1 -C3 alkyl, C1 -C3 heteroalkyl, C1 - C3 haloalkyl, C1 -C3 perhaloalkyl, C1 -C3 perhaloalkoxy, C1 -C3 alkoxy, C1 -C3 haloalkoxy, C1 - C3 alkoxy C1 -C3 alkyl, C1 -C4 acyl, oxo, C1 -C4 acyloxy, carboxyl, amido, cyano, amino, C1 -C3 alkylamino, C1-C3 alkylamino C1 -C3 alkyl, thiol, C1 -C3 alkylthio, C1 -C3 alkylthio C1 -C3 alkyl, C1 -C3 haloalkylthio, C1 -C3 perhaloalkylthio, nitro, C5-C7 aryl, C5-C7 aryl C1 -C3 alkyl, C5-C7 cycloalkyl, C5-C7 cycloalkyl C1 -C3 alkyl, 5-7 membered heterocycloalkyl, 5-7 membered heterocycle C1 -C3 alkyl, 5-7 membered heteroaryl, 5-7 membered heteroaryl C1 -C3 alkyl, C1 - C3 alkylsulfonyl, sulfonamide, C1 -C3 alkylsulfonamido or C1 -C3 alkylsilyloxy.

[0105] In certain embodiments of compounds having a structure of the above-defined Formula B, wherein R³⁰ has a structure of Formula F-1 , R^22A is hydrogen and R^22B is halogen, -NR²⁷R²⁸ or -R^H-R²⁹-R³¹-R³⁰, or R^22B is hydrogen and R^22A is halogen, -NR²⁷R²⁸ or -R^H-R²⁹-R³¹-R³⁰. In certain embodiments of compounds having a structure of the above-defined Formula B, wherein R³⁰ has a structure of Formula F-1 , R^22A or R^22B is -R^H-R²⁹-R³¹-R³⁰. In certain embodiments of the above-defined Formula B, wherein R³⁰ has a structure of Formula F-1 , R^H is -O-C(O)-.

[0106] In certain embodiments of the above-defined Formula B, wherein R³⁰ has a structure of Formula F-1 , when X⁶¹ , X⁶², X⁶³, X⁶⁴ and X⁶⁵ independently is carbon, R⁶¹ , R⁶², R⁶³, R⁶⁴ and R⁶⁵, respectively, independently is two substituents, each of which two substituents

independently is a Group A moiety chosen from hydrogen, halogen, hydroxy, C1 -C3 alkyl, C1 - C3 heteroalkyl, C1 -C3 haloalkyl, C1 -C3 perhaloalkyl, C1 -C3 perhaloalkoxy, C1-C3 alkoxy, C1 - C3 haloalkoxy, C1 -C3 alkoxy C1 -C3 alkyl, acyl, oxo, acyloxy, carboxyl, amido, cyano, amino, C1 -C3 alkylamino, C1 -C3 alkylaminoalkyl, thiol, C1-C3 alkylthio, C1-C3 alkylthio C1-C3 alkyl, C1 -C3 haloalkylthio, C1 -C3 perhaloalkylthio, nitro, C5-C7 aryl, C5-C7 aryl C1 -C3 alkyl, C5-C7 cycloalkyl C1 -C3 alkyl, 5-7 membered heterocycle-C1 -C3 alkyl, C5-C7 cycloalkyl, 5-7 membered heterocycloalkyl, 5-7 membered heteroaryl, 5-7 membered heteroaryl C1 -C3 alkyl, C1 -C3 alkylsulfonyl, sulfonamide, C1-C3 alkylsulfonamido and C1-C3 alkylsilyloxy. Stated another way, when X⁶¹ is carbon, R⁶¹ is two substituents, each of which two substituents independently is a Group A moiety; when X⁶² is carbon, R⁶² is two substituents, each of which two substituents independently is a Group A moiety; when X⁶³ is carbon, R⁶³ is two substituents, each of which two substituents independently is a Group A moiety; when X⁶⁴ is carbon, R⁶⁴ is two substituents, each of which two substituents independently is a Group A moiety; and when X⁶⁵ is carbon, R⁶⁵ is two substituents, each of which two substituents independently is a Group A moiety. In certain of these embodiments, R^22A or R^22B is -R^H-R²⁹-R³¹-R³⁰. In certain of these embodiments, R^H is -O-C(O)-.

[0107] In certain embodiments of the above-defined Formula B, wherein R³⁰ has a structure of Formula F-1 , when X⁶¹ , X⁶², X⁶³, X⁶⁴ and X⁶⁵ independently is nitrogen, R⁶¹ , R⁶², R⁶³, R⁶⁴ and R⁶⁵, respectively, independently is a Group B moiety chosen from hydrogen, C1 -C3 alkyl, C1-C3 heteroalkyl, C1 -C3 haloalkyl, C1 -C3 perhaloalkyl, C1 -C4 acyl, amido, C1 -C3 alkylamino, C1 -C3 alkylamino C1 -C3 alkyl, thiol, C1 -C3 alkylthio, C1 -C3 alkylthio C1 -C3 alkyl, C1 -C3 haloalkylthio, C1 -C3 perhaloalkylthio, C5-C7 aryl, C5-C7 aryl C1 -C3 alkyl, C5-C7 cycloalkyl, C5-C7 cycloalkyl C1 -C3 alkyl, 5-7 membered heterocycloalkyl, 5-7 membered heterocycle C1 -C3 alkyl, 5-7 membered heteroaryl, 5-7 membered heteroaryl C1 -C3 alkyl, C1 -C3 alkylsulfonyl, sulfonamide, C1 -C3 alkylsulfonamido and C1 -C3 alkylsilyloxy. Stated another way, when X⁴¹ is nitrogen, R⁴¹ is a Group B moiety; when X⁴² is nitrogen, R⁴² is a Group B moiety; when X⁴³ is nitrogen, R⁴³ is a Group B moiety; when X⁴⁴ is nitrogen, R⁴⁴ is a Group B moiety; and when X⁴⁵ is nitrogen, R⁴⁵ is a Group B moiety. In certain of these embodiments, R^22A or R^22B is -R^H-R²⁹-R³¹-R³⁰. In certain of these embodiments, R^H is -O-C(O)-.

[0108] In certain embodiments of the above-defined Formula B, wherein R³⁰ has a structure of Formula F-1 , when X⁶¹ , X⁶², X⁶³, X⁶⁴ and X⁶⁵ independently is oxygen or sulfur, R⁶¹, R⁶², R⁶³, R⁶⁴ and R⁶⁵, respectively, independently is not present. Stated another way, when X⁶¹ is oxygen or sulfur, R⁶¹ is not present; when X⁶² is oxygen or sulfur, R⁶² is not present; when X⁶³ is oxygen or sulfur, R⁶³ is not present; when X⁶⁴ is oxygen or sulfur, R⁶⁴ is not present; and when X⁶⁵ is oxygen or sulfur, R⁶⁵ is not present. In certain of these embodiments, R^22A or R^22B is -R^H-R²⁹- R³ⁱ__R ³o | _n certain of these embodiments, R^H is -O-C(O)-.

[0109] In some embodiments, compounds provided herein have a structure of Formula B, or a pharmaceutically acceptable salt thereof, wherein:

R^20A is hydrogen and R^20B is -R²³ or -R^F-R²³, or R^20B is hydrogen and R^20A is -R²³ or -R^F-R²³; R^21A is hydrogen and R^{21 B} is hydroxy, -R^G-R²⁴ -R^G-R²⁴-R²⁵ or -R^G-R²⁴-R²⁶-R²⁵, or R^{21 B} is hydrogen and R^21A is hydroxy, -R^G-R²⁴ -R^G-R²⁴-R²⁵ or -R^G-R²⁴-R²⁶-R²⁵; R^22A is hydrogen and R^22B is halogen, -NR²⁷R²⁸, -R^H-R²⁹-R³⁰ or -R^H-R²⁹-R³¹-R³⁰, or R^22B is hydrogen and R^22A is halogen, -NR²⁷R²⁸, -R^H-R²⁹-R³⁰ or -R^H-R²⁹-R³¹-R³⁰;

n is 1 or 2;

R²⁴ has a structure of Formula C-2:

Formula C-2

wherein:

X⁴⁰, X⁴¹ , X⁴², X⁴³, X⁴⁴ and X⁴⁵ together form an aryl or heteroaryl ring;

zero, one, two or three of X⁴⁰, X⁴¹, X⁴², X⁴³, X⁴⁴ and X⁴⁵ are oxygen, nitrogen or sulfur heteroatoms, and the remaining X⁴⁰, X⁴¹ , X⁴², X⁴³, X⁴⁴ and X⁴⁵ are carbon; and

one, two or three of R⁴¹ , R⁴², R⁴³, R⁴⁴ and R⁴⁵ independently is R²⁶-R²⁵, and each of the remaining R⁴¹, R⁴², R⁴³, R⁴⁴ and R⁴⁵ independently is not present or independently is hydrogen, halogen, hydroxy, C1 -C3 alkyl, C1-C3 heteroalkyl, C1 -C3 haloalkyl, C1 -C3 perhaloalkyl, C1-C3 perhaloalkoxy, C1-C3 alkoxy, C1 -C3 haloalkoxy, C1 -C3 alkoxy C1-C3 alkyl, C1 -C4 acyl, oxo, C1 -C4 acyloxy, carboxyl, amido, cyano, amino, C1-C3 alkylamino, C1-C3 alkylamino C1 -C3 alkyl, thiol, C1 -C3 alkylthio, C1 -C3 alkylthio C1 -C3 alkyl, C1 -C3 haloalkylthio, C1 -C3 perhaloalkylthio, nitro, C5-C7 aryl, C5-C7 aryl C1 -C3 alkyl, C5-C7 cycloalkyl, C5-C7 cycloalkyl C1 -C3 alkyl, 5-7 membered heterocycloalkyl, 5-7 membered heterocycle C1 -C3 alkyl, 5-7 membered heteroaryl, 5-7 membered heteroaryl C1 -C3 alkyl, C1 -C3 alkylsulfonyl, sulfonamide, C1 -C3 alkylsulfonamido or C1 -C3 alkylsilyloxy.

[0110] In certain embodiments of compounds having a structure of the above-defined Formula B, wherein R²⁴ has a structure of Formula C-2, R^22A is hydrogen and R^22B is halogen, -NR²⁷R²⁸ or -R^H-R²⁹-R³¹-R³⁰, or R^22B is hydrogen and R^22A is halogen, -NR²⁷R²⁸ or -R^H-R²⁹-R³¹-R³⁰. In certain embodiments of compounds having a structure of the above-defined Formula B, wherein R²⁴ has a structure of Formula C-2, R^21A or R^{21 B} is - R^G-R²⁴-R²⁶-R²⁵. In certain embodiments of the above-defined Formula B, wherein R²⁴ has a structure of Formula C-2, R^G is -O-C(O)-. [0111] In certain embodiments of the above-defined Formula B, wherein R²⁴ has a structure of Formula C-2, when X⁴¹ , X⁴², X⁴³, X⁴⁴ and X⁴⁵ independently is carbon, R⁴¹, R⁴², R⁴³, R⁴⁴ and R⁴⁵, respectively, independently is a Group A moiety chosen from R²⁶-R²⁵, hydrogen, halogen, hydroxy, C1 -C3 alkyl, C1-C3 heteroalkyl, C1 -C3 haloalkyl, C1 -C3 perhaloalkyl, C1 -C3 perhaloalkoxy, C1-C3 alkoxy, C1 -C3 haloalkoxy, C1 -C3 alkoxy C1-C3 alkyl, acyl, oxo, acyloxy, carboxyl, amido, cyano, amino, C1 -C3 alkylamino, C1 -C3 alkylaminoalkyl, thiol, C1 -C3 alkylthio, C1 -C3 alkylthio C1 -C3 alkyl, C1 -C3 haloalkylthio, C1 -C3 perhaloalkylthio, nitro, C5-C7 aryl, C5- C7 aryl C1 -C3 alkyl, C5-C7 cycloalkyl C1 -C3 alkyl, 5-7 membered heterocycle-C1 -C3 alkyl, C5- C7 cycloalkyl, 5-7 membered heterocycloalkyl, 5-7 membered heteroaryl, 5-7 membered heteroaryl C1 -C3 alkyl, C1 -C3 alkylsulfonyl, sulfonamide, C1-C3 alkylsulfonamido and C1-C3 alkylsilyloxy. Stated another way, when X⁴¹ is carbon, R⁴¹ is a Group A moiety; when X⁴² is carbon, R⁴² is a Group A moiety; when X⁴³ is carbon, R⁴³ is a Group A moiety; when X⁴⁴ is carbon, R⁴⁴ is a Group A moiety; and when X⁴⁵ is carbon, R⁴⁵ is a Group A moiety. In certain of these embodiments, R^21A or R^{21 B} is - R^G-R²⁴-R²⁶-R²⁵. In certain of these embodiments, R^G is - O-C(O)-.

[0112] In certain embodiments of the above-defined Formula B, wherein R²⁴ has a structure of Formula C-2, when X⁴¹ , X⁴², X⁴³, X⁴⁴ and X⁴⁵ independently is nitrogen, R⁴¹ , R⁴², R⁴³, R⁴⁴ and R⁴⁵, respectively, independently is a Group B moiety chosen from hydrogen, C1 -C3 alkyl or C1 - C3 alkyl substituted with hydroxyl, halogen or NR²⁷R²⁸. Stated another way, when X⁴¹ is nitrogen, R⁴¹ is a Group B moiety; when X⁴² is nitrogen, R⁴² is a Group B moiety; when X⁴³ is nitrogen, R⁴³ is a Group B moiety; when X⁴⁴ is nitrogen, R⁴⁴ is a Group B moiety; and when X⁴⁵ is nitrogen, R⁴⁵ is a Group B moiety. In certain of these embodiments, R^21A or R^21B is - R^G-R²⁴- _R ²⁶_R25 I _n certain of these embodiments, R^G is -O-C(O)-.

[0113] In certain embodiments of the above-defined Formula B, wherein R²⁴ has a structure of Formula C-2, when X⁴¹ , X⁴², X⁴³, X⁴⁴ and X⁴⁵ independently is oxygen or sulfur, R⁴¹ , R⁴², R⁴³, R⁴⁴ and R⁴⁵, respectively, independently is not present. Stated another way, when X⁴¹ is oxygen or sulfur, R⁴¹ is not present; when X⁴² is oxygen or sulfur, R⁴² is not present; when X⁴³ is oxygen or sulfur, R⁴³ is not present; when X⁴⁴ is oxygen or sulfur, R⁴⁴ is not present; and when X⁴⁵ is oxygen or sulfur, R⁴⁵ is not present. In certain of these embodiments, R^21A or R^{21 B} is - R^G-R²⁴- _R ²⁶_R25 I _n certain of these embodiments, R^G is -O-C(O)-.

[0114] In some embodiments, compounds provided herein have a structure of Formula B, or a pharmaceutically acceptable salt thereof, wherein:

- R^F-R²³ or -R'-R³⁴; R^21A is hydrogen and R^{21 B} is hydrogen, hydroxyl, -R^G-R²⁴ -R^G-R²⁴-R²⁵ or -R^G-R²⁴-R²⁶-R²⁵, or R^21B is hydrogen and R^21A is hydrogen, hydroxyl, -R^G-R²⁴ -R^G-R²⁴-R²⁵ or -R^G-R²⁴-R²⁶-R²⁵;

R^22A is hydrogen and R^22B is hydrogen, hydroxyl, halogen, -N³, -NR²⁷R²⁸, -R^H-R²⁹, -R^H-R²⁹-R³⁰ or -R^H-R²⁹-R³¹-R³⁰, or R^22B is hydrogen and R^22A is hydrogen, hydroxyl, halogen, -N³, - NR²⁷R²⁸, -R^H-R²⁹, -R^H-R²⁹-R³⁰ or -R^H-R²⁹-R³¹-R³⁰;

n is 1 or 2;

R' is -0-S(0)_n-,— S(0)_n-, -S(0)_n-0-, -S(O) _n-NH-, -NH-C(O)-, or -NH-S(O)-;

n is 1 or 2;

R²³ independently is a C5-C7 cycloalkyl or 5-7 membered heteroaryl containing 2 or more heteroatoms, which independently is optionally substituted by one or more substituents chosen from halogen, hydroxyl, alkyl, alkenyl, alkynyl, heteroalkyl, haloalkyl, perhaloalkyl,

perhaloalkoxy, alkoxy, haloalkoxy, alkoxyalkyl, acyl, oxo, acyloxy, carboxyl, amido, cyano, amino, alkylamino, alkylaminoalkyl, thiol, alkylthio, alkylthioalkyl, haloalkylthio, perhaloalkylthio, nitro, aryl, arylalkyl, cycloalkylalkyl, heterocycle-alkyl, cycloalkyl, heterocycloalkyl, heteroaryl, heteroarylalkyl, alkylsulfonyl, sulfonamide, alkylsulfonamido and alkylsilyloxy;

alkylaminoalkyl, thiol, alkylthio, alkylthioalkyl, haloalkylthio, perhaloalkylthio and nitro; R²⁷ and R²⁸ each independently is -R³²-R³³, or hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, perhaloalkyl, alkoxy, cycloalkyl, aryl, heterocycloalkyl, heterocycle-alkyl or heteroaryl, which independently is optionally substituted with one or more substituents chosen from halogen, hydroxyl, alkyl, alkenyl, alkynyl, heteroalkyl, haloalkyl, perhaloalkyl, perhaloalkoxy, alkoxy, haloalkoxy, alkoxyalkyl, acyl, oxo, acyloxy, carboxyl, amido, cyano, amino, alkylamino, alkylaminoalkyl, thiol, alkylthio, alkylthioalkyl, haloalkylthio, perhaloalkylthio, nitro, aryl, arylalkyl, cycloalkylalkyl, heterocycle-alkyl, cycloalkyl, heterocycloalkyl, heteroaryl, heteroarylalkyl, alkylsulfonyl, sulfonamide, alkylsulfonamido and alkylsilyloxy;

R³⁴ independently is alkyl, alkenyl, alkynyl, heteroalkyl or amino, which independently is optionally substituted by one or more substituents chosen from halogen, hydroxyl, alkyl, alkenyl, alkynyl, heteroalkyl, haloalkyl, perhaloalkyl, perhaloalkoxy, alkoxy, haloalkoxy, alkoxyalkyl, acyl, oxo, acyloxy, carboxyl, amido, cyano, amino, alkylamino, alkylaminoalkyl, thiol, alkylthio, alkylthioalkyl, haloalkylthio, perhaloalkylthio, nitro, aryl, arylalkyl, cycloalkylalkyl, heterocycle- alkyl, cycloalkyl, heterocycloalkyl, heteroaryl, heteroarylalkyl, alkylsulfonyl, sulfonamide, alkylsulfonamido and alkylsilyloxy, with the proviso that when R' is -NH-S(O)-, R³⁴ is not straight-chain alkyl; and

R²⁹ has a structure of Formula C-1 :

Formula C-1

wherein:

X⁴⁰, X⁴¹, X⁴², X⁴³, X⁴⁴ and X⁴⁵ together form an aryl or heteroaryl ring;

one, two or three of R⁴¹, R⁴², R⁴³, R⁴⁴ and R⁴⁵ independently is R³¹-R³⁰, and each of the remaining R⁴¹, R⁴², R⁴³, R⁴⁴ and R⁴⁵ independently is not present or independently is hydrogen, halogen, hydroxy, C1-C3 alkyl, C1-C3 heteroalkyl, C1-C3 haloalkyl, C1-C3 perhaloalkyl, C1-C3 perhaloalkoxy, C1-C3 alkoxy, C1-C3 haloalkoxy, C1-C3 alkoxy C1-C3 alkyl,

C1-C4 acyl, oxo, C1-C4 acyloxy, carboxyl, amido, cyano, amino, C1-C3 alkylamino, C1-C3 alkylamino C1-C3 alkyl, thiol, C1-C3 alkylthio, C1-C3 alkylthio C1-C3 alkyl, C1-C3 haloalkylthio,

C1-C3 perhaloalkylthio, nitro, C5-C7 aryl, C5-C7 aryl C1-C3 alkyl, C5-C7 cycloalkyl, C5-C7 cycloalkyl C1-C3 alkyl, 5-7 membered heterocycloalkyl, 5-7 membered heterocycle C1-C3 alkyl, 5-7 membered heteroaryl, 5-7 membered heteroaryl C1 -C3 alkyl, C1 -C3 alkylsulfonyl, sulfonamide, C1 -C3 alkylsulfonamido or C1 -C3 alkylsilyloxy.

[0115] In certain embodiments of compounds having a structure of Formula B as defined directly above, wherein R²⁹ has the structure of Formula C-1 , each of R^20A, R^20B, R^21A, R^21B, R^F, R^G, R^H, R', R²³, R²⁴, R²⁵, R²⁶, R²⁷, R²⁸, R³⁰, R³¹ , R³², R³³ and n are as set forth above, but R^22A is hydrogen and R^22B is hydrogen, hydroxyl, halogen, N₃, -NR²⁷R²⁸ or -R^H-R²⁹-R³¹-R³⁰, or R^22B is hydrogen and R^22A is hydrogen, hydroxyl, halogen, N₃, -NR²⁷R²⁸ or -R^H-R²⁹-R³¹-R³⁰. In certain embodiments of compounds having a structure of Formula B as defined directly above, wherein R²⁹ has a structure of Formula C-1 , R^22A or R^22B is -R^H-R²⁹-R³¹-R³⁰. In certain embodiments of the above-defined Formula B, wherein R²⁹ has a structure of Formula C-1 , R^H is -O-C(O)-.

[0116] In certain embodiments of the above-defined Formula B, wherein R²⁹ has a structure of Formula C-1 , when X⁴¹ , X⁴², X⁴³, X⁴⁴ and X⁴⁵ independently is carbon, R⁴¹ , R⁴², R⁴³, R⁴⁴ and R⁴⁵, respectively, independently is a Group A moiety chosen from R³¹-R³⁰, hydrogen, halogen, hydroxy, C1 -C3 alkyl, C1-C3 heteroalkyl, C1 -C3 haloalkyl, C1 -C3 perhaloalkyl, C1 -C3 perhaloalkoxy, C1-C3 alkoxy, C1 -C3 haloalkoxy, C1 -C3 alkoxy C1-C3 alkyl, acyl, oxo, acyloxy, carboxyl, amido, cyano, amino, C1 -C3 alkylamino, C1 -C3 alkylaminoalkyl, thiol, C1 -C3 alkylthio, C1 -C3 alkylthio C1 -C3 alkyl, C1 -C3 haloalkylthio, C1 -C3 perhaloalkylthio, nitro, C5-C7 aryl, C5- C7 aryl C1 -C3 alkyl, C5-C7 cycloalkyl C1 -C3 alkyl, 5-7 membered heterocycle-C1 -C3 alkyl, C5- C7 cycloalkyl, 5-7 membered heterocycloalkyl, 5-7 membered heteroaryl, 5-7 membered heteroaryl C1 -C3 alkyl, C1 -C3 alkylsulfonyl, sulfonamide, C1-C3 alkylsulfonamido and C1-C3 alkylsilyloxy. Stated another way, when X⁴¹ is carbon, R⁴¹ is a Group A moiety; when X⁴² is carbon, R⁴² is a Group A moiety; when X⁴³ is carbon, R⁴³ is a Group A moiety; when X⁴⁴ is carbon, R⁴⁴ is a Group A moiety; and when X⁴⁵ is carbon, R⁴⁵ is a Group A moiety. In certain of these embodiments, R^22A or R^22B is -R^H-R²⁹-R³¹-R³⁰. In certain of these embodiments, R^H is - C(O)-.

[0117] In certain embodiments of the above-defined Formula B, wherein R²⁹ has a structure of Formula C-1 , when X⁴¹ , X⁴², X⁴³, X⁴⁴ and X⁴⁵ independently is nitrogen, R⁴¹ , R⁴², R⁴³, R⁴⁴ and R⁴⁵, respectively, independently is a Group B moiety chosen from hydrogen, C1 -C3 alkyl or C1 - C3 alkyl substituted with hydroxyl, halogen or NR²⁷R²⁸. Stated another way, when X⁴¹ is nitrogen, R⁴¹ is a Group B moiety; when X⁴² is nitrogen, R⁴² is a Group B moiety; when X⁴³ is nitrogen, R⁴³ is a Group B moiety; when X⁴⁴ is nitrogen, R⁴⁴ is a Group B moiety; and when X⁴⁵ is nitrogen, R⁴⁵ is a Group B moiety. In certain of these embodiments, R^22A or R^22B is -R^H-R²⁹- R3I_R3O I _n certain of these embodiments, R^H is -C(O)-. [0118] In certain embodiments of the above-defined Formula B, wherein R²⁹ has a structure of Formula C-1 , when X⁴¹, X⁴², X⁴³, X⁴⁴ and X⁴⁵ independently is oxygen or sulfur, R⁴¹ , R⁴², R⁴³, R⁴⁴ and R⁴⁵, respectively, independently is not present. Stated another way, when X⁴¹ is oxygen or sulfur, R⁴¹ is not present; when X⁴² is oxygen or sulfur, R⁴² is not present; when X⁴³ is oxygen or sulfur, R⁴³ is not present; when X⁴⁴ is oxygen or sulfur, R⁴⁴ is not present; and when X⁴⁵ is oxygen or sulfur, R⁴⁵ is not present. In certain of these embodiments, R^22A or R^22B is -R^H-R²⁹- R³I_R³O I _n certain of these embodiments, R^H is -C(O)-.

[0119] In some embodiments, compounds provided herein have a structure of Formula B, or a pharmaceutically acceptable salt thereof, wherein:

- R^F-R²³ or -R'-R³⁴;

R^21A is hydrogen and R^{21 B} is hydrogen, hydroxyl, -R^G-R²⁴ -R^G-R²⁴-R²⁵ or -R^G-R²⁴-R²⁶-R²⁵, or R^21B is hydrogen and R^21A is hydrogen, hydroxyl, -R^G-R²⁴ -R^G-R²⁴-R²⁵ or -R^G-R²⁴-R²⁶-R²⁵;

R^22A is hydrogen and R^22B is hydrogen, hydroxy, halogen, -N³, -NR²⁷R²⁸, -R^H-R²⁹, -R^H-R²⁹-R³⁰ or -R^H-R²⁹-R³¹-R³⁰, or R^22B is hydrogen and R^22A is hydrogen, hydroxy, halogen, -N³, -NR²⁷R²⁸, - R^H-R²⁹, -R^H-R²⁹-R³⁰ or -R^H-R²⁹-R³¹-R³⁰;

n is 1 or 2;

R' is— O— S(O) _n— ,— S(0)_n— ,— S(O) _n— O— , -S(O) _n-NH-, -NH-C(O)-, or -NH-S(O)-;

n is 1 or 2;

R²⁴ and R²⁹ each independently is:

(1) alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl, which independently is optionally substituted by one or more substituents chosen from halogen, hydroxyl, alkyl, alkenyl, alkynyl, heteroalkyl, haloalkyl, perhaloalkyl, perhaloalkoxy, alkoxy, haloalkoxy, alkoxyalkyl, acyl, oxo, acyloxy, carboxyl, amido, cyano, amino, alkylamino, alkylaminoalkyl, thiol, alkylthio, alkylthioalkyl, haloalkylthio, perhaloalkylthio, nitro, aryl, arylalkyl, cycloalkylalkyl, heterocycle-alkyl, cycloalkyl, heterocycloalkyl, heteroaryl, heteroarylalkyl, alkylsulfonyl, sulfonamide, alkylsulfonamido and alkylsilyloxy

or

R²⁷ and R²⁸ each independently is -R³²-R³³, or hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, perhaloalkyl, alkoxy, cycloalkyl, aryl, heterocycloalkyl, heterocycle-alkyl or heteroaryl, which independently is optionally substituted with one or more substituents chosen from halogen, hydroxy, alkyl, alkenyl, alkynyl, heteroalkyl, haloalkyl, perhaloalkyl, perhaloalkoxy, alkoxy, haloalkoxy, alkoxyalkyl, acyl, oxo, acyloxy, carboxyl, amido, cyano, amino, alkylamino, alkylaminoalkyl, thiol, alkylthio, alkylthioalkyl, haloalkylthio, perhaloalkylthio, nitro, aryl, arylalkyl, cycloalkylalkyl, heterocycle-alkyl, cycloalkyl, heterocycloalkyl, heteroaryl, heteroarylalkyl, alkylsulfonyl, sulfonamide, alkylsulfonamido and alkylsilyloxy;

R³⁴ independently is alkyl, alkenyl, alkynyl, heteroalkyl or amino, which independently is optionally substituted by one or more substituents chosen from halogen, hydroxy, alkyl, alkenyl, alkynyl, heteroalkyl, haloalkyl, perhaloalkyl, perhaloalkoxy, alkoxy, haloalkoxy, alkoxyalkyl, acyl, oxo, acyloxy, carboxyl, amido, cyano, amino, alkylamino, alkylaminoalkyl, thiol, alkylthio, alkylthioalkyl, haloalkylthio, perhaloalkylthio, nitro, aryl, arylalkyl, cycloalkylalkyl, heterocycle- alkyl, cycloalkyl, heterocycloalkyl, heteroaryl, heteroarylalkyl, alkylsulfonyl, sulfonamide, alkylsulfonamido and alkylsilyloxy, with the proviso that when R¹ is -NH-S(O)-, R³⁴ is not straight-chain alkyl; and

R³⁰ has a structure of Formula F-1 :

Formula F- 1

wherein:

[0120] In certain embodiments of compounds having a structure of Formula B as defined directly above, wherein R30 has the structure of Formula F-1 , each of R^20A, R^20B, R^21A, R^{21 B}, R^F, R^G, R^H, R¹, R²³, R²⁴, R²⁵, R²⁶, R²⁷, R²⁸, R²⁹, R³¹ , R³², R³³ and n are as set forth above, but R^22A is hydrogen and R^22B is hydrogen, hydroxy, halogen, N₃, -NR²⁷R²⁸ or -R^H-R²⁹-R³¹-R³⁰, or R^22B is hydrogen and R^22A is hydrogen, hydroxy, halogen, N₃, -NR²⁷R²⁸ or -R^H-R²⁹-R³¹-R³⁰. In certain embodiments of compounds having a structure of the above-defined Formula B, wherein R³⁰ has a structure of Formula F-1 , R^22A or R^22B is -R^H-R²⁹-R³¹-R³⁰. In certain embodiments of the above-defined Formula B, wherein R³⁰ has a structure of Formula F-1 , R^H is -O-C(O)-.

[0121] In certain embodiments of the above-defined Formula B, wherein R³⁰ has a structure of Formula F-1 , when X⁶¹ , X⁶², X⁶³, X⁶⁴ and X⁶⁵ independently is carbon, R⁶¹, R⁶², R⁶³, R⁶⁴ and R⁶⁵, respectively, independently is two substituents, each of which two substituents independently is a Group A moiety chosen from hydrogen, halogen, hydroxy, C1 -C3 alkyl, C1 -C3 heteroalkyl, C1 -C3 haloalkyl, C1 -C3 perhaloalkyl, C1 -C3 perhaloalkoxy, C1 -C3 alkoxy, C1 -C3 haloalkoxy, C1 -C3 alkoxy C1 -C3 alkyl, acyl, oxo, acyloxy, carboxyl, amido, cyano, amino, C1 -C3 alkylamino, C1-C3 alkylaminoalkyl, thiol, C1 -C3 alkylthio, C1 -C3 alkylthio C1 -C3 alkyl, C1 -C3 haloalkylthio, C1 -C3 perhaloalkylthio, nitro, C5-C7 aryl, C5-C7 aryl C1 -C3 alkyl, C5-C7 cycloalkyl C1 -C3 alkyl, 5-7 membered heterocycle-C1 -C3 alkyl, C5-C7 cycloalkyl, 5-7 membered heterocycloalkyl, 5-7 membered heteroaryl, 5-7 membered heteroaryl C1 -C3 alkyl, C1 -C3 alkylsulfonyl, sulfonamide, C1-C3 alkylsulfonamido and C1-C3 alkylsilyloxy. Stated another way, when X⁶¹ is carbon, R⁶¹ is two substituents, each of which two substituents independently is a Group A moiety; when X⁶² is carbon, R⁶² is two substituents, each of which two substituents independently is a Group A moiety; when X⁶³ is carbon, R⁶³ is two substituents, each of which two substituents independently is a Group A moiety; when X⁶⁴ is carbon, R⁶⁴ is two substituents, each of which two substituents independently is a Group A moiety; and when X⁶⁵ is carbon, R⁶⁵ is two substituents, each of which two substituents independently is a Group A moiety. In certain of these embodiments, R^22A or R^22B is -R^H-R²⁹-R³¹-R³⁰. In certain of these embodiments, R^H is -O-C(O)-.

[0122] In certain embodiments of the above-defined Formula B, wherein R³⁰ has a structure of Formula F-1 , when X⁶¹ , X⁶², X⁶³, X⁶⁴ and X⁶⁵ independently is nitrogen, R⁶¹ , R⁶², R⁶³, R⁶⁴ and R⁶⁵, respectively, independently is a Group B moiety chosen from hydrogen, C1 -C3 alkyl, C1-C3 heteroalkyl, C1 -C3 haloalkyl, C1 -C3 perhaloalkyl, C1 -C4 acyl, amido, C1 -C3 alkylamino, C1 -C3 alkylamino C1 -C3 alkyl, thiol, C1 -C3 alkylthio, C1 -C3 alkylthio C1 -C3 alkyl, C1 -C3 haloalkylthio, C1 -C3 perhaloalkylthio, C5-C7 aryl, C5-C7 aryl C1 -C3 alkyl, C5-C7 cycloalkyl, C5-C7 cycloalkyl C1 -C3 alkyl, 5-7 membered heterocycloalkyl, 5-7 membered heterocycle C1 -C3 alkyl, 5-7 membered heteroaryl, 5-7 membered heteroaryl C1 -C3 alkyl, C1 -C3 alkylsulfonyl, sulfonamide, C1 -C3 alkylsulfonamido and C1 -C3 alkylsilyloxy. Stated another way, when X⁴¹ is nitrogen, R⁴¹ is a Group B moiety; when X⁴² is nitrogen, R⁴² is a Group B moiety; when X⁴³ is nitrogen, R⁴³ is a Group B moiety; when X⁴⁴ is nitrogen, R⁴⁴ is a Group B moiety; and when X⁴⁵ is nitrogen, R⁴⁵ is a Group B moiety. In certain of these embodiments, R^22A or R^22B is -R^H-R²⁹-R³¹-R³⁰. In certain of these embodiments, R^H is -O-C(O)-.

[0123] In certain embodiments of the above-defined Formula B, wherein R³⁰ has a structure of Formula F-1 , when X⁶¹ , X⁶², X⁶³, X⁶⁴ and X⁶⁵ independently is oxygen or sulfur, R⁶¹, R⁶², R⁶³, R⁶⁴ and R⁶⁵, respectively, independently is not present. Stated another way, when X⁶¹ is oxygen or sulfur, R⁶¹ is not present; when X⁶² is oxygen or sulfur, R⁶² is not present; when X⁶³ is oxygen or sulfur, R⁶³ is not present; when X⁶⁴ is oxygen or sulfur, R⁶⁴ is not present; and when X⁶⁵ is oxygen or sulfur, R⁶⁵ is not present. In certain of these embodiments, R^22A or R^22B is -R^H-R²⁹- R³I_R³O I _n certain of these embodiments, R^H is -O-C(O)-. [0124] In some embodiments, compounds provided herein have a structure of Formula B, or a pharmaceutically acceptable salt thereof, wherein:

5 - R^F-R²³ or -R'-R³⁴;

R^21A is hydrogen and R^{21 B} is hydrogen, hydroxy, -R^G-R²⁴ -R^G-R²⁴-R²⁵ or -R^G-R²⁴-R²⁶-R²⁵, or

R^21B is hydrogen and R^21A is hydrogen, hydroxy, -R^G-R²⁴ -R^G-R²⁴-R²⁵ or -R^G-R²⁴-R²⁶-R²⁵;

R^22A is hydrogen and R^22B is hydrogen, hydroxy, halogen, -N³, -NR²⁷R²⁸, -R^H-R²⁹, -R^H-R²⁹-R³⁰ or

-R^H-R²⁹-R³¹-R³⁰, or R^22B is hydrogen and R^22A is hydrogen, hydroxy, halogen, -N³, -NR²⁷R²⁸, -

10 ²⁹, - RH_ _R29_ _R30 _{o r} __RH__R29__R31__R30.

n is 1 or 2;

15 R' is— O— S(O) _n— ,— S(0)_n— ,— S(O) _n— O— , -S(O) _n-NH-, -NH-C(O)-, or -NH-S(O)-;

n is 1 or 2;

R²³ independently is a C5-C7 cycloalkyl or 5-7 membered heteroaryl containing 2 or more heteroatoms, which independently is optionally substituted by one or more substituents chosen from halogen, hydroxy, alkyl, alkenyl, alkynyl, heteroalkyl, haloalkyl, perhaloalkyl, perhaloalkoxy,

20 alkoxy, haloalkoxy, alkoxyalkyl, acyl, oxo, acyloxy, carboxyl, amido, cyano, amino, alkylamino, alkylaminoalkyl, thiol, alkylthio, alkylthioalkyl, haloalkylthio, perhaloalkylthio, nitro, aryl, arylalkyl, cycloalkylalkyl, heterocycle-alkyl, cycloalkyl, heterocycloalkyl, heteroaryl, heteroarylalkyl, alkylsulfonyl, sulfonamide, alkylsulfonamido and alkylsilyloxy;

R²⁹ independently is:

25 (1) alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl, which

independently is optionally substituted by one or more substituents chosen from halogen, hydroxy, alkyl, alkenyl, alkynyl, heteroalkyl, haloalkyl, perhaloalkyl, perhaloalkoxy, alkoxy, haloalkoxy, alkoxyalkyl, acyl, oxo, acyloxy, carboxyl, amido, cyano, amino, alkylamino, alkylaminoalkyl, thiol, alkylthio, alkylthioalkyl, haloalkylthio, perhaloalkylthio, nitro, aryl, arylalkyl,

30 cycloalkylalkyl, heterocycle-alkyl, cycloalkyl, heterocycloalkyl, heteroaryl, heteroarylalkyl,

alkylsulfonyl, sulfonamide, alkylsulfonamido and alkylsilyloxy

or

(2) cycloalkyl, heterocycloalkyl, aryl or heteroaryl, which independently is optionally substituted by one or more substituents chosen from halogen, hydroxy, alkyl, alkenyl, alkynyl, heteroalkyl,

35 haloalkyl, perhaloalkyl, perhaloalkoxy, alkoxy, haloalkoxy, alkoxyalkyl, acyl, oxo, acyloxy, carboxyl, amido, cyano, amino, alkylamino, alkylaminoalkyl, thiol, alkylthio, alkylthioalkyl, haloalkylthio, perhaloalkylthio, nitro, aryl, arylalkyl, cycloalkylalkyl, heterocycle-alkyl, cycloalkyl, heterocycloalkyl, heteroaryl, heteroarylalkyl, alkylsulfonyl, sulfonamide, alkylsulfonamido and alkylsilyloxy;

R³⁴ independently is alkyl, alkenyl, alkynyl, heteroalkyl or amino, which independently is optionally substituted by one or more substituents chosen from halogen, hydroxy, alkyl, alkenyl, alkynyl, heteroalkyl, haloalkyl, perhaloalkyl, perhaloalkoxy, alkoxy, haloalkoxy, alkoxyalkyl, acyl, oxo, acyloxy, carboxyl, amido, cyano, amino, alkylamino, alkylaminoalkyl, thiol, alkylthio, alkylthioalkyl, haloalkylthio, perhaloalkylthio, nitro, aryl, arylalkyl, cycloalkylalkyl, heterocycle- alkyl, cycloalkyl, heterocycloalkyl, heteroaryl, heteroarylalkyl, alkylsulfonyl, sulfonamide, alkylsulfonamido and alkylsilyloxy, with the proviso that when R' is -NH-S(O)-, R³⁴ is not straight-chain alkyl; and

R²⁴ has a structure of Formula C-2:

Formula C-2

wherein:

[0125] In certain embodiments of compounds having the structure of Formula B as defined directly above, wherein R²⁴ has the structure of Formula C-2, each of R^20A, R^20B, R^21A, R^21B, R^F, R^G, R^H, R¹, R²³, R²⁴, R²⁵, R²⁶, R²⁷, R²⁸, R²⁹, R³⁰, R³¹ , R³², R³³ and n are as set forth above, but R^22A is hydrogen and R^22B is hydrogen, hydroxy, halogen, N₃, -NR²⁷R²⁸ or -R^H-R²⁹-R³¹-R³⁰, or R^22B is hydrogen and R^22A is hydrogen, hydroxy, halogen, N₃, -NR²⁷R²⁸ or -R^H-R²⁹-R³¹-R³⁰. In certain embodiments of compounds having a structure of Formula B as defined directly above, wherein R²⁴ has a structure of Formula C-2, R^21A or R^{21 B} is - R^G-R²⁴-R²⁶-R²⁵. In certain embodiments of the above-defined Formula B, wherein R²⁴ has a structure of Formula C-2, R^G is -O-C(O)-.

[0126] In certain embodiments of the above-defined Formula B, wherein R²⁴ has a structure of Formula C-2, when X⁴¹ , X⁴², X⁴³, X⁴⁴ and X⁴⁵ independently is carbon, R⁴¹, R⁴², R⁴³, R⁴⁴ and R⁴⁵, respectively, independently is a Group A moiety chosen from R²⁶-R²⁵, hydrogen, halogen, hydroxy, C1 -C3 alkyl, C1-C3 heteroalkyl, C1 -C3 haloalkyl, C1 -C3 perhaloalkyl, C1 -C3 perhaloalkoxy, C1-C3 alkoxy, C1 -C3 haloalkoxy, C1 -C3 alkoxy C1-C3 alkyl, acyl, oxo, acyloxy, carboxyl, amido, cyano, amino, C1 -C3 alkylamino, C1 -C3 alkylaminoalkyl, thiol, C1 -C3 alkylthio, C1 -C3 alkylthio C1 -C3 alkyl, C1 -C3 haloalkylthio, C1 -C3 perhaloalkylthio, nitro, C5-C7 aryl, C5- C7 aryl C1 -C3 alkyl, C5-C7 cycloalkyl C1 -C3 alkyl, 5-7 membered heterocycle-C1 -C3 alkyl, C5- C7 cycloalkyl, 5-7 membered heterocycloalkyl, 5-7 membered heteroaryl, 5-7 membered heteroaryl C1 -C3 alkyl, C1 -C3 alkylsulfonyl, sulfonamide, C1-C3 alkylsulfonamido and C1-C3 alkylsilyloxy. Stated another way, when X⁴¹ is carbon, R⁴¹ is a Group A moiety; when X⁴² is carbon, R⁴² is a Group A moiety; when X⁴³ is carbon, R⁴³ is a Group A moiety; when X⁴⁴ is carbon, R⁴⁴ is a Group A moiety; and when X⁴⁵ is carbon, R⁴⁵ is a Group A moiety. In certain of these embodiments, R^21A or R^{21 B} is - R^G-R²⁴-R²⁶-R²⁵. In certain of these embodiments, R^G is - O-C(O)-.

[0127] In certain embodiments of the above-defined Formula B, wherein R²⁴ has a structure of Formula C-2, when X⁴¹ , X⁴², X⁴³, X⁴⁴ and X⁴⁵ independently is nitrogen, R⁴¹ , R⁴², R⁴³, R⁴⁴ and R⁴⁵, respectively, independently is a Group B moiety chosen from hydrogen, C1 -C3 alkyl or C1 - C3 alkyl substituted with hydroxyl, halogen or NR²⁷R²⁸. Stated another way, when X⁴¹ is nitrogen, R⁴¹ is a Group B moiety; when X⁴² is nitrogen, R⁴² is a Group B moiety; when X⁴³ is nitrogen, R⁴³ is a Group B moiety; when X⁴⁴ is nitrogen, R⁴⁴ is a Group B moiety; and when X⁴⁵ is nitrogen, R⁴⁵ is a Group B moiety. In certain of these embodiments, R^21A or R^21B is - R^G-R²⁴- _R ²⁶_R25 I _n certain of these embodiments, R^G is -O-C(O)-.

[0128] In certain embodiments of the above-defined Formula B, wherein R²⁴ has a structure of Formula C-2, when X⁴¹ , X⁴², X⁴³, X⁴⁴ and X⁴⁵ independently is oxygen or sulfur, R⁴¹ , R⁴², R⁴³, R⁴⁴ and R⁴⁵, respectively, independently is not present. Stated another way, when X⁴¹ is oxygen or sulfur, R⁴¹ is not present; when X⁴² is oxygen or sulfur, R⁴² is not present; when X⁴³ is oxygen or sulfur, R⁴³ is not present; when X⁴⁴ is oxygen or sulfur, R⁴⁴ is not present; and when X⁴⁵ is oxygen or sulfur, R⁴⁵ is not present. In certain of these embodiments, R^21A or R^{21 B} is - R^G-R²⁴- _R ²⁶_R25 I _n certain of these embodiments, R^G is -O-C(O)-.

[0129] In some embodiments, compounds provided herein have a structure of Formula B, or a pharmaceutically acceptable salt thereof, wherein:

R^21A is hydrogen and R^{21 B} is hydroxy, -R^G-R²⁴ -R^G-R²⁴-R²⁵ or -R^G-R²⁴-R²⁶-R²⁵, or R^{21 B} is hydrogen and R^21A is hydroxy, -R^G-R²⁴ -R^G-R²⁴-R²⁵ or -R^G-R²⁴-R²⁶-R²⁵;

R^22A is hydrogen and R^22B is halogen, -N₃, -NR²⁷R²⁸, -R^H-R²⁹, -R^H-R²⁹-R³⁰ or -R^H-R²⁹-R³¹-R³⁰, or R^22B is hydrogen and R^22A is halogen, -N₃, -NR²⁷R²⁸, -R^H-R²⁹, -R^H-R²⁹-R³⁰ or -R^H-R²⁹-R³¹-

R³⁰; R^F, R^G and R^H each independently is -0-, -C(O)-, -O-C(O)-, -C(0)-0-, -S-, -S(0)_n-, -O- S(0)_n-, -S(0)_n-0-, -NH-S(0)_n-, -S(O) _n-NH-, -NH-C(O)-, -C(0)-NH-, -NH-C(S)-, -C(S)- NH-, -S-C(S)-, -C(S)-S-, or -C(S)-;

n is 1 or 2;

R²³ has a structure of Formula C-3:

Formula C-3

wherein:

X⁵⁰, X⁵¹, X⁵², X⁵³, X⁵⁴ and X⁵⁵ together form an aryl or heteroaryl ring;

zero, one, two or three of X⁵⁰, X⁵¹, X⁵², X⁵³, X⁵⁴ and X⁵⁵ are oxygen, nitrogen or sulfur heteroatoms, and the remaining X⁵⁰, X⁵¹ , X⁵², X⁵³, X⁵⁴ and X⁵⁵ are carbon; and

R⁵¹, R⁵², R⁵³, R⁵⁴ and R⁵⁵ each independently is not present or independently is hydrogen, halogen, hydroxy, C1-C3 alkyl, C1-C3 heteroalkyl, C1-C3 haloalkyl, C1-C3 perhaloalkyl, C1-C3 perhaloalkoxy, C1-C3 alkoxy, C1-C3 haloalkoxy, C1-C3 alkoxy C1-C3 alkyl, C1-C4 acyl, oxo, C1-C4 acyloxy, carboxyl, amido, cyano, amino, C1-C3 alkylamino, C1-C3 alkylamino C1-C3 alkyl, thiol, C1-C3 alkylthio, C1-C3 alkylthio C1-C3 alkyl, C1-C3 haloalkylthio, C1-C3 perhaloalkylthio, nitro, C5-C7 aryl, C5-C7 aryl C1-C3 alkyl, C5-C7 cycloalkyl, C5-C7 cycloalkyl C1-C3 alkyl, 5-7 membered heterocycloalkyl, 5-7 membered heterocycle C1-C3 alkyl, 5-7 membered heteroaryl, 5-7 membered heteroaryl C1-C3 alkyl, C1-C3 alkylsulfonyl, sulfonamide, C1-C3 alkylsulfonamido or C1-C3 alkylsilyloxy;

R²⁴ is cycloalkyl, heterocycloalkyl, aryl or heteroaryl, which independently is optionally substituted by one or more substituents chosen from halogen, hydroxy, alkyl, alkenyl, alkynyl, heteroalkyl, haloalkyl, perhaloalkyl, perhaloalkoxy, alkoxy, haloalkoxy, alkoxyalkyl, acyl, oxo, acyloxy, carboxyl, amido, cyano, amino, alkylamino, alkylaminoalkyl, thiol, alkylthio, alkylthioalkyl, haloalkylthio, perhaloalkylthio, nitro, aryl, arylalkyl, cycloalkylalkyl, heterocycle- alkyl, cycloalkyl, heterocycloalkyl, heteroaryl, heteroarylalkyl, alkylsulfonyl, sulfonamide, alkylsulfonamido and alkylsilyloxy;

alkylthioalkyl, haloalkylthio, perhaloalkylthio and nitro;

R²⁷ and R²⁸ each independently is -R³²-R³³, or hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, perhaloalkyl, alkoxy, cycloalkyl, aryl, cycloalkyl, heterocycloalkyl, heterocycle-alkyl or heteroaryl, which independently is optionally substituted with one or more substituents chosen from halogen, hydroxy, alkyl, alkenyl, alkynyl, heteroalkyl, haloalkyl, perhaloalkyl, perhaloalkoxy, alkoxy, haloalkoxy, alkoxyalkyl, acyl, oxo, acyloxy, carboxyl, amido, cyano, amino, alkylamino, alkylaminoalkyl, thiol, alkylthio, alkylthioalkyl, haloalkylthio, perhaloalkylthio, nitro, aryl, arylalkyl, cycloalkylalkyl, heterocycle-alkyl, cycloalkyl, heterocycloalkyl, heteroaryl, heteroarylalkyl, alkylsulfonyl, sulfonamide, alkylsulfonamido, alkylsilyloxy, alkylsulfinamido, arylsulfinamido, and arylsulfonamido; and

R²⁹ has a structure of Formula C-1 :

Formula C-1

wherein:

one, two or three of R⁴¹ , R⁴², R⁴³, R⁴⁴ and R⁴⁵ independently is R³¹-R³⁰, and each of the remaining R⁴¹, R⁴², R⁴³, R⁴⁴ and R⁴⁵ independently is not present or independently is hydrogen, halogen, hydroxyl, C1 -C3 alkyl, C1 -C3 heteroalkyl, C1 -C3 haloalkyl, C1 -C3 perhaloalkyl, C1 -C3 perhaloalkoxy, C1-C3 alkoxy, C1 -C3 haloalkoxy, C1 -C3 alkoxy C1-C3 alkyl, C1 -C4 acyl, oxo,

C1 -C4 acyloxy, carboxyl, amido, cyano, amino, C1-C3 alkylamino, C1-C3 alkylamino C1 -C3 alkyl, thiol, C1 -C3 alkylthio, C1 -C3 alkylthio C1 -C3 alkyl, C1 -C3 haloalkylthio, C1 -C3

perhaloalkylthio, nitro, C5-C7 aryl, C5-C7 aryl C1 -C3 alkyl, C5-C7 cycloalkyl, C5-C7 cycloalkyl

C1 -C3 alkyl, 5-7 membered heterocycloalkyl, 5-7 membered heterocycle C1 -C3 alkyl, 5-7 membered heteroaryl, 5-7 membered heteroaryl C1 -C3 alkyl, C1 -C3 alkylsulfonyl, sulfonamide, C1 -C3 alkylsulfonamido or C1 -C3 alkylsilyloxy.

[0130] In certain embodiments of compounds having a structure of the above-defined Formula B, wherein R²³ has a structure of Formula C-3 and R²⁹ has a structure of Formula C-1 , R^22A or R^22B is -R^H-R²⁹-R³¹-R³⁰. In certain embodiments of the above-defined Formula B, wherein R²⁹ has a structure of Formula C-1 , R^H is -O-C(O)-.

[0131] In certain embodiments of compounds having a structure of the above-defined Formula B, wherein R²³ has a structure of Formula C-3 and R²⁹ has a structure of Formula C-1 , when X⁴¹ , X⁴², X⁴³, X⁴⁴ and X⁴⁵ independently is carbon, R⁴¹ , R⁴², R⁴³, R⁴⁴ and R⁴⁵, respectively, independently is a Group A moiety chosen from R³¹-R³⁰, hydrogen, halogen, hydroxy, C1-C3 alkyl, C1 -C3 heteroalkyl, C1 -C3 haloalkyl, C1 -C3 perhaloalkyl, C1 -C3 perhaloalkoxy, C1 -C3 alkoxy, C1 -C3 haloalkoxy, C1-C3 alkoxy C1 -C3 alkyl, acyl, oxo, acyloxy, carboxyl, amido, cyano, amino, C1 -C3 alkylamino, C1 -C3 alkylaminoalkyl, thiol, C1 -C3 alkylthio, C1 -C3 alkylthio C1 -C3 alkyl, C1 -C3 haloalkylthio, C1-C3 perhaloalkylthio, nitro, C5-C7 aryl, C5-C7 aryl C1-C3 alkyl, C5-C7 cycloalkyl C1 -C3 alkyl, 5-7 membered heterocycle-C1 -C3 alkyl, C5-C7 cycloalkyl, 5-7 membered heterocycloalkyl, 5-7 membered heteroaryl, 5-7 membered heteroaryl C1 -C3 alkyl, C1 -C3 alkylsulfonyl, sulfonamide, C1 -C3 alkylsulfonamido and C1-C3 alkylsilyloxy. Stated another way, when X⁴¹ is carbon, R⁴¹ is a Group A moiety; when X⁴² is carbon, R⁴² is a Group A moiety; when X⁴³ is carbon, R⁴³ is a Group A moiety; when X⁴⁴ is carbon, R⁴⁴ is a Group A moiety; and when X⁴⁵ is carbon, R⁴⁵ is a Group A moiety. In certain of these embodiments, R^22A or R^22B is -R^H-R²⁹-R³¹-R³⁰. In certain of these embodiments, R^H is -O-C(O)-.

[0132] In certain embodiments of the above-defined Formula B, wherein R²³ has a structure of Formula C-3 and R²⁹ has a structure of Formula C-1 , when X⁴¹ , X⁴², X⁴³, X⁴⁴ and X⁴⁵ independently is nitrogen, R⁴¹ , R⁴², R⁴³, R⁴⁴ and R⁴⁵, respectively, independently is a Group B moiety chosen from hydrogen, C1-C3 alkyl or C1 -C3 alkyl substituted with hydroxyl, halogen or NR²⁷R²⁸. Stated another way, when X⁴¹ is nitrogen, R⁴¹ is a Group B moiety; when X⁴² is nitrogen, R⁴² is a Group B moiety; when X⁴³ is nitrogen, R⁴³ is a Group B moiety; when X⁴⁴ is nitrogen, R⁴⁴ is a Group B moiety; and when X⁴⁵ is nitrogen, R⁴⁵ is a Group B moiety. In certain of these embodiments, R^22A or R^22B is -R^H-R²⁹-R³¹-R³⁰. In certain of these embodiments, R^H is -O-C(O)-.

[0133] In certain embodiments of compounds having a structure of the above-defined Formula B, wherein R²³ has a structure of Formula C-3 and R²⁹ has a structure of Formula C-1 , when X⁴¹ , X⁴², X⁴³, X⁴⁴ and X⁴⁵ independently is oxygen or sulfur, R⁴¹ , R⁴², R⁴³, R⁴⁴ and R⁴⁵, respectively, independently is not present. Stated another way, when X⁴¹ is oxygen or sulfur, R⁴¹ is not present; when X⁴² is oxygen or sulfur, R⁴² is not present; when X⁴³ is oxygen or sulfur, R⁴³ is not present; when X⁴⁴ is oxygen or sulfur, R⁴⁴ is not present; and when X⁴⁵ is oxygen or sulfur, R⁴⁵ is not present. In certain of these embodiments, R^22A or R^22B is -R^H-R²⁹-R³¹-R³⁰. In certain of these embodiments, R^H is -O-C(O)-.

[0134] In some embodiments, compounds provided herein have a structure of Formula B, or a pharmaceutically acceptable salt thereof, wherein:

R^21A is hydrogen and R^{21 B} is hydroxy, -R^G-R²⁴ _, -R^G-R²⁴-R²⁵ or -R^G-R²⁴-R²⁶-R²⁵, or R^{21 B} is hydrogen and R^21A is hydroxy, -R^G-R²⁴ -R^G-R²⁴-R²⁵ or -R^G-R²⁴-R²⁶-R²⁵;

R³⁰;

n is 1 or 2;

R²³ has a structure of Formula C-3:

Formula C-3

wherein:

X⁵⁰, X⁵¹ , X⁵², X⁵³, X⁵⁴ and X⁵⁵ together form an aryl or heteroaryl ring;

R⁵¹ , R⁵², R⁵³, R⁵⁴ and R⁵⁵ each independently is not present or independently is hydrogen, halogen, hydroxy, C1 -C3 alkyl, C1-C3 heteroalkyl, C1 -C3 haloalkyl, C1 -C3 perhaloalkyl, C1-C3 perhaloalkoxy, C1-C3 alkoxy, C1 -C3 haloalkoxy, C1 -C3 alkoxy C1-C3 alkyl, C1 -C4 acyl, oxo,

C1 -C4 acyloxy, carboxyl, amido, cyano, amino, C1-C3 alkylamino, C1-C3 alkylamino C1 -C3 alkyl, thiol, C1 -C3 alkylthio, C1 -C3 alkylthio C1 -C3 alkyl, C1 -C3 haloalkylthio, C1 -C3 perhaloalkylthio, nitro, C5-C7 aryl, C5-C7 aryl C1 -C3 alkyl, C5-C7 cycloalkyl, C5-C7 cycloalkyl C1-C3 alkyl, 5-7 membered heterocycloalkyl, 5-7 membered heterocycle C1-C3 alkyl, 5-7 membered heteroaryl, 5-7 membered heteroaryl C1-C3 alkyl, C1-C3 alkylsulfonyl, sulfonamide, C1-C3 alkylsulfonamido or C1-C3 alkylsilyloxy;

alkylthioalkyl, haloalkylthio, perhaloalkylthio and nitro;

R³⁰ has a structure of Formula F-1 :

Formula F- 1

wherein:

R⁶¹ , R⁶², R⁶³, R⁶⁴ and R⁶⁵ each represent zero, one or two substituents and each of which substituents independently is hydrogen, halogen, hydroxy, C1 -C3 alkyl, C1-C3 heteroalkyl, C1 - C3 haloalkyl, C1 -C3 perhaloalkyl, C1 -C3 perhaloalkoxy, C1 -C3 alkoxy, C1 -C3 haloalkoxy, C1 - C3 alkoxy C1 -C3 alkyl, C1 -C4 acyl, oxo, C1 -C4 acyloxy, carboxyl, amido, cyano, amino, C1 -C3 alkylamino, C1-C3 alkylamino C1 -C3 alkyl, thiol, C1 -C3 alkylthio, C1 -C3 alkylthio C1 -C3 alkyl, C1 -C3 haloalkylthio, C1 -C3 perhaloalkylthio, nitro, C5-C7 aryl, C5-C7 aryl C1 -C3 alkyl, C5-C7 cycloalkyl, C5-C7 cycloalkyl C1 -C3 alkyl, 5-7 membered heterocycloalkyl, 5-7 membered heterocycle C1 -C3 alkyl, 5-7 membered heteroaryl, 5-7 membered heteroaryl C1 -C3 alkyl, C1 - C3 alkylsulfonyl, sulfonamide, C1 -C3 alkylsulfonamido or C1 -C3 alkylsilyloxy.

[0135] In certain embodiments of compounds having a structure of the above-defined Formula B, wherein R²³ has a structure of Formula C-3 and R³⁰ has a structure of Formula F-1 , R^22A or R^22B is -R^H-R²⁹-R³¹-R³⁰. In certain embodiments of the above-defined Formula B, wherein R³⁰ has a structure of Formula F-1 , R^H is -O-C(O)-.

[0136] In certain embodiments of the above-defined Formula B, wherein R²³ has a structure of Formula C-3 and R³⁰ has a structure of Formula F-1 , when X⁶¹ , X⁶², X⁶³, X⁶⁴ and X⁶⁵ independently is carbon, R⁶¹, R⁶², R⁶³, R⁶⁴ and R⁶⁵, respectively, independently is two substituents, each of which two substituents independently is a Group A moiety chosen from hydrogen, halogen, hydroxy, C1 -C3 alkyl, C1 -C3 heteroalkyl, C1 -C3 haloalkyl, C1 -C3 perhaloalkyl, C1 -C3 perhaloalkoxy, C1 -C3 alkoxy, C1 -C3 haloalkoxy, C1 -C3 alkoxy C1-C3 alkyl, acyl, oxo, acyloxy, carboxyl, amido, cyano, amino, C1 -C3 alkylamino, C1 -C3 alkylaminoalkyl, thiol, C1 -C3 alkylthio, C1-C3 alkylthio C1 -C3 alkyl, C1 -C3 haloalkylthio, C1-C3 perhaloalkylthio, nitro, C5-C7 aryl, C5-C7 aryl C1 -C3 alkyl, C5-C7 cycloalkyl C1 -C3 alkyl, 5-7 membered heterocycle-C1 -C3 alkyl, C5-C7 cycloalkyl, 5-7 membered heterocycloalkyl, 5-7 membered heteroaryl, 5-7 membered heteroaryl C1 -C3 alkyl, C1 -C3 alkylsulfonyl, sulfonamide, C1 -C3 alkylsulfonamido and C1 -C3 alkylsilyloxy. Stated another way, when X⁶¹ is carbon, R⁶¹ is two substituents, each of which two substituents independently is a Group A moiety; when X⁶² is carbon, R⁶² is two substituents, each of which two substituents independently is a Group A moiety; when X⁶³ is carbon, R⁶³ is two substituents, each of which two substituents

independently is a Group A moiety; when X⁶⁴ is carbon, R⁶⁴ is two substituents, each of which two substituents independently is a Group A moiety; and when X⁶⁵ is carbon, R⁶⁵ is two substituents, each of which two substituents independently is a Group A moiety. In certain of these embodiments, R^22A or R^22B is -R^H-R²⁹-R³¹-R³⁰. In certain of these embodiments, R^H is - O-C(O)-.

[0137] In certain embodiments of the above-defined Formula B, wherein R²³ has a structure of Formula C-3 and R³⁰ has a structure of Formula F-1 , when X⁶¹ , X⁶², X⁶³, X⁶⁴ and X⁶⁵ independently is nitrogen, R⁶¹ , R⁶², R⁶³, R⁶⁴ and R⁶⁵, respectively, independently is a Group B moiety chosen from hydrogen, C1-C3 alkyl, C1 -C3 heteroalkyl, C1 -C3 haloalkyl, C1 -C3 perhaloalkyl, C1 -C4 acyl, amido, C1 -C3 alkylamino, C1 -C3 alkylamino C1 -C3 alkyl, thiol, C1 -C3 alkylthio, C1 -C3 alkylthio C1 -C3 alkyl, C1 -C3 haloalkylthio, C1 -C3 perhaloalkylthio, C5-C7 aryl, C5-C7 aryl C1-C3 alkyl, C5-C7 cycloalkyl, C5-C7 cycloalkyl C1 -C3 alkyl, 5-7 membered heterocycloalkyl, 5-7 membered heterocycle C1-C3 alkyl, 5-7 membered heteroaryl, 5-7 membered heteroaryl C1-C3 alkyl, C1-C3 alkylsulfonyl, sulfonamide, C1 -C3 alkylsulfonamido and C1 -C3 alkylsilyloxy. Stated another way, when X⁴¹ is nitrogen, R⁴¹ is a Group B moiety; when X⁴² is nitrogen, R⁴² is a Group B moiety; when X⁴³ is nitrogen, R⁴³ is a Group B moiety; when X⁴⁴ is nitrogen, R⁴⁴ is a Group B moiety; and when X⁴⁵ is nitrogen, R⁴⁵ is a Group B moiety. In certain of these embodiments, R^22A or R^22B is -R^H-R²⁹-R³¹-R³⁰. In certain of these embodiments, R^H is -O-C(O)-.

[0138] In certain embodiments of the above-defined Formula B, wherein R²³ has a structure of Formula C-3 and R³⁰ has a structure of Formula F-1 , when X⁶¹ , X⁶², X⁶³, X⁶⁴ and X⁶⁵ independently is oxygen or sulfur, R⁶¹, R⁶², R⁶³, R⁶⁴ and R⁶⁵, respectively, independently is not present. Stated another way, when X⁶¹ is oxygen or sulfur, R⁶¹ is not present; when X⁶² is oxygen or sulfur, R⁶² is not present; when X⁶³ is oxygen or sulfur, R⁶³ is not present; when X⁶⁴ is oxygen or sulfur, R⁶⁴ is not present; and when X⁶⁵ is oxygen or sulfur, R⁶⁵ is not present. In certain of these embodiments, R^22A or R^22B is -R^H-R²⁹-R³¹-R³⁰. In certain of these

embodiments, R^H is -O-C(O)-.

[0139] In some embodiments, compounds provided herein have a structure of Formula B, or a pharmaceutically acceptable salt thereof, wherein:

R³⁰;

n is 1 or 2;

R²³ has a structure of Formula C-3:

Formula C-3

wherein:

R⁵¹ , R⁵², R⁵³, R⁵⁴ and R⁵⁵ each independently is not present or independently is hydrogen, halogen, hydroxyl, C1 -C3 alkyl, C1 -C3 heteroalkyl, C1 -C3 haloalkyl, C1 -C3 perhaloalkyl, C1 -C3 perhaloalkoxy, C1-C3 alkoxy, C1 -C3 haloalkoxy, C1 -C3 alkoxy C1-C3 alkyl, C1 -C4 acyl, oxo, C1 -C4 acyloxy, carboxyl, amido, cyano, amino, C1-C3 alkylamino, C1-C3 alkylamino C1 -C3 alkyl, thiol, C1 -C3 alkylthio, C1 -C3 alkylthio C1 -C3 alkyl, C1 -C3 haloalkylthio, C1 -C3 perhaloalkylthio, nitro, C5-C7 aryl, C5-C7 aryl C1 -C3 alkyl, C5-C7 cycloalkyl, C5-C7 cycloalkyl C1 -C3 alkyl, 5-7 membered heterocycloalkyl, 5-7 membered heterocycle C1 -C3 alkyl, 5-7 membered heteroaryl, 5-7 membered heteroaryl C1 -C3 alkyl, C1 -C3 alkylsulfonyl, sulfonamide, C1 -C3 alkylsulfonamido or C1 -C3 alkylsilyloxy;

R²⁹ is cycloalkyl, heterocycloalkyl, aryl or heteroaryl, which independently is optionally substituted by one or more substituents chosen from halogen, hydroxy, alkyl, alkenyl, alkynyl, heteroalkyl, haloalkyl, perhaloalkyl, perhaloalkoxy, alkoxy, haloalkoxy, alkoxyalkyl, acyl, oxo, acyloxy, carboxyl, amido, cyano, amino, alkylamino, alkylaminoalkyl, thiol, alkylthio, alkylthioalkyl, haloalkylthio, perhaloalkylthio, nitro, aryl, arylalkyl, cycloalkylalkyl, heterocycle- alkyl, cycloalkyl, heterocycloalkyl, heteroaryl, heteroarylalkyl, alkylsulfonyl, sulfonamide, alkylsulfonamido and alkylsilyloxy;

alkylthioalkyl, haloalkylthio, perhaloalkylthio and nitro;

R²⁴ has a structure of Formula C-2:

Formula C-2

wherein:

one, two or three of R⁴¹ , R⁴², R⁴³, R⁴⁴ and R⁴⁵ independently is R²⁶-R²⁵, and each of the remaining R⁴¹, R⁴², R⁴³, R⁴⁴ and R⁴⁵ independently is not present or independently is hydrogen, halogen, hydroxyl, C1-C3 alkyl, C1-C3 heteroalkyl, C1-C3 haloalkyl, C1-C3 perhaloalkyl, C1-C3 perhaloalkoxy, C1-C3 alkoxy, C1-C3 haloalkoxy, C1-C3 alkoxy C1-C3 alkyl, C1-C4 acyl, oxo, C1-C4 acyloxy, carboxyl, amido, cyano, amino, C1-C3 alkylamino, C1-C3 alkylamino C1-C3 alkyl, thiol, C1-C3 alkylthio, C1-C3 alkylthio C1-C3 alkyl, C1-C3 haloalkylthio, C1-C3

perhaloalkylthio, nitro, C5-C7 aryl, C5-C7 aryl C1-C3 alkyl, C5-C7 cycloalkyl, C5-C7 cycloalkyl C1-C3 alkyl, 5-7 membered heterocycloalkyl, 5-7 membered heterocycle C1-C3 alkyl, 5-7 membered heteroaryl, 5-7 membered heteroaryl C1-C3 alkyl, C1-C3 alkylsulfonyl, sulfonamide, C1-C3 alkylsulfonamido or C1-C3 alkylsilyloxy.

[0140] In certain embodiments of compounds having a structure of the above-defined Formula B, wherein R²³ has a structure of Formula C-3 and R²⁴ has a structure of Formula C-2, R^21A or R^21B is - R^G-R²⁴-R²⁶-R²⁵. In certain embodiments of the above-defined Formula B, wherein R²⁴ has a structure of Formula C-2, R^G is -O-C(O)-.

[0141] In certain embodiments of the above-defined Formula B, wherein R²³ has a structure of Formula C-3 and R²⁴ has a structure of Formula C-2, when X⁴¹, X⁴², X⁴³, X⁴⁴ and X⁴⁵

independently is carbon, R⁴¹, R⁴², R⁴³, R⁴⁴ and R⁴⁵, respectively, independently is a Group A moiety chosen from R²⁶-R²⁵, hydrogen, halogen, hydroxyl, C1-C3 alkyl, C1-C3 heteroalkyl, C1- C3 haloalkyl, C1-C3 perhaloalkyl, C1-C3 perhaloalkoxy, C1-C3 alkoxy, C1-C3 haloalkoxy, C1- C3 alkoxy C1-C3 alkyl, acyl, oxo, acyloxy, carboxyl, amido, cyano, amino, C1-C3 alkylamino, C1-C3 alkylaminoalkyl, thiol, C1-C3 alkylthio, C1-C3 alkylthio C1-C3 alkyl, C1-C3 haloalkylthio, C1-C3 perhaloalkylthio, nitro, C5-C7 aryl, C5-C7 aryl C1-C3 alkyl, C5-C7 cycloalkyl C1-C3 alkyl, 5-7 membered heterocycle-C1-C3 alkyl, C5-C7 cycloalkyl, 5-7 membered heterocycloalkyl, 5-7 membered heteroaryl, 5-7 membered heteroaryl C1-C3 alkyl, C1-C3 alkylsulfonyl, sulfonamide, C1-C3 alkylsulfonamido and C1-C3 alkylsilyloxy. Stated another way, when X⁴¹ is carbon, R⁴¹ is a Group A moiety; when X⁴² is carbon, R⁴² is a Group A moiety; when X⁴³ is carbon, R⁴³ is a Group A moiety; when X⁴⁴ is carbon, R⁴⁴ is a Group A moiety; and when X⁴⁵ is carbon, R⁴⁵ is a Group A moiety. In certain of these embodiments, R^21A or R^21B is - R^G-R²⁴-R²⁶-R²⁵. In certain of these embodiments, R^G is -O-C(O)-.

[0142] In certain embodiments of the above-defined Formula B, wherein R²³ has a structure of Formula C-3 and R²⁴ has a structure of Formula C-2, when X⁴¹, X⁴², X⁴³, X⁴⁴ and X⁴⁵

independently is nitrogen, R⁴¹, R⁴², R⁴³, R⁴⁴ and R⁴⁵, respectively, independently is a Group B moiety chosen from hydrogen, C1-C3 alkyl or C1-C3 alkyl substituted with hydroxyl, halogen or NR²⁷R²⁸. Stated another way, when X⁴¹ is nitrogen, R⁴¹ is a Group B moiety; when X⁴² is nitrogen, R⁴² is a Group B moiety; when X⁴³ is nitrogen, R⁴³ is a Group B moiety; when X⁴⁴ is nitrogen, R⁴⁴ is a Group B moiety; and when X⁴⁵ is nitrogen, R⁴⁵ is a Group B moiety. In certain of these embodiments, R^21A or R^{21 B} is - R^G-R²⁴-R²⁶-R²⁵. In certain of these embodiments, R^G is -O-C(O)-.

[0143] In certain embodiments of the above-defined Formula B, wherein R²³ has a structure of Formula C-3 and R²⁴ has a structure of Formula C-2, when X⁴¹ , X⁴², X⁴³, X⁴⁴ and X⁴⁵ independently is oxygen or sulfur, R⁴¹, R⁴², R⁴³, R⁴⁴ and R⁴⁵, respectively, independently is not present. Stated another way, when X⁴¹ is oxygen or sulfur, R⁴¹ is not present; when X⁴² is oxygen or sulfur, R⁴² is not present; when X⁴³ is oxygen or sulfur, R⁴³ is not present; when X⁴⁴ is oxygen or sulfur, R⁴⁴ is not present; and when X⁴⁵ is oxygen or sulfur, R⁴⁵ is not present. In certain of these embodiments, R^21A or R^{21 B} is - R^G-R²⁴-R²⁶-R²⁵. In certain of these

embodiments, R^G is -O-C(O)-.

[0144] The following tables provide non-limiting examples of some embodiments of the present compounds.

[0145] Table 1 lists the substituents for R^20A, R^20B, R^22A and R^22B of examples of some of the compounds of Formula B where each of the listed embodiments represents six embodiments as follows:

one embodiment wherein the substituents for R^20A, R^20B, R^22A and R^22B are as listed and R^21A is hydrogen and R^{21 B} is hydroxy,

one embodiment wherein the substituents for R^20A, R^20B, R^22A and R^22B are as listed and R^21A is hydroxy and R^21B is hydrogen,

one embodiment wherein the substituents for R^20A, R^20B, R^22A and R^22B are as listed and R^21A is hydrogen and R^{21 B} is p-bromomethylbenzoyl, and

one embodiment wherein the substituents for R^20A, R^20B, R^22A and R^22B are as listed and R^21A is p-bromomethylbenzoyl and R^21B is hydrogen,

one embodiment wherein the substituents for R^20A, R^20B, R^22A and R^22B are as listed and R^21A is hydrogen and R^{21 B} is (4-methylpiperazin-1-yl)p-methylbenzoyl, and

one embodiment wherein the substituents for R^20A, R^20B, R^22A and R^22B are as listed and R^21A is (4-methylpiperazin-1-yl)p-methylbenzoyl and R^{21 B} is hydrogen.

Table 1

[0146] Table 2 lists the substituents for R^20A, R^20B, R^22A and R^22B of examples of some of the compounds of Formula B where each of the listed embodiments represents six embodiments as follows:

one embodiment wherein the substituents for R^20A, R^20B, R^22A and R^22B are as listed and R^21A is hydrogen and R^21B is hydroxy,

one embodiment wherein the substituents for R^20A, R^20B, R^22A and R^22B are as listed and R^21A is hydroxy and R^21B is hydrogen, one embodiment wherein the substituents for R^20A, R^20B, R^22A and R^22B are as listed and R^21A is hydrogen and R^21B is p-bromomethylbenzoyl, and

one embodiment wherein the substituents for R^20A, R^20B, R^22A and R^22B are as listed and R^21A is hydrogen and R^21B is (4-methylpiperazin-1-yl)p-methylbenzoyl, and

one embodiment wherein the substituents for R^20A, R^20B, R^22A and R^22B are as listed and R^21A is (4-methylpiperazin-1-yl)p-methylbenzoyl and R^21B is hydrogen. Table 2

[0147] Table 3 lists the substituents for R^20A, R^20B, R^22A and R^22B of examples of some of the compounds of Formula B where each of the listed embodiments represents six embodiments as follows: one embodiment wherein the substituents for R^20A, R^20B, R^22A and R^22B are as listed and R^21A is hydrogen and R^21B is hydroxy,

one embodiment wherein the substituents for R^20A, R^20B, R^22A and R^22B are as listed and R^21A is hydrogen and R^21B is p-bromomethylbenzoyl, and

one embodiment wherein the substituents for R^20A, R^20B, R^22A and R^22B are as listed and R^21A is (4-methylpiperazin-1-yl)p-methylbenzoyl and R^21B is hydrogen.

Table 3

[0148] Table 4 lists the substituents for R^20A, R^20B, R^22A and R^22B of examples of some of the compounds of Formula B where each of the listed embodiments represents six embodiments as follows:

one embodiment wherein the substituents for R^20A, R^20B, R^22A and R^22B are as listed and R^21A is hydrogen and R^21B is hydroxy, one embodiment wherein the substituents for R^20A, R^20B, R^22A and R^22B are as listed and R^21A is hydroxy and R^21B is hydrogen,

Table 4

[0149] In some embodiments, the compound is a pharmaceutically acceptable salt comprising at least one counter ion chosen from phosphate, hydrochloride, besylate, benzoate, carbonate, chloride, citrate, dihydrochloride, dimaleate, diphosphate, estolate, fumarate, gluconate, malate, maleate, pamoate, stearate, succinate, sulfate, sulfonate, tartrate, tosylate, and valerate. In some embodiments, the counter ion is phosphate. In some embodiments, the counter ion is hydrochloride.

[0150] In the context of a chemical structure, a reference to a chemical structure, or a structure provided as part of a synthetic scheme, a number or letter normally designated as a superscript, for example, the“1” in R¹, or the“L” in R^L, may be referred to as a subscript, for example, Ri or R_L, or without any modification of script, such as, for example, R1 or RL.

[0151] Any definition herein may be used in combination with any other definition to describe a composite structural group. By convention, the trailing element of any such definition is that which attaches to a parent moiety. The composite group alkylamido, for example, would represent an alkyl group attached to a parent molecule through an amido group, and the term alkoxyalkyl would represent an alkoxy group attached to a parent molecule through an alkyl group, for example. [0152] When a group is defined to be“null,” the group is absent. The term“optionally substituted” means the anteceding group may be substituted or unsubstituted. The term “substituted,” as used herein, refers, without limitation, to one or more substituents which can include, for example, substituents independently selected from the following groups or a particular designated set of groups, alone or in combination: lower alkyl, lower alkenyl, lower alkynyl, lower alkanoyl, lower heteroalkyl, lower aryl, lower cycloalkyl, lower heteroaryl, lower heterocycloalkyl, aryl, cycloalkyl, heteroaryl, heterocycloalkyl, lower haloalkyl, lower haloalkenyl, lower haloalkynyl, lower perhaloalkyl, lower perhaloalkoxy, phenyl, aryloxy, lower alkoxy, lower haloalkoxy, oxo, lower acyloxy, carbonyl, carboxyl, lower alkylcarbonyl, lower carboxyester, lower carboxamido, cyano, hydrogen, halogen, hydroxy, amino, lower alkylamino, arylamino, amido, nitro, thiol, lower alkylthio, lower haloalkylthio, lower perhaloalkylthio, arylthio, sulfonate, sulfonic acid, trisubstituted silyl, N₃, SH, SCH₃, C(0)CH₃, C0₂CH₃, C0₂H, pyridinyl, thiophene, furanyl, lower carbamate, and lower urea. Two substituents may be joined together to form a fused five-, six-, or seven-membered carbocyclic, heterocyclic aryl, or heteroaryl ring system having zero to three heteroatoms, for example, forming methylenedioxy or ethylenedioxy. An optionally substituted group may be unsubstituted (e.g., -CH₂CH₃), fully substituted (e.g., - CF₂CF₃), monosubstituted (e.g., -CH₂CH₂F) or substituted at a level anywhere in-between fully substituted and monosubstituted (e.g., -CH₂CF₃). Where substituents are recited without qualification as to substitution, both substituted and unsubstituted forms are encompassed. Where a substituent is qualified as“substituted,” the substituted form is specifically intended. Additionally, different sets of optional substituents to a particular moiety may be defined as needed. An optional substitution often is as defined, sometimes immediately following the phrase,“optionally substituted with.”

[0153] The term R or the term R’, appearing by itself and without a number designation, unless otherwise defined, refers to a moiety chosen from hydrogen, alkyl, cycloalkyl, heteroalkyl, aryl, heteroaryl and heterocycloalkyl, any of which may be optionally substituted. Such R and R’ groups should be understood to be optionally substituted as defined herein. Whether an R group has a number designation or not, every R group, including R, R’ and Rⁿ where n = (1 , 2, 3, ...n), every substituent, and every term should be understood to be independent of every other in terms of selection from a group. Should any variable, substituent, or term (e.g. aryl, heterocycle, R, etc.) occur more than one time in a formula or generic structure, its definition at each occurrence is independent of the definition at every other occurrence. Certain groups may be attached to a parent molecule or may occupy a position in a chain of elements from either end as written. Thus, by way of example only, an asymmetrical group such as -C(0)N(R)- may be attached to a parent moiety at either the carbon or the nitrogen. [0154] The term“acyl,” as used herein, alone or in combination, refers to a carbonyl attached to an alkenyl, alkyl, aryl, cycloalkyl, heteroaryl, heterocycle, or any other moiety where the atom attached to the carbonyl is carbon. Non-limiting examples of acyl groups include formyl, alkanoyl and aroyl.

[0155] An“acetyl” group refers to a -C(0)CH₃ group.

[0156] The term“aliphatic,” as used herein, refers to saturated and partially unsaturated, nonaromatic, straight chain (i.e., unbranched), branched and cyclic (including bicyclic and polycyclic) hydrocarbons which may be optionally substituted with one or more functional groups. In certain embodiments, an aliphatic group contains 1 to 20 carbon atoms, 1 to 10 carbon atoms, 1 to 8 carbon atoms, 1 to 6 carbon atoms or 1 to 3 carbon atoms.

[0157] An“alkylcarbonyl” or“alkanoyl” group refers to an alkyl group attached to a parent molecular moiety through a carbonyl group. Non-limiting examples of such groups include methylcarbonyl and ethylcarbonyl.

[0158] The term“alkenyl,” as used herein, alone or in combination, refers to a straight-chain or branched-chain hydrocarbon radical having one or more double bonds and containing from 2 to 20 carbon atoms. In certain embodiments, an alkenyl includes 2 to 6 carbon atoms. The term “alkenylene” refers to a carbon-carbon double bond system attached at two or more positions such as ethenylene [(-CH=CH-),(-C::C-)]. Non-limiting examples of alkenyl radicals include ethenyl, propenyl, 2-methylpropenyl, 1 ,4-butadienyl and the like. Unless otherwise specified, the term“alkenyl” may include“alkenylene” groups.

[0159] The term“alkoxy,” as used herein, alone or in combination, refers to an alkyl ether radical, where the term alkyl is as defined below. Non-limiting examples of alkyl ether radicals include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, iso-butoxy, sec-butoxy, tert-butoxy, and the like.

[0160] The term“alkyl,” as used herein, alone or in combination, refers to a saturated straight- chain or branched-chain hydrocarbon radical containing from 1 to 20 carbon atoms. The term “straight-chain alkyl” refers to a saturated straight-chain hydrocarbon radical. The term “branched-chain alkyl” refers to a saturated branched-chain hydrocarbon radical. In certain embodiments, an alkyl includes 1 to 10 carbon atoms, 1 to 8 carbon atoms, 1 to 6 carbon atoms or 1 to 3 carbon atoms. Alkyl groups may be optionally substituted as defined herein. Non- limiting examples of alkyl radicals include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, iso-amyl, hexyl, octyl, nonyl and the like.

[0161] The term“alkylene,” as used herein, alone or in combination, refers to a saturated aliphatic group derived from a straight or branched chain saturated hydrocarbon attached at two or more positions, such as methylene (-CH₂-). Unless otherwise specified, the term“alkyl” may include“alkylene” groups.

[0162] The term“alkylamino,” as used herein, alone or in combination, refers to an alkyl group attached to a parent molecular moiety through an amino group. Alkylamino groups include mono- or dialkylated groups, non-limiting examples of which include N-methylamino, N- ethylamino, N,N-dimethylamino, N,N-ethylmethylamino and the like.

[0163] The term“alkylidene,” as used herein, alone or in combination, refers to an alkenyl group in which one carbon atom of the carbon-carbon double bond belongs to the moiety to which the alkenyl group is attached.

[0164] The term“alkylthio,” as used herein, alone or in combination, refers to an alkyl thioether (R-S-) radical where the term alkyl is as defined above and where the sulfur may be singly or doubly oxidized. Non-limiting examples of alkyl thioether radicals include methylthio, ethylthio, n-propylthio, isopropylthio, n-butylthio, iso-butylthio, sec-butylthio, tert-butylthio, methanesulfonyl, ethanesulfinyl, and the like.

[0165] The term“alkynyl,” as used herein, alone or in combination, refers to a straight-chain or branched chain hydrocarbon radical having one or more triple bonds and containing from 2 to 20 carbon atoms. In certain embodiments, an alkynyl includes 2 to 6 carbon atoms. In some embodiments, an alkynyl includes 2 to 4 carbon atoms. The term“alkynylene” refers to a carbon-carbon triple bond attached at two positions such as ethynylene (-C:::C-, -CºC-). Nonlimiting examples of alkynyl radicals include ethynyl, propynyl, hydroxypropynyl, butyn-1-yl, butyn-2-yl, pentyn-1-yl, 3-methylbutyn-1-yl, hexyn-2-yl, and the like. Unless otherwise specified, the term“alkynyl” may include“alkynylene” groups.

[0166] The terms“amido” and“carbamoyl,” as used herein, alone or in combination, refer to an amino group as described below attached to a parent molecular moiety through a carbonyl group, or vice versa. The term“C-amido” as used herein, alone or in combination, refers to a -C(0)N(RR’) group with R and R’ as defined herein or as defined by the specifically enumerated“R” groups designated. The term“N-amido” as used herein, alone or in combination, refers to a RC(0)N(R’)- group, with R and R’ as defined herein or as defined by the specifically enumerated“R” groups designated. The term "acylamino" as used herein, alone or in combination, includes an acyl group attached to a parent moiety through an amino group.

A non-limiting example of an "acylamino" group is acetylamino (CH₃C(0)NH-).

[0167] The term“amino,” as used herein, alone or in combination, refers to -NRR’, where R and R’ are independently chosen from hydrogen, alkyl, alkenyl, alkynyl, acyl, heteroalkyl, aryl, cycloalkyl, heteroaryl, and heterocycloalkyl, any of which may themselves be optionally substituted. Additionally, R and R’ may combine to form heterocycloalkyl or heteroaryl, either of which may be optionally substituted.

[0168] The term "aryl," as used herein, alone or in combination, refers to an aromatic cyclic ring system, or aromatic hydrocarbon ring system, in which all of the atoms that form the covalent structure of the one or more aromatic rings are carbon (referred to herein as an“aryl ring”). The aryl ring may be optionally substituted as defined herein. The ring system may be monocyclic or fused polycyclic, for example, bicyclic or tricylic (containing two or three rings fused together). In certain embodiments, the monocyclic aryl ring is C4-C10, or C5-C9, or C5- C8, or C5-C7, or, in certain embodiments, C5-C6, where these carbon numbers refer to the number of carbon ring member atoms that form the ring system. In some embodiments, the polycyclic ring system is a bicyclic aryl group, where the bicyclic aryl group in some

embodiments is C8-C12, or, for example, C9-C10. In some embodiments, the polycyclic ring system is a tricyclic aryl group, where the tricyclic aryl group is C1 1-C18, or, for example, C12- C16. Non-limiting examples of aryl ring systems include phenyl (monocyclic, C6), naphthyl (bicyclic, C10), anthracenyl (tricyclic, C14) and phenanthryl (tricyclic, C14).

[0169] The term“arylalkenyl” or“aralkenyl,” as used herein, alone or in combination, refers to an aryl group attached to a parent molecular moiety through an alkenyl group.

[0170] The term“arylalkoxy” or“aralkoxy,” as used herein, alone or in combination, refers to an aryl group attached to a parent molecular moiety through an alkoxy group.

[0171] The term“arylalkyl” or“aralkyl,” as used herein, alone or in combination, refers to an aryl group attached to a parent molecular moiety through an alkyl group.

[0172] The term“arylalkynyl” or“aralkynyl,” as used herein, alone or in combination, refers to an aryl group attached to a parent molecular moiety through an alkynyl group. [0173] The term“arylalkanoyl” or“aralkanoyl” or“aroyl,” as used herein, alone or in combination, refers to an acyl radical derived from an aryl-substituted alkanecarboxylic acid, non-limiting examples of which include benzoyl, napthoyl, phenylacetyl, 3-phenylpropionyl (hydrocinnamoyl), 4-phenylbutyryl, (2-naphthyl)acetyl, 4-chlorohydrocinnamoyl, and the like.

[0174] The term“aryloxy” as used herein, alone or in combination, refers to an aryl group attached to a parent molecular moiety through an oxy.

[0175] The terms“benzo” and“benz,” as used herein, alone or in combination, refer to the divalent radical C₆H₄= derived from benzene. Non-limiting examples include benzothiophene and benzimidazole.

[0176] The term“carbamate,” as used herein, alone or in combination, refers to an ester of carbamic acid (-NHCOO-) which may be attached to a parent molecular moiety from either the nitrogen or acid end, and which may be optionally substituted as defined herein.

[0177] The term“O-carbamyl” as used herein, alone or in combination, refers to

a -0C(0)NRR’ group where R and R’ are as defined herein.

[0178] The term“N-carbamyl” as used herein, alone or in combination, refers to a

R0C(0)NR’- group, where R and R’ are defined herein.

[0179] The term“carbonyl,” as used herein, when alone includes formyl [-C(0)H] and in combination includes a -C(O)- group.

[0180] The term“carboxyl” or“carboxy,” as used herein, refers to -C(0)0H or the

corresponding“carboxylate” anion (e.g., in a carboxylic acid salt). An“O-carboxy” group refers to a RC(0)0- group, where R is as defined herein. A“C-carboxy” group refers to a -C(0)0R groups where R is as defined herein.

[0181] The term“cyano,” as used herein, alone or in combination, refers to -CN.

[0182] The terms“cycloalkyl,” and, interchangeably,“carbocycle,” as used herein, alone or in combination, refers to a ring system in which all of the ring member atoms are carbon and at least one of the rings is a saturated or partially unsaturated aliphatic cyclic ring moiety (referred to herein as a“cycloalkyl ring” or“carbocycle ring”). In some embodiments, each cyclic moiety contains from 3 to 12 carbon ring member atoms which may be optionally substituted as defined herein. In some embodiments, a cycloalkyl group contains 3 to 10 carbon ring member atoms. In certain embodiments, a cycloalkyl includes 5 to 7 carbon atoms. In certain embodiments, a cycloalkyl includes 5 to 6 carbon atoms. A cycloalkyl can be a monocyclic or polycyclic, e.g., bicyclic or tricyclic, ring system in which at least one cyclic ring is a cycloalkyl ring. In certain embodiments, the monocyclic cycloalkyl ring is C3-C10, or C5-C9, or C5-C8, or C5-C7, or, in certain embodiments, C5-C6, where these carbon numbers refer to the number of carbon ring member atoms that form the ring system. Polycyclic cycloalkyl ring systems include fused, bridged and spiro-fused rings. Polycyclic cycloalkyl ring systems as defined herein, include ring systems in which one or more cycloalkyl rings is/are fused to one or more aryl rings (benzo- fused cycloalkyl ring systems) and/or other cycloalkyl rings. In some embodiments, all of the rings in a polycyclic cycloalkyl ring system are cycloalkyl rings. In some embodiments, the polycyclic ring system is a bicyclic cycloalkyl group, where the bicyclic cycloalkyl group in some embodiments is C8-C12, or, for example, C9-C10. In some embodiments, the polycyclic ring system is a tricyclic cycloalkyl group, where the tricyclic cycloalkyl group is C1 1-C18, or, for example, C12-C16. Non-limiting examples of such cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, octahydronaphthalene,

decahydronaphthalene, bicyclo[1 , 1 ,1]pentane and the like. Examples of aryl-fused cyclolalkyl ring systems include a benzene ring fused to hydrogenated or partially hydrogenated ring systems, non-limiting examples of which include dihydronaphthalene, tetrahydronaphthalene and indanyl. In polycyclic systems in which a cycloalkyl is fused to an aryl, attachment of the polycycle to the indicated point of attachment on the parent molecule may be through any ring atom of the polycycle rings. In some embodiments of polycyclic cycloalkyls, the polycycle is attached to the indicated point of attachment through a ring member atom of a cycloalkyl ring.

In some embodiments of polycyclic cycloalkyls, the polycycle is attached to the indicated point of attachment through a ring member atom of a ring that is not a cycloalkyl ring, e.g., an aryl ring.

[0183] The term“carbocycle-alkyl” or“cycloalkylalkyl” as used herein, alone or in combination, refers to a carbocycle group attached to a parent molecular moiety through an alkyl group.

[0184] The term“ester,” as used herein, alone or in combination, refers to a carboxy group bridging two moieties linked at carbon atoms.

[0185] The term“ether,” as used herein, alone or in combination, refers to an oxy group bridging two moieties linked at carbon atoms. [0186] The term“halo,” or“halogen,” as used herein, alone or in combination, refers to fluorine, chlorine, bromine, or iodine.

[0187] The term“haloalkoxy,” as used herein, alone or in combination, refers to a haloalkyl group attached to a parent molecular moiety through an oxygen atom.

[0188] The term“haloalkyl,” as used herein, alone or in combination, refers to an alkyl radical having the meaning as defined above where one or more hydrogens are replaced with a halogen. Specifically included are monohaloalkyl, dihaloalkyl and polyhaloalkyl radicals. A monohaloalkyl radical, for example, sometimes include an iodo, bromo, chloro or fluoro atom within the radical. Dihalo and polyhaloalkyl radicals sometimes include two or more of the same halo atoms or a combination of different halo radicals. Non-limiting examples of haloalkyl radicals include fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, pentafluoroethyl, heptafluoropropyl, difluorochloromethyl, dichlorofluoromethyl, difluoroethyl, difluoropropyl, dichloroethyl and dichloropropyl. “Haloalkylene” refers to a haloalkyl group attached at two or more positions. Non-limiting examples include

fluoromethylene (-CFH-), difluoromethylene

[0189] (-CF₂ -), chloromethylene (-CHCI-) and the like.

[0190] The term“heteroaliphatic,” as used herein, refers to an aliphatic moiety, as defined herein, that contains one or more heteroatoms, such as, for example, oxygen, nitrogen, sulfur, phosphorous and/or silicon, e.g., in place of a carbon atom or between carbon atoms. In some embodiments, a heteroaliphatic group contains from one to three heteroatoms chosen from O,

N, and S, and where the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. In certain embodiments, the heteroatom(s) may be placed at any interior position of the heteroaliphatic group. In some embodiments, up to two heteroatoms may be consecutive. In certain embodiments, a heteroaliphatic group includes 2 to 20 carbon atoms, 2 to 10 carbon atoms, 2 to 8 carbon atoms or 2 to 6 carbon atoms.

[0191] The term "heteroalkyl," as used herein, alone or in combination, refers to a saturated or unsaturated, stable straight or branched hydrocarbon chain having the stated number of carbon atoms and one or more heteroatoms, such as, for example, oxygen, nitrogen, sulfur, phosphorous and/or silicon, e.g., in place of a carbon atom. In some embodiments, a heteroalkyl contains from one to three heteroatoms chosen from O, N, and S, and where the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. In certain embodiments, the heteroatom(s) may be placed at any interior position of the heteroalkyl group. In some embodiments, up to two heteroatoms may be consecutive, such as, for example, -CH2-NH-OCH3. In certain embodiments, a heteroalkyl includes 2 to 20 carbon atoms, 2 to 10 carbon atoms, 2 to 8 carbon atoms or 2 to 6 carbon atoms. In some instances, a heteroalkyl contains from 1 to 3 degrees of unsaturation.

Heteroalkyl groups may be optionally substituted as defined herein.

[0192] The term“heteroalkenyl,” as used herein, alone or in combination, refers to an alkenyl moiety, as defined herein, that contains one or more heteroatoms, such as, for example, oxygen, nitrogen, sulfur, phosphorous and/or silicon, e.g., in place of a carbon atom or between carbon atoms. In some embodiments, a heteroalkenyl contains from one to three heteroatoms chosen from O, N, and S, and where the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. In certain embodiments, the heteroatom(s) may be placed at any interior position of the heteroalkenyl group. In some embodiments, up to two heteroatoms may be consecutive. In certain embodiments, a heteroalkenyl includes 2 to 20 carbon atoms, 2 to 10 carbon atoms, 2 to 8 carbon atoms or 2 to 6 carbon atoms.

[0193] The term“heteroalkynyl,” as used herein, alone or in combination, refers to an alkynyl moiety, as defined herein, that contains one or more heteroatoms, such as, for example, oxygen, nitrogen, sulfur, phosphorous and/or silicon, e.g., in place of a carbon atom or between carbon atoms. In some embodiments, a heteroalkynyl contains from one to three heteroatoms chosen from O, N, and S, and where the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. In certain embodiments, the heteroatom(s) may be placed at any interior position of the heteroalkynyl group. In some embodiments, up to two heteroatoms may be consecutive. In certain embodiments, a heteroalkynyl includes 2 to 20 carbon atoms, 2 to 10 carbon atoms, 2 to 8 carbon atoms or 2 to 6 carbon atoms.

[0194] The term "heteroaryl," as used herein, alone or in combination, refers to a cyclic ring system in which at least one of the rings is an aromatic ring in which all ring member atoms are carbon, except for at least one heteroatom (referred to herein as a“heteroaryl ring”), such as, for example, nitrogen, oxygen and sulfur. The heteroaryl ring may be optionally substituted as defined herein. A heteroaryl can be a monocyclic or a fused polycyclic, e.g., bicyclic or tricyclic, ring system in which at least one cyclic ring is an aromatic heteroaryl ring. Polycyclic, e.g., bicyclic and tricyclic, fused heteroaryl ring systems as defined herein include heteroaryl ring systems in which one or more heteroaryl rings is/are fused to one or more aryl rings (which are referred to herein as aryl-fused heteroaryl rings), one or more cycloalkyl rings and/or one or more other heteroaryl rings. In some embodiments, all of the rings in a polycyclic heteroaryl ring system are heteroaryl rings. In certain embodiments, a heteroaryl ring contains at least one atom chosen from O, S, and N. In certain embodiments, a heteroaryl ring is a 3 to 15 membered monocyclic ring. In certain embodiments, a monocyclic heteroaryl group may contain from 4 to 10 ring member atoms, and may have, for example, 1 to 4 heteroatoms in the ring, where the remaining ring member atoms are carbon. In some embodiments, a bicyclic heteroaryl ring may contain from 8 to 15 ring member atoms, and have from 1 to 8 heteroatoms, where the remaining ring member atoms are carbon. In some embodiments, a tricyclic heteroaryl ring may contain from 1 1 to 18 ring member atoms, and have from 1 to 10 heteroatoms, where the remaining ring member atoms are carbon. Non-limiting examples of heteroaryls include pyrrolyl, pyrrolinyl, imidazolyl, pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazolyl, furyl, thienyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, thiadiazolyl, isothiazolyl, indolyl, isoindolyl, indolizinyl, benzimidazolyl, quinolyl, isoquinolyl, quinoxalinyl, quinazolinyl, indazolyl, benzotriazolyl, thienopyridinyl, furopyridinyl, pyrrolopyridinyl and the like. Exemplary bicyclic and tricyclic heteroaryl groups include phenanthrolinyl, dibenzofuranyl, acridinyl, phenanthridinyl, dihydro[1 ,3]oxazolo[4,5-b]pyridinyl, benzothiazolyl, and the like. In polycyclic systems in which a heteroaryl is fused to one or more rings that are not heteroaryl, attachment of the polycycle to the indicated point of attachment on the parent molecule may be through any ring member atom of the polycycle rings. In some embodiments of polycyclic heteroaryls, the polycycle is attached to the indicated point of attachment through a ring member atom of a heteroaryl ring. In some embodiments of monocyclic or polycyclic heteroaryls, the monocyle or polycycle is attached to the indicated point of attachment through a ring member heteroatom of a heteroaryl ring. In some embodiments of polycyclic heteroaryls, the polycycle is attached to the indicated point of attachment through a ring member atom of a ring that is not a heteroaryl ring, e.g., an aryl ring or a cycloalkyl ring. “Heteroaryl” includes sulfones, sulfoxides, N-oxides of tertiary nitrogen ring member atoms, and carbocyclic fused and benzo-fused ring systems. Non-limiting examples of a heteroaryl group may be referred to as an aryl group having one or more carbon atoms substituted with O, NRⁿ, S, SO, S0₂, where “n” denotes any positive integer.

[0195] The term“heteroarylalkyl” as used herein, alone or in combination, refers to an unsubstituted or substituted heteroaryl group attached to a parent molecular moiety through an alkyl group.

[0196] The term“heterocycle-alkyl” as used herein, alone or in combination, refers to a substituted or unsubstituted heterocycle group attached to a parent molecular moiety through an alkyl group. [0197] The terms“heterocycloalkyl” and, interchangeably,“heterocycle,” or“heterocyclic” as used herein, alone or in combination, each refer to a ring system in which at least one of the rings is a saturated or partially unsaturated, heteroaliphatic, nonaromatic cyclic ring moiety in which all of the ring member atoms are carbon, except for at least one heteroatom (referred to herein as a“heterocycloalkyl ring,”“heterocycle ring” or“heterocyclic ring”). The one or more heteroatoms that can be in the ring include, for example, nitrogen, oxygen, sulfur, phosphorous and/or silicon. In some embodiments, the ring heteroatom or heteroatoms is selected from nitrogen, oxygen and sulfur. The heterocycloalkyl ring may be optionally substituted as defined herein. A heterocycloalkyl is a monocyclic or polycyclic, e.g., bicyclic or tricyclic, ring system in which at least one cyclic ring is a heterocycloalkyl ring. Polycyclic heterocycloalkyl ring systems include fused, bridged and spiro-fused rings. Polycyclic heterocycloalkyl ring systems as defined herein, include ring systems in which one or more heterocycloalkyl rings is/are fused to one or more cycloalkyl, aryl, heteroaryl and/or heterocycloalkyl rings. In some embodiments, all of the rings in a polycyclic heterocycloalkyl ring system are heterocycloalkyl rings. In certain embodiments, a hetercycloalkyl includes 1 to 4 heteroatoms as ring member atoms. In some embodiments, a hetercycloalkyl moiety includes 1 to 2 heteroatoms as ring member atoms. In certain embodiments, a hetercycloalkyl moiety includes 3 to 8 ring member atoms in each ring.

In some embodiments, a hetercycloalkyl moiety includes 3 to 7 ring member atoms in each ring. In yet some embodiments, a hetercycloalkyl moiety includes 5 to 6 ring member atoms in each ring. In some embodiments, a heterocycloalkyl can be a 3 to 15 membered nonaromatic ring, or a fused bicyclic, or tricyclic non-aromatic ring, which contains at least one atom chosen from O, S, and N. In certain embodiments, a monocyclic heterocycloalkyl or heterocycle group may contain from 4 to 10 ring member atoms, and may have, for example, 1 to 4 heteroatoms in the ring, where the remaining ring member atoms are carbon. In some embodiments, a bicyclic heterocycloalkyl or heterocycle group may contain from 8 to 15 ring member atoms, and have from 1 to 8 heteroatoms, where the remaining ring member atoms are carbon. In some embodiments, a tricyclic heterocycloalkyl or heterocycle group may contain from 1 1 to 18 ring member atoms, and have from 1 to 10 heteroatoms, where the remaining ring member atoms are carbon. The term also includes fused polycyclic groups where one or more heterocyclic rings are fused with one or more cycloalkyl rings, aryl, heteroaryl and/or other heterocyclic groups. In polycyclic systems in which a heterocycloalkyl ring is fused to one or more rings that are not heterocycloalkyl, attachment of the polycycle to the indicated point of attachment on the parent molecule may be through any ring member atom of the polycycle rings. In some embodiments of polycyclic heterocycloalkyls, the polycycle is attached to the indicated point of attachment through a ring member atom of a heterocycloalkyl ring. In some embodiments of monocyclic or polycyclic heterocycloalkyls, the monocyle or polycycle is attached to the indicated point of attachment through a ring member heteroatom of a heterocycloalkyl ring. In some embodiments of polycyclic heterocycloalkyls, the polycycle is attached to the indicated point of attachment through a ring member atom of a ring that is not a heterocycloalkyl ring, e.g., an aryl ring, heteroaryl ring or a cycloalkyl ring. “Heterocycloalkyl” and“heterocycle” include sulfones, sulfoxides and N-oxides of tertiary nitrogen ring member atoms. Non-limiting examples of heterocycle groups include aziridinyl, azetidinyl, 1 ,3-dioxanyl, 1 ,4-dioxanyl, 1 ,3- dioxolanyl, morpholinyl, piperazinyl, pyrrolidinyl, piperidinyl, thiomorpholinyl, pyranyl, dihydropyridinyl, tetrahydropyridinyl, carabazolyl, xanthenyl, 1 ,3-benzodioxolyl,

dihydroisoquinolinyl, dihydrocinnolinyl, dihydrobenzodioxinyl, isoindolinyl, dihydroisoindolyl and dihydroindolyl, and the like. The heterocycle groups may be optionally substituted unless specifically prohibited. Non-limiting examples of heterocycloalkyl groups may be referred to as cycloalkyl group having one or more carbon atoms substituted with O, NRⁿ, S, SO, S0₂, where n denotes any positive integer.

[0198] The term“hydrazinyl” as used herein, alone or in combination, refers to two amino groups joined by a single bond, i.e., -HN-NH-.

[0199] The term“hydroxy,” as used herein, alone or in combination, refers to -OH.

[0200] The term“hydroxyalkyl,” as used herein, alone or in combination, refers to a hydroxy group attached to a parent molecular moiety through an alkyl group.

[0201] The term“imino,” as used herein, alone or in combination, refers to =N-

[0202] The term“iminohydroxy,” as used herein, alone or in combination, refers to =N(OH) and =N-0-

[0203] The phrase“in the main chain” refers to the longest contiguous or adjacent chain of carbon atoms starting at the point of attachment of a group to the compounds of any one of the formulas disclosed herein.

[0204] The term“isocyanato” refers to a -NCO group.

[0205] The term“isothiocyanato” refers to a -NCS group.

[0206] The phrase“linear chain of atoms” refers to the longest straight chain of atoms independently selected from carbon, nitrogen, oxygen and sulfur. [0207] The term“lower,” as used herein, alone or in a combination, where not otherwise specifically defined, means a moiety containing from 1 to and including 6 carbon atoms. A "lower alkyl," for example, refers to an alkyl containing 1 to 6 carbon atoms, 1 to 5 carbon atoms, 1 to 4 carbon atoms, 1 to 3 carbon atoms, or 1 to 2 carbon atoms (e.g., an alkyl containing 1 , 2, 3, 4, 5 or 6 carbon atoms).

[0208] The term“lower aryl,” as used herein, alone or in combination, means a C4-C6 aryl group, for example, a C5-C6 aryl group. A lower aryl group sometimes is a C4-C6 aryl ring group, or C5-C6 aryl ring group for example, including without limitation, phenyl. The term may also refer to a C8-C10 bicyclic ring aryl group, for example, including without limitation, napthyl. Lower aryl groups, including phenyl or napthyl, may be optionally substituted as provided.

[0209] The term“lower heteroaryl,” as used herein, alone or in combination, means a four- membered, five-membered, or six-membered heteroaryl group. A lower heteroaryl group sometimes is (1) a monocyclic heteroaryl ring comprising five or six ring member atoms, of which between one and four of the ring member atoms may be heteroatoms chosen from O, S, and N, or (2) a bicyclic heteroaryl ring, where each of the fused rings comprises five or six ring member atoms, comprising between them one to four heteroatoms chosen from O, S, and N. Lower heteroaryl groups may be optionally substituted as provided.

[0210] The term“lower cycloalkyl,” as used herein, alone or in combination, means a monocyclic cycloalkyl having between three and six ring member atoms. Non-limiting examples of lower cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. Lower cycloalkyl groups may be optionally substituted as provided.

[0211] The term“lower heterocycloalkyl,” as used herein, alone or in combination, means a monocyclic heterocycloalkyl having between three and six ring member atoms, of which between one and four may be heteroatoms chosen from O, S, and N. Non-limiting examples of lower heterocycloalkyls include pyrrolidinyl, imidazolidinyl, pyrazolidinyl, piperidinyl, piperazinyl, and morpholinyl. Lower heterocycloalkyl groups may be optionally substituted as provided.

[0212] The term“lower amino,” as used herein, alone or in combination, refers to -NRR’, where R and R’ are independently chosen from hydrogen, lower alkyl, and lower heteroalkyl, any of which may be optionally substituted. Additionally, the R and R’ of a lower amino group may combine to form a five- or six-membered heterocycloalkyl, either of which may be optionally substituted. [0213] The terms“mercaptyl” or“mercaptan” as used herein, alone or in combination, refers to an RS- group, where R is as defined herein.

[0214] The term“menthol,” as used herein, refers to 2-isopropyl-5-methylcyclohexanol.

Menthol contains 3 chiral carbons and the term“menthol” encompasses all stereoisomers of the molecule unless specifically stated otherwise herein. For example, isomers of menthol include the (-)-menthol isomer ((1 R, 2S, 5R)-2-isopropyl-5-methylcyclohexanol), (+)-menthol isomer ((1 S, 2R, 5S)-2-isopropyl-5-methylcyclohexanol), (-)-isomenthol isomer ((1 R, 2S, 5S)-2- isopropyl-5-methylcyclohexanol), (+)-isomenthol isomer ((1 S, 2R, 5R)-2-isopropyl-5- methylcyclohexanol), (-)-neomenthol isomer ((1 R, 2R, 5S)-2-isopropyl-5-methylcyclohexanol), (+)-neomenthol isomer ((1 S, 2S, 5R)-2-isopropyl-5-methylcyclohexanol), (-)-neoisomenthol isomer ((1 S, 2S, 5S)-2-isopropyl-5-methylcyclohexanol) and (+)-neoisomenthol isomer ((1 R, 2R, 5R)-2-isopropyl-5-methylcyclohexanol).

[0215] The term“menthyl,” as used herein, refers to a radical derived from menthol. Typically, a menthyl radical can be linked to another chemical group through the oxygen atom of the menthyl group.

[0216] The term“nitro,” as used herein, alone or in combination, refers to -N0₂.

[0217] The terms“oxy” or“oxa,” as used herein, alone or in combination, refer to -0-.

[0218] The term“oxo,” as used herein, alone or in combination, refers to =0.

[0219] The term“partially unsaturated,” as used herein, alone or in combination, refers to a straight-chain, branched-chain or ring moiety that includes at least one double or triple bond and that is not fully saturated. The term“partially unsaturated” when used in reference to a ring moiety means a ring having one or multiple sites of unsaturation but does not include aryl rings or heteroaryl rings as defined herein.

[0220] The term“perhaloalkoxy” refers to an alkoxy group where all of the hydrogen atoms are replaced by halogen atoms.

[0221] The term“perhaloalkyl” as used herein, alone or in combination, refers to an alkyl group where all of the hydrogen atoms are replaced by halogen atoms. [0222] The term“piperitol,” as used herein, refers to p-menth-1-en-3-ol. Piperitol contains 2 chiral carbons and the term“piperitol” encompasses all stereoisomers of the molecule unless specifically stated otherwise herein. For example, isomers of piperitol include (3R, 4R)-piperitol (also referred to as trans-piperitol) and (3S, 4R)-piperitol (also referred to as cis-piperitol).

[0223] The term“ring member atoms,” as used herein, refers to all of the atoms that form the covalent structure of a cyclic ring structure.

[0224] By“saturated” is meant that the carbon-containing group contains no carbon-carbon double or triple bonds.

[0225] The terms“sulfonate,”“sulfonic acid,” and“sulfonic,” as used herein, alone or in combination, refer the -SO3H group and its anion as the sulfonic acid is used in salt formation.

[0226] The term“sulfanyl,” as used herein, alone or in combination, refers to -S-.

[0227] The term“sulfinyl,” as used herein, alone or in combination, refers to

-S(O)-.

[0228] The term“sulfonyl,” as used herein, alone or in combination, refers to -S(0)₂-

[0229] The term“N-sulfonamido” refers to a RS(=0)₂NR’- group with R and R’ as defined herein.

[0230] The term“S-sulfonamido” refers to a -S(=0)₂NRR’, group, with R and R’ as defined herein.

[0231] The terms“thia” and“thio,” as used herein, alone or in combination, refer to a -S- group or an ether where the oxygen is replaced with sulfur. The oxidized derivatives of the thio group, namely sulfinyl and sulfonyl, are included in the definition of thia and thio.

[0232] The term“thiol,” as used herein, alone or in combination, refers to an -SH group.

[0233] The term“thiocarbonyl,” as used herein, when alone includes thioformyl -C(S)H and in combination is a -C(S)- group. [0234] The term“N-thiocarbamyl” refers to an ROC(S)NR’- group, with R and R’ as defined herein.

[0235] The term“O-thiocarbamyl” refers to a -OC(S)NRR’, group with R and R’ as defined herein.

[0236] The term“thiocyanato” refers to a -CNS group.

[0237] The term“trihalomethanesulfonamido” refers to a X₃CS(0)₂NR- group with X is a halogen and R as defined herein.

[0238] The term“trihalomethanesulfonyl” refers to a X₃CS(0)₂- group where X is a halogen.

[0239] The term“trihalomethoxy” refers to a X₃CO- group where X is a halogen.

[0240] The term“trisubstituted silyl,” as used herein, alone or in combination, refers to a silicone group substituted at its three free valences with groups as listed herein under the definition of substituted amino. Non-limiting examples include trimethylsilyl, tert- butyldimethylsilyl, triphenylsilyl and the like.

[0241] The term“ureido,” as used herein, alone or in combination, refers to the univalent radical NH₂CONH- derived from urea. Non-limiting examples include ureidoproprionate and ureidosuccinate.

Synthesis of compounds

[0242] Many compounds provided herein can be synthesized using rapamycin as a starting material. Rapamycin can be prepared synthetically or through fermentation using methods described herein and/or known in the art. Rapamycin is also commercially available from multiple sources.

[0243] In certain embodiments of the compounds provided herein, the methoxy group bonded to the carbon at position 7 of rapamycin is substituted with another moiety. In general, some of the compounds in these embodiments can be synthesized from rapamycin through hydrolysis of the 7-methylether linkage followed by nucleophilic substitution. For example, in some embodiments of the compounds provided herein, the C7 methoxy group is substituted with o,p- dimethoxyphenyl (also referred to herein as DMOP) which can be accomplished by nucleophilic substitution of rapamycin using 1 ,3-dimethoxybenzene. Many of the compounds provided herein that contain o,p-dimethoxyphenyl at the C7 position can be synthesized using 7 (S)- dimethoxyphenyl-rapamycin (also referred to as 7(S)-DMOP-rapamycin) as a starting material.

[0244] In some embodiments of the compounds provided herein, the hydroxyl group at carbon number 40 of rapamycin is substituted with a halogen. Because rapamycin is a relatively large molecule with a number of potential sites for nucleophilic substitution by halogen, such compounds can be produced using site-specific halogenation methods described herein. For example, in a method of synthesis provided herein, rapamycin (or an analog thereof, such as, e.g., 7(S)-dimethoxyphenyl-rapamycin) can be reacted with trifluoromethanesulfonic anhydride followed by reaction with a tetrabutylammonium halide to yield site-directed halogenation at the C40 position.

[0245] In some embodiments of the compounds provided herein, the hydroxyl group at carbon number 40 of rapamycin is substituted with a benzoic acid ester-containing moiety. In general, some of the compounds in these embodiments can be synthesized from rapamycin (or an analog thereof, such as, e.g., 7(S)-dimethoxyphenyl-rapamycin) through an alcohol esterification reaction with a substituted benzoic acid, a dehydrating agent (e.g.,

dicyclohexylcarbodiimide referred to as DCC) and a nucleophilic acylation catalyst (e.g., dimethylaminopyridine referred to as DMAP). Detailed methods for synthesis of the compounds provided herein are described in the specific Examples.

[0246] A non-limiting example of modification is esterification, where a non-limiting example of an ester group is tosyl. A non-limiting example of a substitution reaction is a substitution of OH with a halogen.

[0247] The term“esterification” as used herein refers to chemical reaction conditions used to create an ester.

[0248] The term“reduction” as used herein refers to a chemical redox reaction in which the oxygen atom of a carboxylic group is substituted by two hydrogen atoms, reducing C0₂H to CH₂OH.

[0249] The term“activation of primary OH” as used herein refers to a chemical process in which the primary alcohol is modified or substituted to create a labile group as used in an alkylation reaction. [0250] The term“N-alkylation conditions’ as used herein refers to chemical reaction conditions used to form an N-R bond.

[0251] The term“coupling conditions” as used herein refers to chemical reaction conditions used to create a chemical bond between N and CO as in a peptide bond.

Characterization of compounds

[0252] Compounds described herein, or pharmaceutically acceptable salts thereof, can be characterized for a particular property using a suitable method. Compounds described herein, or pharmaceutically acceptable salts thereof, can be characterized in a number of ways, including, for example, for binding characteristics to a protein and for solubility in water.

Evaluating binding of compounds

[0253] Compounds provided herein can be evaluated for the ability to bind proteins using methods described herein or known in the art. For example, compounds provided herein can be evaluated for binding to one or more FRB proteins, FKBP proteins (e.g., FKBP12) and/or variants of FRB and FKBP proteins. By being“capable of binding”, as in the example of the binding of a compound or multimeric or heterodimeric ligand to a ligand-binding region or multimerizing region, is meant that the compound or ligand binds to a ligand-binding region and that this binding may be detected by an assay method including, but not limited to, a biological assay, a chemical assay, or physical means of detection such as, for example, x-ray

crystallography. In addition, where a ligand or multimeric compound is considered to“not significantly bind” it is meant that there may be minor detection of binding of a ligand or multimeric compound to the ligand binding region, but that this amount of binding, or the stability of binding, is not significantly detectable, and, when occurring in the cells of a functional biological assay, does not produce the activity (e.g., activation of a modified cell or apoptosis) that would result if significant binding of the compound occurred. For example, in transcriptional switch assays of compounds, as described herein, where the compound does not“significantly bind,” upon administration to cells expressing switch component fusion proteins, the number of cells undergoing activation or apoptosis is less than 10, 5, 4, 3, 2 or 1 %.

[0254] The binding affinity of compounds described herein, or pharmaceutically acceptable salts thereof, may be determined by assaying binding to polypeptides, such as, for example, rapamycin-binding polypeptides. In some examples, the polypeptide is a multimerizing region polypeptide (multimeric ligand binding region), such as, for example, an FRB polypeptide, or variant thereof (e.g., FRBL), or an FKBP12 polypeptide, or variant thereof (e.g., FKBP12v36). Methods for measuring binding affinity are described herein and/or known in the art and include, but are not limited to, functional binding assays, such as, for example, measuring an activity associated with binding, or the multimerization of chimeric fusion polypeptides expressed in cells, following treatment of the cells with a compound described herein, or pharmaceutically acceptable salt thereof.

[0255] Some functional assays incorporate secreted alkaline phosphatase (SEAP) as a readily detectable reporter molecule. For example, cells may be transfected or transduced with nucleic acid encoding two separate inducible pro-apoptotic fusion proteins (each fusion containing one of two multimerizing region polypeptides being tested for binding), the cells are contacted with a compound described herein, or a pharmaceutically acceptable salt thereof, and multimerization-induced apoptosis is then measured using a SEAP assay. An example of a pro- apoptotic protein is caspase. Dose-response studies using such an assay can be used to determine binding affinity of a compound for a protein based on IC₅o values determined from the assay results. An example of a SEAP apoptosis-based assay that may be used to determine binding characteristics of a compound to a multimerization region is provided in Example 23.

[0256] Another example of a SeAP assay is one in which cells are transfected or transduced with nucleic acid encoding components of a multimerizing compound-induced SeAP

transcription-based system. In this assay, compounds that effectively bind (and thus dimerize) an FKBP12 (or variant thereof) and FRB (or variant thereof) will, in so doing, activate a“switch” to initiate transcription of a reporter protein (e.g., SeAP). The switch includes a fusion protein of a DNA-binding domain (e.g., of a yeast GAL4 protein such as, e.g., SEQ ID NO: 97 encoded by SEQ ID NO: 23) with one or more (e.g., three) copies of FKBP12 (or a variant thereof). This fusion protein is coexpressed with a fusion of FRB (or variant thereof) and a transcription activator (e.g., the herpes simplex virus (HSV) VP16 protein) that activates transcription only when present near gene promoter elements, but lacks intrinsic DNA-binding activity. These switch components are coexpressed in cells with a reporter gene plasmid containing nucleic acids that are sites to which the DNA-binding domain fusion protein binds. In one example, the reporter plasmid can contain five GAL4-specific DNA recognition sites proximal to the transcriptional start site. Addition of a compound, such as rapamycin or a rapalog, that is capable of binding the FKBP12 and the FRB polypeptides in the fusion proteins, recruits FRB- transcription activator protein to the DNA-binding-FKBP12 fusion protein that is bound to reporter plasmid DNA to drive reporter gene (e.g., SeAP-encoding DNA) expression according to the relative affinity of the compound for the FRB and FKBP12 proteins. Dose-response studies using such an assay can be used to determine binding affinity of a compound for a protein based on ECso values determined from the assay results. An example of a compound- induced SeAP transcription-based assay that may be used to determine binding characteristics of a compound to a multimerization region is provided in Example 18.

Evaluating Immunosuppressive activity of compounds

[0257] Compounds provided herein can be evaluated for immunosuppressive effects using methods known in the art. For example, in vitro methods for measuring immunosuppressive activity include activated lymphocyte or splenocyte proliferation assays (see, e.g., Collinge et al. (2010) J Immunotoxicol 7:357-366; Luengo et al. (1995) Chem Biol 2:471-481). An example of an in vivo method for measuring immunosuppressive activity is the animal model contact hypersensitivity assay (see, e.g., Olson et al. (2007) Int Immunopharmacol 7(6):734-743).

Evaluating antifungal activity of compounds

[0258] There are a number of in vitro assays for antifungal activity of compounds. For example, some assays are based on the inhibition of growth of Candida albicans (see, e.g., Clerya Alvino Leite et al. (2014) Evidence Based Complementary and Alternative Medicine doi.org/10.1 155/2014/378280). Another example of a method for evaluating antifungal activity is an assay for inhibition of germ tube formation of Candida albicans (see, e.g., Brayman and Wilks (2003) Antimicrob Agents Chemother 47(10):3305-3310).

Evaluating antiproliferative activity of compounds

[0259] The antiproliferative activities of compounds can be evaluated using in vitro methods, for example in assays of tumor cell growth, and in vivo methods using animal xenograph models. In one method of determining the proliferation of tumor cells (e.g., human

osteosarcoma cells) over time in the presence of a compound, the mitochondrial metabolic rate of cells is evaluated through detection of the absorbance of cells that have been seeded into 96- well plates, exposed to the compound and treated with MTT and MTS (tetrazolium compounds that are reduced by viable cells to generate a detectable formazan product) (see, e.g., Riss et al. 2013 Cell Viability Assays. In Assay Guidance Manual; Sittampalam et al., eds.;

www.ncbi.nlm.nih.gov/books/NBK144065/). Antiproliferative activity of compounds in vivo can be evaluated, for example, using mouse xenograph models that can be generated through subcutaneous injection of tumor cells into mice. Tumor volumes of control and compound- treated mice can be compared to determine anti-tumor effects of a compound (see, e.g., Zhao et al (2015) JBUON 20(2):588-594). Evaluating solubility of compounds

[0260] By“soluble” or“solubility” is meant the property of a compound to dissolve in water, buffer, or other liquid, which may be measured in terms of mg.mL^-1. Solubility may be assessed with reference to water, or a buffered solution such as, for example, a solution buffered by acetate, phosphate, citrate or other buffering agent suitable for a buffer solution having a pH of 7 or less, 6 or less, 5 or less, or 4 or less. Examples of pharmaceutically acceptable buffers include those provided in Remington: The Science and Practice of Pharmacy, 22 ed., 2013, Pharmaceutical Press, London (Allen, L.V., Jr., ed.). Where the buffer or pH of the liquid is not provided herein, such as when, for example, the solubility of a compound is discussed alone, or by comparison to a control compound such as rapamycin or 7-demethoxy-7(S)-o,p- dimethoxyphenylrapamycin (CMP001), the reference liquid is water. Methods for evaluating the solubility of a compound include, for example, a 96-well plate-based assay in which aqueous suspensions of the compound are vacuum-filtered and the concentration of compound is measured by UV spectrophotometry (Roy et al (2001) Drug Dev Ind Pharm 27(1): 107-109).

Evaluating stability of compounds

[0261] Some rapalogs may have certain properties (e.g., solubility, binding characteristics) that are preferable for some uses of the analogs but may possess other properties, e.g., in vivo stability, that are diminished relative to rapamycin or a rapalog such as CMP001. For pharmaceutical uses, a compound should have suitable pharmacokinetic properties (e.g., good absorption, metabolic clearance rate and bioavailability). Embodiments of some of the compounds provided herein exhibit increased metabolic stability relative to rapamycin and/or certain rapamycin analogs, including, for example, but not limited to, 7-demethoxy-7(S)-o,p- dimethoxyphenylrapamycin (CMP001).

[0262] Methods for evaluating the metabolic stability of a compound are described herein and/or known in the art. Assays using liver microsomes can be used as a rapid in vitro method to evaluate metabolic stability as a reasonably accurate prediction of in vivo, intrinsic hepatic clearance in a live whole organism (e.g., a mammal, such as a human). Because the liver is a major site of drug processing in the body, with a majority of drugs being metabolized through hepatic CYP-mediated mechanisms, liver microsomes contain membrane-bound metabolizing enzymes which makes them useful for in vitro assessment of metabolic stability of compounds. Examples of liver microsome-based assays for compound stability are described by Hill ((2003) Curr Protocols Pharmacol 7(8): 1 -7.8.1 1) and Knights et al. ((2016) Curr Protocols Pharmacol 74(1):7.8.1-7.8.24). In vivo metabolic stability assays of compounds can also be conducted in animal models using methods known in the art (see, e.g., Paoloni et al. [(2010) Rapamycin Pharmacokinetic and Pharmacodynamic Relationships in Osteosarcoma: A

Comparative Oncology Study in Dogs. PLoS ONE 5(6): e1 1013.

doi: 10.1371 /journal.pone.001 1013] and Bouzas et al. [(2010) Upsala J Med Sci 1 15:125-130]).

Methods of using the compounds

Methods of multimerizing chimeric polypeptides

[0263] Certain embodiments of some of the methods provided herein incorporate chemically induced dimerization (CID) for conditional control of one or more proteins. In addition to this technique being inducible, it also is reversible, due to degradation of a labile dimerizing agent or administration of a monomeric competitive inhibitor. In some embodiments, a CID system- based method provided herein uses a compound provided herein. Included in such

embodiments, are compounds provided herein that are analogs of rapamycin that lack some or all of the bioactivity of natural rapamycin while gaining the ability to crosslink a molecule genetically fused to the FK506-binding protein, FKBP12, or variant thereof, with a molecule genetically fused to the FKBP-rapamycin binding domain (FRB), or variant thereof. Multimeric compounds described herein, or pharmaceutically acceptable salts thereof, bind to and multimerize polypeptides that contain multimeric ligand binding regions or multimerizing regions as discussed herein and can be used as the chemical inducer of multimerization in methods provided herein.

[0264] The terms“chimeric,”“fusion” and“chimeric fusion” are used interchangeably herein with reference to a polypeptide containing two or more proteins (or a portion(s) of one or more of the two or more proteins) that have been joined to create a chimeric polypeptide. The two or more proteins (or portions thereof) may be directly joined to each other, wherein a terminal amino acid residue of one protein (or portion thereof) is directly bonded to a terminal amino acid residue of another protein (or portion thereof), or may be joined through one or more intervening elements (e.g., one or more amino acids that are not part of either protein, such as a linker or adapter, or a non-amino acid polymer). For example, a polypeptide that is produced from nucleic acid encoding a fusion of a multimerizing protein (or portion thereof) and another protein (e.g., a DNA-binding protein, transcription activation protein, pro-apoptotic protein or protein component of an immune cell activation pathway), or portion thereof, may be referred to as a chimeric, fusion or chimeric fusion polypeptide. Methods of cell surface protein multimerization

[0265] Provided herein are methods of multimerizing cell surface proteins, e.g., cell surface receptors. The methods include a step of contacting cells expressing fusion proteins containing cell surface proteins, or portions thereof, and a ligand-binding domain that binds to a compound provided herein, or pharmaceutically acceptable salts thereof. In one example of these methods, a cell is transfected or transduced with (1) nucleic acid encoding a fusion of one cell surface protein, or portion thereof, and an FRB protein (or variant thereof) and (2) nucleic acid encoding a fusion of a second cell surface protein (e.g., the same as or different from the cell surface protein fused to an FRB protein), or portion thereof, and an FKBP12 protein (or variant thereof). The cell is then contacted with a compound provided herein, or pharmaceutically acceptable salts thereof, that binds to and multimerizes the FRB protein and FKBP12 protein contained in the fusion proteins (e.g., heterodimers) and may be monitored for particular activities, such as, for example, changes in cell structure, function, protein phosphorylation (e.g., cell surface protein phosphorylation), receptor internalization or cell signaling. Such methods are useful, for example, in dissecting cell signaling pathways and elucidating protein

associations within cells. Methods of generating nucleic acid vectors for expression of fusion proteins, transfecting and transducing cells with the nucleic acids, and monitoring cells for multimerization of fusion proteins and effects thereof are described herein and/or known to those of skill in the art (see, e.g., Song and Hinkle (2005) Mol Endocrinol 19(1 1):2859-2870; Spencer et al. (1993) Science 262:1019-1024; Spencer et al. (1996) Curr Biol 6:839-847 ; Blau et al. ,(1997) Proc Natl Acad Sci USA 94:3076-3081 ; Muthuswamy et al. (1999) Mol Cell Biol 19(10):6845-6857; Otto et al. (2001) Blood 97:3662-3664).

Methods of protein translocation and recruitment to cellular membranes

[0266] Provided herein are methods of intracellular protein translocation and protein recruitment to cellular membranes through multimerization induced by compounds provided herein, or pharmaceutically acceptable salts thereof. In one example of these methods, a cell is transfected or transduced with (1) nucleic acid encoding a fusion of an intracellular protein, or portion thereof, and either an FRB protein (or variant thereof) or FKBP12 protein (or variant thereof) and (2) nucleic acid encoding a fusion of a plasma membrane-targeting myristoylation signal protein and an FKBP12 protein (or variant thereof), if the first fusion is with an FRB protein, or an FRB protein (or variant thereof) if the first fusion is with an FKBP12 protein (or portion thereof). The cell is then contacted with a compound provided herein, or

pharmaceutically acceptable salts thereof, that binds to and multimerizes the FRB protein and FKBP12 protein contained in the fusion proteins and may be monitored for localization of the intracellular protein fusion to the plasma membrane and/or particular activities, such as, for example, cell signal transduction, associated with plasma membrane localization. Such methods are useful, for example, in dissecting cell signaling pathways and protein localization requirements thereof as well as in inducing a reaction at the plasma membrane or other membrane. Methods of generating nucleic acid vectors for expression of fusion proteins, transfecting and transducing cells with the nucleic acids, and monitoring cells for multimerization of fusion proteins and effects (e.g., protein translocation) thereof are described herein and/or known to those of skill in the art (see, e.g., Putyrski and Schultz (2012) FEBS Lett

586(15):2091-2105; van Unen et al (2016) Nature Scientific Reports vol. 6, article number 36825, https://doi.Org/10.1038/srep36825).

[0267] In an example of another method provided herein, a cell is transfected or transduced with (1) nucleic acid encoding a fusion of a nuclear localization signal(NLS)/DNA-binding protein (e.g., GAL4), or portion thereof, and either an FRB protein (or variant thereof) or FKBP12 protein (or variant thereof) and (2) nucleic acid encoding a fusion of a nuclear export signal (NES) protein and an FKBP12 protein (or variant thereof), if the first fusion is with an FRB protein, or an FRB protein (or variant thereof) if the first fusion is with an FKBP12 protein (or portion thereof). The cell is then contacted with a compound provided herein, or

pharmaceutically acceptable salts thereof, that binds to and multimerizes the FRB protein and FKBP12 protein contained in the fusion proteins and may be monitored for localization of the nucleus-targeted protein fusion to the cytoplasm. Such methods are useful in, for example, the identification of nuclear export signals and inducibly shuttling proteins between the nucleus and the cytoplasm. Methods of generating nucleic acid vectors for expression of fusion proteins, transfecting and transducing cells with the nucleic acids, and monitoring cells for multimerization of fusion proteins and effects (e.g., protein translocation) thereof are described herein and/or known to those of skill in the art (see, e.g., Terrillon and Bouvier (2004) EMBO J 23:3950-3961 ; Heo et al. (2006) Science 314(5804): 1458- 1461 ; Klemm et al. (1997) Curr Biol 7:638-644; Bayle et al. (2006) Chem Biol 13:99-107).

Methods of recruiting nucleic acid-binding proteins to nucleic acids

[0268] Provided herein are methods of recruiting nucleic acid-binding proteins to nucleic acids through multimerization induced by compounds provided herein, or pharmaceutically acceptable salts thereof. Examples of nucleic acid-binding proteins include, but are not limited to, transcription factors and splicing regulator proteins (e.g., SR proteins or RS domains thereof; see, e.g., Graveley (2005) RNA 1 1 :355-358). In one example of these methods, a cell is transfected or transduced with (1) nucleic acid encoding a fusion of a DNA binding-domain (including a nuclear localization signal) protein, or portion thereof, and either an FRB protein (or variant thereof) or FKBP12 protein (or variant thereof) and (2) nucleic acid encoding a fusion of a transcription factor activation domain (including a nuclear localization signal) protein and an FKBP12 protein (or variant thereof), if the first fusion is with an FRB protein, or an FRB protein (or variant thereof) if the first fusion is with an FKBP12 protein (or portion thereof). If dimerization-induced transcription in the cell is intended to be from a heterologous DNA vector, the cell is also transfected or transduced with that vector which includes DNA to which the DNA- binding domain of one of the fusion proteins binds. The cell is then contacted with a compound provided herein, or pharmaceutically acceptable salts thereof, that binds to and multimerizes the FRB protein and FKBP12 protein contained in the fusion proteins and may be monitored for transcription of an endogenous or heterologous gene. This method is useful, for example, in pharmacologic control of gene expression in gene therapy and in generating reporter gene transcription-based assays in cells to identify multimerizing ligand-binding proteins that bind to the compound being administered to the cells. Methods of generating nucleic acid vectors for expression of fusion proteins (e.g., containing transcriptional activator and/or repressor proteins), transfecting and transducing cells with the nucleic acids, and monitoring cells for multimerization of fusion proteins and effects thereof (e.g., activation and/or repression of transcription) are described herein and/or known to those of skill in the art (see, e.g., Biggar and Crabtree (2000) J Biol Chem 275(33):25381-25390).

[0269] In another example of these methods provided herein, a cell is transfected or transduced with (1) nucleic acid encoding a fusion of an RNA binding-domain protein, or portion thereof, and either an FRB protein (or variant thereof) or FKBP12 protein (or variant thereof) and (2) nucleic acid encoding a fusion of an mRNA splicing regulator protein (e.g., an SR protein), or RS domain thereof, and an FKBP12 protein (or variant thereof), if the first fusion is with an FRB protein, or an FRB protein (or variant thereof) if the first fusion is with an FKBP12 protein (or portion thereof). The cell is then contacted with a compound provided herein, or pharmaceutically acceptable salts thereof, that binds to and multimerizes the FRB protein and FKBP12 protein contained in the fusion proteins and may be monitored for pre-mRNA splicing and expression. This method is useful, for example, for regulation of protein expression.

Methods of generating nucleic acid vectors for expression of fusion proteins, transfecting and transducing cells with the nucleic acids, and monitoring cells for multimerization of fusion proteins and effects thereof (e.g., transcription and mRNA splicing) are described herein and/ known to those of skill in the art (see, e.g., Rivera et al. (1996) Nat ecf 2:1028-1032; Graveley (2005) RNA 1 1 :355-358).

Ligand-controlled cell switch methods [0270] In some embodiments, the compounds described herein, or pharmaceutically acceptable salts thereof, may be used to dimerize or multimerize chimeric polypeptides that each contain one or more multimerizing regions. This dimerization or multimerization of the chimeric polypeptides expressed in a cell may switch protein function and alter cell physiology.

In some embodiments, the compounds described herein, or pharmaceutically acceptable salts thereof, may be used as small molecule ligands for ligand-controlled cell elimination and/or ligand-controlled cell activation.

Ligand-controlled cell elimination

[0271] The term“cell elimination,” as used herein, refers to a reduction of cell function, viability and/or number. It can refer to a partial or complete reduction of cell function, viability and/or number. For example, a reduction may be at least about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97% or 100% reduction in a cell function, viability and/or number. Chemical Induction of protein dimerization (CID) can be effectively applied to make cellular suicide or apoptosis inducible with a small molecule dimerizing ligand, such as embodiments of compounds provided herein, or pharmaceutically acceptable salts thereof. This technology underlies the“safety switch” incorporated as a gene therapy adjunct in cell transplants. Using this technology, normal cellular regulatory pathways that rely on protein-protein interaction as part of a signaling pathway can be adapted to ligand-dependent, conditional control if a small molecule dimerizing drug is used to control the protein-protein oligomerization event. Induced dimerization of a fusion protein comprising caspase-9 (or a portion thereof) and one or more multimerizing domains (i.e.,“icaspase9/iCasp9/iC9) using a multimerizing ligand can rapidly effect cell death.

[0272] Ligand-controlled cell elimination methods provided herein can include a step of contacting cells expressing fusion proteins containing a pro-apoptotic protein, or a functionally equivalent portion thereof, and a ligand-binding domain that binds to a compound provided herein, with a compound provided herein or a pharmaceutically acceptable salt thereof. In one example of these methods, a cell is transfected or transduced with (1) nucleic acid encoding a fusion of a pro-apoptotic protein, or portion thereof, and an FRB protein (or variant thereof) and (2) nucleic acid encoding a fusion of the pro-apoptotic protein, or portion thereof, and an FKBP12 protein (or variant thereof). In another example of these methods, a cell is transfected or transduced with nucleic acid encoding a fusion of a pro-apoptotic protein, or portion thereof, one or more copies of an FRB protein (or variant thereof) and one or more copies of an FKBP12 protein (or variant thereof). The cell can then be contacted with a compound provided herein, or pharmaceutically acceptable salts thereof, that binds to and multimerizes the FRB protein and FKBP12 protein contained in the fusion proteins (e.g., heterodimers) to induce apoptosis in the cell.

[0273] In some embodiments, a chimeric protein is provided, or a nucleic acid encoding such a protein is provided, or a cell that contains such a protein or nucleic acid, for the purpose of inducing cell death in response to a compound described herein, or a pharmaceutically acceptable salt thereof. In certain embodiments, a chimeric protein contains one or more ligand binding regions, or multimerizing regions, and an apoptosis-inducing polypeptide, such as, for example, a caspase polypeptide, for example, a modified caspase-9 polypeptide that lacks the CARD domain. Contacting the multimerizing region, for example, by contacting a cell that expresses the chimeric polypeptide, with a compound described herein, or a pharmaceutically acceptable salt thereof, leads to multimerization of two or more chimeric caspase polypeptides, which results in apoptosis. This provides the system with temporal control and enhanced specificity. In some embodiments, the cell is an immune cell, e.g., a T cell. In some embodiments, the cell co-expresses a chimeric antigen receptor or a recombinant T cell receptor that recognizes an antigen expressed by a target cell. In some embodiments, the cell co-expresses a chimeric protein that includes one or more ligand-binding regions and a co stimulatory polypeptide, such as, for example, a CD40 and/or MyD88 polypeptide, or portions thereof.

[0274] In some examples, by fusing one or more FRB polypeptides (or variant thereof) and one or more FKBP12 polypeptides (or variant thereof) to a caspase-9 polypeptide, one can stimulate caspase-9 activity in a dimerizer drug-dependent manner. In some embodiments of the methods, in which a cell has been constructed to include a dual switch cell activation-cell elimination system, the ability to induce caspase-9 activity through dimerizer drug exposure can be provided for by fusing one or more FKBP12 polypeptides or polypeptide variants to a caspase-9 polypeptide. Ligand-controlled apoptosis may also be used as an assay to determine the binding of the compounds described herein, or pharmaceutically acceptable salts thereof.

[0275] As used herein, the term "icaspase-9" molecule, polypeptide, or protein is defined as an inducible caspase-9. The term“icaspase-9” embraces icaspase-9 nucleic acids, icaspase-9 polypeptides and/or icaspase-9 expression vectors. The term also encompasses either the natural icaspase-9 nucleotide or amino acid sequence, or a truncated sequence that is lacking the CARD domain. [0276] In some embodiments, where an expression construct encodes a truncated caspase-9 polypeptide, the truncated caspase-9 polypeptide is encoded by the nucleotide sequence of SEQ ID NO 41 , or a functionally equivalent fragment thereof, with or without DNA linkers, or has the amino acid sequence of SEQ ID NO: 1 13, or a functionally equivalent fragment thereof. A functionally equivalent fragment of the caspase-9 polypeptide has substantially the same ability to induce apoptosis as the polypeptide of SEQ ID NO: 1 13, with at least 50%, 60%, 70%, 80%, 90%, or 95% of the activity of the polypeptide of SEQ ID NO: 1 13 . In some embodiments, the expression construct encodes a truncated caspase-9 polypeptide encoded by the caspase-9 nucleotide sequences of pM006 or pM009.

[0277] "Function-conservative variants" of caspase-9, or other proteins discussed herein, are proteins or enzymes in which a given amino acid residue has been changed without altering overall conformation and function of the protein or enzyme, including, but not limited to, replacement of an amino acid with one having similar properties, including polar or non-polar character, size, shape and charge. Conservative amino acid substitutions for many of the commonly known non-genetically encoded amino acids are well known in the art. Conservative substitutions for other non-encoded amino acids can be determined based on their physical properties as compared to the properties of the genetically encoded amino acids.

[0278] As used herein, the term“functionally equivalent,” as it relates to caspase-9, or truncated caspase-9, for example, refers to a caspase-9 polypeptide that stimulates an apoptotic response or a nucleic acid encoding such a caspase-9. “Functionally equivalent" refers, for example, to a caspase-9 polypeptide that is lacking the CARD domain, but is capable of inducing an apoptotic cell response. When the term“functionally equivalent” is applied to other nucleic acids or polypeptides, such as, for example, a multimeric ligand binding region, multimerizing region, or CD3, it refers to fragments, variants, and the like that have the same or similar activity as the reference polypeptides of the methods herein.

[0279] Non-limiting examples of chimeric polypeptides useful for inducing cell death or apoptosis, and related methods for inducing cell death or apoptosis, including expression constructs, methods for constructing vectors, assays for activity or function, multimerization of the chimeric polypeptides by contacting cells that express inducible chimeric polypeptides with a multimerizing agent that binds to the multimerizing region of the chimeric polypeptides both ex vivo and in vivo, administration of expression vectors, cells, or multimeric compounds, and administration of multimeric compounds to subjects who have been administered cells that express the inducible chimeric polypeptides, may also be found in the following patents and patent applications, each of which is incorporated by reference herein in its entirety for all purposes: U.S. Patent Application No. 13/1 12,739, filed May 20, 201 1 , entitled METHODS FOR INDUCING SELECTIVE APOPTOSIS, published Nov. 24, 201 1 , as US201 1-0286980-A1 , issued July 28, 2015 as U.S. Patent 9,089,520; U.S. Patent Application No. 13/792,135, filed Mar. 10, 2013, entitled MODIFIED CASPASE POLYPEPTIDES AND USES THEREOF, published Sept. 1 1 , 2014 as US2014-0255360-A1 , issued Sept. 6, 2016 as U.S. Patent No. 9,434,935, by Spencer et al.; International Patent Application No. PCT/US2014/022004, filed Mar. 7, 2014, published Oct. 9, 2014 as WO2014/16438; U.S. Patent Application No.

14/296,404, filed June 4, 2014, entitled METHODS FOR INDUCING PARTIAL APOPTOSIS USING CASPASE POLYPEPTIDES, published June 2, 2016 as US2016-0151465-A1 , by Slawin et al; International Application No. PCT/US2014/040964 filed June 4, 2014, published as WO2014/197638 on Feb. 5, 2015, by Slawin et al.; U.S. Patent Application No. 14/640,553, filed Mar. 6, 2015, entitled CASPASE POLYPEPTIDES HAVING MODIFIED ACTIVITY AND USES THEREOF, published Nov. 19, 2015 as US2015-0328292-A1 ; International Patent Application No. PCT/US2015/019186, filed Mar. 6, 2015, published Sept. 1 1 , 2015 as

WO2015/134877, by Spencer et al.; U.S. Patent Application No. 14/968,737, filed Dec. 14, 2015, entitled METHODS FOR CONTROLLED ELIMINATION OF THERAPEUTIC CELLS, published June 16, 2016 as US2016-0166613-A1 , by Spencer et al.; International Patent Application No. PCT/US2015/065629 filed Dec. 14, 2015, published June 23, 2016 as

WO2016/100236, by Spencer et al.; U.S. Patent Application No. 14/968,853, filed Dec. 14, 2015, entitled METHODS FOR CONTROLLED ACTIVATION OR ELIMINATION OF

THERAPEUTIC CELLS, published June 23, 2016 as US2016-0175359-A1 , by Spencer et al.; International Patent Application No. PCT/US2015/065646, filed Dec. 14, 2015, published Sept.15, 2016 as WO2016/100241 , by Spencer et al.; U.S. Patent Application No. 15/377,776, filed Dec. 13, 2016, entitled DUAL CONTROLS FOR THERAPEUTIC CELL ACTIVATION OR ELIMINATION, published June 15, 2017 as US2017-0166877-A1., by Bayle et al.; and

International Patent Application No. PCT/US2016/066371 , filed Dec. 13, 2016, published June 22, 2017 as W02017/106185, by Bayle et al., each of which is incorporated by reference herein in its entirety for all purposes. Multimeric compounds described herein, or pharmaceutically acceptable salts thereof, may be used essentially as discussed in examples provided in these publications, and other examples provided herein, to the extent that they refer to multimeric ligands.

Ligand-controlled cell activation

[0280] In some embodiments, a chimeric protein is provided, or a nucleic acid encoding such a protein is provided, or a cell that contains such a protein or nucleic acid is provided, for the purpose of inducing cell activation in response to a compound described herein, or a pharmaceutically acceptable salt thereof. As used herein, the term“activation” with reference to a cell, refers to a process in which a cell is activated or stimulated to perform a cellular function. Cellular functions, include, but are not limited to, cellular responses, gene transcription, growth, division, proliferation, differentiation, signaling, production (e.g., of a polypeptide), interactions or reactions (e.g., binding, enzymatic processes) of cellular elements and secretion. One nonlimiting example of cell activation is the activation of immune cell function. For example, T cell activation includes the process in which binding of a T cell to an antigen leads to proliferation, maturation, cytokine secretion and/or cytotoxin release by the T cell. There can be multiple cellular elements involved in cell activation. For example, co-stimulating polypeptides may be used to enhance the activation of immune cells, e.g., T cells, and of CAR-expressing immune cells, e.g., T cells, against target antigens, which would increase the potency of adoptive immunotherapy.

[0281] Co-stimulation of immune cells refers to the cellular processes that participate in the complete activation and response of immune cells to ensure survival, growth, proliferation, persistence, expansion of immune cells, as well efficient target cell killing. As used herein, the terms“co-stimulatory” or“co-stimulating,” with reference to a protein or polypeptide associated with an immune cell, refers to the involvement or function of a polypeptide in the cellular signaling pathways that can participate in activation of an immune cell, such as, for example, a T-cell. Such pathways include initial activation steps of cell surface receptor and membrane interactions and downstream intracellular protein interactions involved in complete immune cell activation. Often these pathways involve activation of secondary signals including, but not limited to, NFAT, NF-kB, JNK, p38 mitogen-activated protein kinase (MAPK), activator protein 1 (AP1), ERK and/or AKT. Co-stimulatory polypeptides are involved in these processes and include, but are not limited to, receptors and adaptor proteins, such as, e.g., MyD88 and CD40.

[0282] Ligand-controlled cell activation methods provided herein can include a step of contacting cells expressing fusion proteins containing one or more co-stimulatory proteins, or a functionally equivalent portion thereof, and a ligand-binding domain that binds to a compound provided herein, with a compound provided herein or a pharmaceutically acceptable salt thereof. In one example of these methods, a cell is transfected or transduced with (1) nucleic acid encoding a fusion of one or more co-stimulatory proteins, or portion thereof, and an FRB protein (or variant thereof) and (2) nucleic acid encoding a fusion of the one or more costimulatory proteins, or portion thereof, and an FKBP12 protein (or variant thereof). In another example of these methods, a cell is transfected or transduced with nucleic acid encoding a fusion of one or more co-stimulatory proteins, or portion thereof, one or more copies of an FRB protein (or variant thereof) and one or more copies of an FKBP12 protein (or variant thereof). The cell can then be contacted with a compound provided herein, or pharmaceutically acceptable salts thereof, that binds to and multimerizes the FRB protein and FKBP12 protein contained in the fusion proteins (e.g., heterodimers) to induce and/or enhance activation of the cell.

[0283] Contacting the multimerizing region, for example, by contacting a cell that expresses the chimeric fusion protein(s), with a multimeric compound described herein, or a

pharmaceutically acceptable salt thereof, leads to multimerization of two or more chimeric proteins, which leads to activation and/or enhancement or amplification of activation of the cell.

In some embodiments, the cell is a T cell. In some embodiments, the cell co-expresses a chimeric antigen receptor or a recombinant T cell receptor that recognizes an antigen expressed by a target cell. In some embodiments, the cell co-expresses a chimeric protein that includes one or more ligand-binding regions and an apoptosis-inducing polypeptide, such as, for example, a caspase polypeptide, for example, a modified caspase-9 polypeptide that lacks the CARD domain. Contacting cells that express the chimeric co-stimulating polypeptide with a multimeric compound described herein, or a pharmaceutically acceptable salt thereof, results in multimerization of the chimeric proteins, and activation and/or amplification of the immune activity of the cell, which, in some embodiments, leads to increased elimination of target cells.

[0284] Co-stimulating polypeptides provided in the chimeric polypeptides are capable of amplifying the cell-mediated immune response through activation of signaling pathways involved in cell survival and proliferation. Co-stimulating polypeptides may include, but are not limited to, any molecule or polypeptide that activates the NF-kappaB pathway, Akt pathway, and/or p38 pathway. Non-limiting examples of costimulating polypeptides include, for example, members of the tumor necrosis factor receptor (TNFR) family (i.e., CD40, RANK/TRANCE-R, 0X40, 4-1 BB) and CD28 family members (CD28, ICOS), and may also include pattern recognition receptor adapters such as, for example MyD88. Chimeric polypeptides may comprise one, two, three, or more co-stimulating polypeptides or functionally equivalent portions thereof.

[0285] For example, in some embodiments, where an expression construct encodes a CD40 polypeptide, including but not limited to a human CD40 protein, the polypeptide may be a portion of the full-length CD40 polypeptide (also referred to as“truncated” CD40). By “truncated,” is meant that the protein is not full length and may lack, for example, a domain. By “cytoplasmic CD40” or“CD40 lacking the CD40 extracellular domain” is meant a CD40 polypeptide that lacks the CD40 extracellular domain. In some examples, the terms also refer to a CD40 polypeptide that lacks both the CD40 extracellular domain and a portion of, or all of, the CD40 transmembrane domain. In some embodiments, an expression construct encodes a CD40 polypeptide containing the intracellular domain of the CD40 protein. For example, an intracellular domain of a human CD40 polypeptide is encoded by the nucleotide sequence of SEQ ID NO: 33, or a functionally equivalent fragment thereof, with or without DNA linkers, or has an amino acid sequence of SEQ ID NO: 105, or a functionally equivalent fragment thereof.

A functionally equivalent portion of the CD40 polypeptide has substantially the same ability to stimulate intracellular signaling as the polypeptide of SEQ ID NO: 105, with at least 50%, 60%, 70%, 80%, 90%, or 95% of the activity of the polypeptide of SEQ ID NO: 105. In some embodiments, the expression construct encodes an intracellular region of the CD40 polypeptide lacking the extracellular domain and transmembrane domain encoded by the CD40-encoding nucleotide sequences of pM006 (FIG. 6), pM007 (FIG. 7) or pM009 (FIG. 8). By a nucleic acid sequence coding for“truncated CD40” is meant the nucleic acid sequence coding for a truncated CD40 polypeptide, the term may also refer to the nucleic acid sequence including the portion coding for any amino acids added as an artifact of cloning, including any amino acids coded for by the linkers.

[0286] It is understood that where a method or construct refers to a truncated CD40 polypeptide, the method may also be used, or the construct designed to refer to another CD40 polypeptide, such as a full length CD40 polypeptide. Where a method or construct refers to a full length CD40 polypeptide, the method may also be used, or the construct designed to refer to a truncated CD40 polypeptide.

[0287] As used herein, the term“functionally equivalent,” as it relates to CD40, or a portion thereof, for example, refers to a CD40 polypeptide that stimulates a cell-signaling response or a nucleic acid encoding such a CD40 polypeptide. “Functionally equivalent" refers, for example, to a CD40 polypeptide that is lacking an extracellular domain, but is capable of stimulating a cell-signaling response.

[0288] In some embodiments, where an expression construct encodes an MyD88 polypeptide, the polypeptide may be a portion of the full-length MyD88 polypeptide. By MyD88, or MyD88 polypeptide, is meant the polypeptide product of the myeloid differentiation primary response gene 88, for example, but not limited to the human version, cited as NCBI Gene ID 4615. In some embodiments, an expression construct encodes a portion of the MyD88 polypeptide lacking the TIR domain. In some embodiments, the expression construct encodes a portion of the MyD88 polypeptide containing the DD (death domain) or the DD and intermediary domains. By“truncated,” is meant that the protein is not full length and may lack, for example, a domain. For example, a truncated MyD88 is not full length and may, for example, be missing the TIR domain. Examples of a truncated MyD88 polypeptide amino acid sequence are presented as SEQ ID NOS: 102 and 103, or a functionally equivalent fragment thereof. Examples of a truncated MyD88 polypeptide are encoded by the nucleotide sequences of SEQ ID NOS: 30 and 31 , or a functionally equivalent fragment thereof. A functionally equivalent portion of the MyD88 polypeptide has substantially the same ability to stimulate intracellular signaling as the polypeptide of SEQ ID NOS: 102 or 103, with at least 50%, 60%, 70%, 80%, 90%, or 95% of the activity of the polypeptide of SEQ ID NOS: 102 or 103. In some embodiments, the expression construct encodes a portion of an MyD88 polypeptide lacking the TIR domain such as the polypeptide encoded by the MyD88 polypeptide-encoding nucleotide sequence of pM006 (FIG. 6), pM007 (FIG. 7) or pM009 (FIG. 8). By a nucleic acid sequence coding for“truncated MyD88” is meant the nucleic acid sequence coding for a truncated MyD88 polypeptide, the term may also refer to the nucleic acid sequence including the portion coding for any amino acids added as an artifact of cloning, including any amino acids coded for by the linkers.

[0289] It is understood that where a method or construct refers to a truncated MyD88 polypeptide, the method may also be used, or the construct designed to refer to another MyD88 polypeptide, such as a full length MyD88 polypeptide. Where a method or construct refers to a full length MyD88 polypeptide, the method may also be used, or the construct designed to refer to a truncated MyD88 polypeptide.

[0290] In the methods herein, in which a chimeric polypeptide comprises an MyD88 polypeptide (or portion thereof) and a CD40 polypeptide (or portion thereof), the MyD88 polypeptide of the chimeric polypeptide may be located either upstream or downstream from the CD40 polypeptide. In certain embodiments, the MyD88 polypeptide (or portion thereof) is located upstream of the CD40 polypeptide (or portion thereof).

[0291] As used herein, the term“functionally equivalent,” as it relates to MyD88, or a portion thereof, for example, refers to an MyD88 polypeptide that stimulates a cell-signaling response or a nucleic acid encoding such an MyD88 polypeptide. “Functionally equivalent" refers, for example, to an MyD88 polypeptide that is lacking a TIR domain, but is capable of stimulating a cell-signaling response.

[0292] Non-limiting examples of chimeric proteins useful for inducing cell activation, and related methods for inducing cell activation using a multimerizing agent, including expression constructs, methods for constructing vectors, assays for activity or function, multimerization of the chimeric polypeptides by contacting cells that express inducible chimeric polypeptides with a multimerizing agent that binds to the multimerizing region of the chimeric polypeptides both ex vivo and in vivo, administration of expression vectors, cells, or multimerizing agents to subjects, and administration of multimerizing agents to subjects who have been administered cells that express the inducible chimeric polypeptides, may also be found in the following patents and patent applications, each of which is incorporated by reference herein in its entirety for all purposes: U.S. Patent Application No. 14/210,034, filed Mar. 13, 2014, entitled METHODS FOR CONTROLLING T CELL PROLIFERATION, published Sept. 25, 2014 as US2014- 0286987-A1 ; International Patent Application No. PCT/US2014/026734, filed Mar. 13, 2014, published Sept. 25, 2014 as W02014/151960, by Spencer et al.; U.S. Patent Application No. 14/622,018, filed Feb, 13, 2014, entitled METHODS FOR ACTIVATING T CELLS USING AN INDUCIBLE CHIMERIC POLYPEPTIDE, published Feb. 18, 2016 as US2016-0046700-A1 ; International Patent Application No. PCT/US2015/015829, filed Feb. 13, 2015, published Aug. 20, 2015 as WO2015/123527; U.S. Patent Application No. 10/781 ,384, filed Feb. 18, 2004, entitled INDUCED ACTIVATION OF DENDRITIC CELLS, published Oct, 21 , 2004 as US2004- 0209836-A1 , issued June 29, 2008 as U.S. Patent No. 7,404,950, by Spencer et al.;

International Patent Application No. PCT/US2004/004757, filed Feb.18, 2004, published Mar.

24, 2005 as W02004/073641A3; U.S. Patent Application No. 12/445,939, filed Oct. 26, 2010, entitled METHODS AND COMPOSITIONS FOR GENERATING AN IMMUNE RESPONSE BY INDUCING CD40 AND PATTERN RECOGNITION RECEPTORS AND ADAPTORS THEREOF, published Feb. 10, 201 1 as US201 1-0033388-A1 , issued Apr. 8, 2014 as U.S. Patent No.

8,691 ,210, by Spencer et al.; International Patent Application No. PCT/US2007/081963, filed Oct. 19, 2007, published Apr. 24, 2008 as W02008/0491 13; U.S. Patent Application No.

13/763,591 , filed Feb. 8, 2013, entitled METHODS AND COMPOSITIONS FOR GENERATING AN IMMUNE RESPONSE BY INDUCING CD40 AND PATTERN RECOGNITION RECEPTOR ADAPTERS, published Mar. 27, 2014 as US2014-0087468-A1 , issued Apr. 19, 2016 as U.S. Patent No. 9,315,559, by Spencer et al.; International Patent Application No.

PCT/US2009/057738, filed Sept. 21 , 2009, published Mar. 25, 2010 as WO201033949; U.S. Patent Application No.13/087,329, filed Apr. 14, 201 1 , entitled METHODS FOR TREATING SOLID TUMORS, published Nov. 24, 201 1 as US201 1-0287038-A1 , by Slawin et al.;

International Patent Application No. PCT/US201 1/032572, filed April 14, 201 1 , published Oct. 20, 201 1 as WO201 1/130566, by Slawin et al; U.S. Patent Application No. 14/968,853, filed Dec. 14, 2015, entitled METHODS FOR CONTROLLED ACTIVATION OR ELIMINATION OF THERAPEUTIC CELLS, published June 23, 2016 as US2016-0175359-A1 , by Spencer et al.; International Patent Application No. PCT/US2015/065646, filed Dec. 14, 2015, published Sept.15, 2016 as WO2016/100241 , by Spencer et al.; U.S. Patent Application No. 15/377,776, filed Dec. 13, 2016, entitled DUAL CONTROLS FOR THERAPEUTIC CELL ACTIVATION OR ELIMINATION, published June 15, 2017 as US2017-0166877-A1., by Bayle et al.; International Patent Application No. PCT/US2016/066371 , filed Dec. 13, 2016, published June 22, 2017 as W02017/106185, by Bayle et al.; and U.S. Provisional Patent Application No. 62/503,565, filed May 9, 2017, entitled METHODS TO AUGMENT OR ALTER SIGNAL TRANSDUCTION, by Bayle et al., each of which is incorporated by reference herein in its entirety for all purposes. Multimeric compounds described herein, or pharmaceutically acceptable salts thereof, may be used essentially as discussed in examples provided in these publications, and other examples provided herein, to the extent that they refer to multimeric ligands.

Ligand-controlled cell activation/elimination

[0293] In some embodiments, chimeric proteins are provided, or nucleic acid encoding such proteins are provided, or a cell that contains such proteins or nucleic acids, for the purpose of inducing cell activation and cell death (or apoptosis or elimination) in response to one or more compounds described herein (or pharmaceutically acceptable salts thereof). In some embodiments, chimeric proteins designed to provide for inducible cell activation and chimeric proteins designed to provide for inducible cell death bind to different multimerizing agents. In such embodiments, both multimerizing agents may be compounds provided herein, or one multimerizing agent may be a compound provided herein and the other multimerizing agent may be a compound including, but not limited to, a compound described in U.S. patent application no. 62/608,552 (attorney docket no. BEL-2027-PV filed December 20, 2017, and entitled “Multimeric Compounds”), AP1903 (rimiducid; CAS no. 195514-63-7), AP20187 (CAS no. 195514-80-8) or AP1510 (see, e.g., Amara et al (1997) Proc Natl Acad Sci U.S.A. 94:10618- 10623). Embodiments in which chimeric proteins are provided, or nucleic acid encoding such proteins are provided, or a cell that contains such proteins or nucleic acids, for the purpose of enabling cell activation and cell death (or apoptosis or elimination) are referred to as“dual switch” or“dual control” compositions and methods.

[0294] Methods provided herein that include dual switch components can utilize chimeric proteins (and/or nucleic acids encoding such chimeric proteins), and cells expressing chimeric proteins, such as those described as examples for use in ligand-controlled cell activation and ligand-controlled cell apoptosis herein. For example, combinations of chimeric proteins containing co-stimulatory polypeptides (e.g., CD40 and/or MyD88) and chimeric proteins containing a pro-apoptotic polypeptide (e.g., caspase-9), and nucleic acids encoding such chimeric proteins, as well as cells containing such chimeric proteins and/or nucleic acids encoding them, can be used in methods provided herein.

[0295] In some embodiments of dual switch methods provided herein, a compound provided herein serves as one of the multimerizing agents (e.g., for induction of either cell activation or elimination) and a compound described in U.S. patent application no. 62/608,552 is used as another multimerizing agent (e.g., for induction of cell elimination if a compound provided herein is used for induction of cell activation or for induction of cell activation if a compound provided herein is used for cell elimination). In such methods, a compound described in U.S. patent application no. 62/608,552 may be, for example, one having a structure of the following Formula I or Formula II (or pharmaceutically acceptable salts of Formula I or Formula II):

I wherein: Z and Z’ are the same or different and each independently is O, NR¹², -N=, S, SO, SO2 or CH₂;

Y is L, M or Q:

R\ R², R³, and R⁴ are the same or different, and each is independently hydrogen, lower alkyl, heteroalkyl, perhaloalkyl, lower alkoxy, lower cycloalkyl, lower aryl, cycloalkyl, aryl, lower heterocycloalkyl, lower heteroaryl, heterocycloalkyl, or heteroaryl, which independently are optionally substituted with one or more substituents chosen from halogen, hydroxy, alkyl, heteroalkyl, haloalkyl, perhaloalkyl, perhaloalkoxy, alkoxy, haloalkoxy, alkoxyalkyl, acyl, oxo, acyloxy, carboxyl, amido, cyano, amino, alkylamino, alkylaminoalkyl, thiol, alkylthio,

alkylthioalkyl, haloalkylthio, perhaloalkylthio, nitro, aryl, arylalkyl, cycloalkylalkyl, heterocycle- alkyl, cycloalkyl, heterocycloalkyl, heteroaryl, heteroarylalkyl, alkylsulfonyl, sulfonamide, alkylsulfonamido and alkylsilyloxy;

when Y is M, R¹ and R² together with -N-R_L-N- may form a heterocyclic or heteroaryl ring optionally substituted with one or more substituents chosen from halogen, hydroxy, alkyl, heteroalkyl, haloalkyl, perhaloalkyl, perhaloalkoxy, alkoxy, haloalkoxy, alkoxyalkyl, acyl, oxo, acyloxy, carboxyl, amido, cyano, amino, alkylamino, alkylaminoalkyl, thiol, alkylthio,

alkylthioalkyl, haloalkylthio, perhaloalkylthio, nitro, aryl, arylalkyl, cycloalkylalkyl, heterocycle- alkyl, cycloalkyl, heterocycloalkyl, heteroaryl, heteroarylalkyl, alkylsulfonyl, sulfonamide, alkylsulfonamido and alkylsilyloxy; when Y is Q, R¹ and R² together with N⁺ may form a heterocyclic or heteroaryl ring optionally substituted with one or more substituents chosen from halogen, hydroxy, alkyl, heteroalkyl, haloalkyl, perhaloalkyl, perhaloalkoxy, alkoxy, haloalkoxy, alkoxyalkyl, acyl, oxo, acyloxy, carboxyl, amido, cyano, amino, alkylamino, alkylaminoalkyl, thiol, alkylthio, alkylthioalkyl, haloalkylthio, perhaloalkylthio, nitro, aryl, arylalkyl, cycloalkylalkyl, heterocycle-alkyl, cycloalkyl, heterocycloalkyl, heteroaryl, heteroarylalkyl, alkylsulfonyl, sulfonamide, alkylsulfonamido and alkylsilyloxy;

when Y is Q, R³ and R⁴ together with N⁺ may form a heterocyclic or heteroaryl ring optionally substituted with one or more substituents chosen from halogen, hydroxy, alkyl, heteroalkyl, haloalkyl, perhaloalkyl, perhaloalkoxy, alkoxy, haloalkoxy, alkoxyalkyl, acyl, oxo, acyloxy, carboxyl, amido, cyano, amino, alkylamino, alkylaminoalkyl, thiol, alkylthio, alkylthioalkyl, haloalkylthio, perhaloalkylthio, nitro, aryl, arylalkyl, heterocycle-alkyl,

cycloalkylalkyl, cycloalkyl, heterocycloalkyl, heteroaryl, heteroarylalkyl, alkylsulfonyl, sulfonamide, alkylsulfonamido and alkylsilyloxy;

when Y is Q, R¹ and R³ together with -N⁺-R_L-N⁺- may form a heterocyclic or heteroaryl ring optionally substituted with one or more substituents chosen from halogen, hydroxy, alkyl, heteroalkyl, haloalkyl, perhaloalkyl, perhaloalkoxy, alkoxy, haloalkoxy, alkoxyalkyl, acyl, oxo, acyloxy, carboxyl, amido, cyano, amino, alkylamino, alkylaminoalkyl, thiol, alkylthio, alkylthioalkyl, haloalkylthio, perhaloalkylthio, nitro, aryl, arylalkyl, heterocycle-alkyl,

when Y is Q, R² and R⁴ together with -N⁺-RL-N⁺- may form a heterocyclic or heteroaryl ring optionally substituted with one or more substituents chosen from halogen, hydroxy, alkyl, heteroalkyl, haloalkyl, perhaloalkyl, perhaloalkoxy, alkoxy, haloalkoxy, alkoxyalkyl, acyl, oxo, acyloxy, carboxyl, amido, cyano, amino, alkylamino, alkylaminoalkyl, thiol, alkylthio, alkylthioalkyl, haloalkylthio, perhaloalkylthio, nitro, aryl, arylalkyl, heterocycle-alkyl,

Optionally, when Y is Q, one of the groups: R¹, R², R³ and R⁴ may be nonexistent. If one of the groups: R¹, R², R³ or R⁴ is non-existent, and Y is Q, the compound is a monosalt;

R_L is a lower alkylene, alkenylene, alkynylene, acyl, cycloalkyl, or aryl, in which none or one or more carbon atoms are replaced by O, NR¹³, S, SO, S0₂, and which is optionally substituted with hydroxyl, alkoxyl, amino, alkylamino, thiol, thioalkyl, or halogen;

A and A’ are the same or different and each independently are

, thiophene, furan, pyrrole, carbonyl, lower dialkyl ether, lower dialkyl thioether, lower dialkylamino, cyclopropylene, alkanylene, cycloalkanylene, alkenylene, cycloalkenylene, lower alkynylene, lower cycloalkynylene, carbamate, sulfanyl, sulfinyl, sulfonyl, thiocarbonyl, imino, or hydroxyimino, in which independently none or one or more carbon atoms are replaced by O, NR¹⁴, S, SO, S0₂, and which is optionally substituted with hydroxyl, alkoxyl, amino, alkylamino, thiol, thioalkyl, or halogen;

R¹² is hydrogen, lower alkyl, heteroalkyl, perhaloalkyl, lower alkoxy, lower cycloalkyl, lower aryl, cycloalkyl, aryl, lower heterocycloalkyl, lower heteroaryl, heterocycloalkyl, or heteroaryl, which independently are optionally substituted with one or more substituents chosen from halogen, hydroxy, alkyl, heteroalkyl, haloalkyl, perhaloalkyl, perhaloalkoxy, alkoxy, haloalkoxy, alkoxyalkyl, acyl, oxo, acyloxy, carboxyl, amido, cyano, amino, alkylamino, alkylaminoalkyl, thiol, alkylthio, alkylthioalkyl, haloalkylthio, perhaloalkylthio, nitro, aryl, arylalkyl, heterocycle-alkyl, cycloalkylalkyl, cycloalkyl, heterocycloalkyl, heteroaryl, heteroarylalkyl, alkylsulfonyl, sulfonamide, alkylsulfonamido and alkylsilyloxy;

R¹³ is hydrogen, lower alkyl, heteroalkyl, perhaloalkyl, lower alkoxy, lower cycloalkyl, lower aryl, cycloalkyl, aryl, lower heterocycloalkyl, lower heteroaryl, heterocycloalkyl, or heteroaryl, which independently are optionally substituted with one or more substituents chosen from halogen, hydroxy, alkyl, heteroalkyl, haloalkyl, perhaloalkyl, perhaloalkoxy, alkoxy, haloalkoxy, alkoxyalkyl, acyl, oxo, acyloxy, carboxyl, amido, cyano, amino, alkylamino, alkylaminoalkyl, thiol, alkylthio, alkylthioalkyl, haloalkylthio, perhaloalkylthio, nitro, aryl, arylalkyl, heterocycle-alkyl, cycloalkylalkyl, cycloalkyl, heterocycloalkyl, heteroaryl, heteroarylalkyl, alkylsulfonyl, sulfonamide, alkylsulfonamido and alkylsilyloxy;

R¹⁴ hydrogen, lower alkyl, heteroalkyl, perhaloalkyl, lower alkoxy, lower cycloalkyl, lower aryl, cycloalkyl, aryl, lower heterocycloalkyl, lower heteroaryl, heterocycloalkyl, or heteroaryl, which independently are optionally substituted with one or more substituents chosen from halogen, hydroxy, alkyl, heteroalkyl, haloalkyl, perhaloalkyl, perhaloalkoxy, alkoxy, haloalkoxy, alkoxyalkyl, acyl, oxo, acyloxy, carboxyl, amido, cyano, amino, alkylamino, alkylaminoalkyl, thiol, alkylthio, alkylthioalkyl, haloalkylthio, perhaloalkylthio, nitro, aryl, arylalkyl, heterocycle-alkyl, cycloalkylalkyl, cycloalkyl, heterocycloalkyl, heteroaryl, heteroarylalkyl, alkylsulfonyl, sulfonamide, alkylsulfonamido and alkylsilyloxy;

X¹, X², X³, X⁴, X⁵ and X⁶ independently are carbon or nitrogen with the proviso that none, one, two or three of X¹, X², X³, X⁴, X⁵ and X⁶ are nitrogen;

when X², X³, X⁴, X⁵ or X⁶ is carbon, R⁵, R⁶, R⁷, R⁸ or R⁹, respectively, independently is hydrogen, hydroxyl, halogen, C1-C2 alkyl or C1-C2 alkyl substituted with hydroxyl, halogen or NR¹⁰R¹¹;

R¹⁰ and R¹¹ independently are hydrogen or C1-C2 alkyl;

when X², X³, X⁴ or X⁶ is nitrogen, R⁵, R⁶, R⁷, R⁸ or R⁹, respectively, is not present or is hydrogen, C1 -C2 alkyl or C1-C2 alkyl substituted with hydroxyl, halogen or NR¹⁰R¹¹ ;

with the proviso that (i) Y is M or Q when A and A’ are phenyl and Z and Z' are oxygen, or (ii) A and A' are not the same, or one or both of A and A' are not phenyl, or Z and Z' are not the same, or one or both of Z and Z' are not oxygen, when Y is L and R_L is -CH2-CH2-. Expression constructs and cells for use in the methods

[0296] In some embodiments of the methods provided herein, the method involves use of chimeric fusion proteins and/or nucleic acids encoding such proteins, such as, for example, those described herein. In some embodiments, the method involves use of expression vectors or constructs, such as, for example, those described herein, containing nucleic acids encoding such proteins. In some embodiments, the method includes steps of generating such proteins and/or nucleic acids encoding such proteins and/or expression vectors or constructs containing nucleic acids encoding such proteins. In some embodiments, the method involves use of cells, such as, for example, cells described herein. In some embodiments, the method involves use of cells containing chimeric fusion proteins and/or nucleic acids encoding such proteins expressing such proteins and/or expression vectors or constructs containing nucleic acids encoding such proteins. In some embodiments, the method includes steps of generating such cells.

[0297] Expression constructs include, for example, constructs containing nucleic acids encoding chimeric polypeptides comprising one or more multimerizing regions and at least one additional polypeptide, such as, for example, a caspase-9 polypeptide (or portion thereof), or a costimulating polypeptide (or portion(s) thereof), such as, for example, MyD88, CD40, or both MyD88 and CD40 polypeptides. In embodiments of methods provided herein, the chimeric polypeptides expressed from such expression constructs may be contacted by a multimeric compound described herein, or a pharmaceutically acceptable salt thereof.

[0298] In certain embodiments, a chimeric polypeptide may comprise more than one ligand binding domain or multimerizing region. In some embodiments, the chimeric polypeptide may comprise one, two, three, or more ligand binding domains or multimerizing regions. In some embodiments in which a chimeric polypeptide is contacted with a compound provided herein, the selective affinity of the compound for a ligand-binding polypeptide, such as, e.g., FRB (or a variant thereof) and/or FRBP12 (or a variant thereof) permits specific binding of the compound to the chimeric polypeptide without the induction (or diminished induction) of undesired cellular activities and/or side effects in vivo.

[0299] A multimerizing region of a chimeric protein encoded by an expression construct may contain, for example, an FRB polypeptide (or variant thereof) and/or an FKBP12 polypeptide (or variant thereof). Examples of FRB variant polypeptides include, but are not limited to, KLW (T2098L), PLW (K2095P, T2098L), TLW (K2095T, T2098L), KTF (W2101 F), ATF (K2095A, W2101 F), PTF (K2095P, W2101 F), KLF (T2098L, W2101 F), TLF (K2095T, T2098L, W2101 F) and RLF (K2095R, T2098L, W2101 F). An FRB variant KLW is also referred to as FRB_L polypeptide (see, e.g., SEQ ID NO: 79 for an example of an amino acid sequence containing an FRB_L polypeptide). By comparing the KLW variant of SEQ ID NO: 79 with a wild type FRB polypeptide (SEQ ID NO: 77), one can determine the sequence of other FRB variants, including those listed herein. In some examples, an FRB polypeptide variant, or FRB mutant, binds to a ligand, such as a rapamycin analog or a multimeric compound provided herein, or a pharmaceutically acceptable salt thereof, with at least 100 times more affinity than a wild type FRB polypeptide, such as, for example, the wild type FRB polypeptide having the amino acid sequence of SEQ ID NO: 77.

[0300] An example of an FKBP12 variant is FKBP12v36 (SEQ ID NO: 93). The amino acid at position 36 of wild type FKBP12 polypeptide is phenylalanine. FKBP12 polypeptide variants include, but are not limited to, those having amino acid substitutions at position 36, e.g., valine, leucine, isoleucine, and alanine. FKBP12 variants having amino acid substitutions and deletions, such as FKBP12v36, that bind to a multimizer drug, may also be used in methods provided herein.

[0301] In certain embodiments, for example, those involving dual switch compositions and methods, a chimeric polypeptide may be contacted with a multimerizing agent, such as, for example, a compound described in U.S. patent application no. 62/608,552 or a third-generation AP20187/AP1903 CID which has selective affinity for an FKBP12 variant (e.g., FKBP12v36), ligand-binding polypeptide which permits specific binding of the agent to the chimeric polypeptide in vivo without the induction of non-specific side effects through endogenous FKBP12. The human 12 kDa FKBP12 protein with an F36 to V substitution (FKBP12v36), the complete mature coding sequence (amino acids 1-107), provides a binding site for synthetic dimerizer drug AP1903 (see, e.g., Jemal et al. (2008) CA Cancer J Clinic 58:71-96; Scher and Kelly (1993) J Clinic Oncol 1 1 :1566-72 (1993)). In some examples, an FKBP12 polypeptide variant, or FKBP12 mutant, binds to a ligand, such as rimiducid or a compound described in U.S. patent application no. 62/608,552, with at least 100 times more affinity than a wild type FKBP12 polypeptide, such as, for example, the wild type FKBP12 polypeptide having the amino acid sequence of SEQ ID NO: 85. Examples of FKBP12 polypeptide variants, and of methods of using the CID system, include, for example, those discussed in Kopytek et al. ((2000) Chem & Biol 7:313-321), Gestwicki et al. ((2007) Combinatorial Chem & High Throughput Screening 10:667-675), Clackson ((2006) Chem Biol Drug Des 67:440-2) and Clackson (in Chemical Biology: From Small Molecules to Systems Biology and Drug Design (Schreiber et al., eds., Wiley, 2007)). [0302] Multiple copies of the ligand-binding polypeptide may also be used in chimeric proteins encoded by a nucleic acid construct so that higher-order oligomers are induced upon cross- linking by multimerizing ligand. For example, for chimeric proteins designed for multimerization by an embodiment of a multimeric compound provided herein, the ligand-binding portion of the protein can contain one or more FRB proteins (or variant thereof) and one or more FKBP12 proteins (or variants thereof). In some embodiments, multiple ligand binding region encoding polynucleotides that may be present in a plasmid (i) often encode identical ligand binding region polypeptides, (ii) sometimes are identical to the other, and/or (iii) sometimes are not identical to one another (e.g., one or more codons are different (e.g., wobbled) when compared to one another). In some embodiments, inducible chimeric polypeptides contain an F_vF_Vis sequence, which comprises two FKBP12v36 polypeptides. In a non-limiting example, F36V’-FKBP12 is a codon-wobbled version of F36V-FKBP12. It encodes the identical polypeptide sequence as F36V-FKPB12 but has only 62% homology at the nucleotide level. F36V’-FKBP12 was designed to reduce recombination in retroviral vectors (Schellhammer et al. (1997) J Urol 157:1731-1735). F36V’-FKBP12 can be constructed, for example, by a PCR assembly procedure. The transgene contains one copy of F36V’-FKBP12 linked directly to one copy of F36V-FKBP12.

[0303] The transduced signal will normally result from ligand-mediated oligomerization of the chimeric protein molecules, i.e., as a result of oligomerization following ligand binding, although other binding events, for example allosteric activation, can be employed to initiate a signal. The construct of the chimeric protein will vary as to the order of the various domains and the number of repeats of an individual domain.

[0304] An expression construct may or may not encode a membrane-targeting sequence. In some embodiments, the chimeric polypeptide may contain a membrane targeting region. In other embodiments, the chimeric polypeptide does not include a membrane targeting region. Appropriate expression constructs may include a co-stimulating or pro-apoptotic polypeptide region on either side or both sides of one or more ligand binding domains or multimerizing regions in a chimeric fusion protein. In some embodiments, one or more co-stimulating or pro- apoptotic polypeptide regions is provided at a location on the polypeptide that is amino-terminal to the one or more ligand-binding domains or multimerizing regions. In some embodiments, the one or more ligand-binding domains or multimerizing regions is provided at a location on the polypeptide that is amino-terminal to the one or more co-stimulating or pro-apoptotic polypeptide regions. In some embodiments, the one or more ligand-binding domains or multimerizing regions are provided on both sides (i.e., at a location on the polypeptide that is amino-terminal to the one or more co-stimulating or pro-apoptotic polypeptide regions and at a location on the polypeptide that is carboxy-terminal to the one or more co-stimulating or pro-apoptotic polypeptide regions).

[0305] For purposes of the present application, the terms“multimerizing region”

“multimerization region”“ligand binding region” and“multimeric ligand binding region” are interchangeable.

[0306] In some embodiments, a nucleic acid that encodes a chimeric polypeptide may encode a heterologous protein (e.g., heterologous to an apoptosis-inducing polypeptide or costimulating polypeptide), non-limiting examples of which include a marker polypeptide, a chimeric antigen receptor, or a recombinant T cell receptor.

[0307] In some embodiments, the polypeptides that make up the components of a cell-based compound-inducible system for use in the methods provided herein may be expressed separately from the same vector, where each polynucleotide coding for one of the polypeptides is operably linked to a separate promoter. In certain embodiments, a promoter may be operably linked to each of multiple polynucleotides, directing the production of multiple separate RNA transcripts, and thus multiple polypeptides. Therefore, the expression constructs discussed herein may contain at least one, or at least two promoters.

[0308] A heterologous polypeptide, for example, a chimeric antigen receptor, may be linked to an apoptosis-inducing polypeptide or co-stimulating polypeptide via a polypeptide sequence, such as, for example, a cleavable 2A-like sequence. For example, a nucleic acid that encodes a chimeric fusion polypeptide may comprise a polynucleotide that encodes the chimeric fusion polypeptide, a polynucleotide that encodes a 2A-like sequence, and a polynucleotide that encodes a heterologous polypeptide, with one promoter operably linked to the three

polynucleotides. In such embodiments, the polypeptides are separated during translation, resulting in two polypeptides, such as, for example, a chimeric fusion polypeptide that includes a multimerizing region and an additional polypeptide, such as, for example an apoptosis-inducing polypeptide or the co-stimulating polypeptide, and a heterologous polypeptide, such as, for example, a chimeric antigen receptor polypeptide.

[0309] 2A-like sequences, or“cleavable” 2A sequences, are derived from, for example, many different viruses, including, from Thosea asigna. These sequences are sometimes also known as“peptide skipping sequences.” When this type of sequence is placed within a cistron, between two polypeptides that are intended to be separated, the ribosome appears to skip a peptide bond, In the case of Thosea asigna sequence, the bond between the Gly and Pro amino acids is omitted. This leaves two polypeptides, for example, a caspase-9 polypeptide and a marker polypeptide, or a chimeric antigen receptor polypeptide. When this sequence is used, the polypeptide that is encoded 5’ of the 2A sequence may end up with additional amino acids at the carboxy terminus, including the Gly residue and any upstream in the 2A sequence. The polypeptide that is encoded 3’ of the 2A sequence may end up with additional amino acids at the amino terminus, including the Pro residue and any downstream in the 2A sequence. “2A” or “2A-like” sequences are part of a large family of peptides that can cause peptide bond-skipping. Various 2A sequences have been characterized (e.g., F2A, P2A, T2A), and are examples of 2A- like sequences that may be encoded by nucleic acid constructs used in methods provided herein. In certain embodiments, a 2A linker includes the amino acid sequence of SEQ ID NO: 108. In certain embodiments, the 2A linker further includes a GSG amino acid sequence at the amino terminus of the polypeptide, in other embodiments, the 2A linker includes a GSGPR amino acid sequence at the amino terminus of the polypeptide. Thus, by a“2A” sequence, the term may refer to a 2A sequence in an example described herein or may also refer to a 2A sequence as listed herein further comprising a GSG or GSGPR sequence at the amino terminus of the linker.

[0310] In certain embodiments, the chimeric fusion polypeptide and a heterologous polypeptide may be expressed in a cell using two separate vectors encoding the separate polypeptides. The cells may be co-transfected or co-transduced with the vectors, or the vectors may be introduced to the cells at different times.

[0311] In some embodiments, a nucleic acid construct is contained within a viral vector. In certain embodiments, the viral vector is a retroviral vector. In certain embodiments, the viral vector is an adenoviral vector or a lentiviral vector. It is understood that in some embodiments, a cell is contacted with the viral vector ex vivo, and in some embodiments, the cell is contacted with the viral vector in vivo. Thus, an expression construct may be inserted into a vector, for example a viral vector or plasmid. The steps of the methods provided may be performed using any suitable method; these methods include, without limitation, methods of transducing, transforming, or otherwise providing nucleic acid to the cell, described herein.

[0312] The terms“gene expression vector”,“nucleic acid expression vector”, or“expression vector” as used interchangeably herein, generally refer to a nucleic acid molecule (e.g., a plasmid, phage, autonomously replicating sequence (ARS), artificial chromosome, yeast artificial chromosome (e.g., YAC)) that can be replicated in a host cell and be utilized to introduce a gene or genes into a host cell. The genes introduced on the expression vector can be endogenous genes (e.g., a gene normally found in the host cell or organism) or heterologous genes (e.g., genes not normally found in the genome or on extra-chromosomal nucleic acids of the host cell or organism). The genes introduced into a cell by an expression vector can be native genes or genes that have been modified or engineered. The gene expression vector also can be engineered to contain 5’ and 3’ untranslated regulatory sequences that sometimes can function as enhancer sequences, promoter regions and/or terminator sequences that can facilitate or enhance efficient transcription of the gene or genes carried on the expression vector. A gene expression vector sometimes also is engineered for replication and/or expression functionality (e.g., transcription and translation) in a particular cell type, cell location, or tissue type. Expression vectors sometimes include a selectable marker for maintenance of the vector in the host or recipient cell.

[0313] As used herein, the terms“expression construct” and“expression vector” are used interchangeably and generally refer to nucleic acids that include product-encoding nucleic acids, in which part or all of the nucleic acid sequence is capable of being transcribed. The transcript may or may not be translated into a protein. In certain embodiments, expression includes both transcription of nucleic acid and translation of mRNA into a product. In other embodiments, expression only includes transcription of the nucleic acid. Expression vectors can contain a variety of control sequences, which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operatively linked coding sequence in a particular host cell or organism. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well and are discussed infra.

[0314] The term“therapeutic construct” refers to an expression construct or transgene that may be used, for example, in prophylaxis or therapy, such as to treat hyperproliferative diseases or disorders, e.g., cancer. As used herein with reference to a disease, disorder or condition, the terms "treatment", "treat", "treated", or "treating" refer to prophylaxis and/or therapy.

[0315] As used herein, the term“gene” is defined as a functional protein-, polypeptide-, or peptide-encoding unit. As will be understood, this functional term includes genomic sequences, cDNA sequences, and smaller engineered gene segments that express, or are adapted to express, proteins, polypeptides, domains, peptides, fusion proteins and/or mutants.

[0316] As used herein, the term“cDNA” is intended to refer to DNA prepared using messenger RNA (mRNA) as template. The advantage of using a cDNA, as opposed to genomic DNA or DNA polymerized from a genomic, non- or partially-processed RNA template, is that the cDNA primarily contains coding sequences of the corresponding protein. There are times when the full or partial genomic sequence is used, such as where the non-coding regions are required for optimal expression or where non-coding regions such as introns are to be targeted in an antisense strategy.

[0317] As used herein, the term“polynucleotide” is defined as a chain of nucleotides.

Furthermore, nucleic acids are polymers of nucleotides. Thus, nucleic acids and

polynucleotides as used herein are interchangeable. Nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric“nucleotides.” The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR™, and the like, and by synthetic means. Furthermore, polynucleotides include mutations of the polynucleotides, include but are not limited to, mutation of the nucleotides, or nucleosides by methods well known in the art. A nucleic acid may comprise one or more polynucleotides.

[0318] Amino acids other than those indicated as conserved may differ in a protein or enzyme so that the percent protein or amino acid sequence similarity between any two proteins of similar function may vary and can be, for example, at least 70%, at least 80%, at least 90%, and at least 95%, as determined according to an alignment scheme. As referred to herein, "sequence similarity" means the extent to which nucleotide or protein sequences are related. The extent of similarity between two sequences can be based on percent sequence identity and/or conservation. "Sequence identity" herein means the extent to which two nucleotide or amino acid sequences are invariant. "Sequence alignment" means the process of lining up two or more sequences to achieve maximal levels of identity (and, in the case of amino acid sequences, conservation) for the purpose of assessing the degree of similarity. Numerous methods for aligning sequences and assessing similarity/identity are known in the art such as, for example, the Cluster Method, wherein similarity is based on the MEGALIGN algorithm, as well as BLASTN, BLASTP, and FASTA. When using any of these programs, the settings may be selected that result in the highest sequence similarity.

[0319] The term "antigen" as used herein is defined as a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both. [0320] An“antigen recognition moiety” may be any polypeptide or fragment thereof, such as, for example, an antibody fragment variable domain, either naturally-derived, or synthetic, which binds to an antigen. Examples of antigen recognition moieties include, but are not limited to, polypeptides derived from antibodies, such as, for example, single-chain variable fragments (scFv), Fab, Fab’, F(ab’)2, and Fv fragments; polypeptides derived from T Cell receptors, such as, for example, TCR variable domains; and any ligand or receptor fragment that binds to the extracellular cognate protein.

[0321] As used herein, the term "promoter" is defined as a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene.

[0322] In some embodiments, the promoter is a developmental^ regulated promoter. The term“developmentally regulated promoter” as used herein refers to a promoter that acts as the initial binding site for RNA polymerase to transcribe a gene which is expressed under certain conditions that are controlled, initiated by or influenced by a developmental program or pathway. Developmentally regulated promoters often have additional control regions at or near the promoter region for binding activators or repressors of transcription that can influence transcription of a gene that is part of a development program or pathway. Developmentally regulated promoters sometimes are involved in transcribing genes whose gene products influence the developmental differentiation of cells. A developmentally regulated promoter may be used in the nucleic acids of the present application, where it is anticipated that the nucleic acid will be expressed in developmentally differentiated cells.

[0323] The term“immune cells,” as used herein, refers to leukocytes, or white blood cells, of the immune system. Immune cells include lymphocytes, monocytes, macrophages and granulocytes (neutrophils, basophils, eosinophils). Lymphocytes include T cells, B cells and natural killer (NK) cells.

[0324] The term“peripheral blood” as used herein, refers to cellular components of blood (e.g., red blood cells, white blood cells and platelets), which are obtained or prepared from the circulating pool of blood and not sequestered within the lymphatic system, spleen, liver or bone marrow.

[0325] Peripheral blood mononuclear cells (PBMCs) are blood cells with round nuclei and include lymphocytes, monocytes and macrophages. Typically, PMBCs are obtained by density gradient centrifugation of anticoagulated peripheral venous blood using a hydrophilic colloid. [0326] The term "transfection" and“transduction” are interchangeable and refer to the process by which an exogenous nucleic acid sequence is introduced into a eukaryotic host cell.

Transfection (or transduction) can be achieved by any one of a number of means including electroporation, microinjection, gene gun delivery, retroviral infection, lipofection, superfection and the like.

[0327] The term“developmentally differentiated cells”, as used herein refers to cells that have undergone a process, often involving expression of specific developmentally regulated genes, by which the cell evolves from a less specialized form to a more specialized form in order to perform a specific function. Non-limiting examples of developmentally differentiated cells are liver cells, lung cells, skin cells, nerve cells, blood cells, and the like. Changes in developmental differentiation generally involve changes in gene expression (e.g., changes in patterns of gene expression), genetic re-organization (e.g., remodeling or chromatin to hide or expose genes that will be silenced or expressed, respectively), and occasionally involve changes in DNA sequences (e.g., immune diversity differentiation). Cellular differentiation during development can be understood as the result of a gene regulatory network. A regulatory gene and its cis- regulatory modules are nodes in a gene regulatory network that receive input (e.g., protein expressed upstream in a development pathway or program) and create output elsewhere in the network (e.g., the expressed gene product acts on other genes downstream in the

developmental pathway or program).

[0328] As used herein, the term "under transcriptional control,”“operably linked,” or “operatively linked” is defined as the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene. In general, the term“operably linked” is meant to indicate that the promoter sequence is functionally linked to a second sequence, wherein, for example, the promoter sequence initiates and mediates transcription of the DNA corresponding to the second sequence.

Methods of treating and/or of regulating treatment

[0329] Provided herein are methods of treating, preventing and/or delaying the onset of a disease, disorder or condition. Also provided herein are methods of regulating a treatment (e.g., a cell-based treatment) administered for therapy, prevention, and/or delaying the onset of a disease, disorder or condition. In some embodiments, the methods include contacting a cell with a compound, or pharmaceutically acceptable salt thereof, provided herein. In some embodiments, the contacting occurs ex vivo, in vivo and/or in vitro. Diseases, disorders and conditions that can be treated, prevented and/or delayed using methods provided herein include, but are not limited to, microbial (e.g., fungal) infections, restenosis (e.g., as can occur after angioplasty/arterial stent implantation), transplantation rejection, graft versus host disease, cancer, autoimmune disorders and proliferative dysregulation disorders (e.g.,

lymphangiomyomatosis, angiolipomas, neurofibromatosis, Cowden’s syndrome and tuberous sclerosis).

[0330] In some embodiments of the methods of treating, preventing and/or regulating treatment provided herein, a compound provided herein is administered to a subject having or suspected of having, a disease, disorder or condition that is to be treated by administration of the compound or one that treatment of is to be regulated by administration of the compound. In some embodiments, a compound provided herein is administered to a subject susceptible to, or at risk of having, a disease, disorder or condition, in order to prevent or delay the onset of the disease, disorder or condition or to regulate administration of a treatment to prevent or delay the onset of the disease, disorder or condition. In some embodiments of the methods, the cell contacted with a compound provided herein is a cell contained within a subject that had not been removed from the subject.

[0331] In some embodiments of the methods of treating, preventing and/or regulating treatment provided herein, a cell is contacted ex vivo or in vitro with a compound, or

pharmaceutically acceptable salt thereof, provided herein. In some embodiments of the methods, the cell that is contacted with a compound provided herein is one that expresses a chimeric polypeptide containing a ligand-binding region to which the compound binds. In some embodiments of the methods, binding of the compound to the chimeric polypeptide results in multimerization of the chimeric polypeptide.

[0332] Some embodiments of the methods of treating, preventing and/or regulating treatment provided herein include one or more steps of (1) obtaining cells from a subject (e.g., a subject having and/or susceptible to a disease, disorder or condition) being treated (2) transferring (e.g., by transducing or transfecting) nucleic acids encoding one or more chimeric proteins containing one or more domains to which a compound provided herein binds into cells (e.g., into a subject’s or other cells), (3) transferring cells containing heterologous nucleic acids encoding one or more proteins that contain one or more domains to which a compound provided herein binds into a subject having and/or susceptible to a disease, disorder or condition and/or (4) administering a compound, or pharmaceutically acceptable salt thereof, provided herein to a subject containing cells that contain heterologous nucleic acids encoding one or more proteins that contain one or more domains to which a compound provided herein binds. [0333] As used herein, the term“ex vivo” refers to“outside” the body. The terms“ex vivo” and “in vitro” can be used interchangeably herein. In some embodiments of methods provided herein, modified cells that express a chimeric polypeptide are administered to a subject before, or at the same time that, a compound described herein, or a pharmaceutically acceptable salt thereof, is administered to the subject. In some embodiments, a compound described herein, or a pharmaceutically acceptable salt thereof, is administered to a subject, wherein modified cells that express a chimeric polypeptide have been administered to the subject. In such

embodiments, the compound described herein, or a pharmaceutically acceptable salt thereof, is administered to a subject who has received a transfusion or other administration of the modified cells, which can express a chimeric polypeptide containing a multimerizing region that binds to a compound provided herein, or a pharmaceutically acceptable salt thereof. The chimeric polypeptide may contain, for example, an apoptosis-inducing polypeptide, such as caspase-9, or a caspase-9 polypeptide that lacks the CARD domain. In other embodiments, the chimeric polypeptide may contain a polypeptide that activates cell activity, for example, immune activity, such as, for example, a co-stimulating polypeptide.

[0334] Non-limiting examples of cells for use in cell-based treatment or therapy methods provided herein include T cells, tumor infiltrating lymphocytes, natural killer cells, natural killer T cells, or progenitor cells, such as, for example, hematopoietic stem cells, mesenchymal stromal cells, stem cells, pluripotent stem cells, and embryonic stem cells. The cells may be from a donor or may be cells obtained from the subject. The cells may, for example, be used in regeneration, for example, to replace the function of diseased cells. The cells may also be modified to express a heterologous gene so that biological agents may be delivered to specific microenvironments such as, for example, diseased bone marrow or metastatic deposits.

Mesenchymal stromal cells have also been used, for example, to provide immunosuppressive activity, and may be used in the treatment of graft versus host disease and autoimmune disorders.

[0335] By“therapeutic cell” is meant a cell used for cell-based treatment or therapy, that is, a cell administered to a subject to treat or prevent a condition or disease. In some embodiments, there is a need to eliminate, or reduce the number of therapeutic cells in a subject. In certain embodiments, the therapeutic cells express a chimeric polypeptide containing one or more multimerizing regions, for example, an FRB (or variant thereof) polypeptide and/or an FKBP12 polypeptide (or variant thereof), and an apoptosis-inducing polypeptide, e.g., a caspase-9 polypeptide, or portion thereof, and the number of therapeutic cells may be reduced by administering a multimeric compound provided herein, or a pharmaceutically acceptable salt thereof, to the subject.

[0336] The terms“cell,”“cell line,” and“cell culture” as used herein may be used

interchangeably. All of these terms also include their progeny, which are any and all subsequent generations. It is understood that all progeny may not be identical due to deliberate or inadvertent mutations.

[0337] By“obtained or prepared” as, for example, in the case of cells, is meant that the cells or cell culture are isolated, purified, or partially purified from the source, where the source may be, for example, umbilical cord blood, bone marrow, or peripheral blood. The terms may also apply to the case where the original source, or a cell culture, has been cultured and the cells have replicated, and where the progeny cells are now derived from the original source.

[0338] In some embodiments of the treatment methods provided herein, T cells are used to treat various diseases and conditions, and as a part of stem cell transplantation. An adverse event that may occur after haploidentical T cell transplantation is graft versus host disease (GvHD). The terms“graft versus host disease” or“GvHD”, refer to a complication often associated with allogeneic bone marrow transplantation and sometimes associated with transfusions of un-irradiated blood to immunocompromised patients. Graft versus host disease sometimes can occur when functional immune cells in the transplanted marrow recognize the recipient as "foreign" and mount an immunologic response. GvHD can be divided into an acute form and a chronic form. Acute GVHD (aGVHD) often is observed within the first 100 days following transplant or transfusion and can affect the liver, skin, mucosa, immune system (e.g., the hematopoietic system, bone marrow, thymus, and the like), lungs and gastrointestinal tract. Chronic GVHD (cGVHD) often begins 100 days or later post-transplant or transfusion and can attack the same organs as acute GvHD, but also can affect connective tissue and exocrine glands. Acute GvHD of the skin can result in a diffuse maculopapular rash, sometimes in a lacy pattern.

[0339] The likelihood of GvHD occurring increases with the increased number of T cells that are transplanted. This limits the number of T cells that may be infused. By having the ability to selectively remove the infused T cells in the event of GvHD in the patient, a greater number of T cells may be infused, increasing the number to greater than 10⁶, greater than 10⁷, greater than 10⁸, or greater than 10⁹ cells. The number of T cells/kg subject body weight that may be administered may be, for example, from about 1 x 10⁴ T cells/kg subject body weight to about 9 x 10⁷ T cells/kg subject body weight, for example about 1 , 2, 3, 4, 5, 6, 7, 8, or 9 x 10⁴; about 1 , 2, 3, 4, 5, 6, 7, 8, or 9 x 10^s; about 1 , 2, 3, 4, 5, 6, 7, 8, or 9 x 10⁶; or about 1 , 2, 3, 4, 5, 6, 7, 8, or 9 x 10⁷ T cells/kg subject body weight. In other examples, therapeutic cells other than T cells may be used. The number of therapeutic cells/kg body weight that may be administered may be, for example, from about 1 x 10⁴ therapeutic cells/kg body weight to about 9 x 10⁷ therapeutic cells/kg body weight, for example about 1 , 2, 3, 4, 5, 6, 7, 8, or 9 x 10⁴; about 1 , 2,

3, 4, 5, 6, 7, 8, or 9 x 10^s; about 1 , 2, 3, 4, 5, 6, 7, 8, or 9 x 10⁶; or about 1 , 2, 3, 4, 5, 6, 7, 8, or 9 x 10⁷ therapeutic cells/kg body weight.

[0340] The term "unit dose" as it pertains to the inoculum refers to physically discrete units suitable as unitary dosages for mammals, each unit containing a predetermined quantity of pharmaceutical composition calculated to produce the desired immunogenic effect in association with the required diluent. The specifications for the unit dose of an inoculum are dictated by and are dependent upon the unique characteristics of the pharmaceutical composition and the particular immunologic effect to be achieved.

[0341] An "effective amount" of a compound or pharmaceutical composition, generally, is defined as that amount sufficient to detectably and repeatedly achieve the stated desired result, for example, to ameliorate, reduce, minimize or limit the extent of a disease, disorder, condition or symptoms associated with a disease, disorder or condition. Other more rigorous definitions may apply, including elimination, eradication or cure of disease. In some embodiments there may be a step of monitoring biomarkers to evaluate the effectiveness of treatment and to control toxicity.

[0342] For eliminating therapeutic cells, an effective amount of a pharmaceutical composition that contains a compound provided herein, or pharmaceutically acceptable salts thereof, could be the amount that achieves the result of selectively reducing the number of cells that express an inducible chimeric apoptotic polypeptide, such as a chimeric polypeptide that includes a multimerizing region and a caspase-9 polypeptide lacking the CARD domain, such that at least about or greater than 60%, 70%, 80%, 85%, 90%, 95%, or 97% or more of the caspase-9 expressing cells are killed or eliminated. An effective amount of a pharmaceutical composition that contains a compound provided herein for eliminating therapeutic cells could also be the amount that achieves the result of eliminating or reducing an adverse effect, or the toxicity, of the cells in a subject. The term is also synonymous with "sufficient amount."

[0343] For activating the desired activity of therapeutic cells (e.g., immune cells), for example, where the therapeutic cells express an inducible chimeric co-stimulatory polypeptide and a chimeric antigen receptor that binds to a target cell, such as, for example, a tumor cell, an effective amount of a compound provided herein or a pharmaceutical composition that contains a compound described herein, or a pharmaceutically acceptable salt thereof, could be the amount that achieves the selective result of reducing the number of target cells, by at least about, or greater than, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 97%. An effective amount of a pharmaceutical composition that contains a compound provided herein for activating the desired activity of therapeutic cells could also be the amount that achieves a particular measurable result, such as reducing the size and/or number tumors or slowing or halting tumor size and/or number increases in a subject to whom the compound or composition is administered. The term is also synonymous with "sufficient amount."

[0344] For activating the immune activity of therapeutic cells, an effective amount of a compound provided herein or a pharmaceutical composition that contains a compound described herein, or a pharmaceutically acceptable salt thereof may be, for example, the amount that increases or decreases biological activity as measured in a biological assay for immune cell activation, such as, for example, a SeAP assay, or increases or decreases the presence of a biological marker, where the increase or decrease in the biological activity, or the increase or decrease of the biological marker is associated with an activation of immune activity of the cell, by at least, or greater than, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 97%. The term is also synonymous with "sufficient amount."

[0345] A“number of target cells” may refer to an actual number of target cells, such as, for example, in a representative sample. In some examples, this number may be obtained from a sample taken before administration of a compound described herein, or a pharmaceutically acceptable salt thereof, and from a sample taken following administration of the compound.

The sample may be of any appropriate tissue or bodily fluid that might provide a representative sampling of the number of target cells. In some examples, the term may refer to the size of a tumor, or the number of tumors present in an organ or tissue. In this example, the number of target cells is considered to be reduced where the size of the tumor, or the number of tumors is reduced following administration of the compound.

[0346] The effective amount for any particular application can vary depending on such factors as the disease or condition being treated, the particular composition being administered, the size of the subject, and/or the severity of the disease or condition, and the inducible chimeric polypeptide. The effective amount of a particular composition provided herein can be empirically determined. For example, initial dosing may be based on parameters such as a subject’s weight and condition, mode of administration, and pharmacokinetic properties of a compound including, half-life, volume of distribution and clearance. Monitoring of serum levels of a compound and clinical status of the subject can be employed during administration of a compound to establish and adjust therapeutic doses and the time course of administration.

[0347] The terms“contacted” and“exposed,” when applied to a cell, tissue or organism, are used herein to discuss the process by which a compound, pharmaceutical composition and/or another agent, such as for example a chemotherapeutic or radiotherapeutic agent, are delivered to a target cell, tissue or organism or are placed in direct juxtaposition with the target cell, tissue or organism. To achieve cell killing or stasis, a compound or pharmaceutical composition and/or additional agent(s) are delivered to one or more cells in a combined amount effective to kill the cell(s) or prevent them from dividing. By“kill” or“killing” as in a percent of cells killed, is meant the death of a cell through apoptosis, as measured using any method known for measuring apoptosis, and, for example, using the assays discussed herein, such as, for example the SeAP assays or T cell assays discussed herein. The term may also refer to cell ablation.

[0348] The administration of a compound or pharmaceutical composition may precede, be co current with and/or follow the other agent(s) by intervals ranging from minutes to weeks. In embodiments where the compound, pharmaceutical composition and other agent(s) are applied separately to a cell, tissue or organism, one would generally ensure that a significant period of time did not expire between the times of each delivery, such that the compound, pharmaceutical composition and agent(s) would still be able to exert an advantageously combined effect on the cell, tissue or organism. For example, in such instances, it is contemplated that one may contact the cell, tissue or organism with two, three, four or more modalities substantially simultaneously (i.e., within less than about a minute) with the compound or pharmaceutical composition. In other aspects, one or more agents may be administered within from

substantially simultaneously, about 1 minute, to about 24 hours to about 7 days to about 1 to about 8 weeks or more, and any range derivable therein, prior to and/or after administering an expression vector. Yet further, various combination regimens of pharmaceutical composition provided herein and one or more agents may be employed.

Optimized and personalized therapeutic treatment

[0349] The administration of the compounds provided herein, or pharmaceutically acceptable salts thereof, may be optimized based on, for example, the disease or condition being treated or prevented, the subject’s health, or other physical characteristics of the subject, or the desired outcome. Provided herein is an example of treatment of patients with a multimerizing agent, following the induction of Graft vs Host Disease, where therapeutic cells that express a chimeric polypeptide comprising an apoptosis-inducing polypeptide have been administered to the patient.

[0350] The induction of apoptosis after administration of multimerizing agent may be optimized by determining the stage of a negative side effect of the therapeutic cells, such as Graft vs Host Disease, or the number of undesired therapeutic cells that remain in the patient. Similarly, the activation of immune activity, such as activating cells that express a chimeric antigen receptor in addition to the inducible chimeric polypeptide, may be optimized by determining the number of target cells remaining in the subject, or by measuring a marker of immune activity, such as, for example, the secretion of certain cytokines or other markers.

[0351] For example, determining that a patient has GvHD, and the stage of the GvHD, provides an indication to a clinician that it may be necessary to induce caspase-9 associated apoptosis by administering a compound provided herein, or a pharmaceutically acceptable salt thereof. In another example, determining that a patient has a reduced level of GvHD after treatment with the compound described herein, or a pharmaceutically acceptable salt thereof, may indicate to the clinician that no additional dose of the compound is needed. Similarly, after treatment with the compound described herein, or a pharmaceutically acceptable salt thereof, determining that the patient continues to exhibit GvHD symptoms, or suffers a relapse of GvHD may indicate to the clinician that it may be necessary to administer at least one additional dose of the compound. The term“dosage” is meant to include both the amount of the dose and the frequency of administration, such as, for example, the timing of the next dose.

[0352] In other embodiments, following administration of therapeutic cells, for example, therapeutic cells which express a chimeric antigen receptor in addition to an inducible caspase- 9 polypeptide, in the event of a need to reduce the number of modified cells or in vivo modified cells, a compound provided herein, or a pharmaceutically acceptable salt thereof, may be administered to the patient. In these embodiments, the methods may comprise determining the presence or absence of a negative symptom or condition, such as Graft vs Host Disease, or off target toxicity, and administering a dose of a compound provided herein. The methods may further involve monitoring the symptom or condition and administering an additional dose of the compound described herein, or a pharmaceutically acceptable salt thereof, in the event the symptom or condition persists. This monitoring and treatment schedule may continue while the therapeutic cells that express chimeric antigen receptors or chimeric signaling molecules remain in the patient. [0353] In other embodiments, following administration of therapeutic cells, for example, therapeutic cells which express a chimeric antigen receptor in addition to an inducible co stimulating polypeptide that activates immune cells, in order to induce the activity of the cell to reduce the number of target cells, such as tumor cells, a compound described herein, or a pharmaceutically acceptable salt thereof, may be administered to the patient. In these embodiments, the methods involve determining the number of target cells, and administering a dose of the compound described herein, or a pharmaceutically acceptable salt thereof, to reduce the number of the target cells. The methods may further comprise monitoring a symptom or condition associated with the presence of the target cells and administering an additional dose of the compound described herein, or a pharmaceutically acceptable salt thereof, in the event the symptom or condition persists. For example, the tumor load, tumor burden, amount of tumor cells, concentration of tumor cells, size of tumors, amount of cancerous or precancerous cells, concentration of cancerous or precancerous cells in the subject may be determined, and a dose of the compound described herein, or a

pharmaceutically acceptable salt thereof is administered to reduce the number or concentration of tumor, cancerous, or precancerous cells.

[0354] An indication of adjusting or maintaining a subsequent drug dose, such as, for example, a subsequent dose of the compound described herein, or a pharmaceutically acceptable salt thereof, and/or the subsequent drug dosage, can be provided in any convenient manner. An indication may be provided in tabular form (e.g., in a physical or electronic medium) in some embodiments. For example, graft versus host disease observed symptoms may be provided in a table, and a clinician may compare the symptoms with a list or table of stages of the disease. Or, for example, the tumor load, tumor burden, amount of tumor cells,

concentration of tumor cells, size of tumors, amount of cancerous or precancerous cells, concentration of cancerous or precancerous cells in the subject may be provided in a table. The clinician then can identify from the table an indication for subsequent drug dose. For example, this information can be provided to a computer (e.g., entered into computer memory by a user or transmitted to a computer via a remote device in a computer network), and software in the computer can generate an indication for adjusting or maintaining a subsequent drug dose, and/or provide the subsequent drug dose amount.

[0355] Once a subsequent dose is determined based on the indication, a clinician may administer the subsequent dose or provide instructions to adjust the dose to another person or entity. The term "clinician" as used herein refers to a decision maker, and a clinician is a medical professional in certain embodiments. A decision maker can be a computer or a displayed computer program output in some embodiments, and a health service provider may act on the indication or subsequent drug dose displayed by the computer. A decision maker may administer the subsequent dose directly (e.g., infuse the subsequent dose into the subject) or remotely (e.g., pump parameters may be changed remotely by a decision maker).

[0356] In some examples, a dose, or multiple doses of a compound described herein, or a pharmaceutically acceptable salt thereof, may be administered before clinical manifestations of GvHD, or other symptoms, such as CRS symptoms, are apparent. In this example, cell therapy is terminated before the appearance of negative symptoms. In other embodiments, such as, for example, hematopoietic cell transplant for the treatment of a genetic disease, the therapy may be terminated after the transplant has made progress toward engraftment, but before clinically observable GvHD, or other negative symptoms, can occur. In other examples, a compound described herein, or a pharmaceutically acceptable salt thereof, may be administered to eliminate the modified cells in order to eliminate on target/off-tumor cells, such as, for example, healthy B cells co-expressing the B cell-associated target antigen.

Kits and Combinations

[0357] Also provided herein are kits and combinations containing nucleic acids, cells, proteins and/or compounds. A non-limiting example of a use of the kits and combinations is in methods provided herein, including, for example, methods that incorporate chemically induced multimerization (e.g., CID) for conditional control of one or more proteins, methods for treating, preventing and/or delaying the onset of a disease, disorder or condition, and methods for regulating treatments used in treating, preventing and/or delaying the onset of a disease, disorder or condition.

[0358] In one embodiment, a kit or combination provided herein contains a compound provided herein. . In one embodiment, a kit or combination provided herein contains a compound of Formula B as described herein. In certain embodiments, a kit or combination containing a compound of Formula B as described herein also contains instructions, e.g., written material, for using the compound, for example in methods of chemically inducing

multimerization (e.g., CID) for conditional control of one or more proteins, methods for treating, preventing and/or delaying the onset of a disease, disorder or condition, and methods for regulating treatments used in treating, preventing and/or delaying the onset of a disease, disorder or condition. In some such embodiments, the instructions include instructions for administering the compound to a cell and/or a subject. In certain embodiments, the instructions include instructions for administering the compound to a cell and/or a subject in order to activate or eliminate a cell ex vivo and/or in vivo. In certain embodiments, the kit or combination provided herein contains a compound provided herein, e.g., a compound of Formula B as described herein, and a compound described in U.S. Patent application no. 62/608,552, such as Compound A or a compound of Formula I or II, which are also described herein. In some embodiment of such kits or combinations, the compound provided herein (e.g., a compound of Formula B) and a compound described in U.S. Patent application no. 62/608,552 (e.g.,

Compound A or a compound of Formula I or II) are present in the kit or combination in separate containers. In certain embodiments, a kit or combination containing a compound of Formula B as described herein and a compound described in U.S. Patent application no. 62/608,552 also contains instructions, e.g., written material, for using the compounds, for example in methods of chemically inducing multimerization (e.g., CID) for conditional control of one or more proteins, methods for treating, preventing and/or delaying the onset of a disease, disorder or condition, and methods for regulating treatments used in treating, preventing and/or delaying the onset of a disease, disorder or condition. In some such embodiments, the instructions include instructions for administering the compounds to a cell and/or a subject. In certain embodiments, the instructions include instructions for administering the compounds to a cell and/or a subject in order to activate and/or eliminate a cell ex vivo and/or in vivo. In some embodiments, the instructions specify that one of either a compound provided herein (e.g., a compound of Formula B) or a compound described in U.S. Patent application no. 62/608,552 is administered to a cell and/or a subject and that the other compound is administered to a cell and/or subject only under certain conditions. Such conditions include, for example, but are not limited to, cytokine storms, tumor lysis syndrome, cytokine release syndrome, macrophage activation syndrome, serious adverse events associated with therapeutic cell (e.g., CAR T cell) treatment and conditions in which it may be desired to reduce the number of, or eliminate, certain cells (e.g., cells containing nucleic acids encoding a polypeptide to which the compound binds and/or a chimeric polypeptide containing a compound-binding polypeptide and a cell elimination polypeptide, e.g., a pro-apoptotic polypeptide such as a caspase).

[0359] In one embodiment, a kit or combination provided herein contains nucleic acids encoding one or more chimeric polypeptides, cells containing nucleic acids encoding one or more chimeric polypeptides, and/or one or more chimeric polypeptides. In certain

embodiments, the one or more chimeric polypeptides includes one or more of any of the chimeric polypeptides described herein and/or in references incorporated herein. In certain aspects, one of the one or more chimeric polypeptides includes one or more polypeptides that bind to a compound provided herein. For example, in one embodiment, one of the one or more chimeric polypeptides includes an FRB wild type or variant protein (or portion thereof), e.g., a human protein, and/or an FKBP12 wild type or variant protein (or portion thereof), e.g., a human protein. In one embodiment, one of the one or more chimeric polypeptides includes an FRB wild type or variant protein (or portion thereof), e.g., a human protein, fused to a cell activation or cell elimination polypeptide and/or an FKBP12 wild type or variant protein (or portion thereof), e.g., a human protein, fused to a cell activation or cell elimination polypeptide. In certain embodiments, the cell activation polypeptide can be one that is involved in stimulating proliferation and/or survival of a cell (e.g., an immune cell), such as, for example, a co stimulatory protein as described herein. The cell activation polypeptide, in some embodiments, can include a CD40 protein (or portion thereof, including a human protein) and/or an MyD88 protein (or portion thereof, including a human protein). In some embodiments, a cell activation protein can be a chimeric protein, e.g., an MC protein or fusion of a CD40 protein (or portion thereof) and an MyD88 protein (or portion thereof). In certain embodiments, the cell elimination polypeptide can be one that is involved in apoptosis or death of a cell (e.g., an immune cell), such as a pro-apoptotic protein as described herein. The cell elimination polypeptide, in some embodiments, can include a caspase protein (or portion thereof, including a human protein).

[0360] In a certain embodiment, a kit or combination provided herein contains nucleic acids encoding two or more chimeric polypeptides, cells containing nucleic acids encoding two or more chimeric polypeptides, and/or two or more chimeric polypeptides. In certain aspects, at least one of the two or more chimeric polypeptides includes one or more polypeptides that bind to a compound provided herein. In some embodiments, at least one of the two or more chimeric polypeptides includes one or more polypeptides that bind to a compound provided herein, and at least one other of the two or more chimeric polypeptides includes one or more polypeptides that bind to a compound that binds to an FKBP12v36 protein (e.g., a human FKBP12v36), e.g., rimiducid or a compound described in U.S. patent application no. 62/608,552, such as

Compound A or a compound of Formula I or II, which are also described herein. For example, in one embodiment, one of the two or more chimeric polypeptides includes an FRB wild type or variant protein (or portion thereof), e.g., a human protein, and/or an FKBP12 wild type or variant protein (or portion thereof), e.g., a human protein, and another of the two or more chimeric polypeptides includes an FKBP12v36 protein or portion thereof (e.g., a human variant protein).

In one embodiment, one of the two or more chimeric polypeptides includes an FRB wild type or variant protein (or portion thereof), e.g., a human protein, fused to a cell activation or cell elimination polypeptide and/or an FKBP12 wild type or variant protein (or portion thereof), e.g., a human protein, fused to a cell activation or cell elimination polypeptide and another of the two or more chimeric polypeptides includes an FKBP12v36 protein, e.g., a human variant protein, fused to a cell activation or cell elimination polypeptide. In one aspect of such embodiments, if the polypeptide to which an FRB and/or FKBP12 is fused is a cell elimination polypeptide, then the polypeptide to which an FKBP12v36 is fused is a cell activation polypeptide. In another aspect of such embodiments, if the polypeptide to which an FRB and/or FKBP12 is fused is a cell activation polypeptide, then the polypeptide to which an FKBP12v36 is fused is a cell elimination polypeptide. In certain embodiments, the cell activation polypeptide can be one that is involved in stimulating proliferation and/or survival of a cell (e.g., an immune cell), such as, for example, a co-stimulatory protein as described herein. The cell activation polypeptide, in some embodiments, can include a CD40 protein (or portion thereof, including a human protein) and/or an MyD88 protein (or portion thereof, including a human protein). In some embodiments, a cell activation protein can be a chimeric protein, e.g., an MC protein or fusion of a CD40 protein (or portion thereof) and an MyD88 protein (or portion thereof). In certain embodiments, the cell elimination polypeptide can be one that is involved in apoptosis or death of a cell (e.g., an immune cell), such as a pro-apoptotic protein as described herein. The cell elimination polypeptide, in some embodiments, can include a caspase protein (or portion thereof, including a human protein).

[0361] In some embodiments of a kit or combination provided herein containing nucleic acids encoding one or more chimeric polypeptides, cells containing nucleic acids encoding one or more chimeric polypeptides, and/or one or more chimeric polypeptides, including embodiments such as described in the preceding paragraphs, the kit or combination also includes a compound provided herein. In some of these embodiments, the compound is a compound of Formula B as described herein. In some of these embodiments, the kit or combination also includes a compound provided herein, e.g., a compound of Formula B as described herein, and a compound described in U.S. Patent application no. 62/608,552, such as Compound A or a compound of Formula I or II, which are also described herein.

[0362] In certain embodiments, a kit or combination containing nucleic acids encoding one or more chimeric polypeptides, cells containing nucleic acids encoding one or more chimeric polypeptides, and/or one or more chimeric polypeptides, including embodiments such as described in the preceding paragraphs, the kit or combination also contains instructions, e.g., written material, for using the nucleic acids, cells, chimeric polypeptides and/or compounds, for example in methods of chemically inducing multimerization (e.g., CID) for conditional control of one or more proteins, methods for treating, preventing and/or delaying the onset of a disease, disorder or condition, and methods for regulating treatments used in treating, preventing and/or delaying the onset of a disease, disorder or condition.

Pharmaceutical compositions and formulations

[0363] A compound provided herein can be prepared as a pharmaceutically acceptable salt. As used herein, the term "pharmaceutically acceptable salt" refers to a derivative of the disclosed compounds where the parent compound is modified by making acid or base salts thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. Pharmaceutically acceptable salts include conventional non-toxic salts or quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, and the like. In certain examples, conventional non-toxic salts include those derived from bases, such as potassium hydroxide, sodium hydroxide, ammonium hydroxide, caffeine, various amines, and the like.

[0364] A pharmaceutically acceptable salt can be prepared from a parent compound, which contains a basic or acidic moiety, by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, for example, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, PA, p. 1418 (1985), the disclosure of which is hereby incorporated by reference herein in its entirety for all purposes.

[0365] The term "pharmaceutically acceptable" or“pharmacologically acceptable” as used herein refers to compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for administration to humans or animals and suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit/risk ratio. Moreover, for human administration, preparations may meet sterility, pyrogenicity, and general safety and purity standards as required by FDA Office of Biologies standards.

[0366] A compound provided herein often is a stable compound and often has a stable structure in a composition provided. The terms "stable compound" and "stable structure" are meant to indicate a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent. Stable compounds are contemplated herein for use in treatment methods described. [0367] In some embodiments, provided herein are pharmaceutical compositions comprising a compound described herein, or a pharmaceutically acceptable salt thereof. In addition to a compound or pharmaceutically acceptable salt thereof, a pharmaceutical composition can include one or more of a pharmaceutically acceptable excipient, carrier, solvent, diluent, isotonic agent, buffering agent, stabilizer, preservative, vaso-constrictive agent, antibacterial agent, antifungal agent, and the like, for example. Non-limiting examples of solvents, and diluents include water, saline, dextrose, ethanol, glycerol, oil, and the like. Examples of isotonic agents include sodium chloride, dextrose, mannitol, sorbitol, lactose, and the like. Useful stabilizers include gelatin, albumin, and the like. A pharmaceutically-acceptable carrier includes any and all solvents, dispersion media, coatings, stabilizing agents, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorption delaying agents, and the like. Carrier(s) generally are compatible with other components of the compound pharmaceutical composition and not deleterious to a subject when administered. A carrier often is sterile and pyrogen-free, and selected based on the mode of administration used, and a carrier utilized often is approved, or will be approved, by an appropriate government agency that oversees development and use of pharmaceuticals. A pharmaceutical composition can include, in certain embodiments, a compatible pharmaceutically acceptable (i.e., sterile or non-toxic) liquid, semisolid, or solid diluent that serves as a pharmaceutical vehicle, excipient, or medium. A diluent can include water, saline, dextrose, ethanol, glycerol, and the like, for example. An isotonic agent can include sodium chloride, dextrose, mannitol, sorbitol, and lactose, among others. A stabilizer can include albumin, among others. A pharmaceutical composition can include, in some embodiments, an antibiotic or preservative, including, for example, gentamicin, merthiolate, or chlorocresol. In some embodiments, an excipient or carrier is chosen from polyethylene glycol (PEG), polysorbate, ethanol, glycerol, glycerin, sorbitol, glucose, sucrose, dimethylacetamide, triacetin, dimethylsulfoxide (DMSO), and an oil, such as a vegetable oil, and combinations thereof. In some embodiments, an excipient or carrier is selected from the group consisting of polyethylene glycol (PEG), polysorbate, ethanol, glycerol, glycerin, sorbitol, glucose, sucrose, dimethylacetamide, triacetin, dimethylsulfoxide (DMSO), and an oil, such as a vegetable oil, and combinations thereof. Various sustained release systems for drugs have also been devised and can be applied to a compound described herein. See, for example, U.S. Patent No. 5,624,677, the methods of which are incorporated herein by reference in its entirety for all purposes.

[0368] In some embodiments, a pharmaceutical composition is a liquid composition. In some embodiments, a pharmaceutical composition is provided as a dry powder composition. In some embodiments, a pharmaceutical composition is in a liposomal composition, sometimes as a micro-emulsion. In some embodiments, a pharmaceutical composition is a spray dried composition. In some embodiments, a pharmaceutical composition comprises a pharmaceutically acceptable co-polymer. In some embodiments, the co-polymer is chosen from, or selected from the group consisting of, poly(vinyl alcohol), poly(vinyl pyrrolidone), hypromellose, acetate, and succinate, and combinations thereof.

[0369] In the case of sterile powders for the preparation of sterile injectable solutions, preparation methods sometimes utilized are vacuum drying and the freeze-drying techniques, which yield a powder of a compound described herein or pharmaceutically acceptable salt thereof in addition to any additional desired ingredient present in the previously sterile-filtered solutions. In some embodiments, the compounds described herein, or pharmaceutically acceptable salts thereof, are provided in a spray dried form. A dry powder or spray dried powder may be provided for shipping of the compound described herein, or a pharmaceutically acceptable salt thereof. A dry powder or spray dried powder may be dissolved in water, buffered water, saline or buffered saline with or without a co-solvent, for use.

[0370] For spray drying, a feed solution comprising a compound described herein can include a co-polymer, non-limiting examples of which include poly(vinyl alcohol), poly(vinyl pyrrolidone), hypromellose acetate succinate, and combinations thereof. A dry powder formulation also can contain a co-polymer in some embodiments.

[0371] Water or saline used for preparing a pharmaceutical composition may be buffered or not buffered. Non-limiting examples of saline solutions that can be used to prepare a pharmaceutical composition include lactated Ringer's solution, acetated Ringer's solution, intravenous sugar solutions (e.g., 5% dextrose in normal saline (D5NS), 10% dextrose in normal saline (D10NS), 5% dextrose in half-normal saline (D5HNS) and 10% dextrose in half-normal saline (D10HNS)). Non-limiting example of buffered saline solutions and related solutions include phosphate buffered saline (PBS), TRIS-buffered saline (TBS), Hank's balanced salt solution (HBSS), Earle's balanced salt solution (EBSS), standard saline citrate (SSC), HEPES- buffered saline (HBS), and Gey's balanced salt solution (GBSS).

[0372] A compound described herein, or pharmaceutically acceptable salt thereof, can be provided in a pharmaceutical dosage form. A pharmaceutical dosage form can include a sterile aqueous solution or dispersion or sterile powder containing a compound described herein or pharmaceutically acceptable salt thereof, which are adapted for the extemporaneous preparation of sterile solutions or dispersions, and optionally encapsulated in liposomes. The ultimate dosage form sometimes is a sterile fluid and stable under the conditions of manufacture and storage. A liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, saline, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. An isotonic agent, for example, a sugar, buffer or sodium chloride is included in some embodiments. Prolonged absorption of an injectable composition can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin. Sterile solutions often are prepared by incorporating an active compound in a required amount in an appropriate solvent, sometimes with one or more of the other ingredients enumerated above, followed by filter sterilization.

[0373] In some embodiments, a liquid pharmaceutical composition has an 80%

(weight/weight) or greater amount of a compound described herein or a pharmaceutically acceptable salt thereof in water. In some embodiments, the concentration of a compound described herein in a liquid composition is about 0.1-25 % (weight/weight), and sometimes about 0.5-10 % (weight/weight). The concentration in a semi-solid or solid composition such as a gel or a powder sometimes is about 0.1-5 % (weight/weight), and sometimes about 0.5-2.5 % (weight/weight).

[0374] In some embodiments, a compound is provided at 0.4 mg/kg per dose, for example at a concentration of 5 mg/ml_. Vials or other containers may be provided containing the compound at, for example, a volume per vial of about 0.25 ml to about 10 ml, for example, about 0.25, 0.5, 1 , 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 ml, for example, about 2 ml.

[0375] A compound described herein or pharmaceutically acceptable salt thereof can be formulated in combination with one or more other pharmaceutically active agents. The one or more other agents can include, without limitation, another compound described herein, an anticell proliferative agent (e.g., chemotherapeutic), an anti-inflammatory agent, or an antigen.

Administration of compounds

[0376] Upon formulation, solutions and solid forms of compounds described herein, or pharmaceutically acceptable salts thereof, can be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations can be administered in a variety of dosage forms, dependent on the method of administration. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

[0377] A compound described herein can be formulated as a pharmaceutical composition and administered to a mammalian host, such as a human patient or nonhuman animal, in a variety of forms adapted to the chosen route of administration. The terms "patient" or“subject” are interchangeable, and, as used herein include, but are not limited to, an organism or animal; a mammal, including, e.g., a human, non-human primate (e.g., monkey), mouse, pig, cow, goat, rabbit, rat, guinea pig, hamster, horse, monkey, sheep, or other non-human mammal; a nonmammal, including, e.g., a non-mammalian vertebrate, such as a bird (e.g., a chicken or duck) or a fish, and a non-mammalian invertebrate.

[0378] The active compositions may include classic pharmaceutical preparations.

Administration of these compositions can be by any common route so long as the target tissue is available via that route. Non-limiting examples of administration routes include oral, nasal, buccal, rectal, vaginal, topical, orthotopic, intradermal, intravitreal, instillation (e.g., bladder instillation, intravesical administration), parenteral, subcutaneous, intravascular, intramuscular, intraperitoneal, intrathecal or intravenous injection or infusion. In certain embodiments, the compounds described herein, or pharmaceutically acceptable salts thereof, are administered by intravenous injection or infusion. Such compositions would normally be administered as pharmaceutically acceptable compositions, discussed herein.

[0379] In certain embodiments, a composition described herein is administered in conjunction with locally applied ultrasound, electromagnetic radiation or electroporation or other electrically based drug delivery technique, local chemical abrasion, or local physical abrasion.

[0380] Useful dosages of compounds can be determined by comparing their in vitro activity, and in vivo activity in animal models. It is understood that methods are available for the extrapolation of effective dosages in mice, and other animals, to humans.

[0381] The amount of the compound, or an active salt or derivative thereof, required for use in treatment varies not only with a particular salt selected but also with the route of administration, the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician. In general a suitable dose sometimes is in the range of from about 0.1 to about 100 mg/kg, e.g., from about 0.1 to about 50 mg/kg of subject body weight per day, from about 0.1 to about 10 mg per kilogram subject body weight of the recipient per day, such as from about 0.2 to 4 mg/kg of subject body weight per day, and often is in the range of 0.3 to 1 mg/kg of subject body weight per day, such as, for example, about 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, or 10 mg/kg subject body weight per day. A compound may be conveniently administered in unit dosage form, and for example, contain 5 to 1000 mg, or 10 to 750 mg, or 50 to 500 mg of a compound described herein or pharmaceutically acceptable salt thereof per unit dosage form.

A compound described herein or pharmaceutically acceptable salt thereof can be administered to achieve peak plasma concentrations of an active compound of from about 0.01 to about 100 pM, about 0.5 to about 75 pM, about 1 to 50 pM, or about 2 to about 30 pM. Such

concentrations may be achieved, for example, by the intravenous injection of a 0.05 to 5% solution of a compound described herein or pharmaceutically acceptable salt thereof, optionally in saline, or orally administered as a bolus containing about 1-100 mg of a compound described herein or pharmaceutically acceptable salt thereof. Desirable blood levels may be maintained by continuous infusion to provide about 0.01-5.0 mg/kg/hr or by intermittent infusions containing about 0.4-15 mg/kg of the compound described herein or pharmaceutically acceptable salt thereof. A desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. A sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations; such as multiple inhalations from an insufflator or by application of a plurality of drops into the eye.

[0382] In some examples, a compound described herein or pharmaceutically acceptable salt thereof may be prepared and administered according to methods used to prepare and administer rapamycin or rapalog, an example of which is described hereafter.

Administration of a compound and other compounds

[0383] In some embodiments, compounds described herein, or pharmaceutically acceptable salts thereof, can be formulated in the same or a similar formulation as for rapamycin or rapalogs thereof. Formulations described hereafter can be used for injection or intravenous administration, and sometimes for another route of administration. An example of a formulation and administration of temsirolimus, an analog of sirolimus (rapamycin) is as follows.

[0384] Temsirolimus is essentially insoluble in water and soluble in alcohol. Temsirolimus is manufactured by Pfizer and provided in a kit for injection as Torisel (NDC 0008-1 179-01) which is maintained at a temperature between 2°C and 8°C, protected from light. The kit contains a single-use vial of 25 mg/ml temsirolimus as a non-aqueous sterile solution (containing dehydrated alcohol (39.5% w/v), dl-alpha-tocopherol (0.075% w/v), propylene glycol (50.3% w/v) and anhydrous citric acid (0.0025% w/v)) and a separate vial containing 1.8 ml of diluent (polysorbate 80 (40.0% w/v), polyethylene glycol 400 (42.8% w/v) and dehydrated alcohol (19.9%)). Two dilutions of the temsirolimus are conducted in preparing the compound for intravenous administration. First, the vial of temsirolimus is mixed with the vial of diluent yielding a solution of 10 mg/ml of temsirolimus. The mixture is then diluted with 0.9% sodium chloride injection, USP, to provide a total of 25 mg in an appropriate volume (e.g., 250 ml) for infusion. Administration of the diluted solution should be completed within 6 hours of dilution. Thirty minutes prior to each Torisel infusion, prophylactic treatment via intravenous

administration of an antihistamine (25-50 mg diphenhydramine) is recommended. Torisel is administered as an intravenous infusion over a 30-60 minute period using non-DEHP, non-PVC administration materials with a filter, once weekly for treatment of advanced renal cell carcinoma. The treatment period is until disease progression or unacceptable toxicity occurs. The mean temsirolimus Cmax in whole blood has been reported as 585 ng/ml and mean AUC in blood as 1627 ng.h/ml with Cmax typically occurring at completion of infusion. Following a 25 mg intravenous dose in cancer patients, sirolimus (the main active metabolite of temsirolimus) AUC was reported as 2.7-fold that of temsirolimus AUC, which was attributed primarily to the longer half-life of sirolimus.

[0385] Components of a pharmaceutical composition often depend on its intended administration route, non-limiting examples of which are provided hereafter.

Intravenous administration

[0386] A compound described herein, or a pharmaceutically acceptable salt thereof, may be formulated in an aqueous solution, in water, saline, in the presence or absence of buffers, stabilizers, nontoxic surfactants, excipients, carriers, and the like, as discussed herein. Nonlimiting examples of buffers, such as, for example weak buffers that may be provided in the solution include acetate, phosphate, citrate, and the like, for example, for compound-containing solutions in which the pH is less than pH 4, pH 5, pH 6, or pH 7. In some embodiments, compounds described herein, or pharmaceutically acceptable salts thereof, are formulated in a 0.1 % glycerol solution. In some embodiments, a compound described herein is formulated in water, saline or related solution with 0.05, 0.1 , 0.15, 0.2, 0.25, 0.3, 0.35, or 0.4% glycerol. In some embodiments, a compound is formulated in water, saline or a related solution with 0.5,

0.1 , 0.15, 0.2, 0.25 0.3, 0.35, or 0.4% ethanol. Administration by injection

[0387] Pharmaceutical forms suitable for injectable use include forms suitable for intravenous administration discussed herein, modified as appropriate for injections, and also include pharmaceutical forms in sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. An injectable formulation often is sterile and is fluid to the extent that easy syringeability exists. It is stable under the conditions of manufacture and storage and is preserved against the contaminating action of microorganisms, such as bacteria and fungi.

[0388] An injectable formulation sometimes includes a carrier, which can be a solvent, excipient, or dispersion medium. Fluidity of an injectable formulation can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be effected by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In certain examples, isotonic agents, for example, sugars or sodium chloride may be included. Prolonged absorption of an injectable compositions can be effected by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin. The pharmaceutical form may comprise a co-polymer such as, for example, a co-polymer chosen from group poly(vinyl alcohol), poly(vinyl pyrrolidone), and hypromellose acetate succinate. The pharmaceutical form may comprise a co-polymer such as, for example, a co-polymer selected from the group consisting of poly(vinyl alcohol), poly(vinyl pyrrolidone), and hypromellose acetate succinate.

Oral administration

[0389] Compounds described herein, or pharmaceutically acceptable salts thereof, described herein may be enclosed in hard or soft shell gelatin capsules, may be compressed into tablets, or may be incorporated directly with the food of the patient's diet. For oral therapeutic administration, an active compound may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations sometimes contain at least 0.1 % of active compound. The percentage of the compositions and preparations may be varied and sometimes are about 2% to about 60% of the weight of a given unit dosage form. The amount of active compound in such therapeutically useful compositions is such that an effective dosage level will be obtained. [0390] Tablets, troches, pills, capsules, and the like may also contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring may be added. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like. A syrup or elixir may contain the active compound, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained-release preparations and devices.

Topical administration

[0391] For embodiments pertaining to topical administration, a compound described herein or pharmaceutically acceptable salt thereof may be applied in pure form, e.g., when in liquid form. However, it is generally desirable to administer a compound as a composition or formulation, in combination with an acceptable carrier, which may be a solid or a liquid. Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like. Useful liquid carriers include water, alcohols or glycols or water-alcohol/glycol blends, or phospholipids in propylene glycol/ethylene glycol, in which the present compounds can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. A composition sometimes includes a diluent and sometimes an adjuvant, carrier (e.g., assimilable, editable), buffer, preservative and the like. Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use. The resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user. [0392] The examples set forth below illustrate certain embodiments and do not limit the technology.

[0393] Example 1: Synthesis of 7(S)-Dimethoxyphenyl-Rapamycin Rapamycin

[0394] Synthesis of 7(S)-dimethoxyphenyl-rapamycin (also referred to herein as (S)-DMOP- rapamycin and CMP001) can be carried out by methods provided herein or known in the art (see, e.g., Luengo et al. (1994) J Org Chem 59:6512-6513). Some methods of synthesizing 7(S)-dimethoxyphenyl-rapamycin begin with a solution of rapamycin. Rapamycin can be prepared synthetically according to any available methods, including, but not limited to, methods such as those described in Nicolaou et al. ((1993) J Am Chem Soc 1 15:1449), Hayward et al.

((1993) J Am Chem Soc 1 15:9345), Romo et al. ((1993) J Am Chem Soc 1 15:7906),

Danishefsky et al. ((1993) J Am Chem Soc 1 15: 9345), Smith et al. ((1995) J Am Chem Soc 117:5407) and Maddess et al. ((2007) Angew Chem Inti Ed 46:591-597).

[0395] Rapamycin can also be obtained through isolation and purification of the compound produced by natural sources, for example, Streptomyces hygroscopicus or Streptomyces rapamycinicus (e.g., ATCC accession nos. 29253 and 27438; MTCC accession no. 5681). Examples of methods of obtaining rapamycin from Streptomyces hygroscopicus include, but are not limited to, those described in U.S. Patent 6,649,595, U.S. Patent 3,929,992, U.S. Patent 9,365,880 and Baby Rani et al. ((2013) Pak J Biol Sci 16(5):219-225 and (210) Prep Biochem Biotechnol 43(6):539-550). Rapamycin is also commercially available through multiple sources such as, for example, LC Laboratories (Woburn, MA, catalog #R-5000).

[0396] Epimers of rapamycin and rapalogs can be prepared through isolation from a mixture of compounds in the R and S configurations using chromatographic methods. Synthetic methods can also be used to directly prepare mixtures of rapamycin epimers that predominantly contain rapamycin having an optical configuration at a particular position in the molecule relative to the natural product rapamycin (FIG. 1) . For example, US Patent nos. 5,525,610 and 7,622,477 describe methods of producing 40-epimers, i.e., 40(S)-rapamycin (referred to therein as a 42-epimer) of rapamycin and U.S. Patent no. 7,196,192 describes methods of producing 29-epimers, i.e., 29(R)-rapamycin (referred to therein as a 28-epimer) of rapamycin. 7(S)-Dimethoxyphenyl-Rapamycin (CMP001) and

7(R)-Dimethoxyphenyl-Rapamycin (CMP002)

[0397] 7(S)-Dimethoxyphenyl-rapamycin was synthesized from rapamycin through hydrolysis of the 7-methylether linkage followed by nucleophilic substitution as follows. To a solution of rapamycin (1 eq) dissolved in dry dichloromethane and cooled down to -40 °C, trifluoracetic acid (5eq) was added dropwise. After the mixture was being stirred for 15 min., 1 ,3- dimethoxybenzene (3eq) was added dropwise and the stirring continued for 4 hrs. at -40°C. Sodium bicarbonate solution was added to quench the reaction and the mixture was left to warm up to room temperature. Phases were separated, organic phase was washed with brine and dried over sodium sulfate. Product was separated on flash column chromatography using gradient dichloromethane:acetone 0-15% as the eluent, and the fractions containing 7 (S)- dimethoxyphenyl-rapamycin (also referred to as (S)-DMOP-rapamycin and CMP001) were collected. Separately, subsequent fractions were collected containing 7(R)-dimethoxyphenyl- rapamycin (CMP002).

[0398] Example 2: Synthesis of 40-(R)-p-Bromomethylbenzoyl-Rapamycin

[0399] To a solution of rapamycin (1 eq) in dry dichloromethane at 0°C, DCC (1.3eq) was added followed by p-bromomethyl-benzoic acid (1.1 eq) and dimethylaminopyridine (1 eq). The reaction mixture was let to warm up to room temperature, while the reaction progress was monitored by TLC. After the reaction was completed, ethyl acetate was added and the precipitated dicyclohexylurea was filtered off. The mixture was purified by flash column chromatography using dichloromethane:methanol 0-10% gradient to obtain 40-(R)-p- bromomethylbenzoyl-rapamycin (also referred to as CMP015).

[0400] Example 3: Synthesis of 7(S)-Dimethoxyphenyl-40-(R)-p-(Bromomethyl)-Benzoyl- Rapamycin

[0401] To a solution of (S)-DMOP-rapamycin (1 eq) in dry dichloromethane at 0°C, DCC (1.3eq) was added followed by p-bromomethyl-benzoic acid (1.1 eq) and dimethylaminopyridine (1 eq). The reaction mixture was let to warm up to room temperature, while the reaction progress was monitored by TLC. After the reaction was completed, ethyl acetate was added and the precipitated dicyclohexylurea was filtered off. The mixture was purified by flash column chromatography using dichloromethane:methanol 0-10% gradient to obtain 7 (S)- dimethoxyphenyl-40-(R)-p-(bromomethyl)-benzoyl-rapamycin (also referred to as CMP025). [0402] Example 4: Synthesis of 40-(R)-((4-Methylpiperazin-1-yl)p-Methylbenzoyl)-Rapamycin

[0403] To a solution of rapamycin (1 eq) in dry dichloromethane at 0°C, DCC (1.3eq) was added followed by p-(N-methyl-piperazine)methyl-benzoic acid (1.1 eq) and

dimethylaminopyridine (1 eq). The reaction mixture was let to warm up to room temperature, while the reaction progress was monitored by TLC. After the reaction was completed, ethyl acetate was added and the precipitated dicyclohexylurea was filtered off. The filtrate was washed with sodium bicarbonate, water and brine to obtain crude product. The final purification was performed on flash column chromatography using dichloromethane:methanol 0-20% gradient as an eluent to obtain 40-(R)-((4-methylpiperazin-1-yl)p-methylbenzoyl)-rapamycin (also referred to herein as CMP012).

[0404] Example 5: Synthesis of 7(S)-Dimethoxyphenyl-40-(R)-p-((N-Methyl-Piperazine)- Methyl) -Benzoyl-Rapamycin

[0405] Two exemplary methods for synthesizing 7(S)-dimethoxyphenyl-40-(R)-p-((N-methyl- piperazine)-methyl)-benzoyl-rapamycin are shown in Scheme 1 and are as follows:

Method A

[0406] To a solution of (S)-DMOP-rapamycin (1 eq) in dry dichloromethane at 0°C, DCC (1.3eq) was added followed by p-bromomethyl-benzoic acid (1.1 eq) and dimethylaminopyridine (1.5eq). The reaction mixture was let to warm up to room temperature, while the reaction progress was monitored by TLC. After the reaction was completed, ethyl acetate was added and the precipitated dicyclohexylurea was filtered off. The filtrate was washed with sodium bicarbonate, water and brine to obtain crude product. The final purification was performed on flash column chromatography using dichloromethane:methanol 0-20% gradient as an eluent to obtain 7(S)-dimethoxyphenyl-40-(R)-p-(bromomethyl)-benzoyl-rapamycin (CMP025).

[0407] To a solution of 7(S)-dimethoxyphenyl-40-(R)-p-(bromomethyl)-benzoyl-rapamycin (1 eq) in dry dichloromethane piperazine was added (2.2eq) dropwise and the reaction mixture was kept stirring at room temperature for 1 hr. The reaction mixture was washed with sodium bicarbonate, water and brine to obtain crude product. Final purification was performed on flash column chromatography using dichloromethane:methanol 0-20% gradient as an eluent to obtain 7(S)-dimethoxyphenyl-40-(R)-p-((N-methyl-piperazine)-methyl)-benzoyl-rapamycin (also referred to herein as 7-(S)-DMOP-40-(R)-benzoylpiperazine-rapamycin and CMP017). Method B

[0408] To a solution of (S)-DMOP-rapamycin (1 eq) in dry dichloromethane at 0°C, DCC (1.3eq) was added followed by p-(N-methyl-piperazine)methyl-benzoic acid (1.1 eq) and dimethylaminopyridine (1.5eq). The reaction mixture was let to warm up to room temperature, while the reaction progress was monitored by TLC. After the reaction was completed, ethyl acetate was added and the precipitated dicyclohexylurea was filtered off. The filtrate was washed with sodium bicarbonate, water and brine to obtain crude product. The final purification was performed on flash column chromatography using dichloromethane:methanol 0-20% gradient as an eluent to obtain 7(S)-dimethoxyphenyl-40-(R)-p-((N-methyl-piperazine)-methyl)- benzoyl-rapamycin (also referred to herein as 7-(S)-DMOP-40-(R)-benzoylpiperazine-rapamycin and CMP017).

[0409] Scheme 1

[0410] 7-(S)-dimethyoxyphenyl-40-(R)-p-((N-methyl-piperazine)-methyl)-benzoyl-rapamycin (CMP017) was dissolved in MeOH. 2eq. of 1 M HCI in MeOH was added while the reaction mixture was vigorously stirred at room temperature. Solvent was evaporated, and the residual methanol was removed by co-evaporation with toluene. The product was dried on oil vacuum pump overnight. The same method was applied to obtain di-maleinic salt. The conversion to a protonated form of CMP017 is depicted in Scheme 2.

[0411] Scheme 2

[0412] Example 6: Synthesis of 40-(S)-lodo-Rapamycin

[0413] To a solution of rapamycin (1 eq) and 2,6-lutidine (5eq) in dry dichloromethane stirred at 0°C, trifluoromethanesulfonic anhydride (3eq) was added dropwise. The mixture was stirred at 0°C for 2 hrs. The reaction was quenched by water, and the phases were separated. The organic phase was washed with sodium bicarbonate and brine and dried over sodium sulfate. Sodium sulfate was filtered off and subsequently tetrabutylammonium iodide (1 3eq) was added to the crude solution of 40-(R)-trifluoromethanesulfonyl-rapamycin, while vigorously stirring at room temperature. After the reaction was completed, the mixture was washed with water and brine and dried over sodium sulfate. The product was purified on flash column chromatography using dichloromethane:methanol 0-10% gradient as an eluent, to obtain 40-(S)-iodo-rapamycin (also referred to herein as CMP006).

[0414] Additional compounds that can be prepared from 40-(R)-trifluoromethanesulfonyl- rapamycin include: 40-(S)-fluoro-rapamycin (also referred to herein as CMP003), 40-(S)-chloro- rapamycin (also referred to herein as CMP004), 40-(S)-bromo-rapamycin (also referred to herein as CMP005) and 40-(S)-azido-rapamycin (also referred to herein as CMP016). These four additional compounds can be synthesized using the method described for synthesis of 40- (S)-iodo-rapamycin by substituting tetrabutylammonium iodide used in the process with tetrabutylammonium fluoride (to generate CMP003), tetrabutylammonium chloride (to generate CMP004), tetrabutylammonium bromide (to generate CMP005) or tetrabutylammonium azide (to generate CMP016). Scheme 3 depicts exemplary methods for synthesizing CMP003, CMP004, CMP005, CMP006 and CMP016.

[0415] Scheme 3

CMP003 CMP004 CMP005 CMP006

[0416] Example 7: Synthesis of 7(S)-Dimethoxyphenyl-40-(S)-lodo-Rapamycin

[0417] To a solution of (S)-DMOP-rapamycin (1 eq) and 2,6-lutidine (5eq) in dry

dichloromethane stirred at 0°C, trifluoromethanesulfonic anhydride (3eq) was added dropwise. The mixture was stirred at 0°C for 2 hrs. The reaction was quenched by water, and the phases were separated. The organic phase was washed with sodium bicarbonate and brine and dried over sodium sulfate. Sodium sulfate was filtered off and tetrabutylammonium iodide (1 3eq) was added to this solution of crude 7-(S)-dimethoxyphenyl-40-(R)-trifluoromethanesulfonyl- rapamycin, while the reaction mixture was vigorously stirred at room temperature. After the reaction was completed, the mixture was washed with water and brine and dried over sodium sulfate. The product was purified on flash column chromatography using dichloromethane:methanol 0-10% gradient as an eluent, to obtain 7-(S)-dimethoxyphenyl-40- (S)-iodo-rapamycin (also referred to herein as CMP009 and 40-(S)-iodo-(S)-DMOP-rapamycin).

[0418] An exemplary method for synthesizing 7(S)-dimethoxyphenyl-40-(S)-iodo-rapamycin is shown in Scheme 4. Also depicted in Scheme 4 are additional compounds that can be prepared from 7-(S)-dimethoxyphenyl-40-(R)-trifluoromethanesulfonyl-rapamycin: 7 (S)- dimethoxyphenyl-40-(S)-fluoro-rapamycin (also referred to herein as CMP007), 7 (S)- dimethoxyphenyl-40-(S)-chloro-rapamycin (also referred to herein as CMP008) and 7 (S)- dimethoxyphenyl-40-(S)-azide-rapamycin (also referred to herein as CMP010). Additionally, 7(S)-dimethoxyphenyl-40-(S)-bromo-rapamycin can be synthesized from 7-(S)- dimethoxyphenyl-40-(R)-trifluoromethanesulfonyl-rapamycin. These four additional compounds can be synthesized using the method described for synthesis of 7(S)-dimethoxyphenyl-40-(S)- iodo-rapamycin by substituting tetrabutylammonium iodide used in the process with

tetrabutylammonium fluoride (to generate CMP007), tetrabutylammonium chloride (to generate CMP008), tetrabutylammonium bromide (to generate 7(S)-dimethoxyphenyl-40-(S)-bromo- rapamycin) or tetrabutylammonium azide (to generate CMP010).

[0419] Scheme 4

[0420] Example 8: Synthesis of 7(S)-tBu-(R)-Sulfinamido-Rapamycin

[0421] The chemical structure of 7-(S)-tBu-(R)-sulfinamido-rapamycin is as follows:

[0422] 7-(S)-tBu-(R)-sulfinamido-rapamycin was synthesized from rapamycin through hydrolysis of the 7-methylether linkage followed by nucleophilic substitution as follows. To a solution of rapamycin (1 eq) dissolved in dry dichloromethane and cooled down to -40 °C, trifluoracetic acid (5eq) was added dropwise. After the mixture was being stirred for 15 min., a solution of (R)-tert-butyl-sulfinamide (3eq) in dry dichloromethane was added dropwise and the stirring continued for 4 hrs at -40°C. Sodium bicarbonate solution was added to quench the reaction and the mixture was left to warm up to room temperature. Phases were separated, organic phase was washed with brine and dried over sodium sulfate. Product was separated on flash column chromatography using gradient dichloromethane:acetone 0-15% as the eluent, and the fractions containing 7-(S)-tBu-(R)-sulfinamido-rapamycin (also referred to as CMP020) were collected. Separately, subsequent fractions were collected containing 7-(R)-tBu-(R)- sulfinamido-rapamycin (CMP021).

[0423] Example 9: Synthesis of 7(S)-lmidazole-Rapamycin

[0424] The chemical structure of 7-(S)-imidazole-rapamycin is as follows:

[0425] 7-(S)-imidazole-rapamycin was synthesized from rapamycin through hydrolysis of the 7-methylether linkage followed by nucleophilic substitution as follows. To a solution of rapamycin (1 eq) dissolved in dry dichloromethane and cooled down to -40 °C, trifluoracetic acid (5eq) was added dropwise. After the mixture was being stirred for 15 min., a solution of (3eq) imidazole in dry dichloromethane was added dropwise and the stirring continued for 4 hrs at - 40°C. Sodium bicarbonate solution was added to quench the reaction and the mixture was left to warm up to room temperature. Phases were separated, organic phase was washed with brine and dried over sodium sulfate. Product was separated on flash column chromatography using gradient dichloromethane:acetone 0-15% as the eluent, and the fractions containing 7- (S)-imidazole-rapamycin (also referred to as CMP018) were collected, followed by the separate collection of fractions of 7-(R)-imidazole-rapamycin (CMP019). [0426] Example 10: Synthesis of 7 (S)-Menthol-Rapamycin

[0427] The chemical structure of 7-(S)-menthol-rapamycin is as follows:

[0428] 7-(S)-menthol-rapamycin was synthesized from rapamycin through hydrolysis of the 7- methylether linkage followed by nucleophilic substitution as follows. To a solution of rapamycin (1 eq) dissolved in dry dichloromethane and cooled down to -40 °C, trifluoracetic acid (5eq) was added dropwise. After the mixture was being stirred for 15 min., a solution of menthol (3eq) in dry dichloromethane was added dropwise and the stirring continued for 4 hrs at -40°C. Sodium bicarbonate solution was added to quench the reaction and the mixture was left to warm up to room temperature. Phases were separated, organic phase was washed with brine and dried over sodium sulfate. Product was separated on flash column chromatography using gradient dichloromethane:acetone 0-15% as the eluent, and the fractions containing 7-(S)-menthol- rapamycin (also referred to as CMP026) were collected.

[0429] Example 11: Synthesis of 7(S)-Piperitol-Rapamycin

[0430] The chemical structure of 7-(S)-piperitol-rapamycin is as follows:

[0431] 7-(S)-piperitol-rapamycin was synthesized from rapamycin through hydrolysis of the 7- methylether linkage followed by nucleophilic substitution as follows. To a solution of rapamycin (1 eq) dissolved in dry dichloromethane and cooled down to -40 °C, trifluoracetic acid (5eq) was added dropwise. After the mixture was being stirred for 15 min., a solution of piperitol (3eq) in dry dichloromethane was added dropwise and the stirring continued for 4 hrs at -40°C. Sodium bicarbonate solution was added to quench the reaction and the mixture was left to warm up to room temperature. Phases were separated, organic phase was washed with brine and dried over sodium sulfate. Product was separated on flash column chromatography using gradient dichloromethane:acetone 0-15% as the eluent, and the fractions containing 7-(S)-piperitol- rapamycin (also referred to as CMP027) were collected. [0432] Example 12: Synthesis of 7(S)-Dimethoxyphenyl-29,40-di-(R)-p-(Bromomethyl)- Benzoyl-Rapamycin

[0433] The chemical structure of 7(S)-dimethoxyphenyl-29,40-di-(R)-p-(bromomethyl)- benzoyl-rapamycin is as follows:

[0434] To a solution of (S)-DMOP-rapamycin (1 eq) in dry dichloromethane at 0°C, DCC (3.3eq) was added followed by p-bromomethyl-benzoic acid (3eq) and dimethylaminopyridine (3.5eq). The reaction mixture was let to warm up to room temperature, while the reaction progress was monitored by TLC. After the reaction was completed, ethyl acetate was added and the precipitated dicyclohexylurea was filtered off. The filtrate was washed with sodium bicarbonate, water and brine to obtain crude product. The final purification was performed on flash column chromatography using dichloromethane:methanol 0-5% gradient as an eluent to obtain 7(S)-dimethoxyphenyl-29,40-di-(R)-p-(bromomethyl)-benzoyl-rapamycin (also referred to as CMP028).

[0435] Example 13: Synthesis of 40-(S)-Amino-Rapamycin [0436] The chemical structure of 40-(S)-amino-rapamycin is as follows:

[0437] 40-(S)-azido-rapamycin (CMP016) was dissolved in tetrahydrofurane and

triphenylphosphine (1.1 eq) was added, followed by water (1 eq). The reaction mixture was vigorously stirred at room temperature overnight. Solvent was evaporated and the product was purified on flash column chormatography, using dichloromethane:methanol 0-20% gradient as an eluent to yield 40-(S)-amino-rapamycin (also referred to as CMP01 1).

[0438] Example 14: Synthesis of 7-(S)-Dmethoxyphenyl-40-(S)-Amino-Rapamycin

[0439] The chemical structure of 7-(S)-dimethoxyphenyl-40-(S)-amino-rapamycin is as follows:

[0440] 7-(S)-dimethoxyphenyl-40-(S)-azido-rapamycin (CMP010) was dissolved in tetrahydrofurane and triphenylphosphine (1.1 eq) was added, followed by water (1 eq). The reaction mixture was vigorously stirred at room temperature overnight. Solvent was evaporated and the product was purified on flash column chormatography, using dichloromethane:methanol 0-20% gradient as an eluent to yield 7-(S)-dimethoxyphenyl-40-(S)-amino-rapamycin (also referred to as CMP024).

[0441] Example 15: Synthesis of 40-(R)-N-(3-(4-Methylpiperazin-1-yl)Propyl)- Rapamycinamine

[0442] The chemical structure of 40-(R)-N-(3-(4-methylpiperazin-1-yl)propyl)-rapamycinamine is as follows:

[0443] To a cooled down to 0°C solution of 40-(S)-iodo-rapamycin (CMP006) in

tetrahydrofuran, a 1 M solution of 3-(4-methylpiperazin-1-yl)propylamine in THF was added slowly. The reaction mixture was stirred for 15 min. Solvent was evaporated and the product was purified on flash column chromatography using chloroform :methanol 0-30% gradient as an eluent to yield 40-(R)-N-(3-(4-methylpiperazin-1-yl)propyl)-rapamycinamine (also referred to as CMP014). [0444] Example 16: Synthesis of 7(S)-Urea-Rapamycin

[0445] The chemical structure of 7-(S)-urea-rapamycin is as follows:

[0446] 7-(S)-urea-rapamycin was synthesized from rapamycin through hydrolysis of the 7- methylether linkage followed by nucleophilic substitution as follows. To a solution of rapamycin (1 eq) dissolved in dry dichloromethane and cooled down to -40 °C, trifluoracetic acid (5eq) was added dropwise. After the mixture was being stirred for 15 min., a solution of (3eq) urea in dry dichloromethane was added dropwise and the stirring continued for 4 hrs at -40°C. Sodium bicarbonate solution was added to quench the reaction and the mixture was left to warm up to room temperature. Phases were separated, organic phase was washed with brine and dried over sodium sulfate. Product was separated on flash column chromatography using gradient dichloromethane:acetone 0-15% as the eluent, and the fractions containing 7-(S)-urea- rapamycin (also referred to as CMP022) were collected.

[0447] Example 17: Synthesis of 7 (S) -Dimethoxyphenyl-29-40-di-p-((N-Methyl-Piperazine) - Methyl) -Benzoyl-Rapamycin

[0448] The chemical structure of 7(S)-dimethoxyphenyl-29-40-di-p-((N-methyl-piperazine)- methyl)-benzoyl-rapamycin is as follows:

[0449] To a solution of (S)-DMOP-rapamycin (1 eq) in dry dichloromethane at 0°C, DCC (3.3eq) was added followed by p-(N-methyl-piperazine)methyl-benzoic acid (3eq) and dimethylaminopyridine (3.5eq). The reaction mixture was let to warm up to room temperature, while the reaction progress was monitored by TLC. After the reaction was completed, ethyl acetate was added and the precipitated dicyclohexylurea was filtered off. The filtrate was washed with sodium bicarbonate, water and brine to obtain crude product. The final purification was performed on flash column chromatography using dichloromethane:methanol 0-20% gradient as an eluent to obtain 7(S)-dimethoxyphenyl-29-40-di-p-((N-methyl-piperazine)- methyl)-benzoyl-rapamycin (also referred to as CMP023).

[0450] Example 18: Transcriptional Switch Assay of Compounds [0451] A compound-dependent, cell-based transcriptional switch assay was used to determine the efficacy of the binding of compounds provided herein to FKBP12 and FRB variants to evaluate the allele specificity of the binding for mutant FRB domains. In this assay, compounds that effectively bind (and thus dimerize) FKBP12 and the selected FRB allele will, in so doing, activate a“switch” to initiate transcription of a reporter protein (secreted alkaline phosphatase (SeAP)). The switch includes two fusion proteins: (1) a fusion protein (referred to as GAL4- FKBP3) of a polypeptide containing the DNA binding domain of a yeast GAL4 protein (amino acids 1-147 of the GAL4 protein; see SEQ ID NO: 98) with three copies of wild-type human FKBP12 and (2) a fusion of a human FRB allele and a polypeptide containing the transcriptional activation domain of the herpes simplex virus (HSV-1) VP16 protein. The VP16 protein activates transcription only when present near gene promoter elements, but lacks intrinsic DNA binding activity. The transcriptional activation domain of VP16 has been located to the carboxy-terminal 78 amino acids (i.e., residues 413-491 ; see, e.g., Shen et al. (1996) J Biol Chem 271 :4827- 4837) of the full-length protein (see, e.g., Genbank accession no. AL018645). These switch components were coexpressed in cells with a SeAP reporter gene plasmid containing five GAL4-specific DNA recognition sites proximal to the transcriptional start site. Addition of a compound, such as rapamycin or a rapalog, that is capable of binding FKBP12 and the FRB allele, recruits FRB-VP16 to GAL4-FKBP3 bound to reporter plasmid DNA to drive SeAP expression according to the relative affinity of the compound for the FRB allele.

Plasmids

FRB-VP16 fusion plasmid

[0452] A plasmid containing DNA encoding an FRB protein (wild-type or mutant) linked to DNA encoding a polypeptide containing the transcriptional activation domain of the herpes simplex virus (HSV-1) VP16 protein (nucleotide SEQ ID NO: 22; amino acid SEQ ID NO: 96) was constructed for expression of one component of the switch system. An exemplary plasmid containing DNA encoding a protein fusion of the wild type human FRB (nucleotide SEQ ID NO: 2; amino acid SEQ ID NO: 78) and a polypeptide containing the transcriptional activation domain of the herpes simplex virus (HSV-1) VP16 protein is referred to as pM001. In this plasmid, DNA encoding the FRB-VP16 fusion protein is contained within an expression vector described previously (see, e.g., US Patent 5,512,661 and Takebe et al. (1988) Mol Cell Biol 8:466), which includes a SRa promoter (containing the SV40 early promoter and R segment and part of the U5 sequence of the long terminal repeat of human T-cell leukemia virus type 1 ; SEQ ID NO: 65) for expression of the fusion protein. Nucleic acid encoding a Flag epitope (nucleotide SEQ ID NO: 66; amino acid SEQ ID NO: 136) and a linker peptide (nucleotide SEQ ID NO: 67; amino acid SEQ ID NO: 137) is located 3’ of the promoter and 5’ of nucleic acid encoding the FRB-VP16 fusion protein, which includes a linker peptide (nucleotide SEQ ID NO: 21 ; amino acid SEQ ID NO: 138) between the FRB- and VP16-encoding nucleic acids.

[0453] A plasmid, referred to as pM002, for expression of DNA encoding a protein fusion of the KLW mutant of human FRB (amino acid SEQ ID NO: 80 encoded by nucleotide SEQ ID NO: 16) and a polypeptide containing the transcriptional activation domain of the herpes simplex virus (HSV-1) VP16 protein, was constructed and is essentially identical to pM001 except that it contains a leucine codon in place of a threonine codon at amino acid 74 of FRB (which is amino acid position 2098 in the full mTOR protein and amino acid 75 of the FRB protein encoded by pM002). A similar vector, referred to as pM003, for the expression of DNA encoding a protein fusion of the TLW mutant of human FRB (amino acid SEQ ID NO: 82 encoded by nucleotide SEQ ID NO: 18) and a polypeptide containing the transcriptional activation domain of the herpes simplex virus (HSV-1) VP16 protein was constructed and is essentially identical to pM002 except that it contains a threonine codon in place of a leucine codon at amino acid 71 of FRB (which is amino acid position 2095 in the full mTOR protein and amino acid 72 of the FRB protein encoded by pM003).

GAL4 DNA binding domain-FKBP12 fusion plasmid

[0454] Plasmid pM004 contains polynucleotides encoding three copies of human FKBP12 domain referred to as FKBP12-1 , FKBP12-2 and FKBP12-3 (nucleotide SEQ ID NOS: 6, 8 and 10, respectively). The polypeptides encoded by the FKBP12-1 , FKBP12-2 and FKBP12-3 encoding polynucleotides are provided in SEQ ID NOS: 87, 89 and 90, respectively. The FKBP12-1 , FKBP12-2 and FKBP12-3 encoding polynucleotides are fused in succession with short linker-encoding polynucleotides positioned between (1) polynucleotides encoding

FKBP12-1 and FKBP12-2 (linker nucleotide sequence SEQ ID NO: 7 and amino acid sequence SEQ ID NO: 88) and (2) polynucleotides encoding FKBP12-2 and FKBP12-3 (linker nucleotide sequence SEQ ID NO: 9 and amino acid sequence SEQ ID NO: 91). The polynucleotide encoding the three FKBP12 polypeptides is fused at the 5’ end to a polynucleotide encoding the amino-terminal 147 amino acids of a yeast GAL4 protein (i.e., the DNA-binding domain of the GAL4 protein: nucleotide SEQ ID NO: 24; amino acid SEQ ID NO: 98) via a polynucleotide encoding a short linker peptide (nucleotide SEQ ID NO: 25; amino acid SEQ ID NO: 99). The polynucleotide encoding the GAL4-FKBP12 fusion protein is contained in the same general plasmid format as for the pM001 plasmid for expression in cells.

SeAP reporter expression plasmid [0455] Plasmid pM005 (also referred to as GAL4-SeAP) contains DNA encoding a secreted alkaline phosphatase, referred to as SeAP (nucleotide SEQ ID NO: 28; amino acid SEQ ID NO: 100), linked at its 5’ end to a minimal IL-2 promoter (SEQ ID NO: 27) which is in turn linked at its 5’ end to multiple copies of a DNA recognition sequence (SEQ ID NO: 26) to which the GAL4 DNA-binding domain protein (which is encoded by DNA in pM004) binds.

[0456] When the GAL4 DNA-binding domain protein binds the recognition sequence of this reporter plasmid in the nucleus of the cell that has been transfected with the plasmid, and an effective compound is administered to the transfected cell, the compound will bind the FRB- VP16 fusion protein in the nucleus and also bind the FKBP12 portion of the GAL4 DNA-binding domain fusion. This dimerization serves to recruit the VP16 transcription activator into proximity of the plasmid DNA in such a way as to activate transcription of the SeAP-encoding DNA, via the promoter in the plasmid.

Cell transfection

[0457] HEK293 cells (1 x 10⁶) were seeded on a 100-mm tissue culture dish in 10 mL DMEM4500, supplemented with glutamine, penicillin/streptomycin and 10% fetal calf serum. After 16-30 hours incubation, cells were transfected using Novagen’s GeneJuice® protocol. Briefly, for each transfection, 0.5 mL OptiMEM reduced serum medium (GIBCO™) was pipeted into a 1.5-mL microcentrifuge tube and 15 pL GeneJuice reagent added followed by 5 sec. vortexing. Samples were rested 5 minutes to settle the GeneJuice suspension. DNA (5 pg total) was added to each tube and mixed by pipetting up and down four times. Samples were allowed to rest for 5 minutes for GeneJuice-DNA complex formation and the suspension added dropwise to one dish of HEK293 cells. A typical transfection contains 1 pg pM005, 2 pg pM004 and 2 pg FRB-VP16 (pM001 , pM002 or pM003).

Assay method

[0458] 24 hours following transfection, the HEK293 cells were split to 96-well plates and incubated with dilutions of rapamycin or a compound as provided herein. Briefly, 100 pL complete media was added to each well of a 96-well flat-bottom plate. Compound (or rapamycin) was diluted in media in tubes to a concentration 4X the top concentration in the gradient to be placed on the plate. 100 pL of compound or rapamycin was added to each of four wells on the far right of the plate (assays are thereby performed in quadruplicate). 100 pL from each drug-containing well was then transferred to the adjacent well and the cycle repeated 10 times to produce a serial two-fold step gradient. The last wells were untreated and serve as a control for basal reporter activity. Transfected HEK293 cells were then trypsinized, washed with complete media, suspended in media and 100 pl_ aliquoted to each well containing compound and to control wells containing rapamycin, or to which no compound or rapamycin had been added. Cells were incubated 16-24 hours.

[0459] 16-24 hours after addition of compound (or rapamycin), 96-well plates were wrapped to prevent evaporation and incubated at 65°C for 2 hours to inactivate endogenous and serum phosphatases while the heat-stable SeAP reporter remained. 100-pL media samples from each well were loaded into individual wells of a 96-well assay plate with black sides (Greiner). 100 mI_ of a solution of 1.0 M 4-methylumbelliferyl phosphate (4-MUP) in 1.0 M diethanolamine carbonate at pH 10.0 was added to each sample well to provide for a final concentration of 0.5 M for both. The samples were incubated for 4 to 16 hours. Hydrolysis of 4-MUP by SEAP produces a fluorescent product, which can be easily measured. Phosphatase activity was measured by fluorescence with excitation at 355 nm and emission at 460 nm. Data was transferred to a Microsoft Excel spreadsheet for tabulation and graphed with GraphPad Prism.

Assay results

Comparison of results of assays for binding of the TLW mutant of FRB

[0460] Transcriptional switch assays employing the TLW double-mutant of FRB (K2095T, T2098L) fusion with a portion of HSV VP16 or the KTW wild type FRB-VP16 fusion were used to compare the binding activities of rapamycin and 7(S)-dimethoxyphenyl-rapamycin (C001). As shown in FIG. 3, the SeAP activity profiles for rapamycin-treated cells expressing the FRB-VP16 fusion and cells expressing the FRB(TLW)-VP16 fusion were similar. This result indicates that rapamycin bound to the TLW mutant of FRB and dimerized it with FKBP12 with only somewhat altered affinity relative to rapamycin binding to wild type FRB and dimerizing it with FKBP12. In contrast, there was almost no SeAP activity in cells expressing wild type FRB when treated with CMP001 , although there was significant SeAP activity in cells expressing the TLW mutant of FRB when treated with CMP001 (EC50 of about 1.71 nM). These results demonstrate the FRB allele selectivity of the binding affinity of CMP001.

Comparison of results of assays for binding of wild type FRB

[0461] Transcriptional switch assays employing KTW wild type FRB fusion with a portion of HSV VP16 were used to compare the binding activities of rapamycin, 7(S)-dimethoxyphenyl- rapamycin and some of the compounds having different moieties at the C40 position of compounds provided herein (CMP01 1 , CMP012, CMP013, CMP014 and CMP015). Figure 4 shows the SeAP activity profiles of transfected assay cells treated with the compounds. As shown in FIG. 4, the SeAP activities of cells treated with compound CMP001 (7 (S)- dimethoxyphenyl-rapamycin), CMP013 (containing bromomethoxybenzoyl groups at positions C40 and C29) or CMP014 (containing an aminopropyl methylpiperazine group at position C40) were lower than the SeAP activities of cells treated with rapamycin. In contrast, the SeAP activities of cells treated with CMP015 (containing one bromomethoxybenzoyl group at position C40) or CMP01 1 (containing an amino group at position C40) reached similar maximum values as cells treated with rapamycin, although at higher concentrations than required for rapamycin. The SeAP activity profile for cells treated with CMP012 (containing a (4-methylpiperazin-1-yl)p- methylbenzoyl group) was similar to that for cells treated with rapamycin. This result indicates that CMP012 has FRB binding- and FRB/FKBP12-dimerizing characteristics that are similar to those of rapamycin but also has a structure that may provide increased solubility as compared to rapamycin and 7(S)-dimethoxyphenyl-rapamycin (CMP001). Compounds with characteristics similar to CMP012 can also be modified at other positions on the molecule, e.g., C7, which can alter the FRB allele specificity with limited effect on the binding to wildtype FKBP12.

Comparision of results of assays for binding of a KLW mutant of FRB

[0462] The EC50 values based on results of transcriptional assays of compounds as compared to EC50 values based on assays of rapamycin are provided in Table 5. The table lists the average EC50 values from multiple assays calculated based on graphs of SeAP activity plotted against compound concentration (nM) and thus are EC50 values for SeAP activities. These EC50 values provide an indication of the efficacies of binding to FRB (KLW) and dimerization of the mutant FRB and FKBP12 as also compared to the efficacies of binding to wildtype FRB and dimerization of wildtype FRB and FKBP12.

*ND = not determined

[0463] Example 19: Comparison of the Effects of Rapamycin and 7(S)-Dimethoxyphenyl- Rapamycin on TORC1 Function

[0464] To compare the effects of rapamycin and 7(S)-dimethoxyphenyl-rapamycin (CMP001) on TORC1 function, cells were separately treated with increasing concentrations of these compounds and examined for the presence of phosphorylated S6 protein, a downstream target in the mTOR signaling pathway. Activation of mTOR, the protein kinase contained in the TORC1 complex, by, for example, growth factors, results in phosphorylation and activation of effector proteins, including S6KI and 4E-BP1 which are regulators of mRNA translation. S6K1 , which is a regulator of cell growth, in turn phosphorylates ribosomal protein S6 which results in enhanced mRNA translation. Thus, S6 phosphorylation is an indicator of activation and functioning of the mTOR signaling pathway in cells. Phosphorylation assay methods

[0465] Primary human peripheral blood mononuclear cells (PBMCs; 1-2 x 10^® cells) in complete media containing IL-7 and IL-15 were stimulated with anti-CD3 and anti-CD28 antibodies precoated to wells of tissue culture plates for approximately 72 hrs. The cells were then cultured overnight in the presence of rapamycin or 7(S)-dimethoxyphenyl-rapamycin (CMP001) at several concentrations (0 nM, 1 nM and 10 nM rapamycin or 1 nM, 5 nM, 10 nM, 50 nM, 100 nM, 500 nM and 1000 nM CMP001). Following the culture period, cells were lysed and the lysates were resolved by SDS-PAGE and western blot probing with antibodies against elF4EBP, ribosomal protein S6 and pS6 (i.e. , S6 that has been phosphorylated at serine residues targeted by the TORC1 pathway). Vinculin was also detected using anti-vinculin antibodies in order to compare the amount of protein loaded into each lane of the SDS-PAGE gel.

Western blot analysis

[0466] Following overnight culture, cells were lysed in 500 mI of RIPA buffer (0.01 M Tris-HCI, pH 8.0/140 mM NaCI/1 % Triton X-100/1 mM phenylmethylsulfonyl fluoride/1 % sodium deoxycholate/0.1 % SDS) with Halt™ Protease Inhibitor Cocktail. The lysates were collected and lysed on ice for 30 min. After pelleting cell debris, protein concentrations from overlying supernatants were measured in 96-well plates with BCA™ Protein Assay as recommended by the manufacturer. 30 pg of proteins were boiled in Laemmli sample buffer (Bio-Rad, Hercules, CA) with 2.5% 2-mercaptoethanol for 5 min at 95°C before being separated by Criterion TGX 10% Tris/glycine protein gel. Membranes were probed with 1/1000 rabbit polyclonal antibodies specific for human elF4EBP, ribosomal protein S6 and pS6 followed by 1/10,000 IRdye680- conjugated goat anti-rabbit IgG F(ab')2 secondary antibody or IRdye800-conjugated goat anti mouse secondary antibody (Licor). Protein bands were detected using Supersignal West Femto chemiluminescent substrate. To ensure equivalent sample loading, blots were stripped at 65°C for 1 hour with Restore PLUS Western Blot Stripping Buffer before labeling with 1/10,000 rabbit anti-vinculin polyclonal antibody. Unless otherwise stated, all the reagents were obtained from Thermo Scientific.

Assay results

[0467] The results of assays for phosphorylation of S6 in human PBMCs treated with anti-CD3 and anti-CD28 antibodies to activate the mTOR signaling pathway in the presence and absence of rapamycin or 7(S)-dimethoxyphenyl-rapamycin (CMP001) are shown in FIG. 5. As shown in the third and fourth lanes (from the left side) of the western blot in FIG. 5, treatment of the activated cells with 1 and 10 nM rapamycin (“RAP”) resulted in no detectable phosphorylated S6 protein (pS6), although unphosphorylated S6 was present in the cells. Furthermore, there was a downward shift in the mobility of 4E-BP1 in the cells which is indicative of a lack of phosphorylation and thus increased mobility of the protein. These results are in contrast to those obtained with activated cells that had not been treated with rapamycin (second lane of the blot) where phosphorylated S6 (pS6) is clearly detectable and the 4E-BP1 protein migrates to higher position in the gel. Lanes 5-1 1 (from the left side) of the gel show the results of blots of lysates of activated cells treated with increasing concentrations of CMP001. These results are similar to those obtained for activated cells that were not treated with rapamycin or CMP001 (shown in the second lane from the left side of the blot). These results in total demonstrate that rapamycin has an inhibitory effect on the mTOR signaling pathway through dimerization of FKBP12 and FRB in mTOR, whereas CMP001 has no such inhibitory effect, even at high concentrations. This is consistent with results of transcriptional switch assays (see FIG. 3 and FIG. 4) showing that CMP001 has little or no ability to bind wildtype FRB.

Example 20: Development of Chimeric Antigen Receptor (CAR) T-Cell Systems Incorporating

Compound-Inducible Control Systems for Cell Activation and/or Elimination

[0468] Recombinant CAR T cells that co-express nucleic acids encoding a first generation CAR, a rapamycin/rapalog- or a rimiducid (or other compound, such as described in U.S. patent application no. 62/608, 552)-inducible chimeric truncated MyD88/CD40 polypeptide (iMC) and a rapamycin/rapalog- or a rimiducid (or other compound, such as described in U.S. patent application no. 62/608, 552)-inducible caspase 9 (iC9) protein were generated using

compositions and methods as described in this example. In one type of chimeric antigen receptor (CAR), the variable heavy (VH) and light (VL) chains for a tumor-specific monoclonal antibody are fused in-frame with the CD3 zeta (z) chain from the T cell receptor complex. The VH and VL are generally connected together using a flexible glycine-serine linker, and then attached to the transmembrane domain by a spacer (e.g., derived from CD8a) to extend the scFv away from the cell surface so that it can interact with tumor antigens. Following transduction, T cells now express the CAR on their surface, and upon contact and ligation with a tumor antigen, signal through the CD3 z chain inducing cytotoxicity and cellular activation. To provide for inducible enhanced co-stimulation and activation of the CAR T cells, nucleic acids encoding an iMC fusion protein can also be introduced into the cells. In order to be able to eliminate the activated CAR T cells, if necessary, nucleic acid encoding an inducible safety switch protein, e.g., iC9, can be introduced into the cells. Plasmid construction

[0469] Two multicistronic retroviruses were used to transduce cells for the coexpression of a human epidermal growth factor 2 receptor (HER2)-specific CAR, iMC and iC9 in the transduced cells. In one example, the retrovirus vectors, referred to as pM006 (FIG. 6) or pM007 (FIG. 7), were used for cell transduction. These two vectors each provide an iRMC fusion proteinencoding nucleic acid construct, and a HER2-specific CAR-encoding nucleic acid. pM006 also provides the iC9 fusion protein. pM007 is co-transduced with pM008 that provides the iC9 fusion protein.

[0470] Plasmid pM006 contains, in the 5’ to 3’ direction, nucleic acid encoding:

(1) a fusion protein containing a KLW mutant of human FRB having a Thr2098Leu substitution (FRBL; SEQ ID NO: 81 encoded by SEQ ID NO: 17) fused, through a 2-amino acid linker peptide (SEQ ID NO: 83 encoded by SEQ ID NO: 19), to a wild type human FKBP12 polypeptide (SEQ ID NO: 86 encoded by SEQ ID NO: 4) fused to a truncated human MyD88 polypeptide (the amino terminal 172 amino acids of MyD88 containing the DD domain and intermediary domain; SEQ ID NO: 102 encoded by SEQ ID NO: 30) fused, through a 2-amino acid linker peptide (SEQ ID NO: 83 encoded by SEQ ID NO: 19), to a portion of a human CD40 polypeptide (the carboxy terminal 62 amino acids, i.e. , amino acids 216-277 of CD40; SEQ ID NO: 105 encoded by SEQ ID NO: 33) (the entire fusion protein is termed MC-Rap or iRMC),

(2) a 3-amino acid linker peptide (SEQ ID NO: 106 encoded by SEQ ID NO: 34),

(3) a P2A self-cleaving peptide (SEQ ID NO: 108 encoded by SEQ ID NO: 36) to provide for separation of the fusion protein from the protein encoded by the DNA 3’ of the P2A-encoding DNA in the plasmid,

(4) a 5-amino acid linker peptide (SEQ ID NO: 109 encoded by SEQ ID NO: 37),

(5) a mutant human FKBP12 protein (FKBP12(F36V) also known as FKBP12v36, F_V36, FKBPv, or F_v; SEQ ID NO: 1 1 1 encoded by SEQ ID NO: 39) in which the phenylalanine at amino acid position 36 (or 37 if the initial methionine of the protein is counted) is substituted by a valine which is fused, through an 8-amino acid linker (SEQ ID NO: 1 12 encoded by SEQ ID NO: 40) to a portion of human caspase 9 polypeptide (Acaspase9 which contains amino acids 135-416 of caspase 9; SEQ ID NO: 113 encoded by SEQ ID NO: 41) (the entire fusion protein is termed iC9),

(6) a 5-amino acid linker (SEQ ID NO: 1 15 encoded by SEQ ID NO: 43),

(7) a T2A self-cleaving peptide (SEQ ID NO: 1 16 encoded by SEQ ID NO: 44) to provide for separation of the iC9 fusion protein from the protein encoded by the DNA 3’ of the T2A- encoding DNA in the plasmid,

(8) a 4-amino acid linker (SEQ ID NO: 1 17 encoded by SEQ ID NO: 46), (9) a membrane signal peptide (SEQ ID NO: 1 18 encoded by SEQ ID NO: 47) fused to light (Y55; SEQ ID NO: 120 encoded by SEQ ID NO: 49) and heavy (F109; SEQ ID NO: 122 encoded by SEQ ID NO: 51) chain variable regions of anti-HER2 monoclonal antibody 4D5 (with an intervening 15-amino acid flexible glycine-serine linker, i.e., flex peptide (SEQ ID NO: 121 encoded by SEQ ID NO: 50) between the chains) fused, through a 2-amino acid linker (SEQ ID NO: 123 encoded by SEQ ID NO: 52), to a human CD34 epitope peptide (amino acids 30-45 of CD34; SEQ ID NO: 128 encoded by SEQ ID NO: 57 which is fused to an alpha stalk region of human CD8 (amino acids 141-182 of CD8; SEQ ID NO: 129 encoded by SEQ ID NO: 58) which is fused to the transmembrane domain of human CD8 (amino acids 183-219 of CD8; SEQ ID NO: 130 encoded by SEQ ID NO: 59) which is fused to a portion of human Oϋ3z (amino acids 83-194 of Oϋ3z isoform X2; SEQ ID NO: 132 encoded by SEQ ID NO: 61) through a 2-amino acid linker (SEQ ID NO: 91 encoded by SEQ ID NO: 9).

[0471] Plasmid pM007 contains, in the 5’ to 3’ direction, nucleic acid encoding:

(1) a fusion protein containing a KLW mutant of human FRB having a Thr2098Leu substitution (FRB_L; SEQ ID NO: 81 encoded by SEQ ID NO: 17) fused, through an 8-amino acid linker peptide (SEQ ID NO: 84 encoded by SEQ ID NO: 20), to a wild type human FKBP12 polypeptide (SEQ ID NO: 86 encoded by SEQ ID NO: 5) fused, through a 5-amino acid linker peptide (SEQ ID NO: 92 encoded by SEQ ID NO: 1 1), to a truncated human MyD88 polypeptide (the amino terminal 172 amino acids of MyD88 containing the DD domain and intermediary domain; SEQ ID NO: 103 encoded by SEQ ID NO: 31) fused, through a 2-amino acid linker peptide (SEQ ID NO: 83 encoded by SEQ ID NO: 19), to a portion of a human CD40 polypeptide (the carboxy terminal 62 amino acids, i.e., amino acids 216-277 of CD40; SEQ ID NO: 105 encoded by SEQ ID NO: 33) (the entire fusion protein is termed MC-Rap or iRMC),

(2) a 6-amino acid linker peptide (SEQ ID NO: 107 encoded by SEQ ID NO: 35),

(4) a 2-amino acid linker peptide (SEQ ID NO: 1 10 encoded by SEQ ID NO: 38),

(5) a membrane signal peptide (SEQ ID NO: 1 19 encoded by SEQ ID NO: 48) fused to light (Y55; SEQ ID NO: 120 encoded by SEQ ID NO: 49) and heavy (F109; SEQ ID NO: 122 encoded by SEQ ID NO: 51) chain variable regions of anti-HER2 monoclonal antibody 4D5 (with an intervening 15-amino acid flexible glycine-serine linker, i.e., flex peptide (SEQ ID NO: 121 encoded by SEQ ID NO: 50) between the chains) fused, through a 2-amino acid linker (SEQ ID NO: 123 encoded by SEQ ID NO: 52), to a human CD34 epitope peptide (amino acids 30-45 of CD34; SEQ ID NO: 128 encoded by SEQ ID NO: 57) which is fused to an alpha stalk region of human CD8 (amino acids 141-182 of CD8; SEQ ID NO: 129 encoded by SEQ ID NO: 58) which is fused to the transmembrane domain of human CD8 (amino acids 183-219 of CD8; SEQ ID NO: 130 encoded by SEQ ID NO: 59) which is fused to a portion of human Oϋ3z (amino acids 83-194 of Oϋ3z isoform X2; SEQ ID NO. 132 encoded by SEQ ID NO: 61) through a 2-amino acid linker (SEQ ID NO: 91 encoded by SEQ ID NO: 9).

[0472] Plasmids pM006 and pM007 include nucleic acid encoding a human CD8

transmembrane sequence for incorporation into the chimeric antigen receptor protein molecule. The nucleic acid encoding the transmembrane domain is positioned in the plasmid such that it will be expressed in transduced cells as a fusion with the extracellular domain of the CAR. The chimeric antigen receptor encoded by pM006 and pM007 also includes a human CD8 alpha stalk, that is, an extracellular region of amino acids between the extracellular domain and the transmembrane domain.

[0473] Plasmids pM006 and pM007 further include nucleic acid encoding a human CD34 minimal epitope polypeptide to aid in sorting cells transduced with the plasmid. The nucleic acid encoding the CD34 minimal epitope is incorporated upstream of nucleic acid encoding at a human CD8 stalk polypeptide such that the encoded epitope is positioned at the amino terminal position of the CD8 stalk.

[0474] Plasmid pM008 (FIG. 10) includes a polynucleotide encoding a fusion protein that includes a mutant human FKBP12 protein (FKBP12(F36V) also known as FKBP12v36, FV36, FKBPv, or Fv; SEQ ID NO: 11 1 encoded by SEQ ID NO: 39) in which the phenylalanine at amino acid position 36 (or 37 if the initial methionine of the protein is counted) is substituted by a valine which is fused, through an 8-amino acid linker (SEQ ID NO: 1 12 encoded by SEQ ID NO: 40) to a truncated version of the human caspase 9 polypeptide (Acaspase9 which contains amino acids 135-416 of caspase 9; SEQ ID NO: 1 13 encoded by SEQ ID NO: 41). The entire fusion protein is referred to as iC9.

[0475] Another plasmid that can be used to develop CAR T-cell systems incorporating compound-inducible control systems for cell activation and/or elimination is pM009 (FIG. 8).

This vector provides one iMC fusion protein-encoding nucleic acid construct, one iC9 fusion protein-encoding nucleic acid construct and one HER2-specific CAR-encoding construct.

However, the iMC fusion-protein-encoding nucleic acid construct contains two copies of the FKBP-encoding nucleic acid and two copies of the KLW mutant FRB-encoding nucleic acid which are present as an FRB_L-FKBP12 fusion protein-encoding DNA and an FKBP12-FRB_L fusion protein-encoding DNA fused to the 5’ and 3’ end, respectively, of the MyD88-CD40 fusion protein-encoding DNA. This vector provides one iMC fusion protein-encoding nucleic acid construct, one iC9 fusion protein-encoding nucleic acid construct and one HER2-specific CAR- encoding construct.

[0476] Plasmid pM009 contains, in the 5’ to 3’ direction, nucleic acid encoding:

(1) a fusion protein containing a KLW mutant of human FRB having a Thr2098Leu substitution (FRB_L; SEQ ID NO: 81 encoded by SEQ ID NO: 17) fused, through a 2-amino acid linker peptide (SEQ ID NO: 83 encoded by SEQ ID NO: 19), to a wild type human FKBP12 polypeptide (SEQ ID NO: 86 encoded by SEQ ID NO: 4) fused to a truncated human MyD88 polypeptide (the amino terminal 172 amino acids of MyD88 containing the DD domain and intermediary domain; SEQ ID NO: 102 encoded by SEQ ID NO: 30) fused, through a 2-amino acid linker peptide (SEQ ID NO: 83 encoded by SEQ ID NO: 19), to a portion of a human CD40 polypeptide (the carboxy terminal 62 amino acids, i.e. , amino acids 216-277 of CD40; SEQ ID NO: 105 encoded by SEQ ID NO: 33), (the entire fusion protein is termed MC-Rap or iRMC),

(2) a 3-amino acid linker peptide (SEQ ID NO: 106 encoded by SEQ ID NO: 34),

(4) a 5-amino acid linker peptide (SEQ ID NO: 109 encoded by SEQ ID NO: 37),

(6) a 5-amimo acid linker (SEQ ID NO: 1 15 encoded by SEQ ID NO: 43),

(8) a 4-amino acid linker (SEQ ID NO: 1 17 encoded by SEQ ID NO: 46),

(9) a membrane signal peptide (SEQ ID NO: 1 18 encoded by SEQ ID NO: 47) fused to heavy (SEQ ID NO: 124 encoded by SEQ ID NO: 53) and light chain (SEQ ID NO: 126 encoded by SEQ ID NO: 55) variable regions of anti-HER2 monoclonal antibody FRP5 (with an intervening 15-amino acid flexible glycine-serine linker, i.e., flex peptide (SEQ ID NO: 125 encoded by SEQ ID NO: 54) between the chains) fused, through a 2-amino acid linker (SEQ ID NO: 127 encoded by SEQ ID NO: 56), to a human CD34 epitope peptide (amino acids 30-45 of CD34; SEQ ID NO: 128 encoded by SEQ ID NO: 57) which is fused to an alpha stalk region of human CD8 (amino acids 141-182 of CD8; SEQ ID NO: 129 encoded by SEQ ID NO: 58) which is fused to the transmembrane domain of human CD8 (amino acids 183-219 of CD8; SEQ ID NO: 130 encoded by SEQ ID NO: 59) which is fused to a portion of human Oϋ3z (amino acids 83-194 of Oϋ3z isoform X2; SEQ ID NO: 132 encoded by SEQ ID NO: 61) through a 2-amino acid linker (SEQ ID NO: 91 encoded by SEQ ID NO: 9).

Cell transfection and transduction

Production of retroviruses

[0477] 2.5 - 3.0 x 10⁶ HEK 293T cells were seeded on a 100-mm tissue culture dish in 8 mL IMDM, supplemented with glutamine, 1% penicillin/streptomycin and 10% fetal calf serum. After 16-30 hours incubation, cells were transfected using Novagen’s GeneJuice® protocol. Briefly, for each transfection, 0.5 mL OptiMEM reduced serum medium (GIBCO™) was pipeted into a 1.5-mL microcentrifuge tube and 30 pL GeneJuice reagent added followed by 5 sec. vortexing. Samples were rested 5 minutes to settle the GeneJuice suspension. DNA (15 pg total) was added to each tube and mixed by pipetting up and down four times. Samples were allowed to rest for 5 minutes for GeneJuice-DNA complex formation and the suspension added dropwise to one dish of 293T cells. A typical transfection included these plasmids to produce replication incompetent retrovirus: 3.75 pg plasmid containing gag-pol (pEQ-PAM3(-E)), 2.5 pg plasmid containing viral envelope (e.g., RD114 or RDF) and 3.75 pg retroviral vector containing gene of interest.

Transduction of peripheral blood mononuclear cells (PBMCs)

[0478] Human PBMCs (1-2 x 10⁶ cells) were stimulated with anti-CD3 and anti-CD28 antibodies precoated to wells of tissue culture plates. 24 hours after plating, 15 ng/mL IL-7 and 5 ng/mL IL-15 were added to the culture. On day 2 or 3, supernatant containing retrovirus from transfected 293T cells was filtered at 0.45 pm and centrifuged on non-tissue culture treated plates precoated with retronectin (~10 pi per well in 1 ml of PBS per 1 cm² of surface). Plates were centrifuged at 2000 g for 2 hours at room temperature. CD3/CD28 blasts were resuspended at ~2.5x10⁵ cells/ml in complete media, supplemented with 15 ng/mL IL-7 and 5 ng/mL IL-15 and centrifuged on the plate at 1000 x g for 10 minutes at room temperature. After 3-4 days incubation, cells were counted and transduction efficiency measured by flow cytometry using the appropriate marker antibodies (transduced T cells were marked with the Q-bend 10 (Q) epitope derived from CD34). CAR expression was analyzed by flow cytometry using antibody, QBend-10 (Biolegend), specific for an epitope derived from human CD34 incorporated into the extracellular portion of a 1^st generation CAR-z, and T cell viability was assessed using a Nexelon Cellometer with the cells stained with acridine orange and propidium iodide cells. Cells were maintained in complete media supplemented with 15 ng/mL IL-7 and 5 ng/mL IL-15 refed cells every 2-3 days with fresh media and 15 ng/mL IL-7 and 5 ng/mL IL-15 and split as needed to expand the cells.

[0479] The transduced PBMCs were also transduced with pM010 to stably label the cells with nuclear-localized RFP protein (Fig 14). This enabled fluorescence-based evaluation of cell proliferation over time. Approximately 24 hours after the PMBCs had been transduced with pM006 and pM007, the cells were transferred to plates containing retrovirus that contained pM010 for cotransduction. Retrovirus containing pM010 was produced using the retrovirus production methods as described in this Example. Briefly, HEK 293T cells were transfected with the following plasmids to produce replication incompetent retrovirus: 3.75 pg plasmid containing gag-pol (pEQ-PAM3(-E)), 2.5 pg plasmid containing viral envelope (e.g., RD1 14 or RDF) and 3.75 pg pM010. Plasmid pM010 contains the following polynucleotides in the 5’ to 3’ direction: polynucleotide encoding an SP163 translation enhancer (SEQ ID NO: 139 encoded by SEQ ID NO: 68), a polynucleotide encoding a linker polypeptide (SEQ ID NO: 69), a polynucleotide encoding red fluorescent protein (RFP; SEQ ID NO: 140 encoded by SEQ ID NO: 70), a polynucleotide encoding a linker peptide (SEQ ID NO: 141 encoded by SEQ ID NO: 71) and a polynucleotide encoding three nuclear localization sequences fused in succession (SEQ ID NO: 142 encoded by SEQ ID NO: 72).

[0480] Methods discussed herein, including, but not limited to, methods for constructing vectors, transfecting, transducing or transforming cells, assays for activity or function, administration to patients and methods for monitoring patients may also be found in the following patents and patent applications, which are hereby incorporated by reference herein in their entirety.

[0481] U.S. Patent Application Serial Number 15/377,776, titled DUAL CONTROLS FOR THERAPEUTIC CELL ACTIVATION OR ELIMINATION, filed December 13, 2016; U.S. Patent Application Serial Number 14/210,034, titled METHODS FOR CONTROLLING T CELL PROLIFERATION, filed March 13, 2014; U.S. Patent Application Serial Number 13/1 12,739, filed May 20, 201 1 , issued as U.S. Patent 9,089,520, July 28, 2015, and entitled METHODS FOR INDUCING SELECTIVE APOPTOSIS; U.S. Patent Application Serial Number 14/622,018, filed February 13, 2014, titled METHODS FOR ACTIVATING T CELLS USING AN INDUCIBLE CHIMERIC POLYPEPTIDE; U.S. Patent application Serial Number 13/1 12,739, filed May 20,

201 1 , titled METHODS FOR INDUCING SELECTIVE APOPTOSIS; U.S. Patent Application Serial Number 13/792, 135, filed March 10, 2013, titled MODIFIED CASPASE POLYPEPTIDES AND USES THEREOF; U.S. Patent Application Serial Number 14/296,404, filed June 4, 2014, titled METHODS FOR INDUCING PARTIAL APOPTOSIS USING CASPASE POLYPEPTIDES; U.S. Provisional Patent Application Serial Number 62/044,885, filed September 2, 2014, and U.S. Patent Application 14/842,710, filed September 1 , 2015, each titled COSTIMULATION OF CHIMERIC ANTIGEN RECEPTORS BY MyD88 AND CD40 POLYPEPTIDES; U.S. Patent Application Serial Number 14/640,554, filed 6 March, 2015, titled CASPASE POLYPEPTIDES HAVING MODIFIED ACTIVITY AND USES THEREOF; U.S. Patent Number 7,404,950, issued June 29, 2008, to Spencer, D. et al., U.S. Patent applications 12/445,939 by Spencer, D., et al., filed October 26, 2010; U.S. Patent application 12/563,991 by Spencer, D., et al., filed

September 21 , 2009; 13/087,329 by Slawin, K., et al., filed April 14, 201 1 ; 13/763,591 by Spencer, D., et al., filed February 8, 2013; and International Patent Application Number PCT/US2014/022004, filed 7 March 2014, published as PCT/US2014/022004 on 9 October 2014, titled MODIFIED CASPASE POLYPEPTIDES AND USES THEREOF.

[0482] Example 21: Fluorescence-Based Assay Evaluation of Effects of Compounds on Proliferation of CAR T Cells and OE19 Cells

Assay methods

[0483] PMBCs that had been transduced with retrovirus containing pM006 and pM007 and the red fluorescent protein-encoding plasmid pM010 were evaluated for proliferation, activation and effects on ERBB2 (HER2)-expressing human OE19 esophageal tumor cells in a coculture assay at a 1 :15 effector to target (E:T) ratio. The OE19 tumor cells had been stably labeled through transduction with retrovirus containing nucleic acid encoding GFP-FFluc (a Green Fluorescent Protein, such as Aequorea GFP, see, e.g., Genbank Accession no. AAN41637 and SEQ ID NO: 147, fused with firefly luciferase, see, e.g., Genbank Accession no. BAL46512 and SEQ ID NO: 148) contained within an SFG retroviral vector (see, e.g., Riviere et al. (1995) Proc. Natl. Acad. Sci. U.S. A. 92: 6733-6737) to provide an in vivo cell marker. To set up the coculture, 4000 OE- 19 cells were seeded per well of 96-well plates in 100 pi of CTL medium without IL-2 for at least 4 hours to allow tumor cells to adhere. Transduced PBMCs, after being allowed to rest for at least 7 days in culture post transduction, were seeded according to a 1 :15 E:T ratio to the OE19-GFP-containing 96-well plates. A compound, or rapalog (CMP001), can also be added to the culture to reach 300 mI total volume per well. Each plate was set up in duplicates, one plate to monitor with the IncuCyte cell imaging system (Essen Bioscience) and one plate for supernatant collection for ELISA assay on day 2. The plates were centrifuged for 5 min at 400 x g and placed inside the IncuCyte (Essen Bioscience, Dual Color Model 4459) to monitor red fluorescence (and green fluorescence of OE19 cells labeled with GFP-Ffluc) every 2-3 hours for a total of 7 days at 10x objective. On day 7, PMBC-RFP cells were analyzed using the“Red Object Count (1/well)” metric. Also on day 7, compound (0 or 10 nM) was added to each well of the coculture and placed back in the IncuCyte to monitor cell proliferation. On day 8, OE19- GFP cells were analyzed using the“Total Green Object Integrated Intensity” metric. Each condition was performed at least in duplicates and each well was imaged at 4 different locations. The fluorescence values measured for the cells provided an indication of the number of cells at each time point in the assay.

[0484] PMBCs transduced with nucleic acid encoding the human HER2-5rbaίίo-ΰ03z chimeric antigen receptor (but lacking nucleic acid encoding a co-stimulatory MC fusion protein or an iC9 cell elimination fusion protein) and untransduced PMBCs were used as control cells in the assay.

Assay results

[0485] FIG. 9 (A and B) shows the results of fluorescence assays of the cocultured transduced PMBCs (i.e. , T cells) and the OE19-GFP-fluc cells over time using the IncuCyte cell imaging system. In FIG. 9(A), the graph of green fluorescence measurements of OE19 cells shows that OE19 cells cocultured with untransduced T cells (Ctrl T cells) continued to increase in numbers over time after about 24 hours. OE19 cells cocultured with CAR T cells also increased in number over the same time period but to lower levels and at a slower rate than OE19 cells cocultured with control T cells. OE19 cells cocultured with untreated CAR T cells (designated“CAR” in FIG. 9) or untreated CAR T cells that were also transduced with iMC- and iC9-encoding nucleic acids (designated“DS-CAR-T” in FIG. 9) showed similar growth curves, whereas OE19 cells cocultured with CAR T cells in the presence of 5 nM CMP001 grew slightly faster and to somewhat greater levels than those cells. Growth of OE19 cells cocultured in the presence of 5 nM CMP001 with DS-CAR-T cells was significantly inhibited compared to the proliferation of OE19 cells cocultured under all other tested conditions. This result demonstrates that CAR T cells expressing the iMC co-stimulatory FKBP12-FRB_L-MyD88-CD40 fusion proteins, when exposed to a rapalog that binds FKBP12 and FRBL, are activated through dimerization-induced intracellular signaling for enhanced tumor cell killing relative to uninduced DS-CAR-T cells and first generation CAR T cells.

[0486] The graph of red fluorescence measurements of cocultured cells shown in FIG. 9(B) reveals limited growth and proliferation of CAR T cells (in the presence and absence of CMP001) and DS-CAR-T cells in the absence of CMP001 , and no proliferation of control T cells. In contrast, DS-CAR-T cells treated with CMP001 exhibited significantly greater growth rates and proliferation over time. This result demonstrates that CAR T cells expressing the iMC co stimulatory FKBP12-FRB_L-MyD88-CD40 fusion proteins, when exposed to a rapalog that binds FKBP12 and FRB_L, are also stimulated through dimerization-induced intracellular signaling for enhanced growth relative to uninduced DS-CAR-T cells and first generation CAR T cells.

[0487] FIG. 9 also shows the results of ELISA assays designed to measure cytokine (IFN-g in FIG. 9(C) and IL-2 in FIG. 9(D)) levels of coculture (E:T of 1 :5) supernatants collected after two days of culturing. Cytokine levels were essentially undetectable in supernatants of cultures of control T cells and CAR T cells in the presence or absence of CMP001. Relatively small amounts of IFN-g and IL-2 were detected in supernatants of DS-CAR-T cell cultures in the absence of CMP001. In comparison, the levels of both cytokines in DS-CAR-T cell cultures in the presence of CMP001 were several fold higher than in the absence of CMP001. These results demonstrate that induction of multimerization of iMC fusion proteins by a rapalog activates signaling pathways in DS-CAR-T cells that lead to NFAT and NF-kB activation.

[0488] Example 22: Evaluation of Proliferation and Compound-Induced Activation of CAR T Cells in Mice

[0489] In order to evaluate the effect of compounds on the proliferation of, and tumor cell- inhibiting efficacy of, CAR T cells transduced with nucleic acid encoding an iMC (FKBP12-FRB_L- MyD88-CD40) fusion protein and a cell elimination iC9 (FKBP12v36-Acaspase 9) fusion protein in vivo, transduced cells were infused into immunodeficient mice engrafted with tumor cells.

The CAR T cells and the tumor cells used in the experiments were also transduced with bioluminescent protein-encoding nucleic acids to facilitate non-invasive visualization of the cells in whole animals using in vivo imaging system (IVIS) technology. Additionally, cytokine levels of serum from the mice were measured following CAR T cell infusion.

Development of dual switch CAR T cells

[0490] Dual switch CAR T cells used for in vivo evaluation were generated through transduction of human PMBC cells with retrovirus containing plasmids encoding iMC and iC9 proteins using methods as described herein and/or in patent applications incorporated herein by reference thereto.

Plasmid construction [0491] Two multicistronic retroviruses were used in transducing cells for the coexpression of a human epidermal growth factor 2 receptor (HER2)-specific CAR, iMC and iC9 in the transduced cells. In one example, two retrovirus vectors, referred to as pM007 (FIG. 7) and pM008 (FIG.

10) were used for cell transduction. These two vectors provide one copy of an iMC fusion protein-encoding nucleic acid construct (from pM007), one iC9 fusion protein-encoding nucleic acid construct (from pM008) and one copy of a HER2-specific CAR-encoding nucleic acid (from pM007).

[0492] Vector pM007 is described in Example 20. Vector pM008 contains, in the 5’ to 3’ direction, nucleic acid encoding:

(1) a fusion protein containing an FKBP12v36 mutant of human FKBP12 having an F36 to V substitution (SEQ ID NO: 94 encoded by SEQ ID NO: 13) fused, through a 6-amino acid linker peptide (SEQ ID NO: 95 encoded by SEQ ID NO: 14), to a portion of human caspase 9 polypeptide (Acaspase9 which contains amino acids 135-416 of caspase 9; SEQ ID NO: 1 14 encoded by SEQ ID NO: 42) (the entire fusion protein is termed iC9),

(2) a T2A self-cleaving peptide (SEQ ID NO: 1 16 encoded by SEQ ID NO: 45) to provide for separation of the fusion protein from the protein encoded by the DNA 3’ of the T2A-encoding DNA in the plasmid, and

(3) a non-signaling portion of human CD19 (amino acids 1-333 of CD19; SEQ ID NO:

131 encoded by SEQ ID NO: 60) that served as a surface marker.

Cell transfection and transduction

Production of retroviruses

[0493] Retroviruses containing pM008 were produced in HEK 293T cells transfected with the plasmids using Novagen’s GeneJuice® protocol as described in the previous Example. A typical transfection included these plasmids to produce replication incompetent retrovirus: 3.75 pg plasmid containing gag-pol (pEQ-PAM3(-E)), 2.5 pg plasmid containing viral envelope (e.g., RD1 14 or RDF) and 3.75 pg retroviral vector containing gene of interest.

Transduction of PBMCs

[0494] Human PBMCs were transduced with retroviruses containing nucleic acids encoding the (HER2)-specific CAR and the iMC and iC9 fusion proteins using methods as described in the previous Example. Transduction efficiency was measured by flow cytometry using the appropriate marker antibodies (transduced T cells were marked with the Q-bend 10 (Q) epitope derived from CD34). CAR expression was analyzed by flow cytometry using antibody, QBend- 10 (Biolegend), specific for an epitope derived from human CD34 incorporated into the extracellular portion of a 1^st generation CAR-z, and T cell viability was assessed using a Nexelon Cellometer with the cells stained with acridine orange and propidium iodide cells. Cells were maintained in complete media supplemented with 15 ng/mL IL-7 and 5 ng/mL IL-15 refed cells every 2-3 days with fresh media and 15 ng/mL IL-7 and 5 ng/mL IL-15 and split as needed to expand the cells.

[0495] The transduced PBMCs were also transduced with pM01 1 encoding a chimera of amino acids 1-218 of mKusabiraOrange2 and amino acids 4-31 1 of Renilla luciferase (RLuc8.6- 535) to stably label the cells with a bioluminescent protein. This enabled bioluminescence- based evaluation of cell proliferation over time in mice to which the transduced cells were administered. Approximately 24 hours after the PMBCs had been transduced with pM007 and pM008, the cells were transferred to plates containing retrovirus that contained pM01 1 for cotransduction. Retrovirus containing pM01 1 was produced using the retrovirus production methods as described in the previous Example. Briefly, HEK 293T cells were transfected with the following plasmids to produce replication incompetent retrovirus: 3.75 pg plasmid containing gag-pol (pEQ-PAM3(-E)), 2.5 pg plasmid containing viral envelope (e.g., RD1 14 or RDF) and 3.75 pg pM01 1. Plasmid pM01 1 contains the following nucleic acids in the 5’ to 3’ direction: nucleic acid encoding amino acids 1-218 of mKusabiraOrange2 (SEQ ID NO: 143 encoded by SEQ ID NO: 73), nucleic acid encoding a 2-amino acid linker peptide (SEQ ID NO: 144 encoded by SEQ ID NO: 74) and nucleic acid encoding amino acids 4-31 1 of Renilla luciferase (RLuc8.6- 535; SEQ ID NO: 145 encoded by SEQ ID NO: 75).

Transduction of OE19 cells

[0496] Human OE19 cells were obtained from American Type Culture Collection (ATCC) and transduced with retrovirus containing nucleic acid encoding firefly luciferase (GFP Flue). This enabled bioluminescence-based evaluation of tumor cell proliferation over time in mice in which the transduced OE19 cells were engrafted. The OE19 tumor cells had been stably labeled through transduction with retrovirus containing nucleic acid encoding GFP-FFluc (a Green Fluorescent Protein, such as Aequorea GFP, see, e.g., Genbank Accession no. AAN41637 and SEQ ID NO: 147, fused with firefly luciferase, see, e.g., Genbank Accession no. BAL46512 and SEQ ID NO: 148) contained within an SFG retroviral vector (see, e.g., Riviere et al. (1995) Proc. Natl. Acad. Sci. U.S.A. 92: 6733-6737) to provide an in vivo cell marker. Development of animal models

[0497] FIG. 1 1 (left side) shows a schematic depiction of the timeline for the animal model development protocol. Immunodeficient NOD scid gamma (NOD.CgPrkdc^scic' ll2rg^{imil ?,}/SzJ; NSG) mice (available, e.g., from The Jackson Laboratory, Bar Harbor, ME) were engrafted with the transduced OE19 cells (1.75 x10⁶ cells) by subcutaneous injection using PBS as a vehicle. Four days later, the mice received 5 x10⁶ transduced PMBCs (designated CAR T in FIG. 1 1) via intravenous infusion using PBS as a vehicle. Beginning the day after infusion with transduced PMBCs, compound, e.g., CMP001 (i.e.,“Go” drug), in 5% polyethylene glycol 400 (PEG400) and 5% polyoxyethylene sorbitan molooleate (i.e., polysorbate 80 or Tween 80) was given intraperitoneally and weekly thereafter at 1 mg/kg body weight.

Imaging of treated mice

[0498] The treated mice were subjected to MS imaging using an MS Spectrum in vivo imaging system (Perkin Elmer) on day 0 and about weekly thereafter. Mice were imaged for firefly luciferase- or Renilla luciferase-derived bioluminescence. The mice were anesthetized with isofluorane and injected with the appropriate luciferase substrate by an intraperitoneal (i.p.) route in the lower abdomen. For imaging of firefly luciferase, 100 pi D-luciferin (15 mg/ml stock solution in PBS) was the substrate administered i.p. For imaging of Renilla luciferase, coelenterazine (RediJect Coelentrazine (150 mg/mL, Perkin Elmer); 100 mI per mouse) was the substrate administered i.p. After 10 minutes the animals were transferred from the anesthesia chamber to the MS platform. Images were acquired from the dorsal and ventral sides, and bioluminescence imaging (BLI) quantitated and documented with Living Image software (MS Imaging Systems).

Results of imaging of treated mice

[0499] Figures 1 1 and 12 provide photographs of luciferase-derived bioluminescence imaging of NGS mice that had been engrafted with OE19 tumor cells (transduced with nucleic acid encoding GFP FFluc) and infused with CAR T cells (PMBCs transduced with retrovirus containing plasmids encoding iRMC and iC9 proteins and ONL Rluc), which are designated as “iRMC-GoCAR-T + iC9” in the figures. Luciferase-derived bioluminescence imaging of control mice is also shown in the figures. The control mice (designated“iC9” in the figures) had been engrafted with OE19 tumor cells (transduced with nucleic acid encoding GFP FFluc) and infused with PMBCs transduced with retrovirus containing nucleic acid encoding the iC9 protein

(pM008) and ONL Rluc, but lacking nucleic acid encoding iRMC and a chimeric antigen receptor. Control mice had also been administered a vehicle (5% PEG400 and 5% polysorbate 80) instead of CMP001 on the day after CAR T cell infusion and weekly thereafter. Figures 1 1 and 12 show the results of Renilla luciferase-derived bioluminenscence and firefly luciferase-derived bioluminescence, respectively.

[0500] As shown in FIG. 1 1 (right side), no Renilla luciferase-derived bioluminescence was detected in the control mice. All control mice expired by the 55^th day post cell infusion. Mice that had been engrafted with transduced OE19 tumor cells and then infused with CAR T cells (transduced with retrovirus containing plasmids encoding iRMC and iC9 proteins and ONL Rluc) and administered vehicle instead of CMP001 also did not exhibit any Renilla luciferase-derived bioluminescence, and 2 of the 4 animals expired by the 55^th day post cell infusion. In contrast, mice that had been engrafted with transduced OE19 tumor cells and then infused with CAR T cells (transduced with retrovirus containing plasmids encoding iRMC and iC9 proteins and ONL Rluc) and administered CMP001 on the day after cell infusion and weekly thereafter were alive and exhibited Renilla luciferase-derived bioluminesce by the 15^th day post cell infusion and continuing into the 55^th day post cell infusion.

[0501] As shown in FIG. 12 (left side), all of the control mice and vehicle-treated iRMC- GoCAR-T + iC9 mice exhibited increasing amounts of firefly luciferase-derived bioluminescence with time post OE19 cell engraftment and CAR T cell infusion. In contrast, although the

CM P001 -treated iRMC-GoCAR-T + iC9 mice showed some bioluminescence post OE19 cell engraftment through day 16 post CAR T cell infusion, the bioluminescence diminished by day 16 post CAR T cell infusion and was no longer detectable by day 22 post CAR T cell infusion. FIG. 12 (right side) shows the results of Kaplan-Meier analysis from the in vivo assay of the control and iRMC-GoCAR-T + iC9 mice that were imaged as shown in FIG. 1 1 and the left side of FIG. 12.

[0502] The results shown in FIG. 1 1 reveal that CAR T cells co-expressing 8hίί-I-^2/ΰ03z CAR, iRMC and iC9 fusion proteins (“iRMC-GoCAR-T + iC9”) are able to survive and proliferate for an extended period of time in vivo in tumor cell-engrafted NGS mice, but only in the presence of an effective FKBP12-FRB_L-dimerizing compound, e.g., CMP001 . The results shown in FIG. 12 confirm that control (“ic9”) and dual switch (“iRMC-GoCAR-T + iC9”) mice received the OE19 tumor cells expressing firefly luciferase. However, only the dual switch (“iRMC-GoCAR-T + iC9”) mice receiving an effective FKBP12-FRB -dimerizing compound, e.g., CMP001 , were able to eradicate the tumor cells and remain essentially tumor cell-free for at least 51 days post CAR T cell infusion. Accordingly, this example demonstrates that intracellular approximation of co-stimulatory MyD88 and CD40 polypeptides through the binding, by an effective dimerizing compound (e.g., CMP001), of FKBP12 and a variant FRB (e.g.,

FRB_l) proteins fused to the co-stimulatory polypeptides in an immune cell expressing a CAR enables persistent growth and proliferation of the immune cell in vivo. This example further demonstrates that such proliferating immune cells are able to effectively inhibit and kill tumor cells recognized by the CAR in vivo.

Evaluation of cytokine levels of mice infused with CAR T cells

[0503] Serum was collected from the control mice and mice infused with CAR T cells coexpressing 3hίί-HER2/ΰ03z CAR, iRMC and iC9 fusion proteins (“iRMC-GoCAR-T + iC9”) on the 9^th day following cell infusion. The relative levels of 29 cytokines were assessed in the serum samples using a multiplex assay system (LUMINEX): IFN-g, IL-5, IP-10, GMCSF, TNF-a, IL-6, VEGF, IL-13, IL-3, IL-2, TNF-b, MCP-1 , MIP-1 a, Eotaxin, IL-17A, GCSF, IL-4, IL-8, IL-1 RA, MIP-1 b, IL-7, IL-10, IL-12p70, IFN-a2, IL1-a, IL1 - b, EGF, IL-12p40 and IL-15. As shown in FIG. 13, the levels of all the measured cytokines in serum from control mice were low (as indicated by the darker blue and violet shading on the relative color-based scale in the figure). The levels of about half of the measured cytokines in serum from iRMC-GoCAR-T + iC9 mice that were administered vehicle instead of compound (CMP001) were somewhat higher than the levels of the same cytokines in the control mice serum, but were still relatively low. In contrast, the levels of a substantial portion of the cytokines measured in serum from the iRMC-GoCAR-T + iC9 mice that were administered dimerizing compound (e.g., CMP001) were detectably higher than the levels of the same cytokines in the serum from the vehicle-treated mice (as indicated by the lighter blue and violet shading and the pink shading on the relative color-based scale in the figure). These results are consistent with the results of the imaging analyses that revealed the persistent proliferation and activation of CAR T cells coexpressing 3hίί-HER2/ΰ03z CAR, iRMC and iC9 fusion proteins in mice treated with dimerizing compound (e.g., CMP001). The increased cytokine production in the cells in the presence of dimerizing compound enhanced the growth and proliferation of the cells and lead to further increases in cytokine release.

[0504] Example 23: SeAP Reporter Apoptosis-Based Compound Binding Assays

[0505] The ability of compounds to bind to human FKBP12 and FRB proteins and/or variants thereof can be evaluated using an apoptosis-based assay that uses secreted alkaline phosphatase (SeAP) as a reporter molecule (see, e.g., MacCorkle et al (1998) Proc Natl Acad Sci 95: 3655-3660 ). In this assay, cells are co-transfected with expression plasmids encoding inducible chimeric caspase-9 polypeptides and an SRa promoter-driven SEAP-encoding DNA (SeAP reporter plasmid). For example, one of the chimeric caspase-9 polypeptide-encoding plasmids can contain DNA encoding an FKBP12 protein fused to caspase 9 (or a portion thereof) and the other chimeric caspase-9 polypeptide-encoding plasmid can contain DNA encoding FRB or a variant FRB protein fused to caspase 9 (or portion thereof). Transfected cells are then contacted with the compound being evaluated and, after a period of time, the transfected cell culture supernatant is assayed for SeAP activity. The SeAP activity can be compared to the SeAP activity of the supernatant of transfected cells that have not been treated with compound. If a compound binds to and dimerizes the chimeric fusion proteins, the dimerized caspase proteins are activated and trigger apoptosis in the cells. Apoptosis of the cells is indicated by a reduction in the SeAP activity of cells treated with a binding compound compared to the SeAP activity of untreated cells. IC50 values for compound-induced reduction of SeAP activity (indicative of apoptosis) can be calculated from dose-response studies of the compounds in this assay. The following is an example protocol for conducting the assay.

Cell line maintenance and transfection

Assay methods

[0506] Twenty-four to forty-eight hours after addition of compound, ~100 pi of supernatants are harvested into a 96-well plate and assayed for SEAP activity as described, for example, in MacCorkle et al. ((1998) Proc Natl Acad Sci U S A 95(7):3655-3660) and in Spencer et al.

((1996) Curr Biol 6(7):839-847). Briefly, after 65°C heat denaturation for 45 minutes to reduce background caused by endogenous (and serum-derived) alkaline phosphatases that are sensitive to heat, 5 pi of supernatants are added to 95 mI of PBS and added to 100 mI of substrate buffer, containing 1 mI of 100 mM 4-methylumbelliferyl phosphate (4-MUP; Sigma, St. Louis, MO) and re-suspended in 2 M diethanolamine. Hydrolysis of 4-MUP by SeAP produces a fluorescent metabolite with excitation/emission (355/460 nm), which can be easily measured. Assays are performed in black opaque 96-well plates to minimize fluorescence leakage between wells. Since SeAP is used as a marker for cell viability, reduced SeAP reading corresponds with increased icaspase-9 activities, which corresponds to greater induction of the chimeric caspase-9 polypeptide following treatment with the test compound. Compounds that result in reduced SeAP readings correspond to greater binding of the compound to the multimerizing region.

[0507] Example 24: Liver Microsome Assay for Compound Stability Methods

[0508] The in vitro stability of compounds can be evaluated in liver microsome-based assays using methods described herein and/or known in the art. Liver microsomes can be prepared according to known methods and are commercially available (e.g., from XenoTech). Liver microsomes (0.5 mg/ml) in a reaction mixture containing potassium phosphate (pH 7.4;

100mM), magnesium chloride (5 mM) and compound (1 mM) were equilibrated in a shaking water bath at 37°C for 3 minutes. A control compound, testosterone, was run simultaneously with the test compound in a separate reaction. The reaction was initiated by the addition of NADPH cofactor (1 mM), and the mixture was incubated in a shaking water bath at 37°C. Aliquots (100 pi) were withdrawn at 0, 10, 20, 30 and 60 minutes. At each time point, withdrawn samples were immediately combined with ice-cold 50/50 acetonitrile (ACN)/H₂0 (400 pi) containing 0.1 % formic acid and internal standard to terminate the reaction. The samples were then mixed and centrifuged to precipitate proteins. All samples were assayed by LC-MS/MS using electrospray ionization. The peak area response ratio (PARR) to internal standard was compared to the PARR at time 0 to determine the percent remaining at each time point. Half- lives were calculated using GraphPad software, fitting to a single-phase exponential decay equation.

Results

[0509] The results of liver microsome assays of CMP001 using microsomes from five different species are shown in Table 6.

Table 6: Stability of CMP001 in Liver Microsomes from Different Species

aWhen the calculated half-life is longer than the duration of the experiment, the half-life is expressed as > the longest incubation time. Then, if the calculated half-life is <2x the duration of the experiment, the calculated half-life is listed in parentheses.

intrinsic clearance (Clint) was calculated based on Cl_int=k/P, where k is the elimination rate constant and P is the protein concentration in the incubation.

[0510] The results of liver microsome assays of compounds using human microsomes are shown in Table 7. Table 7: Stability of Compounds in Human Liver Microsomes

intrinsic clearance (CU) was calculated based on Cl_int=k/P, where k is the elimination rate constant and P is the protein concentration in the incubation.

[0511] The half-life for the testosterone control assay using human microsomes was 7.2 min (Cl_mt was 0.191 ml/min/mg protein) and the acceptable half-life range was <41 min.

[0512] Example 25: Synthesis of (1R, 1'R)-((((ethane-1,2-diylbis(methylazanediyl))bis(ethane- 2, 1-diyl))bis(oxy))bis(3, 1 -phenylene))bis(3-(3,4-dimethoxyphenyl) ropane-1 , 1-diyl) (2S,2'S)- bis(1-((S)-2-(3,4,5-trimethoxyphenyl)butanoyl)piperidine-2-carboxylate)

[0513] Certain embodiments of compositions (e.g., combinations) and methods, for example, ligand-controlled cell activation/elimination (dual switch) methods, provided herein, include compounds and/or polypeptides that bind to compounds, described in U.S. patent application no. 62/608,552. In general, certain embodiments of the compounds described in U.S. patent application no. 62/608,552 may be synthesized using, for example, the following methods. Methods 1 , 2, and 3 are examples of methods that may be used to synthesize compounds of Formula I or II, as shown herein and in U.S. Patent application no. 62/608,552, where Y is L or M.

Formula I

Formula II

Method 1 : Reduction of Diamide (12)

t-butyl bromoacetae

Method 2: Reduction of Acid (6), followed by alkylation of diamine

[0514] GAct¹ and GAct² are groups that may be used in the process of alkylation to activate the carbon atom to which the group is attached. The group can be removed at certain conditions, as required in the process, and subsequently substituted by, for example, but not limited to, -NHR¹R^LNHR² or, for example, but not limited to, NR¹R^LNR² Non-limiting examples of GAct include halogen, OTs (O-tosyl), OMs, or OTf. Method 3: Reduction of Acid, followed by alkylation of diamine

Method 3 is a modification of Method 2.

[0515] (1 R,TR)-((((ethane-1 ,2-diylbis(methylazanediyl))bis(ethane-2,1-diyl))bis(oxy))bis(3, 1- phenylene))bis(3-(3,4-dimethoxyphenyl)propane-1 ,1-diyl) (2S,2'S)-bis(1-((S)-2-(3,4,5- trimethoxyphenyl)butanoyl)piperidine-2-carboxylate), also referred to as Compound A, has the following structure:

[0516] An example of a method of preparation of (1 R,TR)-((((ethane-1 ,2- diylbis(methylazanediyl))bis(ethane-2,1-diyl))bis(oxy))bis(3,1-phenylene))bis(3-(3,4- dimethoxyphenyl)propane-1 ,1-diyl) (2S,2'S)-bis(1-((S)-2-(3,4,5- trimethoxyphenyl)butanoyl)piperidine-2-carboxylate) according to Method 3 is as follows.

[0517] A suspension of LiAlhU (1.41 g, 37.18 mmol, 0.2 vol.) in THF (tetrahydrofuran) (74 mL, 9.0 vol.) was cooled to 0 °C. To this suspension was added a solution of [3[(7R)-3-(3,4- dimethoxyphenyl)-1- hydroxyphenyl]phenoxy]-acetic acid (6, 8.05 g, 23.24 mmol; compounds 6 and 14 may be prepared as described in Example 26) in THF (60 mL, 7.5 vol.) while maintaining the internal temperature below 20 °C. The reaction was stirred for a further 2 hours at room temperature. [IPC TLC (in process check: thin layer chromatography) (Si0₂: EtOAc/Hexane 50:50) 6: Rf 0.15, 19: Rf 0.26] Upon completion, the reaction mixture was cooled to 0 °C and water (1.5 mL) was added dropwise followed by 15% NaOH aq. (1.5 mL) and water (3 mL) while maintaining the internal temperature below 30 °C. The quenched reaction was then diluted with TBME (tert-Butyl methyl ether) (100 mL, 12.0 vol.) and stirring continued for 30 minutes. The reaction mixture was dried with MgS0₄, filtered and solvent was removed by rotary evaporation. The crude product 3-(3,4-dimethoxyphenyl)-1-[3-(2-hydroxyethoxy)phenyl]propan-1-one 19 (7.10 g, 21.38 mmol, 92% yield) was obtained as a colorless oil and was used directly in the next step without further purification.

[0518] 3-(3,4-dimethoxyphenyl)-1-[3-(2-hydroxyethoxy)phenyl]propan-1-one (19, 68.6 g, 206.5 mmol) was dissolved in DCM (Dichloromethane) (590 mL, 8.5 vol.). Triethylamine (43.2 mL, 309.8 mmol, 1.5 eq.) was added followed by 4-toluenesulfonyl chloride (39.4 g, 206.5 mmol, 1.0 eq.). The reaction mixture was stirred at room temperature for 18 hours. [IPC TLC (Si0₂:

EtOAc/Hexane 50:50) 19: Rf 0.26, 20-OTs: Rf 0.52, TsCI: Rf 0.76] Upon completion, the DCM was removed by rotary evaporation and exchanged for EtOAc (500 mL, 7.1 vol.). The organic layer was washed with HCI aq. 1 M (3 x 100 mL, 1.4 vol.), NaHC0₃ sat. aq. (2 x 10 mL, 1.4 vol.), brine (70 mL, 1.0 vol.), dried over MgS0₄ then filtered and solvent was removed by rotary evaporation. The crude residue was purified by BIOTAGE (340 g SNAP Ultra column; 30-90% EtOAc in Hexane) to provide 2-{3-[3-(3,4- dimethoxyphenyl)propanoyl]phenoxy}ethyl 4- methylbenzenesulfonate, 20-OTs (63.6 g, 130.7 mmol, 63% yield) as a colorless oil. This material was used directly in the following step.

[0519] 2-{3-[3-(3,4-dimethoxyphenyl)propanoyl]phenoxy}ethyl 4-methylbenzenesulfonate 20- OTs (3.93 g, 8.08 mmol) and L/,L/'-dimethyl-ethylenediamine (0.36 g, 4.04 mmol, 0.5 eq.) were dissolved in MeCN (acetonitrile) (20 mL, 5.0 vol.). To this solution was added K₂C0₃ (2.23 g, 16.16 mmol, 2.0 eq.) and Kl (0.67 g, 4.04 mmol, 0.5 eq.). The reaction mixture was heated to 70 °C and stirred for 16 hours. [IPC TLC (Si0₂: EtOAc/Hexane 50:50) 20-OTs: Rf 0.26, 13-(CH₂)₂-: Rf baseline]. Upon completion, MeCN was removed by rotary evaporation and exchanged for EtOAc (50 mL, 12.0 vol.). The organic layer was washed with water (2 x 20 mL, 5.0 vol.) and NaHC0₃ sat. aq. (2 x 20 mL, 5.0 vol.). The desired product was then extracted into the aqueous layer with HCI 1 M aq. (2 x 30 mL, 7.5 vol.) and the aqueous phase was washed with EtOAc (2 x 20 mL, 5.0 vol.). The aqueous phase was then basified with NaHC0₃ sat. aq. (90 mL, 22.5 vol.) and extracted with EtOAc (3 x 30 mL, 7.5 vol.). The organic phase was then washed with brine (20 mL, 5.0 vol.), dried over MgS0₄, filtered and solvent was removed by rotary evaporation to provide 13-(CH₂)₂- (2.75 g, 3.83 mmol, 95% yield). The crude material was used directly in the next step without further purification.

[0520] 13-(CH₂)₂- (2.47 g, 3.45 mmol) and 14 (2.77 g, 7.59 mmol, 2.2 eq.) were dissolved in DCM (35 mL, 14.0 vol.) and the solution cooled to -15 °C. To the reaction mixture was added EDCI (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide) (1.59 g, 8.28 mmol, 2.4 eq.) followed by DMAP (4-Dimethylaminopyridine) (1.86 g, 15.18 mmol, 4.4 eq.). The reaction was stirred overnight at -15 °C. After 24 hours the internal temperature of the reaction was increased to -4 °C and the reaction left to stir for a further 24 hours. [IPC TLC (Si0₂: MeOH/DCM 5:95) 15- (CH₂)₂-: Rf 0.15, 14: Rf 0.37, Compound A: Rf 0.66] Upon completion, the reaction was quenched by addition of 5M HCI sol. (10 mL, 4.0 vol.) to the reaction mixture at -4 °C. DCM was then removed by vacuum distillation while maintaining the internal temperature below 20 °C.

The residue was diluted with EtOAc (50 mL, 20.0 vol.) and the organic layer washed sequentially with 1 M HCI sol. (2 x 10 mL, 4.0 vol.), NaHC0₃ sol. (10 mL, 4.0 vol.) and brine (10 mL, 4.0 vol.). The organic layer was dried with MgS0₄, filtered and solvent removed by rotary evaporation. The crude residue was purified by BIOTAGE (50 g SNAP Ultra, 0- 10% MeOH in DCM) to provide Compound A (2.09 g, 1.48 mmol, 43% yield) as an amorphous white solid.

MS: (+ESI): calculated for C₈oHi₀₆N₄Oi₈ [M+H]⁺ 141 1.7575, found 141 1.7982. [0521] Example 26: Preparation of Intermediate Compounds in the synthesis of (1R, 1'R)- ((((ethane-1 ,2-diylbis(methylazanediyl))bis(ethane-2, 1 -diyi))bis(oxy))bis(3, 1-phenylene))bis(3- (3, 4-dimethoxyphenyl) propane- 7, 1-diyl) (2S,2'S)-bis( 1-((S)-2-(3, 4, 5- trimethoxyphenyl)butanoyl)piperidine-2-carboxylate)

[0522] The present Example provides additional information regarding methods that may be used to synthesize Compound A, and certain intermediate compounds that may be used in the synthesis scheme.

[0523] Synthetic Routes to Common Intermediates 6, 9 and 11 (Scheme 5):

[0524] The synthesis of 6 involves five chemical conversions and one chiral upgrade (see Method 1 , herein); Claisen-Schmidt condensation between 1 and 2, reduction of the 3 double bond by transfer hydrogenation, alkylation of the 4 intermediate followed by asymmetric reduction of 5 and hydrolysis provides crude 6. Diastereomeric salt formation of crude 6 with (-)- cinchonidine followed by recrystallization and acidification yields 6 with chemical purity >98% and chiral purity >99.8%.

Scheme 5

[0525] Method:

[0526] 3,4-Dimethoxybenzaldehyde (1 , 39.9 g, 0.24 mol) and 3'-hydroxyacetophenone (2,

32.7 g, 0.24 mol, 1.0 eq.) were dissolved in EtOH (320 mL, 8.0 vol.) and the solution was cooled to 10 °C. A solution of KOH (53.9 g, 0.96 mol, 4.0 eq.) in water (200 mL, 5.1 vol.) was added slowly to the reaction mixture while maintaining the internal temperature below 25 °C. The reaction mixture was left to stir for 16 hours at 20 °C. [I PC TLC (Si0₂: EtOAc/Hexane 50:50) 1 : Rf 0.55, 2: Rf 0.45, 3: Rf 0.33.] Upon completion, the reaction was cooled to 0 °C and 37% HCI solution (102 mL, 2.6 vol.) was slowly added until pH 3 while maintaining the internal temperature below 25 °C. Dl water (250 mL, 6.3 vol.) was added and the resulting slurry stirred for 3 hours at 20 °C. The product was filtered, washed with water (3 x 80 mL), and dried under vacuum at 40 °C for 72 hours to constant mass. The product 3,4-dimethoxy-3’-hydroxychalone 3 (60.0 g, 0.21 mol, 88% yield) was obtained as a yellow powder.

[0527] HPLC (High-performance liquid chromatography) analysis shows 3 (9.54 min) exhibits a weak negative absorption at 250-280 nm. Residual 1 (4.03 min) and 2 (4.58 min) were occasionally observed but could be effectively purged in the following step.

[0528] 3,4-dimethoxy-3’-hydroxychalone (3, 42.8 g, 150.5 mmol) and 10% Pd/C 50% w/w wet (4.2 g, 10 wt%, 0.1 vol.) were suspended in MeOH (213 mL, 5.0 vol.). NH₄OCHO (14.2 g, 225.8 mmol, 1.50 eq.) was added in one portion. The slurry was heated to 55 °C for 1.5 hours. [IPC TLC (Si0₂: EtOAc/Hexane 50:50) 3: Rf 0.41 , 4: Rf 0.52.]. Upon completion the reaction was cooled to 45 °C and filtered, while hot, through celite and rinsed forward with MeOH (60 mL, 1.5 vol.). The combined filtrate was reheated to 50 °C to ensure a clear solution. Dl water (291 mL, 6.8 vol.) was added over 1 hour while keeping internal temperature at 50 °C. [NOTE: Significant precipitation had occurred after 50% of the addition]. The resulting slurry was cooled to 20 °C over a period of 3 hours and left to stir for a further 12 hours. The product was filtered, washed with Dl water (2 x 80 mL, 2.0 vol.) and dried under vacuum at 40 °C for 48 hours to constant mass. The product 3-(3,4-dimethyoxyphenyl)-1-(3-hydroxyphenyl)-1-propanone 4 (36.6 g, 0.13 mol, 85% yield) was obtained as a colorless powder.

[0529] HPLC analysis showed 4 (6.87 min) and two minor impurities < 1% by area.

[0530] 3-(3,4-dimethyoxyphenyl)-1 -(3-hydroxyphenyl)-1-propanone (4, 40.1 g, 0.14 mol) and K₂C0₃ powder (29.0 g, 210.0 mmol, 1.50 eq.) were suspended in acetone (400 mL, 10.0 vol.) at 20 °C. To the reaction mixture was added tert- butyl bromoacetate (27.9 mL, 189 mmol, 1.35 eq.) over 10 minutes. The resulting suspension was heated to 55 °C for 12 hours. [IPC TLC (Si0₂: EtOAc/Hexane 50:50) 4: Rf 0.57, 5: Rf 0.73] Upon completion, the reaction was cooled to 20 °C, filtered and the washed forward with acetone (20 mL, 0.5 vol.). The acetone was removed by rotary evaporation at 40 °C and co-evaporated with TBME (2 x 200 mL, 5.0 vol.). The residue was dissolved in TBME (88 mL, 2.2 vol.) and filtered through a silica plug (10 g, 0.3 vol.) and washed forward with TBME (56 mL, 1.4 vol.). The combined filtrate was stirred for 12 hours at 20 °C then cooled further to 0 °C for a further 2 hours prior to filtration. The filter cake was then washed with 20% TBME in Heptane (40 mL, 1.0 vol.) and dried under vacuum at 20 °C for 24 hours to constant mass. The product 3[3-(3,4-dimethyoxyphenyl)-1- oxopropyl]phenoxy]-acetic acid, 1 ,1-dimethylester 5 (39.8 g, 99.4 mmol, 71 % yield) was obtained as a colorless crystalline powder.

[0531] HPLC analysis showed crystalline 5 (6.72 min) to be excellent purity with no detectable impurities.

[0532] 3[3-(3,4-dimethyoxyphenyl)-1-oxopropyl]phenoxy]-acetic acid, 1 , 1-dimethylester (5,

31.60 g, 78.91 mmol) was dissolved in THF (170 mL, 5.4 vol.) and cooled to -40 °C. A solution of (+)-DIPCI [62.5 wt% in Hexanes] (121.50 g, 140.8 mL, 236.74 mmol, 3.0 eq.) was added over 15 minutes ensuring the reaction remain below -30 °C. After complete addition the reaction was stirred for a further 1 hour, then allowed to warm to -15 °C over 1 hour, then to room

temperature over a further 1 hour. [IPC TLC (Si0₂: EtOAc/Hexane 50:50) 5: Rf 0.65, Intermediate ester: Rf 0.49, 6: Rf 0.15] Upon completion the reaction was cooled to 0 °C and water (63 mL, 2.0 vol.) added slowly while maintaining the internal temperature below 20 °C.

The mixture was stirred for 15 minutes, separated and the organic layer evaporated to dryness. The resulting oil was dissolved in MeOH (63 mL, 2.0 vol.), cooled to 10 °C and 5M NaOH aq.

(76 mL, 2.4 vol.) added slowly while maintaining the temperature below 25 °C. The reaction was stirred vigorously for 30 minutes at 20°C until complete hydrolysis to the acid was observed by TLC. The reaction was concentrated to remove MeOH. 5M HCI aq. (54 mL, 1.7 vol.) was added to the oil to pH 7. After 30 minutes of agitation the aqueous layer was then washed with EtOAc (2 x 60 mL, 1.8 vol.). 5M HCI aq. (16 mL, 0.5 vol.) was then added to the aqueous solution to pH 2. The mixture was diluted with water (63 mL, 2.0 vol.) and heptane (22 mL, 0.7 vol.). The mixture was agitated for 16 hours at 20 °C. The resulting slurry was filtered and washed with water (63 mL, 2.0 vol.) and the crude product dried under vacuum at 40 °C for 24 hours to constant mass. The product [3[(1 R)-3-(3,4-dimethoxyphenyl)-1-hydroxyphenyl]phenoxy]-acetic acid 6 (16.95 g, 48.93 mmol, 62% yield, 97% e.e.) was obtained as a lumpy white solid.

[0533] HPLC analysis shows desired (R)-6 (10.98 min) and undesired (S)-6 (9.73 min).

[0534] Alternate Synthetic Route to Intermediate 6

Scheme 6

[0535] To improve chiral purity of intermediate 6, the product was converted into a

diastereomeric salt, in order to separate the two diastereomers. Following enrichment of the R salt, the salt is converted to an enriched product 6, with about 99.5% chiral purity. An initial attempt at the salt formation and recrystallization was based on patent (PCT Patent Application No: PCT/US2012/022642, filed January 26, 2012, PCT Patent Application Publication No: WO2012103279A2, published August 2, 2012, Li, F., et al.,“Methods and compositions for the synthesis of multimerizing agents”, which is hereby incorporated by reference herein in its entirety) and provided a 37% isolated yield. Subsequent efforts to combine the initial salt formation with the crystallization gave >80% yields, however <99.6% chiral purity was achieved. By adjusting the volumes of isopropanol used during the scale-up, a thick slurry was formed, which was effectively removed from the vessel by re-charging the alcoholic filtrate. Eventually, the isolated product was obtained in the expected yield. Protocol

[0536] [3[(1 R)-3-(3,4-dimethoxyphenyl)-1-hydroxyphenyl]phenoxy]-acetic acid (6, 210 g,

0.60 mol) and (-)-cinchonidine (178 g, 0.60 mol, 1.0 eq.) were dissolved in EtOAc (5.2 L, 25.0 vol.) and heated to 50 °C for 1 hour. The reaction mixture was cooled to 20 °C and agitated for a further 16 hours. The product was isolated by filtration and washed with EtOAc (0.7 L, 3.5 vol.). The product was dried under vacuum at 40 °C for 16 hours to constant mass. The product C6 (383 g, 0.60 mol, 99% yield) was obtained as a white powder. C6 (cinchonidine salt) (383 g, 0.60 mol) was re-dissolved in /PrOH (3.5 L, 9.0 vol. relative to C6) and heated to 50 °C. Once fully dissolved the solution was allowed to cool to 20 °C over 3 hours and stirred for a further 12 hours at 20 °C. The solid was filtered and washed with EtOAc (0.8 L, 2.0 vol.) then dried under vacuum at 40 °C to constant mass for 16 hours. The product C6 (345 g, 0.54 mol, 89% yield over 2 steps) was obtained as a colorless powder.

Analysis

[0537] A small sample was subject to acidification, extraction and isolation to provide an IPC sample prior to acidification of the bulk material. HPLC analysis revealed a 99.8% chiral purity, in line with specification for the GMP starting material that would be used for the manufacture of

Compound A.

[0538] HPLC analysis showed desired (R)-6 (10.67 min) and undesired (S)-6 (9.46 min).

Scheme 7

[0539] An alternative method improves chiral purity of the R form of 6 involved acidification of C6 followed by extraction to provide 6. Post extraction and solvent switch from

dichloromethane (DCM) into isopropanol, the solution was charged into water leading to the immediate precipitation of the product. After stirring overnight, the mobile product slurry was easily filtered and isolated in the expected yield.

Protocol

[0540] [3[(1 R)-3-(3,4-dimethoxyphenyl)-1-hydroxyphenyl]phenoxy]-acetic acid (-)-cinchonidine salt (C6, 6.49 g, 10.12 mmol) was dissolved in DCM (20 mL, 3.0 vol.) and water (26 mL, 4.0 vol.) at 20 °C. 5M HCI aq. (10 mL, 1.6 vol.) was then added and the biphasic mixture stirred for 1 hour at 20 °C. The organic layer was separated and washed with 1 M HCI aq. (2 x 7 mL, 1.0 vol.) and brine (7 mL, 1.0 vol.). The DCM was removed by rotary evaporation and then co distilled with /PrOH (2 x 20 mL, 3.0 vol.) to 7 mL total volume. The thick /PrOH solution was added slowly to a separate vessel containing Dl water (80 mL, 12.0 vol.), seeded with pure 6 and stirred for 16 hours. The product was filtered and washed with Dl water (2 x 13 mL, 2.0 vol.) and then dried under vacuum at 40 °C to constant mass for 16 hours. The product [3[(1 R)-3- (3,4-dimethoxyphenyl)-1-hydroxyphenyl]phenoxy]-acetic acid 6 (2.89 g, 8.36 mmol, 83% yield, 99.5% e.e.) was obtained as a lumpy white powder. [NOTE: The chiral purity is not degraded once 6 has been subject to chiral upgrade.]

Analysis

[0541] HPLC analysis showed desired (R)-6 (11.06 min) and undesired (S)-6 (9.84 min). [0542] Synthetic Route to Common Intermediate 14 (Scheme 8):

[0543] The synthesis of GMP (Good Manufacturing Practice) intermediate 14 involves two chemical transformations and one purification (Scheme 8); Amide coupling between 9 and 11 followed by hydrolysis of the methyl ester provides crude 14. Recrystallization of crude 14 from ethanol yields 14 with chemical purity >99.0% and chiral purity >99.5%.

Scheme 8

[0544] Method:

[0545] (2S)-2-(3,4,5-Trimethoxyphenyl)butanoic acid 9 (93.0 g, 0.37 mol), Methyl (2S)- piperidinecarboxylate hydrochloride 11 (69.0 g, 0.38 mol, 1.1 eq.) and 2-chloro-1- methylpyridinium iodide (1 17.0 g, 0.46 mol, 1.3 eq.) were dissolved in DCM (720 mL, 8.0 vol.) and cooled to 12 °C. To the reaction mixture was added a solution of triethylamine (1 11.0 g, 1.10 mol, 3.0 eq.) in DCM (180 mL, 2.0 vol.) while maintaining the internal temperature below 25 °C. The reaction was then allowed to stir for 3-4 hours at 22 °C. [IPC HPLC (C18: ACN/H2O 10-90%, 20 min, 1 mL/min) 9: rrt 0.53 min, Intermediate ester: rrt 0.69, 14: rrt 0.58] Upon completion, the DCM was removed by rotary evaporation and exchanged for MeOH (225 mL, 2.5 vol.). To the methanolic solution was added a solution of UOH.H2O (77.0 g, 1.83 mol, 5.0 eq.) in Dl water (225 mL, 2.5 vol.) and the reaction was stirred for 12 hours at 22 °C. Upon completion, as determined by IPC, additional water (180 mL, 2.0 vol.) was added and the solution is vacuum distilled to remove methanol. EtOAc (450 mL, 5.0 vol.) was added and the biphasic solution stirred vigorously for 10 minutes after which the phases were separated. The aqueous layer containing the product was removed and the organic phase was washed again with water (180 mL, 2.0 vol.) and 10% NaHC0₃ sol. (360 mL, 4.0 vol.). The combined aqueous layers were cooled to 4 °C. A solution of 5M HCI was slowly added until the pH 4. The resulting slurry was stirred at 4 °C for 3 hours then the solid was collected by vacuum filtration. The crude 14 (1 17.0 g, 0.32 mol) was dissolved in EtOH (1080 mL, 12.0 vol.), heated and maintained at 50 °C for 30 minutes. The ethanolic solution was then cooled to 0 °C over 6 hours. The resulting crystalline solid is collected by vacuum filtration and washed with cold EtOH (90 mL, 1.0 vol.). The product 14 (87.5 g, 0.24 mol, 66% yield) was obtained a colorless crystalline solid.

[0546] Reverse phase HPLC analysis showed crystalline 14 (1 1.64 min) to be excellent purity with no detectable impurities. Chiral HPLC analysis shows crystalline 14 (32.58 min) to be excellent purity with no detectable chiral impurities.

[0547] Synthetic Route to Compound A (Scheme 9):

[0548] The synthesis of Compound A involves two further transformations of the 6 fragment prior to dimerization and coupling with 14 (Scheme 9); Reduction of 6 and tosylation of the resulting alcohol 6-OH followed by dimerization with dimethyl diaminoethylene to provide 13* (where R¹ and R² are methyl, R_L is -CH2-CH2-). Extraction of 13* into the aqueous phase provides a strategy for purification. Finally, amide coupling between 13* and 14 yields

Compound A which is then subject to chromatographic purification.

Scheme 9

[0549] Method:

[0550] A suspension of UAIH₄ (1.41 g, 37.18 mmol, 0.2 vol.) in THF (74 mL, 9.0 vol.) was cooled to 0 °C. To this suspension was added a solution of [3[(1 R)-3-(3,4-dimethoxyphenyl)-1- hydroxyphenyl]phenoxy]-acetic acid (6, 8.05 g, 23.24 mmol) in THF (60 mL, 7.5 vol.) while maintaining the internal temperature below 20 °C. The reaction was stirred for a further 2 hours at room temperature. [IPC TLC (Si0₂: EtOAc/Hexane 50:50) 6: Rf 0.15, 19: Rf 0.26] Upon completion, the reaction mixture was cooled to 0 °C and water (1.5 mL) was added dropwise followed by 15% NaOH aq. (1.5 mL) and water (3 mL) while maintaining the internal temperature below 30 °C. The quenched reaction was then diluted with TBME (100 mL, 12.0 vol.) and stirring continued for 30 minutes. The reaction mixture was dried with MgS0₄, filtered and solvent removed by rotary evaporation. The crude product 3-(3,4-dimethoxyphenyl)-1-[3-(2- hydroxyethoxy)phenyl]propan-1-one 19 (7.10 g, 21 .38 mmol, 92% yield) was obtained as a colorless oil and was used directly in the next step without further purification.

[0551] Reverse phase HPLC analysis showed crude 19 (8.71 min) to be excellent purity with no major impurities. The material is carried through directly into the following step.

[0552] 3-(3,4-dimethoxyphenyl)-1-[3-(2-hydroxyethoxy)phenyl]propan-1-one (19, 68.6 g, 206.5 mmol) was dissolved in DCM (590 mL, 8.5 vol.). Triethylamine (43.2 mL, 309.8 mmol, 1.5 eq.) was added followed by 4-toluenesulfonyl chloride (39.4 g, 206.5 mmol, 1.0 eq.). The reaction mixture was stirred at room temperature for 18 hours. [IPC TLC (Si0₂: EtOAc/Hexane 50:50) 19: Rf 0.26, 20: Rf 0.52, TsCI: Rf 0.76] Upon completion, the DCM was removed by rotary evaporation and exchanged for EtOAc (500 mL, 7.1 vol.). The organic layer was washed with HCI aq. 1 M (3 x 100 mL, 1.4 vol.), NaHC0₃ sat. aq. (2 x 10 mL, 1.4 vol.), brine (70 mL, 1.0 vol.), dried over MgS0₄ then filtered and solvent removed by rotary evaporation. The crude residue was purified by Biotage (340 g SNAP Ultra column; 30-90% EtOAc in Hexane) to provide 2-{3- [3-(3,4-dimethoxyphenyl)propanoyl]phenoxy}ethyl 4-methylbenzenesulfonate, 20 (63.6 g,

130.7 mmol, 63% yield) as a colorless oil. This material was used directly in the following step.

[0553] Reverse phase HPLC analysis showed crude 20 (24.36 min) to be 88% purity with several small impurities present even after chromatographic purification.

[0554] 2-{3-[3-(3,4-dimethoxyphenyl)propanoyl]phenoxy}ethyl 4-methylbenzenesulfonate 20 (3.93 g, 8.08 mmol) and L/,L/'-dimethyl diaminoethylene (0.36 g, 4.04 mmol, 0.5 eq.) were dissolved in MeCN (20 mL, 5.0 vol.). To this solution were added K₂C0₃ (2.23 g, 16.16 mmol,

2.0 eq.) and Kl (0.67 g, 4.04 mmol, 0.5 eq.). The reaction mixture was heated to 70 °C and stirred for 16 hours. [IPC TLC (Si0₂: EtOAc/Hexane 50:50) 20: Rf 0.26, 13* Rf baseline] 13* refers to compound 13 of method 3, where R_L is -CH₂-CH₂-. Upon completion, MeCN was removed by rotary evaporation and exchanged for EtOAc (50 mL, 12.0 vol.). The organic layer was washed with water (2 x 20 mL, 5.0 vol.) and NaHC0₃ sat. aq. (2 x 20 mL, 5.0 vol.). The desired product was then extracted into the aqueous layer with HC1 1 M aq. (2 x 30 mL, 7.5 vol.) and the aqueous phase was washed with EtOAc (2 x 20 mL, 5.0 vol.). The aqueous phase was then basified with NaHC0₃ sat. aq. (90 mL, 22.5 vol.) and extracted with EtOAc (3 x 30 mL, 7.5 vol.). The organic phase was then washed with brine (20 mL, 5.0 vol.), dried over MgS0₄, filtered and solvent removed by rotary evaporation to provide 13* (2.75 g, 3.83 mmol, 95% yield). The crude material was used directly in the next step without further purification.

[0555] Reverse phase HPLC analysis shows crude 13* (15.62 min) to be 79% purity with a lot of minor impurities and two significant impurities around 5% (9.72 min and 20.16 min). This crude material was used directly in the next step.

[0556] 13* (2.47 g, 3.45 mmol) and 14 (2.77 g, 7.59 mmol, 2.2 eq.) were dissolved in DCM (35 mL, 14.0 vol.) and the solution cooled to -15 °C. To the reaction mixture was added EDCI (1.59 g, 8.28 mmol, 2.4 eq.) followed by DMAP (1.86 g, 15.18 mmol, 4.4 eq.). The reaction was stirred overnight at -15 °C. After 24 hours the internal temperature of the reaction was increased to -4 °C and the reaction left to stir for a further 24 hours. [IPC TLC (Si0₂: MeOH/DCM 5:95) 13*: Rf 0.15, 14: Rf 0.37, Compound A: Rf 0.66] Compound A may also be referred to as 15*, which indicates 15 as provided in Method 3, where R¹ and R² are methyl, R_L is -CH2-CH2-. Upon completion, the reaction was quenched by addition of 5M HCI sol. (10 mL, 4.0 vol.) to the reaction mixture at -4 °C. DCM was then removed by vacuum distillation while maintaining the internal temperature below 20 °C. The residue was diluted with EtOAc (50 mL, 20.0 vol.) and the organic layer washed sequentially with 1 M HCI sol. (2 x 10 mL, 4.0 vol.), NaHC0₃ sol. (10 mL, 4.0 vol.) and brine (10 mL, 4.0 vol.). The organic layer was dried with MgSC , filtered and solvent removed by rotary evaporation. The crude residue was purified by Biotage (50 g SNAP Ultra, 0-10% MeOH in DCM) to provide Compound A (2.09 g, 1.48 mmol, 43% yield) as an amorphous white solid. DCC denotes L/,L/'-Dicyclohexylcarbodiimide.

[0557] Reverse phase HPLC analysis shows Compound A (29.06 min) after one chromatographic purification. The major impurity (28.02 min) making up 8% corresponds to the mono-coupled intermediate. Further HPLC and LCMS method development will be needed to establish the diastereomeric purity.

[0558] Example 27: Examples of Particular Nucleic Acid and Amino Acid Sequences

Table 8: Nucleic Acid Sequences

Table 9: Amino Acid Sequences

[0559] Example 28: Representative Embodiments [0560] A1. A compound having a structure of the following Formula A:

Formula A or a pharmaceutically acceptable salt thereof, wherein:

R²⁰ is hydrogen, -R²³ or -R^F-R²³;

R²¹ is hydrogen, hydroxy, -R^G-R²⁴ -R^G-R²⁴-R²⁵ or -R^G-R²⁴-R²⁶-R²⁵;

R²² is halogen, -NR²⁷R²⁸, -R^H-R²⁹-R³⁰ or -R^H-R²⁹-R³¹-R³⁰;

R^F, R^G and R^H each independently is -0-, -C(O)-, -O-C(O)-, -C(0)-0-, -S-, -S(0)_n-, -O- S(0)_n-, -S(0)_n-0-, -NH-S(0)_n-, -S(O) _n-NH-, -NH-C(O)-, -C(0)-NH-, -NH-C(S)-, -C(S)- NH-, -S-C(S)-, -C(S)-S-, or -C(S)-; n is 1 or 2;

R²³ independently is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, aryl, heterocycloalkyl or heteroaryl, which independently is optionally substituted by one or more substituents chosen from halogen, hydroxy, alkyl, alkenyl, alkynyl, heteroalkyl, haloalkyl, perhaloalkyl, perhaloalkoxy, alkoxy, haloalkoxy, alkoxyalkyl, acyl, oxo, acyloxy, carboxyl, amido, cyano, amino, alkylamino, alkylaminoalkyl, thiol, alkylthio, alkylthioalkyl, haloalkylthio, perhaloalkylthio, nitro, aryl, arylalkyl, cycloalkylalkyl, heterocycle-alkyl, cycloalkyl, heterocycloalkyl, heteroaryl, heteroarylalkyl, alkylsulfonyl, sulfonamide, alkylsulfonamido, alkylsilyloxy, alkylsulfinamido, arylsulfinamido, and arylsulfonamido;

R²⁷ and R²⁸ each independently is -R³²-R³³, or hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, perhaloalkyl, alkoxy, cycloalkyl, aryl, heterocycloalkyl, heterocycle-alkyl or heteroaryl, which independently is optionally substituted with one or more substituents chosen from halogen, hydroxy, alkyl, alkenyl, alkynyl, heteroalkyl, haloalkyl, perhaloalkyl, perhaloalkoxy, alkoxy, haloalkoxy, alkoxyalkyl, acyl, oxo, acyloxy, carboxyl, amido, cyano, amino, alkylamino, alkylaminoalkyl, thiol, alkylthio, alkylthioalkyl, haloalkylthio, perhaloalkylthio, nitro, aryl, arylalkyl, cycloalkylalkyl, heterocycle-alkyl, cycloalkyl, heterocycloalkyl, heteroaryl, heteroarylalkyl, alkylsulfonyl, sulfonamide, alkylsulfonamido, alkylsilyloxy, alkylsulfinamido, arylsulfinamido and arylsulfonamido, with the proviso that R²⁷ and R²⁸ are not both hydrogen when -R^F is -O- and R²³ is alkyl.

[0561] A1.1. The compound of embodiment A1 , wherein R²² is halogen, -NR²⁷R²⁸ or -R^H-

R²⁹_ R31— R30

[0562] A2. A compound having a structure of the following Formula B:

Formula B or a pharmaceutically acceptable salt thereof, wherein:

R^21A is hydrogen and R^{21 B} is hydroxy, -R^G-R²⁴ -R^G-R²⁴-R²⁵ or -R^G-R²⁴-R²⁶-R²⁵, or R^{21 B} is hydrogen and R^21A is hydroxy, -R^G-R²⁴ _, -R^G-R²⁴-R²⁵ or -R^G-R²⁴-R²⁶-R²⁵;

R²⁷ and R²⁸ each independently is -R³²-R³³, or hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, perhaloalkyl, alkoxy, cycloalkyl, aryl, heterocycloalkyl, heterocycle-alkyl or heteroaryl, which independently is optionally substituted with one or more substituents chosen from halogen, hydroxy, alkyl, alkenyl, alkynyl, heteroalkyl, haloalkyl, perhaloalkyl, perhaloalkoxy, alkoxy, haloalkoxy, alkoxyalkyl, acyl, oxo, acyloxy, carboxyl, amido, cyano, amino, alkylamino, alkylaminoalkyl, thiol, alkylthio, alkylthioalkyl, haloalkylthio, perhaloalkylthio, nitro, aryl, arylalkyl, cycloalkylalkyl, heterocycle-alkyl, cycloalkyl, heterocycloalkyl, heteroaryl, heteroarylalkyl, alkylsulfonyl, sulfonamide, alkylsulfonamido, alkylsilyloxy, alkylsulfinamido, arylsulfinamido, and arylsulfonamido, with the proviso that R²⁷ and R²⁸ are not both hydrogen when -R^F is -O- and R²³ is alkyl.

[0563] A2.1. The compound of embodiment A2, wherein R^22A is hydrogen and R^22B is halogen, -NR²⁷R²⁸ or -R^H-R²⁹-R³¹-R³⁰, or R^22B is hydrogen and R^22A is halogen, -NR²⁷R²⁸ or -R^H-R²⁹-

R³¹ R³⁰.

[0564] A2.2. The compound of embodiment A1 , A1 .1 , A2 or A2.1 , wherein R^F, R^G and R^H each independently is - 0-, -C(O)-, -O-C(O)- or -C(0)-0-

[0565] A2.3. The compound of embodiment A1 , wherein R²² is halogen, -R^H-R²⁹-R³⁰ or -R^H- R²⁹-R³ⁱ_R³o and wherein if R²² is -R^H-R²⁹-R³⁰, R²⁹ is substituted with halogen and/or R³⁰ is halogen, or if R²² is -R^H-R²⁹-R³¹-R³⁰, R²⁹ is substituted with halogen and/or R³⁰ is halogen and/or R³¹ is substituted with halogen.

[0566] A2.4. The compound of embodiment A2.3, wherein R²⁰ is hydrogen or -R^F-R²³.

[0567] A2.5. The compound of embodiment A2.4, wherein R²⁰ is -OCH₃.

[0568] A2.6. The compound of embodiment A2, wherein R^22A is hydrogen and R^22B is halogen, -R^H-R²⁹-R³⁰ or -R^H-R²⁹-R³¹-R³⁰ and wherein if R^22B is -R^H-R²⁹-R³⁰, R²⁹ is substituted with halogen and/or R³⁰ is halogen, or if R^22B is -R^H-R²⁹-R³¹-R³⁰, R²⁹ is substituted with halogen and/or R³⁰ is halogen and/or R³¹ is substituted with halogen.

[0569] A2.7. The compound of embodiment A2, wherein R^22B is hydrogen and R^22A is halogen, -R^H-R²⁹-R³⁰ or -R^H-R²⁹-R³¹-R³⁰ and wherein if R^22A is -R^H-R²⁹-R³⁰, R²⁹ is substituted with halogen and/or R³⁰ is halogen, or if R^22A is -R^H-R²⁹-R³¹-R³⁰, R²⁹ is substituted with halogen and/or R³⁰ is halogen and/or R³¹ is substituted with halogen.

[0570] A2.8. The compound of embodiment A2.6 or A2.7, wherein R^20A is hydrogen and R^20B is hydrogen or -R^F-R²³ or wherein R^20B is hydrogen and R^20A is hydrogen or -R^F-R²³. [0571] A2.9. The compound of embodiment A2.8, wherein R^20A is hydrogen and R^20B is - OCH3 or wherein R^20B is hydrogen and R^20A is -OCH3.

[0572] A2.10. The compound of embodiment A1 , wherein R²² is NR²⁷R²⁸ and R²⁸ is or is substituted with heterocycloalkyl or is substituted with heterocycle-alkyl.

[0573] A2.11. The compound of embodiment A1 , wherein R²² is -R^H-R²⁹-R³⁰ or -R^H-R²⁹- R³¹-R³⁰ and R²⁹ and/or R³⁰ is or is substituted with heterocycloalkyl or is substituted with heterocycle-alkyl.

[0574] A2.12. The compound of embodiment A2, wherein R^22A is hydrogen, R^22B is NR²⁷R²⁸ and R²⁸ is or is substituted with heterocycloalkyl or is substituted with heterocycle-alkyl.

[0575] A2.13. The compound of embodiment A2, wherein R^22B is hydrogen, R^22A is NR²⁷R²⁸ and R²⁸ is or is substituted with heterocycloalkyl or is substituted with heterocycle-alkyl.

[0576] A2.14. The compound of embodiment A2, wherein R^22A is hydrogen, R^22B is -R^H-R²⁹- R³⁰ or -R^H-R²⁹-R³¹-R³⁰ and R²⁹ and/or R³⁰ is or is substituted with heterocycloalkyl or is substituted with heterocycle-alkyl.

[0577] A2.15. The compound of embodiment A2, wherein R^22B is hydrogen, R^22A is -R^H-R²⁹- R³⁰ or -R^H-R²⁹-R³¹-R³⁰ and R²⁹ and/or R³⁰ is or is substituted with heterocycloalkyl or is substituted with heterocycle-alkyl.

[0578] A2.16. The compound of any one of embodiments A2.10 - A2.15, wherein the heterocycloalkyl or heterocycle-alkyl comprises one or more nitrogen atoms.

[0579] A3. The compound of embodiment A2, wherein R^22A is hydrogen and R^22B is halogen, NR²⁷R²⁸, -R^H-R²⁹-R³⁰ or -R^H-R²⁹-R³¹-R³⁰.

[0580] A3.1 The compound of embodiment A2.1 , wherein R^22A is hydrogen and R^22B is halogen, NR²⁷R²⁸, or -R^H-R²⁹-R³¹-R³⁰.

[0581] A4. The compound of any one of embodiments A1 , A2 and A3, wherein R²², R^22A or R^22B is - R^H-R²⁹-R³¹-R³⁰. [0582] A5. The compound of any one of embodiments A1-A4, wherein R^H is -O-C(O)-.

[0583] A5.1. The compound of any one of embodiments A1-A2.9, A2.11 andA2.14-A5, wherein R²⁹ is C5-C7 cycloalkyl, 5-7 membered heterocycloalkyl, C5-C7 aryl or 5-7 membered heteroaryl, which independently is optionally substituted by one or more substituents chosen from halogen, hydroxy, C1-C3 alkyl, C1-C3 heteroalkyl, C1-C3 haloalkyl, C1-C3 perhaloalkyl, C1-C3 perhaloalkoxy, C1-C3 alkoxy, C1-C3 haloalkoxy, C1-C3 alkoxy C1-C3 alkyl, C1-C4 acyl, oxo, C1-C4 acyloxy, carboxyl, amido, cyano, amino, C1-C3 alkylamino, C1-C3 alkylamino C1- C3 alkyl, thiol, C1-C3 alkylthio, C1-C3 alkylthio C1-C3 alkyl, C1-C3 haloalkylthio, C1-C3 perhaloalkylthio, nitro, C5-C7 aryl, C5-C7 aryl C1-C3 alkyl, C5-C7 cycloalkyl, C5-C7 cycloalkyl C1-C3 alkyl, 5-7 membered heterocycloalkyl, 5-7 membered heterocycle C1-C3 alkyl, 5-7 membered heteroaryl, 5-7 membered heteroaryl C1-C3 alkyl, C1-C3 alkylsulfonyl, sulfonamide, C1-C3 alkylsulfonamido and C1-C3 alkylsilyloxy.

[0584] A6. The compound of embodiment A4, A5 or A5.1 , wherein R²⁹ is a C5-C7 aryl or a 5- 7 membered heteroaryl.

[0585] A7. The compound of embodiment A6, wherein R²⁹ has a structure of Formula C-1 :

Formula C-1

wherein:

zero, one, two or three of X⁴⁰, X⁴¹, X⁴², X⁴³, X⁴⁴ and X⁴⁵ are oxygen, nitrogen or sulfur heteroatoms, and the remaining X⁴⁰, X⁴¹, X⁴², X⁴³, X⁴⁴ and X⁴⁵ are carbon; and

C1-C3 perhaloalkylthio, nitro, C5-C7 aryl, C5-C7 aryl C1-C3 alkyl, C5-C7 cycloalkyl, C5-C7 cycloalkyl C1-C3 alkyl, 5-7 membered heterocycloalkyl, 5-7 membered heterocycle C1-C3 alkyl, 5-7 membered heteroaryl, 5-7 membered heteroaryl C1-C3 alkyl, C1-C3 alkylsulfonyl, sulfonamide, C1-C3 alkylsulfonamido or C1-C3 alkylsilyloxy.

[0586] A8. The compound of embodiment A7, wherein R²⁹ has a structure of Formula D-1 :

Formula D-1 wherein one, two or three of R⁴¹, R⁴², R⁴³, R⁴⁴ and R⁴⁵ independently is R³¹-R³⁰, and each of the remaining R⁴¹, R⁴², R⁴³, R⁴⁴ and R⁴⁵ independently is hydrogen, halogen, hydroxy, C1-C3 alkyl, C1-C3 heteroalkyl, C1-C3 haloalkyl, C1-C3 perhaloalkyl, C1-C3 perhaloalkoxy, C1-C3 alkoxy, C1-C3 haloalkoxy, C1-C3 alkoxy C1-C3 alkyl, C1-C4 acyl, oxo, C1-C4 acyloxy, carboxyl, amido, cyano, amino, C1-C3 alkylamino, C1-C3 alkylamino C1-C3 alkyl, thiol, C1-C3 alkylthio, C1-C3 alkylthio C1-C3 alkyl, C1-C3 haloalkylthio, C1-C3 perhaloalkylthio, nitro, C5-C7 aryl, C5-C7 aryl C1-C3 alkyl, C5-C7 cycloalkyl, C5-C7 cycloalkyl C1-C3 alkyl, 5-7 membered heterocycloalkyl, 5-7 membered heterocycle C1-C3 alkyl, 5-7 membered heteroaryl, 5-7 membered heteroaryl C1-C3 alkyl, C1-C3 alkylsulfonyl, sulfonamide, C1-C3 alkylsulfonamido or C1-C3 alkylsilyloxy.

[0587] A9. The compound of embodiment A7, wherein R²⁹ has a structure of Formula E-1 :

Formula E-1 wherein R⁴³ is -R³¹-R³⁰.

[0588] A10. The compound of any one of embodiments A1-A9, wherein R³¹ is a C1-C3 alkyl linker. [0589] A1 1. The compound of embodiment A10, wherein R³¹ is a methylene linker or ethylene linker.

[0590] A12. The compound of any one of embodiments A1-A1 1 , wherein R³⁰ is a C5-C7 cycloalkyl, 5-7 membered heterocycloalkyl, C5-C7 aryl or 5-7 membered heteroaryl.

[0591] A12.1. The compound of any one of embodiments A1-A12, wherein R³⁰ is a 5-7 membered heterocycloalkyl or a 5-7 membered heteroaryl.

[0592] A12.2. The compound of any one of embodiments A1-A12.1 , wherein R³⁰ is a 5-7 membered heterocycloalkyl.

[0593] A13. The compound of embodiment A12.2, wherein R³⁰ has a structure of Formula F- 1 :

Formula F-1 wherein:

X⁶⁰, X⁶¹, X⁶², X⁶³, X⁶⁴ and X⁶⁵ together form a heterocycloalkyl ring;

one, two or three of X⁶⁰, X⁶¹ , X⁶², X⁶³, X⁶⁴ and X⁶⁵ are oxygen, nitrogen or sulfur heteroatoms, and the remaining X⁶⁰, X⁶¹, X⁶², X⁶³, X⁶⁴ and X⁶⁵ are carbon; and

R⁶¹, R⁶², R⁶³, R⁶⁴ and R⁶⁵ each represent zero, one or two substituents and each of which substituents independently is hydrogen, halogen, hydroxy, C1-C3 alkyl, C1-C3 heteroalkyl, C1- C3 haloalkyl, C1-C3 perhaloalkyl, C1-C3 perhaloalkoxy, C1-C3 alkoxy, C1-C3 haloalkoxy, C1- C3 alkoxy C1-C3 alkyl, C1-C4 acyl, oxo, C1-C4 acyloxy, carboxyl, amido, cyano, amino, C1-C3 alkylamino, C1-C3 alkylamino C1-C3 alkyl, thiol, C1-C3 alkylthio, C1-C3 alkylthio C1-C3 alkyl, C1-C3 haloalkylthio, C1-C3 perhaloalkylthio, nitro, C5-C7 aryl, C5-C7 aryl C1-C3 alkyl, C5-C7 cycloalkyl, C5-C7 cycloalkyl C1-C3 alkyl, 5-7 membered heterocycloalkyl, 5-7 membered heterocycle C1-C3 alkyl, 5-7 membered heteroaryl, 5-7 membered heteroaryl C1-C3 alkyl, C1- C3 alkylsulfonyl, sulfonamide, C1-C3 alkylsulfonamido or C1-C3 alkylsilyloxy.

[0594] A14. The compound of embodiment A13, wherein R³⁰ has a structure of Formula G-1 :

Formula G-1 wherein:

R⁶¹, R⁶², R⁶⁴ and R⁶⁵ each represent two substituents, each of which substituents

independently is hydrogen, halogen, hydroxy, C1-C3 alkyl, C1-C3 heteroalkyl, C1-C3 haloalkyl, C1-C3 perhaloalkyl, C1-C3 perhaloalkoxy, C1-C3 alkoxy, C1-C3 haloalkoxy, C1-C3 alkoxy C1- C3 alkyl, C1-C4 acyl, oxo, C1-C4 acyloxy, carboxyl, amido, cyano, amino, C1-C3 alkylamino, C1-C3 alkylamino C1-C3 alkyl, thiol, C1 -C3 alkylthio, C1-C3 alkylthio C1-C3 alkyl, C1-C3 haloalkylthio, C1-C3 perhaloalkylthio, nitro, C5-C7 aryl, C5-C7 aryl C1-C3 alkyl, C5-C7 cycloalkyl, C5-C7 cycloalkyl C1-C3 alkyl, 5-7 membered heterocycloalkyl, 5-7 membered heterocycle C1-C3 alkyl, 5-7 membered heteroaryl, 5-7 membered heteroaryl C1-C3 alkyl, C1- C3 alkylsulfonyl, sulfonamide, C1-C3 alkylsulfonamido or C1-C3 alkylsilyloxy; and

R⁶³ is hydrogen, C1-C3 alkyl, C1-C3 heteroalkyl, C1-C3 haloalkyl, C1-C3 perhaloalkyl, C1-C4 acyl, amido, C1-C3 alkylamino, C1-C3 alkylamino C1-C3 alkyl, amino, thiol, C1-C3 alkylthio, C1-C3 alkylthio C1-C3 alkyl, C1-C3 haloalkylthio, C1-C3 perhaloalkylthio, C5-C7 aryl, C5-C7 aryl C1-C3 alkyl, C5-C7 cycloalkyl, C5-C7 cycloalkyl C1-C3 alkyl, 5-7 membered

heterocycloalkyl, 5-7 membered heterocycle C1-C3 alkyl, 5-7 membered heteroaryl, 5-7 membered heteroaryl C1-C3 alkyl, C1-C3 alkylsulfonyl, sulfonamide, C1-C3 alkylsulfonamido or C1-C3 alkylsilyloxy.

[0595] A14.1. The compound of embodiment A13, wherein R³⁰ has a structure of Formula H- 1 :

Formula H-1 wherein: R⁶¹, R⁶³, R⁶⁴ and R⁶⁵ each represent two substituents, each of which substituents

R⁶² is hydrogen, C1-C3 alkyl, C1-C3 heteroalkyl, C1-C3 haloalkyl, C1-C3 perhaloalkyl, C1-C4 acyl, amido, C1-C3 alkylamino, C1-C3 alkylamino C1-C3 alkyl, amino, thiol, C1-C3 alkylthio, C1-C3 alkylthio C1-C3 alkyl, C1-C3 haloalkylthio, C1-C3 perhaloalkylthio, C5-C7 aryl, C5-C7 aryl C1-C3 alkyl, C5-C7 cycloalkyl, C5-C7 cycloalkyl C1-C3 alkyl, 5-7 membered

[0596] A15. The compound of embodiment A14, wherein R³⁰ has a structure of Formula J-1 :

Formula J-1 wherein R⁶³ is hydrogen, C1-C3 alkyl, C1-C3 heteroalkyl, C1-C3 haloalkyl or C1-C3

perhaloalkyl.

[0597] A16. The compound of any one of embodiments A1 , A2 and A3, wherein R²², R^22A or R^22B is -NR²⁷R²⁸.

[0598] A17. The compound of embodiment A16, wherein R²⁷ or R²⁸ is hydrogen.

[0599] A17.1. The compound of embodiment A16, wherein R²⁷ is hydrogen and R²⁸ is C1 -C3 alkyl, C1-C3 heteroalkyl, C1-C3 haloalkyl, C1-C3 perhaloalkyl, C1-C4 acyl, amido, C1-C3 alkylamino, C1-C3 alkylamino C1-C3 alkyl, amino, thiol, C1-C3 alkylthio, C1-C3 alkylthio C1-C3 alkyl, C1-C3 haloalkylthio, C1-C3 perhaloalkylthio, C5-C7 aryl, C5-C7 aryl C1-C3 alkyl, C5-C7 cycloalkyl, C5-C7 cycloalkyl C1-C3 alkyl, 5-7 membered heterocycloalkyl, 5-7 membered heterocycle C1-C3 alkyl, 5-7 membered heteroaryl, 5-7 membered heteroaryl C1-C3 alkyl, C1- C3 alkylsulfonyl, sulfonamide, C1-C3 alkylsulfonamido or C1-C3 alkylsilyloxy.

[0600] A18. The compound of embodiment A16, wherein R²⁷ is hydrogen and R²⁸ is -R³²-R³³.

[0601] A19. The compound of embodiment A18, wherein R³² is a C1-C4 alkyl linker.

[0602] A20. The compound of embodiment A18 or A19, wherein R³² is an unsubstituted C3 alkyl linker.

[0603] A21. The compound of any one of embodiments A18-A20, wherein R³³ is a C5-C7 cycloalkyl or a 5-7 membered heterocycloalkyl.

[0604] A21.1. The compound of any one of embodiments A18-A20, wherein R³³ is a 5-7 membered heteroaryl or a 5-7 membered heterocycloalkyl.

[0605] A22. The compound of embodiment A21 or A21.1 , wherein R³³ is a 5-7 membered heterocycloalkyl.

[0606] A23. The compound of embodiment A22, wherein R³³ has a structure of Formula F-2:

Formula F-2 wherein:

R⁶¹, R⁶², R⁶³, R⁶⁴ and R⁶⁵ each represent zero, one or two substituents, each of which substituents independently is hydrogen, halogen, hydroxy, C1-C3 alkyl, C1-C3 heteroalkyl, C1- C3 haloalkyl, C1-C3 perhaloalkyl, C1-C3 perhaloalkoxy, C1-C3 alkoxy, C1-C3 haloalkoxy, C1- C3 alkoxy C1-C3 alkyl, C1-C4 acyl, oxo, C1-C4 acyloxy, carboxyl, amido, cyano, amino, C1-C3 alkylamino, C1-C3 alkylamino C1-C3 alkyl, thiol, C1-C3 alkylthio, C1-C3 alkylthio C1-C3 alkyl, C1-C3 haloalkylthio, C1-C3 perhaloalkylthio, nitro, C5-C7 aryl, C5-C7 aryl C1-C3 alkyl, C5-C7 cycloalkyl, C5-C7 cycloalkyl C1-C3 alkyl, 5-7 membered heterocycloalkyl, 5-7 membered heterocycle C1-C3 alkyl, 5-7 membered heteroaryl, 5-7 membered heteroaryl C1-C3 alkyl, C1- C3 alkylsulfonyl, sulfonamide, C1-C3 alkylsulfonamido or C1-C3 alkylsilyloxy.

[0607] A24. The compound of embodiment A23, wherein R³³ has a structure of Formula G-2:

Formula G-2 wherein:

R⁶¹, R⁶², R⁶⁴ and R⁶⁵ each represent two substituents, each of whichsubstituents independently is hydrogen, halogen, hydroxy, C1-C3 alkyl, C1-C3 heteroalkyl, C1-C3 haloalkyl, C1-C3 perhaloalkyl, C1-C3 perhaloalkoxy, C1-C3 alkoxy, C1-C3 haloalkoxy, C1-C3 alkoxy C1-C3 alkyl, C1-C4 acyl, oxo, C1-C4 acyloxy, carboxyl, amido, cyano, amino, C1-C3 alkylamino, C1-C3 alkylamino C1-C3 alkyl, thiol, C1-C3 alkylthio, C1-C3 alkylthio C1-C3 alkyl, C1-C3 haloalkylthio, C1-C3 perhaloalkylthio, nitro, C5-C7 aryl, C5-C7 aryl C1-C3 alkyl, C5-C7 cycloalkyl, C5-C7 cycloalkyl C1-C3 alkyl, 5-7 membered heterocycloalkyl, 5-7 membered heterocycle C1-C3 alkyl, 5-7 membered heteroaryl, 5-7 membered heteroaryl C1 -C3 alkyl, C1-C3 alkylsulfonyl, sulfonamide, C1-C3 alkylsulfonamido or C1-C3 alkylsilyloxy; and

[0608] A24.1. The compound of embodiment A23, wherein R³³ has a structure of Formula H- 2:

Formula H-2 wherein:

R⁶¹, R⁶³, R⁶⁴ and R⁶⁵ each represent two substituents, each of which substituents

[0609] A25. The compound of embodiment A24, wherein R³³ has a structure of Formula J-2:

Formula J-2 wherein R⁶³ is hydrogen, C1-C3 alkyl, C1-C3 heteroalkyl, C1-C3 haloalkyl or C1-C3

perhaloalkyl. [0610] A26. The compound of any one of embodiments A1 -A25, wherein R²⁴ is a C5-C7 cycloalkyl, 5-7 membered heterocycloalkyl, C5-C7 aryl or 5-7 membered heteroaryl, which independently is optionally substituted by one or more substituents chosen from halogen, hydroxy, C1 -C3 alkyl, C1-C3 heteroalkyl, C1 -C3 haloalkyl, C1 -C3 perhaloalkyl, C1 -C3 perhaloalkoxy, C1-C3 alkoxy, C1 -C3 haloalkoxy, C1 -C3 alkoxy C1-C3 alkyl, C1 -C4 acyl, oxo, C1 -C4 acyloxy, carboxyl, amido, cyano, amino, C1-C3 alkylamino, C1-C3 alkylamino C1 -C3 alkyl, thiol, C1 -C3 alkylthio, C1 -C3 alkylthio C1 -C3 alkyl, C1 -C3 haloalkylthio, C1 -C3 perhaloalkylthio, nitro, C5-C7 aryl, C5-C7 aryl C1 -C3 alkyl, C5-C7 cycloalkyl, C5-C7 cycloalkyl C1 -C3 alkyl, 5-7 membered heterocycloalkyl, 5-7 membered heterocycle C1 -C3 alkyl, 5-7 membered heteroaryl, 5-7 membered heteroaryl C1 -C3 alkyl, C1 -C3 alkylsulfonyl, sulfonamide, C1 -C3 alkylsulfonamido and C1 -C3 alkylsilyloxy.

[0611] A26.1 . The compound of any one of embodiments A2-A26, wherein R^{21 B} is hydrogen and R^21A is hydroxy, -R^G-R²⁴, - R^G-R²⁴-R²⁵ or - R^G-R²⁴-R²⁶-R²⁵.

[0612] A27. The compound of any one of embodiments A1 -A26, wherein R²¹ , R^21A or R^{21 B} is - R^G— R²⁴— R²⁶— R²⁵.

[0613] A28. The compound of any one of embodiments A1 -A27, wherein R^G is -O-C(O)-.

[0614] A29. The compound of embodiment A27 or A28, wherein R²⁴ is a C5-C7 aryl or a 5-7 membered heteroaryl.

[0615] A30. The compound of embodiment A29, wherein R²⁴ has a structure of Formula C-2:

Formula C-2

wherein:

one, two or three of R⁴¹ , R⁴², R⁴³, R⁴⁴ and R⁴⁵ independently is R²⁶-R²⁵, and each of the remaining R⁴¹, R⁴², R⁴³, R⁴⁴ and R⁴⁵ independently is not present or independently is hydrogen, halogen, hydroxy, C1 -C3 alkyl, C1 -C3 heteroalkyl, C1 -C3 haloalkyl, C1 -C3 perhaloalkyl, C1-C3 perhaloalkoxy, C1-C3 alkoxy, C1-C3 haloalkoxy, C1-C3 alkoxy C1-C3 alkyl, C1-C4 acyl, oxo, C1-C4 acyloxy, carboxyl, amido, cyano, amino, C1-C3 alkylamino, C1-C3 alkylamino C1-C3 alkyl, thiol, C1-C3 alkylthio, C1-C3 alkylthio C1-C3 alkyl, C1-C3 haloalkylthio, C1-C3 perhaloalkylthio, nitro, C5-C7 aryl, C5-C7 aryl C1-C3 alkyl, C5-C7 cycloalkyl, C5-C7 cycloalkyl C1-C3 alkyl, 5-7 membered heterocycloalkyl, 5-7 membered heterocycle C1-C3 alkyl, 5-7 membered heteroaryl, 5-7 membered heteroaryl C1 -C3 alkyl, C1-C3 alkylsulfonyl, sulfonamide, C1-C3 alkylsulfonamido or C1-C3 alkylsilyloxy.

[0616] A31. The compound of embodiment A30, wherein R²⁴ has a structure of Formula D-2:

Formula D-2 wherein one, two or three of R⁴¹ , R⁴², R⁴³, R⁴⁴ and R⁴⁵ independently is R²⁶-R²⁵, and each of the remaining R⁴¹ , R⁴², R⁴³, R⁴⁴ and R⁴⁵ independently is hydrogen, halogen, hydroxy, C1-C3 alkyl, C1-C3 heteroalkyl, C1-C3 haloalkyl, C1-C3 perhaloalkyl, C1-C3 perhaloalkoxy, C1-C3 alkoxy, C1-C3 haloalkoxy, C1-C3 alkoxy C1-C3 alkyl, C1-C4 acyl, oxo, C1-C4 acyloxy, carboxyl, amido, cyano, amino, C1-C3 alkylamino, C1-C3 alkylamino C1-C3 alkyl, thiol, C1-C3 alkylthio, C1-C3 alkylthio C1-C3 alkyl, C1-C3 haloalkylthio, C1-C3 perhaloalkylthio, nitro, C5-C7 aryl, C5-C7 aryl C1-C3 alkyl, C5-C7 cycloalkyl, C5-C7 cycloalkyl C1-C3 alkyl, 5-7 membered heterocycloalkyl, 5-7 membered heterocycle C1-C3 alkyl, 5-7 membered heteroaryl, 5-7 membered heteroaryl C1-C3 alkyl, C1-C3 alkylsulfonyl, sulfonamide, C1-C3 alkylsulfonamido or C1-C3 alkylsilyloxy.

[0617] A32. The compound of embodiment A31 , wherein R²⁴ has a structure of Formula E-2:

Formula E-2 wherein R⁴³ is -R²⁶-R²⁵.

[0618] A33. The compound of any one of embodiments A1 -A32, wherein R²⁶ is a C1-C3 alkyl linker.

[0619] A34. The compound of embodiment A33, wherein R²⁶ is a methylene linker.

[0620] A35. The compound of any one of embodiments A1 -A34, wherein R²⁵ is a 5-7 membered heterocycloalkyl.

[0621] A36. The compound of embodiment A35, wherein R²⁵ has a structure of Formula F-3:

Formula F-3 wherein:

R⁶¹ , R⁶², R⁶³, R⁶⁴ and R⁶⁵ each represent zero, one or two substituents, each of which substituents independently is hydrogen, halogen, hydroxy, C1 -C3 alkyl, C1 -C3 heteroalkyl, C1 - C3 haloalkyl, C1 -C3 perhaloalkyl, C1 -C3 perhaloalkoxy, C1 -C3 alkoxy, C1 -C3 haloalkoxy, C1 - C3 alkoxy C1 -C3 alkyl, C1 -C4 acyl, oxo, C1 -C4 acyloxy, carboxyl, amido, cyano, amino, C1 -C3 alkylamino, C1-C3 alkylamino C1 -C3 alkyl, thiol, C1 -C3 alkylthio, C1 -C3 alkylthio C1 -C3 alkyl, C1 -C3 haloalkylthio, C1 -C3 perhaloalkylthio, nitro, C5-C7 aryl, C5-C7 aryl C1 -C3 alkyl, C5-C7 cycloalkyl, C5-C7 cycloalkyl C1 -C3 alkyl, 5-7 membered heterocycloalkyl, 5-7 membered heterocycle C1 -C3 alkyl, 5-7 membered heteroaryl, 5-7 membered heteroaryl C1 -C3 alkyl, C1 - C3 alkylsulfonyl, sulfonamide, C1 -C3 alkylsulfonamido or C1 -C3 alkylsilyloxy.

[0622] A37. The compound of embodiment A36, wherein R²⁵ has a structure of Formula G-3:

Formula G-3 wherein:

[0623] A37.1. The compound of embodiment A37, wherein R²⁵ has a structure of Formula H- 3:

Formula H-3 wherein: R⁶¹, R⁶³, R⁶⁴ and R⁶⁵ each represent two substituents, each of which substituents

[0624] A38. The compound of embodiment A37, wherein R²⁵ has a structure of Formula J-3:

Formula J-3 wherein R⁶³ is hydrogen, C1-C3 alkyl, C1-C3 heteroalkyl, C1-C3 haloalkyl or C1-C3

perhaloalkyl.

[0625] A39. The compound of any one of embodiments A1 , A1.1 , A2.2, A2.3, A2.10, A2.1 1 , A2.16 and A4-A38, wherein R²⁰ is -R²³.

[0626] A39.1. The compound of any one of embodiments A2, A2.6, A2.7 and A2.12-A38, wherein R^20A is hydrogen and R^20B is -R²³, or wherein R^20B is hydrogen and R^20A is -R²³.

[0627] A39.2. The compound of embodiment A39.1 , wherein R^20A is hydrogen and R^20B is - R²³. [0628] A39.3. The compound of any one of embodiments A1 , A1 .1 , A2.2, A2.3, A2.10, A2.1 1 , A2.16 and A4-A38, wherein R²⁰ is -R^F-R²³.

[0629] A39.4. The compound of any one of embodiments A2, A2.6, A2.7 and A2.12-A38, wherein R^20A is hydrogen and R^20B is -R^F-R²³, or wherein R^20B is hydrogen and R^20A is -R^F-R²³.

[0630] A39.5. The compound of embodiment A39.4, wherein R^20A is hydrogen and R^20B is - R^F-R²³.

[0631] A39.6. The compound of any one of embodiments A1 -A38, A39.3, A39.4 and A39.5, wherein R^F is -0-, -C(O)-, -O-C(O)- or -C(0)-0-

[0632] A39.7. The compound of any one of embodiments A1 -A38, A39.3, A39.4, A39.5 and A39.6, wherein R^F is -0-.

[0633] A39.8. The compound of any one of embodiments A1 -A38, A39.3, A39.4 and A39.5, wherein R^F is -NH-S(O)-.

[0634] A39.9. The compound of any one of embodiments A1 -A26.1 and A28-A38, wherein R²¹ is hydroxy; R^21A is hydrogen and R^21B is hydroxy; or R^21A is hydroxy and R^{21 B} is hydrogen.

[0635] A40. The compound of any one of embodiments A1 -A39.8, wherein R²³ is a C3-C10 alkyl, C5-C7 aryl or 5-7 membered heteroaryl, which independently is optionally substituted by one or more substituents chosen from halogen, hydroxy, C1 -C3 alkyl, C1 -C3 heteroalkyl, C1 -C3 haloalkyl, C1 -C3 perhaloalkyl, C1 -C3 perhaloalkoxy, C1 -C3 alkoxy, C1-C3 haloalkoxy, C1-C3 alkoxy C1 -C3 alkyl, C1 -C4 acyl, oxo, C1 -C4 acyloxy, carboxyl, amido, cyano, amino, C1 -C3 alkylamino, C1-C3 alkylamino C1 -C3 alkyl, thiol, C1 -C3 alkylthio, C1 -C3 alkylthio C1 -C3 alkyl, C1 -C3 haloalkylthio, C1 -C3 perhaloalkylthio, nitro, C5-C7 aryl, C5-C7 aryl C1 -C3 alkyl, C5-C7 cycloalkyl, C5-C7 cycloalkyl C1 -C3 alkyl, 5-7 membered heterocycloalkyl, 5-7 membered heterocycle C1 -C3 alkyl, 5-7 membered heteroaryl, 5-7 membered heteroaryl C1 -C3 alkyl, C1 - C3 alkylsulfonyl, sulfonamide, C1 -C3 alkylsulfonamido and C1-C3 alkylsilyloxy.

[0636] A41 . The compound of any one of embodiments A1 -A39.9, wherein R²³ is a C3-C10 alkyl, C5-C7 aryl, C5-C7 cycloalkyl, or 5-7 membered heteroaryl. [0637] A42. The compound of embodiment A41 , wherein R²³ has a structure of Formula C-3:

Formula C-3

wherein:

R⁵¹, R⁵², R⁵³, R⁵⁴ and R⁵⁵ each independently is not present or independently is hydrogen, halogen, hydroxy, C1-C3 alkyl, C1-C3 heteroalkyl, C1-C3 haloalkyl, C1-C3 perhaloalkyl, C1-C3 perhaloalkoxy, C1-C3 alkoxy, C1-C3 haloalkoxy, C1-C3 alkoxy C1-C3 alkyl, C1-C4 acyl, oxo, C1-C4 acyloxy, carboxyl, amido, cyano, amino, C1-C3 alkylamino, C1-C3 alkylamino C1-C3 alkyl, thiol, C1-C3 alkylthio, C1-C3 alkylthio C1-C3 alkyl, C1-C3 haloalkylthio, C1-C3 perhaloalkylthio, nitro, C5-C7 aryl, C5-C7 aryl C1-C3 alkyl, C5-C7 cycloalkyl, C5-C7 cycloalkyl C1-C3 alkyl, 5-7 membered heterocycloalkyl, 5-7 membered heterocycle C1-C3 alkyl, 5-7 membered heteroaryl, 5-7 membered heteroaryl C1-C3 alkyl, C1-C3 alkylsulfonyl, sulfonamide, C1-C3 alkylsulfonamido or C1-C3 alkylsilyloxy.

[0638] A43. The compound of embodiment A42, wherein R²³ has a structure of Formula D-3:

Formula D-3 wherein R⁵¹, R⁵², R⁵³, R⁵⁴ and R⁵⁵ each independently is hydrogen, halogen, hydroxy, C1-C3 alkyl, C1-C3 heteroalkyl, C1-C3 haloalkyl, C1-C3 perhaloalkyl, C1-C3 perhaloalkoxy, C1-C3 alkoxy, C1-C3 haloalkoxy, C1-C3 alkoxy C1-C3 alkyl, C1-C4 acyl, oxo, C1-C4 acyloxy, carboxyl, amido, cyano, amino, C1-C3 alkylamino, C1-C3 alkylamino C1-C3 alkyl, thiol, C1-C3 alkylthio, C1-C3 alkylthio C1-C3 alkyl, C1-C3 haloalkylthio, C1-C3 perhaloalkylthio, nitro, C5-C7 aryl, C5-C7 aryl C1-C3 alkyl, C5-C7 cycloalkyl, C5-C7 cycloalkyl C1-C3 alkyl, 5-7 membered heterocycloalkyl, 5-7 membered heterocycle C1-C3 alkyl, 5-7 membered heteroaryl, 5-7 membered heteroaryl C1-C3 alkyl, C1-C3 alkylsulfonyl, sulfonamide, C1-C3 alkylsulfonamido or C1-C3 alkylsilyloxy.

[0639] A43.1. The compound of embodiment A42, wherein R²³ has a structure of Formula E- 3:

Formula E-3 wherein R⁵¹ and R⁵³ each independently is C1-C3 alkyl, C1-C3 heteroalkyl, C1 -C3 haloalkyl, C1-C3 perhaloalkyl, C1-C3 perhaloalkoxy, C1-C3 alkoxy, C1-C3 haloalkoxy or C1-C3 alkoxy C1-C3 alkyl.

[0640] A43.2. The compound of embodiment A43.1 , wherein R⁵¹ and R⁵³ each are methoxy.

[0641] A43.3. The compound of any one of embodiments A1-A41 , wherein R²³ is a C3-C10 alkyl.

[0642] A43.4. The compound of embodiment A43.3, wherein R²³ is a C3 or C4 alkyl.

[0643] A43.5. The compound of embodiment A43.4, wherein R²³ is isopropyl or isobutyl.

[0644] A43.6. The compound of any one of embodiments A1 , A2 and A3-A49.9, wherein R²³ is a C1-C2 alkyl.

[0645] A43.7. The compound of embodiment A43.6, wherein R²³ is methyl.

[0646] A43.8. The compound of any one of embodiments A43.3-A43.7, wherein R^F is -NH- S(O)-.

[0647] A43.9. The compound of any one of embodiments A43.3-A43.7, wherein R^F is -0-. [0648] A43.10. The compound of any one of embodiments A43.3-A43.7, wherein R^F is not present.

[0649] A43.1 1 . The compound of any one of embodiments A1-A2.4, A2.6-A2.8, A2.10-A41 , wherein R²³ is a saturated C5-C7 cycloalkyl or a C5-C7 cycloalkyl comprising one degree of unsaturation.

[0650] A43.12. The compound of embodiment A43.1 1 , wherein R²³ is a saturated C6 cycloalkyl or a C6 cycloalkyl comprising one degree of unsaturation.

[0651] A43.13. The compound of embodiment A43.1 1 or A43.12, wherein R²³ comprises one or two C1-C4 alkyl substituents.

[0652] A43.14. The compound of any one of embodiments A43.1 1-A43.13, wherein R^F is not present.

[0653] A43.15. The compound of any one of embodiments A43.1 1-A43.13, wherein R^F is - NH-S(O)-.

[0654] A43.16. The compound of any one of embodiments A43.1 1-A43.13, wherein R^F is -O-

[0655] A43.17. The compound of embodiment A43.16, wherein -R^F-R²³ together is p-menth- 1-en-3-ol.

[0656] A43.18. The compound of embodiment A43.16, wherein -R^F-R²³ together is 2- isopropyl-5-methylcyclohexanol.

[0657] A43.19. The compound of any one of embodiments A1-A39.9, wherein R²³ is a 5-7 membered heterocycle.

[0658] A43.20. The compound of embodiment A43.19, wherein R²³ is a 5 membered heterocycle.

[0659] A43.21 . The compound of embodiment A43.19 or A43.20, wherein R²³ comprises one, two or three nitrogen ring atoms. [0660] A43.22. The compound of embodiment A43.21 , wherein R²³ is imidazole.

[0661] A43.23. The compound of any one of embodiments A43.19-A43.20, wherein R^F is not present.

[0662] A44. The compound of any one of embodiments A1 -A2.9, A2.1 1 , A2.14-A17, A18- A43.23, wherein:

R²², R^22A or R^22B is halogen, -NR²⁷R²⁸ or-R^H-R²⁹-R³¹-R³⁰;

R^H is -O-C(O)-;

R²⁷ is hydrogen and R²⁸ is -R³²-R³³;

R²⁹ is C5-C7 aryl or 5-7 membered heteroaryl;

R³¹ is a C1 -C3 alkyl linker;

R³⁰ is a 5-7 membered heterocycloalkyl;

R³² is a C1 to C4 alkyl linker; and

R³³ is a 5-7 membered heterocycloalkyl.

[0663] A44.1 . The compound of embodiment A44, wherein R²², R^22A or R^22B is halogen.

[0664] A44.2. The compound of embodiment A44.1 , wherein the halogen independently is fluoro, chloro, bromo or iodo.

[0665] A45. The compound of embodiment A44, wherein:

R²², R^22A or R^22B is -NR²⁷R²⁸.

[0666] A46. The compound of embodiment A44, wherein:

R²², R^22A or R^22B is -R^H-R²⁹-R³¹-R³⁰;

R^H is -O-C(O)-;

R²⁹ is C5-C7 aryl or 5-7 membered heteroaryl;

R³¹ is a C1 -C3 alkyl linker; and

R³⁰ is a 5-7 membered heterocycloalkyl.

[0667] A47. The compound of embodiment A44 or A46, wherein R³¹ is a methylene linker or ethylene linker.

[0668] A48. The compound of any one of embodiments A44, A46 or A47, wherein R²⁹ has a structure of Formula E-1 and R⁴³ is R³¹-R³⁰. [0669] A49. The compound of any one of embodiments A44 and A46-A48, wherein:

R³⁰ has a structure of Formula G-1 or Formula H-1 ; and

R⁶¹, R⁶², R⁶³, R⁶⁴ and R⁶⁵ each represent one or two substituents, which one or two substituents each independently is hydrogen or C1-C3 alkyl.

[0670] A50. The compound of any one of embodiments A44 and A46-A49, wherein:

R³⁰ has a structure of Formula J-1 ; and

R⁶³ is hydrogen or C1-C3 alkyl.

[0671] A51. The compound of any one of embodiments A44-A50, wherein:

R^F is -0-, -NH-S(O)-, or is not present; and

R²³ is C1-C2 alkyl, C3-C10 alkyl, C5-C7 aryl, C5-C7 cycloalkyl or 5-7 membered heteroaryl.

[0672] A52. The compound of any one of embodiments A44-A51 , wherein:

R²³ has a structure of Formula D-3; and

R⁵¹, R⁵², R⁵³, R⁵⁴ and R⁵⁵ each independently is hydrogen, halogen, hydroxyl, C1-C3 alkyl, C1-C3 heteroalkyl, C1-C3 haloalkyl, C1-C3 perhaloalkyl, C1-C3 perhaloalkoxy, C1-C3 alkoxy, C1-C3 haloalkoxy or C1-C3 alkoxy C1-C3 alkyl.

[0673] A53. The compound of any one of embodiments A44-A52, wherein:

wherein R²³ has a structure of Formula E-3; and

R⁵¹ and R⁵³ each independently is C1-C3 alkyl or C1-C3 alkoxy.

[0674] A54. The compound of any one of embodiments A44-A53, wherein:

wherein R²³ has a structure of Formula E-3; and

R⁵¹ and R⁵³ each are methoxy.

[0675] A55. The compound of any one of embodiments A44-A51 , wherein R²³ is C1 -C2 alkyl.

[0676] A56. The compound of embodiment A55, wherein R²³ is methyl.

[0677] A57. The compound of any one of embodiments A44-A50, wherein R²¹ is hydroxy; R^21A is hydrogen and R^21B is hydroxy; or R^21A is hydroxy and R^21B is hydrogen.

[0678] A58. The compound of any one of embodiments A44-A51 , wherein R²³ is a C3-C10 alkyl. [0679] A59. The compound of embodiment A58, wherein R²³ is a C3 or C4 alkyl.

[0680] A60. The compound of embodiment A59, wherein R²³ is isopropyl or isobutyl.

[0681] A61. The compound of any one of embodiments A58-A60, wherein R^F is -NH-S(O)-.

[0682] A62. The compound of any one of embodiments A58-A60, wherein R^F is -0-.

[0683] A63. The compound of any one of embodiments A58-A60, wherein R^F is not present.

[0684] A64. The compound of any one of embodiments A44-A51 , wherein R²³ is a saturated C5-C7 cycloalkyl or a C5-C7 cycloalkyl comprising one degree of unsaturation.

[0685] A65. The compound of embodiment A64, wherein R²³ is a saturated C6 cycloalkyl or a C6 cycloalkyl comprising one degree of unsaturation.

[0686] A66. The compound of embodiment A64 or A65, wherein R²³ comprises one or two C1-C4 alkyl substituents.

[0687] A67. The compound of any one of embodiments A64-A66, wherein R^F is not present.

[0688] A68. The compound of any one of embodiments A64-A66, wherein R^F is -NH-S(O)-.

[0689] A69. The compound of any one of embodiments A64-A66, wherein R^F is -0-.

[0690] A70. The compound of embodiment A69, wherein -R^F-R²³ together is p-menth-1-en- 3-ol.

[0691] A71. The compound of embodiment A69, wherein -R^F-R²³ together is 2-isopropyl-5- methylcyclohexanol.

[0692] A72. The compound of any one of embodiments A44-A50, wherein R²³ is a 5-7 membered heterocycle.

[0693] A73. The compound of embodiment A72, wherein R²³ is a 5 membered heterocycle. [0694] A74. The compound of embodiment A72 or A73, wherein R²³ comprises one, two or three nitrogen ring atoms.

[0695] A75. The compound of embodiment A74, wherein R²³ is imidazole.

[0696] A76. The compound of any one of embodiments A72-A75, wherein R^F is not present.

[0697] A77. The compound of embodiment A15, wherein:

R²², R^22A or R^22B is -R^H-R²⁹-R³¹-R³⁰;

R^H is -O-C(O)-;

R²⁹ is C5-C7 aryl or 5-7 membered heteroaryl; and

R³¹ is a C1-C3 alkyl linker.

[0698] A78. The compound of embodiment A77, wherein:

R²¹, R^21A or R^21B is -R^G-R²⁴-R²⁶-R²⁵ ;

R^G is -O-C(O)-;

R²⁴ is C5-C7 aryl or 5-7 membered heteroaryl; and

R²⁶ is a C1-C3 alkyl linker.

[0699] A79. The compound of embodiment A78, wherein R²⁵ has a structure of Formula J-3:

Formula J-3 wherein R⁶³ is hydrogen, C1-C3 alkyl, C1-C3 heteroalkyl, C1-C3 haloalkyl or C1-C3 perhaloalkyl. [0700] B1. A compound having a structure of the following Formula A:

or a pharmaceutically acceptable salt thereof, wherein:

R²⁰ is -R²³ or -R^F-R²³;

R²¹ is hydrogen, hydroxy or -R^G-R³⁴;

R²² is -R^H-R³⁵;

n is 1 or 2;

R²³ independently is C3-C10 alkyl, C3-C10 heteroalkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl, which independently is optionally substituted by one or more substituents chosen from halogen, hydroxy, alkyl, alkenyl, alkynyl, heteroalkyl, haloalkyl, perhaloalkyl, perhaloalkoxy, alkoxy, haloalkoxy, alkoxyalkyl, acyl, oxo, acyloxy, carboxyl, amido, cyano, amino, alkylamino, alkylaminoalkyl, thiol, alkylthio, alkylthioalkyl, haloalkylthio, perhaloalkylthio, nitro, aryl, arylalkyl, cycloalkylalkyl, heterocycle-alkyl, cycloalkyl, heterocycloalkyl, heteroaryl, heteroarylalkyl, alkylsulfonyl, sulfonamide, alkylsulfonamido and alkylsilyloxy, with the proviso that if R²³ is aryl, R^F is not -0-; and

[0701] B1.1 The compound of embodiment B, wherein R²³ independently is C3-C10 alkyl, cycloalkyl, heterocycloalkyl or heteroaryl, which independently is optionally substituted by one or more substituents chosen from halogen, hydroxy, alkyl, alkenyl, alkynyl, heteroalkyl, haloalkyl, perhaloalkyl, perhaloalkoxy, alkoxy, haloalkoxy, alkoxyalkyl, acyl, oxo, acyloxy, carboxyl, amido, cyano, amino, alkylamino, alkylaminoalkyl, thiol, alkylthio, alkylthioalkyl, haloalkylthio, perhaloalkylthio, nitro, aryl, arylalkyl, cycloalkylalkyl, heterocycle-alkyl, cycloalkyl,

heterocycloalkyl, heteroaryl, heteroarylalkyl, alkylsulfonyl, sulfonamide, alkylsulfonamido and alkylsilyloxy.

[0702] B2. A compound having a structure of the following Formula B:

Formula B or a pharmaceutically acceptable salt thereof, wherein:

R^21A is hydrogen and R^{21 B} is hydrogen, hydroxy or -R^G-R³⁴, or R^{21 B} is hydrogen and R^21A is hydrogen, hydroxy or -R^G-R³⁴;

n is 1 or 2;

R²³ independently is C3-C10 alkyl, C3-C10 heteroalkyl, cycloalkyl, aryl, heterocycloalkyl or heteroaryl, which independently is optionally substituted by one or more substituents chosen from halogen, hydroxy, alkyl, alkenyl, alkynyl, heteroalkyl, haloalkyl, perhaloalkyl, perhaloalkoxy, alkoxy, haloalkoxy, alkoxyalkyl, acyl, oxo, acyloxy, carboxyl, amido, cyano, amino, alkylamino, alkylaminoalkyl, thiol, alkylthio, alkylthioalkyl, haloalkylthio, perhaloalkylthio, nitro, aryl, arylalkyl, cycloalkylalkyl, heterocycle-alkyl, cycloalkyl, heterocycloalkyl, heteroaryl, heteroarylalkyl, alkylsulfonyl, sulfonamide, alkylsulfonamido and alkylsilyloxy, with the proviso that if R²³ is aryl, R^F is not -0-; and

[0703] B2.1 The compound of embodiment B2, wherein R²³ independently is C3-C10 alkyl, cycloalkyl, heterocycloalkyl or heteroaryl, which independently is optionally substituted by one or more substituents chosen from halogen, hydroxy, alkyl, alkenyl, alkynyl, heteroalkyl, haloalkyl, perhaloalkyl, perhaloalkoxy, alkoxy, haloalkoxy, alkoxyalkyl, acyl, oxo, acyloxy, carboxyl, amido, cyano, amino, alkylamino, alkylaminoalkyl, thiol, alkylthio, alkylthioalkyl, haloalkylthio, perhaloalkylthio, nitro, aryl, arylalkyl, cycloalkylalkyl, heterocycle-alkyl, cycloalkyl,

[0704] B2.2. The compound of any of embodiments B1 , B1.1 , B2 and B2.1 , wherein R^F, R^G and R^H each independently is -0-, -C(O)-, -O-C(O)- or -C(0)-0-

[0705] B3. The compound of any of embodiments B1 , B1.1 , B2, B2.1 and B2.2, wherein R²⁰ is -R²³.

[0706] B3.1. The compound of any of embodiments B2, B2.1 and B2.2, wherein R^20A is hydrogen and R^20B is -R²³, or wherein R^20B is hydrogen and R^20A is -R²³.

[0707] B3.2. The compound of embodiment B3.1 , wherein R^20A is hydrogen and R^20B is -R²³.

[0708] B3.3. The compound of any of embodiments B1 , B1.1 and B2.2, wherein R²⁰ is -R^F- R²³.

[0709] B3.4. The compound of any of embodiments B2, B2.1 and B2.2, wherein R^20A is hydrogen and R^20B is -R^F-R²³, or wherein R^20B is hydrogen and R^20A is -R^F-R²³.

[0710] B3.5. The compound of embodiment B3.4, wherein R^20A is hydrogen and R^20B is -R^F- R²³.

[0711] B3.6. The compound of any one of embodiments B1 , B1.1 , B2, B2.1 , B2.2, B3.3, B3.4 and B3.5, wherein R^F is -0-, -C(O)-, -O-C(O)- or -C(0)-0- [0712] B3.7. The compound of any one of embodiments B1 , B1.1 , B2, B2.1 , B2.2, B3.3, B3.4, B3.5 and B3.6, wherein R^F is -0-.

[0713] B3.8. The compound of any one of embodiments B1 , B1.1 , B2, B2.1 , B2.2, B3.3, B3.4, B3.5 and B3.6, wherein R^F is -NH-S(O)-.

[0714] B4. The compound of any one of embodiments B1-B3.8, wherein R²³ is a C3-C10 alkyl, C5-C7 cylcloalkyl, C5-C7 aryl or 5-7 membered heteroaryl, which independently is optionally substituted by one or more substituents chosen from halogen, hydroxy, C1-C3 alkyl, C1-C3 heteroalkyl, C1-C3 haloalkyl, C1-C3 perhaloalkyl, C1-C3 perhaloalkoxy, C1-C3 alkoxy, C1-C3 haloalkoxy, C1-C3 alkoxy C1-C3 alkyl, C1 -C4 acyl, oxo, C1-C4 acyloxy, carboxyl, amido, cyano, amino, C1-C3 alkylamino, C1-C3 alkylamino C1-C3 alkyl, thiol, C1-C3 alkylthio, C1-C3 alkylthio C1-C3 alkyl, C1-C3 haloalkylthio, C1-C3 perhaloalkylthio, nitro, C5-C7 aryl, C5-C7 aryl C1-C3 alkyl, C5-C7 cycloalkyl, C5-C7 cycloalkyl C1-C3 alkyl, 5-7 membered heterocycloalkyl, 5- 7 membered heterocycle C1-C3 alkyl, 5-7 membered heteroaryl, 5-7 membered heteroaryl C1- C3 alkyl, C1-C3 alkylsulfonyl, sulfonamide, C1-C3 alkylsulfonamido and C1-C3 alkylsilyloxy.

[0715] B4.1. The compound of any one of embodiments B1-B4, wherein R²³ is a C3-C10 alkyl.

[0716] B5. The compound of embodiment B4.1 , wherein R²³ is a C3 or C4 alkyl.

[0717] B6. The compound of embodiment B5, wherein R²³ is isopropyl or isobutyl.

^[0718] B7 _The compound of any one of embodiments B1-B4, wherein R²³ is a C5-C7 cycloalkyl, C5-C7 aryl or 5-7 membered heteroaryl.

[0719] B8. The compound of embodiment B7, wherein R²³ has a structure of Formula C-3:

Formula C-3 wherein:

each R⁵¹, R⁵², R⁵³, R⁵⁴ and R⁵⁵ independently is not present or independently is hydrogen, halogen, hydroxy, C1-C3 alkyl, C1-C3 heteroalkyl, C1-C3 haloalkyl, C1-C3 perhaloalkyl, C1-C3 perhaloalkoxy, C1-C3 alkoxy, C1-C3 haloalkoxy, C1-C3 alkoxy C1-C3 alkyl, C1-C4 acyl, oxo, C1-C4 acyloxy, carboxyl, amido, cyano, amino, C1-C3 alkylamino, C1-C3 alkylamino C1-C3 alkyl, thiol, C1-C3 alkylthio, C1-C3 alkylthio C1-C3 alkyl, C1-C3 haloalkylthio, C1-C3 perhaloalkylthio, nitro, C5-C7 aryl, C5-C7 aryl C1-C3 alkyl, C5-C7 cycloalkyl, C5-C7 cycloalkyl C1-C3 alkyl, 5-7 membered heterocycloalkyl, 5-7 membered heterocycle C1-C3 alkyl, 5-7 membered heteroaryl, 5-7 membered heteroaryl C1-C3 alkyl, C1-C3 alkylsulfonyl, sulfonamide, C1-C3 alkylsulfonamido or C1-C3 alkylsilyloxy.

[0720] B9. The compound of embodiment B8, wherein R²³ has a structure of Formula D-3:

[0721] B10. The compound of embodiment B9, wherein R²³ has a structure of Formula E-3:

Formula E-3 wherein each R⁵¹ and R⁵³ independently is C1-C3 alkyl, C1-C3 heteroalkyl, C1-C3 haloalkyl, C1-C3 perhaloalkyl, C1-C3 perhaloalkoxy, C1-C3 alkoxy, C1-C3 haloalkoxy or C1-C3 alkoxy C1-C3 alkyl.

[0722] B1 1. The compound of embodiment B10, wherein R⁵¹ and R⁵³ each are methoxy.

[0723] B12. The compound of any one of embodiments B1-B1 1 , wherein R^F is -NH-S(O)-, - 0-, or is not present.

[0724] B13. The compound of any one of embodiments B1-B4, wherein R²³ is a saturated C5-C7 cycloalkyl or a C5-C7 cycloalkyl comprising one degree of unsaturation.

[0725] B14. The compound of embodiment B13, wherein R²³ is a saturated C6 cycloalkyl or a C6 cycloalkyl comprising one degree of unsaturation.

[0726] B15. The compound of embodiment B13 or B14, wherein R²³ comprises one or two C1-C4 alkyl substituents.

[0727] B16. The compound of any one of embodiments B13-B15, wherein R^F is not present.

[0728] B17. The compound of any one of embodiments B13-B15, wherein R^F is -NH-S(O)-.

[0729] B18. The compound of any one of embodiments B13-B15, wherein R^F is -0-.

[0730] B19. The compound of embodiment B18, wherein -R^F-R²³ together is p-menth-1-en-

3-ol.

[0731] B20. The compound of embodiment B18, wherein -R^F-R²³ together is 2-isopropyl-5- methylcyclohexanol. [0732] B21. The compound of any one of embodiments B1-B4, wherein R²³ is a 5-7 membered heterocycle.

[0733] B22. The compound of embodiment B21 , wherein R²³ is a 5 membered heterocycle.

[0734] B23. The compound of embodiment B21 or B22, wherein R²³ comprises one, two or three nitrogen ring atoms.

[0735] B24. The compound of embodiment B23, wherein R²³ is imidazole.

[0736] B25. The compound of any one of embodiments B21-B24, wherein R^F is not present.

[0737] B26. The compound of any one of embodiments B1-B3, wherein R²¹ is hydroxy; R^21A is hydrogen and R^21B is hydroxy; or R^21A is hydroxy and R^21B is hydrogen.

[0738] B27. The compound of any one of embodiments B1-B26, wherein each R^G and R^H independently is -O- or -O-C(O)-.

[0739] B28. The compound of any one of embodiments B1-B27, wherein R^H is -O-C(O)- and R³⁵ is a C3-C6 alkyl substituted with one or more hydroxy substituents.

[0740] B29. The compound of embodiment B28, wherein R³⁵ is a C3-C5 alkyl substituted with two hydroxy substituents.

[0741] B30. The compound of embodiment B29, wherein R³⁵ is a C4 alkyl substituted with two hydroxy substituents.

[0742] B31. The compound of embodiment B30, wherein R³⁵ is dihydroxy tert-butyl.

[0743] B32. The compound of any one of embodiments B1-B31 , wherein R^H is -O- and R³⁵ is a C2-C6 alkyl substituted with one or more hydroxy substituents.

[0744] B33. The compound of embodiment B32, wherein R³⁵ is a C2-C3 alkyl substituted with one hydroxy substituent.

[0745] B34. The compound of embodiment B28, wherein R³⁵ is hydroxy ethyl. [0746] C1. A compound having a structure of the following Formula A:

Formula A or a pharmaceutically acceptable salt thereof, wherein:

R²⁰ is -R²³, -R^F-R²³ or -R'-R³⁴

R²² is hydrogen, hydroxy, halogen, -N³, -NR²⁷R²⁸, -R^H-R²⁹, -R^H-R²⁹-R³⁰ or -R^H-R²⁹-R³¹-R³⁰;

n is 1 or 2;

R' is -0-S(0)_n-, -S(0)_n-, -S(0)_n-0-, -S(O) _n-NH-, -NH-C(O)-, or -NH-S(O)-;

n is 1 or 2;

R²⁴ and R²⁹ each independently is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl,

heterocycloalkyl, aryl or heteroaryl, which independently is optionally substituted by one or more substituents chosen from halogen, hydroxy, alkyl, alkenyl, alkynyl, heteroalkyl, haloalkyl, perhaloalkyl, perhaloalkoxy, alkoxy, haloalkoxy, alkoxyalkyl, acyl, oxo, acyloxy, carboxyl, amido, cyano, amino, alkylamino, alkylaminoalkyl, thiol, alkylthio, alkylthioalkyl, haloalkylthio, perhaloalkylthio, nitro, aryl, arylalkyl, cycloalkylalkyl, heterocycle-alkyl, cycloalkyl,

heterocycloalkyl, heteroaryl, heteroarylalkyl, alkylsulfonyl, sulfonamide, alkylsulfonamido and alkylsilyloxy;

R³⁴ independently is alkyl, alkenyl, alkynyl, heteroalkyl or amino, which independently is optionally substituted by one or more substituents chosen from halogen, hydroxy, alkyl, alkenyl, alkynyl, heteroalkyl, haloalkyl, perhaloalkyl, perhaloalkoxy, alkoxy, haloalkoxy, alkoxyalkyl, acyl, oxo, acyloxy, carboxyl, amido, cyano, amino, alkylamino, alkylaminoalkyl, thiol, alkylthio, alkylthioalkyl, haloalkylthio, perhaloalkylthio, nitro, aryl, arylalkyl, cycloalkylalkyl, heterocycle- alkyl, cycloalkyl, heterocycloalkyl, heteroaryl, heteroarylalkyl, alkylsulfonyl, sulfonamide, alkylsulfonamido and alkylsilyloxy, with the proviso that when R' is -NH-S(O)-, R³⁴ is not straight-chain alkyl.

[0747] C1.1. The compound of embodiment C1 , wherein R²² is hydrogen, hydroxy, halogen, - N³, -NR²⁷R²⁸, -R^H-R²⁹, -R^H-R²⁹-R³⁰ or -R^H-R²⁹-R³¹-R³⁰ [0748] C1.2. The compound of embodiment C1 or C1.1 , wherein R²⁴ and R²⁹ each independently is cycloalkyl, heterocycloalkyl, aryl or heteroaryl, which independently is optionally substituted by one or more substituents chosen from halogen, hydroxy, alkyl, alkenyl, alkynyl, heteroalkyl, haloalkyl, perhaloalkyl, perhaloalkoxy, alkoxy, haloalkoxy, alkoxyalkyl, acyl, oxo, acyloxy, carboxyl, amido, cyano, amino, alkylamino, alkylaminoalkyl, thiol, alkylthio, alkylthioalkyl, haloalkylthio, perhaloalkylthio, nitro, aryl, arylalkyl, cycloalkylalkyl, heterocycle- alkyl, cycloalkyl, heterocycloalkyl, heteroaryl, heteroarylalkyl, alkylsulfonyl, sulfonamide, alkylsulfonamido and alkylsilyloxy.

[0749] C2. A compound having a structure of the following Formula B:

or a pharmaceutically acceptable salt thereof, wherein:

- R^F-R²³ or -R'-R³⁴;

R^21A is hydrogen and R^{21 B} is hydrogen, hydroxy, -R^G-R²⁴ -R^G-R²⁴-R²⁵ or -R^G-R²⁴-R²⁶-R²⁵, or R^21B is hydrogen and R^21A is hydrogen, hydroxy, -R^G-R²⁴ -R^G-R²⁴-R²⁵ or -R^G-R²⁴-R²⁶-R²⁵;

n is 1 or 2; R' is— 0-S(0)n-,— S(0)n-,— S(O) _n—0— , -S(O) _n-NH-, -NH-C(O)-, or -NH-S(O)-;

n is 1 or 2;

R²⁷ and R²⁸ each independently is -R³²-R³³, or hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, perhaloalkyl, alkoxy, cycloalkyl, aryl, heterocycloalkyl, heterocycle-alkyl or heteroaryl, which independently is optionally substituted with one or more substituents chosen from halogen, hydroxy, alkyl, alkenyl, alkynyl, heteroalkyl, haloalkyl, perhaloalkyl, perhaloalkoxy, alkoxy, haloalkoxy, alkoxyalkyl, acyl, oxo, acyloxy, carboxyl, amido, cyano, amino, alkylamino, alkylaminoalkyl, thiol, alkylthio, alkylthioalkyl, haloalkylthio, perhaloalkylthio, nitro, aryl, arylalkyl, cycloalkylalkyl, heterocycle-alkyl, cycloalkyl, heterocycloalkyl, heteroaryl, heteroarylalkyl, alkylsulfonyl, sulfonamide, alkylsulfonamido and alkylsilyloxy; and R³⁴ independently is alkyl, alkenyl, alkynyl, heteroalkyl or amino, which independently is optionally substituted by one or more substituents chosen from halogen, hydroxy, alkyl, alkenyl, alkynyl, heteroalkyl, haloalkyl, perhaloalkyl, perhaloalkoxy, alkoxy, haloalkoxy, alkoxyalkyl, acyl, oxo, acyloxy, carboxyl, amido, cyano, amino, alkylamino, alkylaminoalkyl, thiol, alkylthio, alkylthioalkyl, haloalkylthio, perhaloalkylthio, nitro, aryl, arylalkyl, cycloalkylalkyl, heterocycle- alkyl, cycloalkyl, heterocycloalkyl, heteroaryl, heteroarylalkyl, alkylsulfonyl, sulfonamide, alkylsulfonamido and alkylsilyloxy, with the proviso that when R' is -NH-S(O)-, R³⁴ is not straight-chain alkyl.

[0750] C2.1 . The compound of embodiment C2, wherein R^22A is hydrogen and R^22B is hydrogen, hydroxy, halogen, -N³, -NR²⁷R²⁸ or -R^H-R²⁹-R³¹-R³⁰, or R^22B is hydrogen and R^22A is hydrogen, hydroxy, halogen, -N³, -NR²⁷R²⁸ or -R^H-R²⁹-R³¹-R³⁰

[0751] C2.2. The compound of embodiment C2 or C2.1 , wherein R²⁴ and R²⁹ each independently is cycloalkyl, heterocycloalkyl, aryl or heteroaryl, which independently is optionally substituted by one or more substituents chosen from halogen, hydroxy, alkyl, alkenyl, alkynyl, heteroalkyl, haloalkyl, perhaloalkyl, perhaloalkoxy, alkoxy, haloalkoxy, alkoxyalkyl, acyl, oxo, acyloxy, carboxyl, amido, cyano, amino, alkylamino, alkylaminoalkyl, thiol, alkylthio, alkylthioalkyl, haloalkylthio, perhaloalkylthio, nitro, aryl, arylalkyl, cycloalkylalkyl, heterocycle- alkyl, cycloalkyl, heterocycloalkyl, heteroaryl, heteroarylalkyl, alkylsulfonyl, sulfonamide, alkylsulfonamido and alkylsilyloxy.

[0752] C2.3. The compound of any one of embodiments C1 -C2, wherein R^F, R^G and R^H each independently is -0-, -C(O)-, -O-C(O)-, -NH-S(0)_n- or -C(0)-0-.

[0753] C3. The compound of any one of embodiments C1 , C1 .1 , C1.2 and C2.3, wherein R²⁰ is -R²³.

[0754] C3.1 . The compound of any one of embodiments C2, C2.1 , C2.2 and C2.3, wherein R^20A is hydrogen and R^20B is -R²³, or wherein R^20B is hydrogen and R^20A is -R²³.

[0755] C3.2. The compound of embodiment C3.1 , wherein R^20A is hydrogen and R^20B is -R²³.

[0756] C3.3. The compound of any one of embodiments C1 , C1 .1 , C1 .2 and C2.3, wherein R²⁰ is -R^F-R²³. [0757] C3.4. The compound of embodiment C2, C2.1 , C2.2 and C2.3, wherein R^20A is hydrogen and R^20B is -R^F-R²³, or wherein R^20B is hydrogen and R^20A is -R^F-R²³.

[0758] C3.5. The compound of embodiment C3.4, wherein R^20A is hydrogen and R^20B is -R^F- R²³.

[0759] C3.6. The compound of any one of embodiments C1 , C1.1 , C1.2, C2, C2.1 , C2.2, C2.3, C3.3, C3.4 and C3.5, wherein R^F is -0-, -C(O)-, -O-C(O)-, -NH-S(0)_n- or -C(0)-0-.

[0760] C3.7. The compound of any one of embodiments C1 , C1.1 , C1.2, C2, C2.1 , C2.2, C2.3, C3.3, C3.4, C3.5 and C3.6, wherein R^F is -0-.

[0761] C3.8. The compound of any one of embodiments C1 , C1.1 , C1.2, C2, C2.1 , C2.2, C2.3, C3.3, C3.4, C3.5 and C3.6, wherein R^F is -NH-S(O)-.

[0762] C4. The compound of any one of embodiments C1-C3.8, wherein R²³ is a C5-C7 cylcloalkyl or 5-7 membered heteroaryl containing two or more heteroatoms, which

independently is optionally substituted by one or more substituents chosen from halogen, hydroxy, C1-C3 alkyl, C1-C3 heteroalkyl, C1-C3 haloalkyl, C1-C3 perhaloalkyl, C1 -C3 perhaloalkoxy, C1-C3 alkoxy, C1-C3 haloalkoxy, C1-C3 alkoxy C1-C3 alkyl, C1-C4 acyl, oxo, C1-C4 acyloxy, carboxyl, amido, cyano, amino, C1-C3 alkylamino, C1-C3 alkylamino C1-C3 alkyl, thiol, C1-C3 alkylthio, C1-C3 alkylthio C1-C3 alkyl, C1-C3 haloalkylthio, C1-C3 perhaloalkylthio, nitro, C5-C7 aryl, C5-C7 aryl C1-C3 alkyl, C5-C7 cycloalkyl, C5-C7 cycloalkyl C1-C3 alkyl, 5-7 membered heterocycloalkyl, 5-7 membered heterocycle C1-C3 alkyl, 5-7 membered heteroaryl, 5-7 membered heteroaryl C1-C3 alkyl, C1-C3 alkylsulfonyl, sulfonamide, C1-C3 alkylsulfonamido and C1-C3 alkylsilyloxy.

[0763] C5. The compound of any one of embodiments C1-C4, wherein R²³ is a saturated C5- C7 cycloalkyl or a C5-C7 cycloalkyl comprising one degree of unsaturation.

[0764] C6. The compound of embodiment C5, wherein R²³ is a saturated C6 cycloalkyl or a C6 cycloalkyl comprising one degree of unsaturation.

[0765] C7. The compound of embodiment C5 or C6, wherein R²³ comprises one or two C1- C4 alkyl substituents.

[0766] C8. The compound of any one of embodiments C5-C7, wherein R^F is not present. [0767] C9. The compound of any one of embodiments C5-C7, wherein R^F is -NH-S(O)-.

[0768] C10. The compound of any one of embodiments C5-C7, wherein R^F is -0-.

[0769] C11. The compound of embodiment C10, wherein -R^F-R²³ together is p-menth-1-en-

3-ol.

[0770] C12. The compound of embodiment C10, wherein -R^F-R²³ together is 2-isopropyl-5- methylcyclohexanol.

[0771] C13. The compound of any one of embodiments C1-C4, wherein R²³ is a 5-7 membered heterocycle containing 2 or more heteroatoms.

[0772] C14. The compound of embodiment C13, wherein R²³ is a 5-membered heterocycle.

[0773] C15. The compound of embodiment C13 or C14, wherein R²³ comprises two or three nitrogen ring atoms.

[0774] C16. The compound of embodiment C15, wherein R²³ is imidazole.

[0775] C17. The compound of any one of embodiments C13-C16, wherein R^F is not present.

[0776] C17.1 The compound of embodiment C1 or C1.1 , wherein R²⁰ is R'- R³⁴.

[0777] C17.2. The compound of embodiment C17.1 , wherein R' is -NH-S(O)-.

[0778] C17.3. The compound of embodiment C17.2, wherein R³⁴ is tert-butyl.

[0779] C17.4. The compound of embodiment C17.1 , wherein R' is -NH-C(O)-.

[0780] C17.5. The compound of embodiment C17.4, wherein R³⁴ is amino.

[0781] C17.6. The compound of any one of embodiments C2, C2.1 , C2.2 and C2.3, wherein R^20A is hydrogen and R^20B is R'- R³⁴. [0782] C17.7. The compound of any one of embodiments C2, C2.1 , C2.2 and C2.3, wherein R^20B is hydrogen and R^20A is R'- R³⁴.

[0783] C17.8. The compound of C17.4 or C17.5, wherein R' is -NH-S(O)-.

[0784] C17.9. The compound of C17.6, wherein R³⁴ is tert-butyl.

[0785] C18. The compound of any one of embodiments C2-C17.7, wherein R^22A is hydrogen and R^22B is hydrogen, hydroxyl, halogen, -N³, -NR²⁷R²⁸ or -R^H-R²⁹-R³¹-R³⁰.

[0786] C19. The compound of any one of embodiments C1-C18, wherein R²², R^22A or R^22B is halogen.

[0787] C20. The compound of embodiment C19, wherein the halogen independently is iodo, chloro, bromo or fluoro.

[0788] C21. The compound of any one of embodiments C1-C18, wherein R²², R^22A or R^22B is -N³.

[0789] C22. The compound of any one of embodiments C1-C18, wherein R²², R^22A or R^22B is

_R^H_R29_p31_p30

[0790] C23. The compound of any one of embodiments C1-C22, wherein R^H is -O-C(O)-.

[0791] C24. The compound of any one of embodiments C1-C23, wherein R²⁹ is C5-C7 cycloalkyl, 5-7 membered heterocycloalkyl, C5-C7 aryl or 5-7 membered heteroaryl, which independently is optionally substituted by one or more substituents chosen from halogen, hydroxy, C1-C3 alkyl, C1-C3 heteroalkyl, C1-C3 haloalkyl, C1-C3 perhaloalkyl, C1 -C3 perhaloalkoxy, C1-C3 alkoxy, C1-C3 haloalkoxy, C1-C3 alkoxy C1-C3 alkyl, C1-C4 acyl, oxo, C1-C4 acyloxy, carboxyl, amido, cyano, amino, C1-C3 alkylamino, C1-C3 alkylamino C1-C3 alkyl, thiol, C1-C3 alkylthio, C1-C3 alkylthio C1-C3 alkyl, C1-C3 haloalkylthio, C1-C3 perhaloalkylthio, nitro, C5-C7 aryl, C5-C7 aryl C1-C3 alkyl, C5-C7 cycloalkyl, C5-C7 cycloalkyl C1-C3 alkyl, 5-7 membered heterocycloalkyl, 5-7 membered heterocycle C1-C3 alkyl, 5-7 membered heteroaryl, 5-7 membered heteroaryl C1-C3 alkyl, C1-C3 alkylsulfonyl, sulfonamide, C1-C3 alkylsulfonamido and C1-C3 alkylsilyloxy. [0792] C25. The compound of embodiment C24, wherein R²⁹ is a C5-C7 aryl or a 5-7 membered heteroaryl.

[0793] C26. The compound of embodiment C25, wherein R²⁹ has a structure of Formula C-1 :

Formula C-1

wherein:

one, two or three of R⁴¹ , R⁴², R⁴³, R⁴⁴ and R⁴⁵ independently is R³¹-R³⁰, and each of the remaining R⁴¹, R⁴², R⁴³, R⁴⁴ and R⁴⁵ independently is not present or independently is hydrogen, halogen, hydroxy, C1 -C3 alkyl, C1 -C3 heteroalkyl, C1 -C3 haloalkyl, C1 -C3 perhaloalkyl, C1 -C3 perhaloalkoxy, C1 -C3 alkoxy, C1 -C3 haloalkoxy, C1 -C3 alkoxy C1-C3 alkyl, C1 -C4 acyl, oxo, C1-C4 acyloxy, carboxyl, amido, cyano, amino, C1 -C3 alkylamino, C1-C3 alkylamino C1 -C3 alkyl, thiol, C1 -C3 alkylthio, C1 -C3 alkylthio C1 -C3 alkyl, C1 -C3 haloalkylthio, C1 -C3 perhaloalkylthio, nitro, C5-C7 aryl, C5-C7 aryl C1 -C3 alkyl, C5-C7 cycloalkyl, C5-C7 cycloalkyl C1 -C3 alkyl, 5-7 membered heterocycloalkyl, 5-7 membered heterocycle C1 -C3 alkyl, 5-7 membered heteroaryl, 5-7 membered heteroaryl C1 -C3 alkyl, C1 -C3 alkylsulfonyl, sulfonamide, C1 -C3 alkylsulfonamido or C1 -C3 alkylsilyloxy.

[0794] C27. The compound of embodiment C26, wherein R²⁹ has a structure of Formula D-1 :

Formula D-1 wherein one, two or three of R⁴¹ , R⁴², R⁴³, R⁴⁴ and R⁴⁵ independently is R³¹-R³⁰, and each of the remaining R⁴¹ , R⁴², R⁴³, R⁴⁴ and R⁴⁵ independently is hydrogen, halogen, hydroxy, C1 -C3 alkyl, C1 -C3 heteroalkyl, C1 -C3 haloalkyl, C1 -C3 perhaloalkyl, C1 -C3 perhaloalkoxy, C1 -C3 alkoxy, C1 -C3 haloalkoxy, C1-C3 alkoxy C1 -C3 alkyl, C1 -C4 acyl, oxo, C1 -C4 acyloxy, carboxyl, amido, cyano, amino, C1 -C3 alkylamino, C1 -C3 alkylamino C1 -C3 alkyl, thiol, C1 -C3 alkylthio, C1 -C3 alkylthio C1 -C3 alkyl, C1-C3 haloalkylthio, C1 -C3 perhaloalkylthio, nitro, C5-C7 aryl, C5-C7 aryl C1 -C3 alkyl, C5-C7 cycloalkyl, C5-C7 cycloalkyl C1 -C3 alkyl, 5-7 membered heterocycloalkyl, 5-7 membered heterocycle C1-C3 alkyl, 5-7 membered heteroaryl, 5-7 membered heteroaryl C1-C3 alkyl, C1-C3 alkylsulfonyl, sulfonamide, C1 -C3 alkylsulfonamido or C1 -C3 alkylsilyloxy.

[0795] C28. The compound of embodiment C26, wherein R²⁹ has a structure of Formula E-1 :

Formula E-1 wherein R⁴³ is -R³¹-R³⁰.

[0796] C29. The compound of any one of embodiments C1 -C28, wherein R³¹ is a C1 -C3 alkyl linker.

[0797] C30. The compound of embodiment C29, wherein R³¹ is a methylene linker or ethylene linker.

[0798] C31 . The compound of any one of embodiments C1 -C30, wherein R³⁰ is a C5-C7 cycloalkyl, 5-7 membered heterocycloalkyl, C5-C7 aryl or 5-7 membered heteroaryl.

[0799] C32. The compound of any one of embodiments C1 -C31 , wherein R³⁰ is a 5-7 membered heterocycloalkyl or a 5-7 membered heteroaryl.

[0800] C33. The compound of any one of embodiments C1 -C32, wherein R³⁰ is a 5-7 membered heterocycloalkyl.

[0801] C34. The compound of embodiment C33, wherein R³⁰ has a structure of Formula F-1 :

Formula F-1 wherein:

[0802] C35. The compound of embodiment C34, wherein R³⁰ has a structure of Formula G-1 :

Formula G-1 wherein:

[0803] C36. The compound of embodiment C35, wherein R³⁰ has a structure of Formula H-1 :

Formula H-1 wherein:

heterocycloalkyl, 5-7 membered heterocycle C1-C3 alkyl, 5-7 membered heteroaryl, 5-7 membered heteroaryl C1-C3 alkyl, C1-C3 alkylsulfonyl, sulfonamide, C1-C3 alkylsulfonamido or C1-C3 alkylsilyloxy. [0804] C37. The compound of embodiment C36, wherein R³⁰ has a structure of Formula J-1 :

Formula J-1 wherein R⁶³ is hydrogen, C1-C3 alkyl, C1-C3 heteroalkyl, C1-C3 haloalkyl or C1-C3 perhaloalkyl.

[0805] C38. The compound of any one of embodiments C1-C18, wherein R²², R^22A or R^22B is -NR²⁷R²⁸.

[0806] C39. The compound of embodiment C38, wherein R²⁷ or R²⁸ is hydrogen, or R²⁷ and R²⁸ are hydrogen.

[0807] C40. The compound of embodiment 39, wherein R²⁷ is hydrogen and R²⁸ is C1-C3 alkyl, C1-C3 heteroalkyl, C1-C3 haloalkyl, C1-C3 perhaloalkyl, C1-C4 acyl, amido, C1-C3 alkylamino, C1-C3 alkylamino C1-C3 alkyl, amino, thiol, C1-C3 alkylthio, C1-C3 alkylthio C1-C3 alkyl, C1-C3 haloalkylthio, C1-C3 perhaloalkylthio, C5-C7 aryl, C5-C7 aryl C1-C3 alkyl, C5-C7 cycloalkyl, C5-C7 cycloalkyl C1-C3 alkyl, 5-7 membered heterocycloalkyl, 5-7 membered heterocycle C1-C3 alkyl, 5-7 membered heteroaryl, 5-7 membered heteroaryl C1-C3 alkyl, C1- C3 alkylsulfonyl, sulfonamide, C1-C3 alkylsulfonamido or C1-C3 alkylsilyloxy.

[0808] C41. The compound of embodiment C38 or C39, wherein R²⁷ is hydrogen and R²⁸ is - R³²-R³³.

[0809] C42. The compound of embodiment C41 , wherein R³² is a C1-C4 alkyl linker.

[0810] C43. The compound of embodiment C41 or C42, wherein R³² is an unsubstituted C3 alkyl linker.

[0811] C44. The compound of any one of embodiments C41-C43, wherein R³³ is a C5-C7 cycloalkyl or a 5-7 membered heterocycloalkyl. [0812] C45. The compound of any one of embodiments C41-C44, wherein R³³ is a 5-7 membered heteroaryl or a 5-7 membered heterocycloalkyl.

[0813] C46. The compound of embodiment C44 or C45, wherein R³³ is a 5-7 membered heterocycloalkyl.

[0814] C47. The compound of embodiment C46, wherein R³³ has a structure of Formula F-2:

Formula F-2 wherein:

[0815] C48. The compound of embodiment C47, wherein R³³ has a structure of Formula G-2:

Formula G-2 wherein:

[0816] C49. The compound of embodiment C48, wherein R³³ has a structure of Formula H-2:

Formula H-2 wherein:

[0817] C50. The compound of embodiment C49, wherein R³³ has a structure of Formula J-2:

perhaloalkyl.

[0818] C51. The compound of any one of embodiments C1-C50, wherein R²⁴ is a C5-C7 cycloalkyl, 5-7 membered heterocycloalkyl, C5-C7 aryl or 5-7 membered heteroaryl, which independently is optionally substituted by one or more substituents chosen from halogen, hydroxy, C1-C3 alkyl, C1-C3 heteroalkyl, C1-C3 haloalkyl, C1-C3 perhaloalkyl, C1 -C3 perhaloalkoxy, C1-C3 alkoxy, C1-C3 haloalkoxy, C1-C3 alkoxy C1-C3 alkyl, C1-C4 acyl, oxo, C1-C4 acyloxy, carboxyl, amido, cyano, amino, C1-C3 alkylamino, C1-C3 alkylamino C1-C3 alkyl, thiol, C1-C3 alkylthio, C1-C3 alkylthio C1-C3 alkyl, C1-C3 haloalkylthio, C1-C3 perhaloalkylthio, nitro, C5-C7 aryl, C5-C7 aryl C1-C3 alkyl, C5-C7 cycloalkyl, C5-C7 cycloalkyl C1-C3 alkyl, 5-7 membered heterocycloalkyl, 5-7 membered heterocycle C1-C3 alkyl, 5-7 membered heteroaryl, 5-7 membered heteroaryl C1 -C3 alkyl, C1 -C3 alkylsulfonyl, sulfonamide, C1 -C3 alkylsulfonamido and C1 -C3 alkylsilyloxy.

[0819] C52. The compound of any one of embodiments C2-C51 , wherein R^{21 B} is hydrogen and R^21A is hydroxy, -R^G-R²⁴ - R^G-R²⁴-R²⁵ or - R^G-R²⁴-R²⁶-R²⁵.

[0820] C53. The compound of any one of embodiments C1 -C52, wherein R²¹ , R^{21 B} or R^{21 B} is - R^G-R²⁴-R²⁶-R²⁵.

[0821] C54. The compound of any one of embodiments C1 -C53, wherein R^G is -O-C(O)-.

[0822] C55. The compound of embodiment C53 or C54, wherein R²⁴ is a C5-C7 aryl or a 5-7 membered heteroaryl.

[0823] C56. The compound of embodiment C55, wherein R²⁴ has a structure of Formula C-2:

Formula C-2

wherein:

one, two or three of R⁴¹ , R⁴², R⁴³, R⁴⁴ and R⁴⁵ independently is R²⁶-R²⁵, and each of the remaining R⁴¹, R⁴², R⁴³, R⁴⁴ and R⁴⁵ independently is not present or independently is hydrogen, halogen, hydroxy, C1 -C3 alkyl, C1 -C3 heteroalkyl, C1 -C3 haloalkyl, C1 -C3 perhaloalkyl, C1 -C3 perhaloalkoxy, C1 -C3 alkoxy, C1 -C3 haloalkoxy, C1 -C3 alkoxy C1-C3 alkyl, C1 -C4 acyl, oxo, C1-C4 acyloxy, carboxyl, amido, cyano, amino, C1 -C3 alkylamino, C1-C3 alkylamino C1 -C3 alkyl, thiol, C1 -C3 alkylthio, C1 -C3 alkylthio C1 -C3 alkyl, C1 -C3 haloalkylthio, C1 -C3 perhaloalkylthio, nitro, C5-C7 aryl, C5-C7 aryl C1 -C3 alkyl, C5-C7 cycloalkyl, C5-C7 cycloalkyl C1 -C3 alkyl, 5-7 membered heterocycloalkyl, 5-7 membered heterocycle C1 -C3 alkyl, 5-7 membered heteroaryl, 5-7 membered heteroaryl C1 -C3 alkyl, C1 -C3 alkylsulfonyl, sulfonamide, C1 -C3 alkylsulfonamido or C1 -C3 alkylsilyloxy.

[0824] C57. The compound of embodiment C56, wherein R²⁴ has a structure of Formula D-2:

Formula D-2 wherein one, two or three of R⁴¹ , R⁴², R⁴³, R⁴⁴ and R⁴⁵ independently is R²⁶-R²⁵, and each of the remaining R⁴¹ , R⁴², R⁴³, R⁴⁴ and R⁴⁵ independently is hydrogen, halogen, hydroxy, C1 -C3 alkyl, C1 -C3 heteroalkyl, C1 -C3 haloalkyl, C1 -C3 perhaloalkyl, C1 -C3 perhaloalkoxy, C1 -C3 alkoxy, C1 -C3 haloalkoxy, C1-C3 alkoxy C1 -C3 alkyl, C1 -C4 acyl, oxo, C1 -C4 acyloxy, carboxyl, amido, cyano, amino, C1 -C3 alkylamino, C1 -C3 alkylamino C1 -C3 alkyl, thiol, C1 -C3 alkylthio, C1 -C3 alkylthio C1 -C3 alkyl, C1-C3 haloalkylthio, C1 -C3 perhaloalkylthio, nitro, C5-C7 aryl, C5-C7 aryl C1 -C3 alkyl, C5-C7 cycloalkyl, C5-C7 cycloalkyl C1 -C3 alkyl, 5-7 membered heterocycloalkyl, 5-7 membered heterocycle C1-C3 alkyl, 5-7 membered heteroaryl, 5-7 membered heteroaryl C1-C3 alkyl, C1-C3 alkylsulfonyl, sulfonamide, C1 -C3 alkylsulfonamido or C1 -C3 alkylsilyloxy.

[0825] C58. The compound of embodiment C57, wherein R²⁴ has a structure of Formula E-2:

Formula E-2 wherein R⁴³ is -R²⁶-R²⁵.

[0826] C59. The compound of any one of embodiments C1 -C58, wherein R²⁶ is a C1 -C3 alkyl linker.

[0827] C60. The compound of embodiment C59, wherein R²⁶ is a methylene linker.

[0828] C61 . The compound of any one of embodiments C1 -C60, wherein R²⁵ is a 5-7 membered heterocycloalkyl. [0829] C62. The compound of embodiment C61 , wherein R²⁵ has a structure of Formula F-3:

Formula F-3 wherein:

[0830] C63. The compound of embodiment C62, wherein R²⁵ has a structure of Formula G-3:

Formula G-3 wherein:

independently is hydrogen, halogen, hydroxy, C1-C3 alkyl, C1-C3 heteroalkyl, C1-C3 haloalkyl, C1-C3 perhaloalkyl, C1-C3 perhaloalkoxy, C1-C3 alkoxy, C1-C3 haloalkoxy, C1-C3 alkoxy C1- C3 alkyl, C1-C4 acyl, oxo, C1-C4 acyloxy, carboxyl, amido, cyano, amino, C1-C3 alkylamino, C1-C3 alkylamino C1-C3 alkyl, thiol, C1-C3 alkylthio, C1-C3 alkylthio C1-C3 alkyl, C1-C3 haloalkylthio, C1-C3 perhaloalkylthio, nitro, C5-C7 aryl, C5-C7 aryl C1-C3 alkyl, C5-C7 cycloalkyl, C5-C7 cycloalkyl C1-C3 alkyl, 5-7 membered heterocycloalkyl, 5-7 membered heterocycle C1-C3 alkyl, 5-7 membered heteroaryl, 5-7 membered heteroaryl C1-C3 alkyl, C1- C3 alkylsulfonyl, sulfonamide, C1-C3 alkylsulfonamido or C1-C3 alkylsilyloxy; and

[0831] C64. The compound of embodiment C33, wherein R²⁵ has a structure of Formula H-3:

Formula H-3 wherein:

R⁶² is hydrogen, C1-C3 alkyl, C1-C3 heteroalkyl, C1-C3 haloalkyl, C1-C3 perhaloalkyl, C1-C4 acyl, amido, C1-C3 alkylamino, C1-C3 alkylamino C1-C3 alkyl, amino, thiol, C1-C3 alkylthio, C1-C3 alkylthio C1-C3 alkyl, C1-C3 haloalkylthio, C1-C3 perhaloalkylthio, C5-C7 aryl, C5-C7 aryl C1-C3 alkyl, C5-C7 cycloalkyl, C5-C7 cycloalkyl C1-C3 alkyl, 5-7 membered heterocycloalkyl, 5-7 membered heterocycle C1-C3 alkyl, 5-7 membered heteroaryl, 5-7 membered heteroaryl C1-C3 alkyl, C1-C3 alkylsulfonyl, sulfonamide, C1-C3 alkylsulfonamido or C1-C3 alkylsilyloxy.

[0832] C65. The compound of embodiment C64, wherein R²⁵ has a structure of Formula J-3:

perhaloalkyl.

[0833] C66. The compound of any one of embodiments C17.1 , C17.6 and C17.7, wherein R' is -NH-C(O)-.

[0834] C67. The compound of embodiment C66, wherein R³⁴ is amino.

[0835] C68. The compound of embodiment C67, wherein R²², R^22A or R^22B is hydroxy.

[0836] DO. A composition comprising a compound of any one of embodiments A1-A76, B1- B34, C1-C65 and E1-E23 and a pharmaceutically acceptable carrier or diluent.

[0837] D1. A method for administering a compound, comprising:

administering a compound of any one of embodiments A1-A76, B1-B34, C1-C65 and E1-E23 to a composition comprising cells, wherein the cells contain a polynucleotide that encodes a polypeptide comprising a domain that binds to a compound of any one of embodiments A1-A76, B1-B34, C1-C65 and E1-E23.

[0838] D2. The method of embodiment D2, wherein the compound is administered to the composition comprising the cells ex vivo.

[0839] D3. A method of administration, comprising: administering a composition comprising a compound of any one of embodiments A1- A76, B1-B34, C1-C65 and E1-E23 to a subject in need thereof.

[0840] D4. The method of embodiment D3, wherein cells in the subject contain a

polynucleotide that encodes a polypeptide comprising a domain that binds to a compound of any one of embodiments A1-A76, B1-B34 and C1-C65.

[0841] D5. The method of embodiment D3, comprising:

modifying cells ex vivo with a polynucleotide that encodes a polypeptide comprising a domain that binds to a compound of any one of embodiments A1-A76, B1-B34, C1-C65 and E1- E23, thereby generating modified cells;

administering the modified cells to a subject; and

administering a composition comprising a compound of any one of embodiments A1- A76, B1-B34, C1-C65 and E1-E23 to the subject.

[0842] D6. A method for treating a subject having a medical condition, comprising:

administering a composition comprising a compound of any one of embodiments A1- A76, B1-B34, C1-C65 and E1-E23 to a subject in need thereof in an amount effective to treat the medical condition.

[0843] D6.1. A method for regulating treatment of a medical condition, comprising:

administering a composition comprising a compound of any one of embodiments A1- A76, B1-B34, C1-C65 and E1-E23 to a subject being treated for a medical condition, thereby regulating the treatment thereof.

[0844] D6.2. The method of embodiment D6.1 , wherein the medical condition is associated with cancer, graft versus host disease, cytokine storms, tumor lysis syndrome, cytokine release syndrome, macrophage activation syndrome and/or an adverse event in connection with therapeutic cell treatment.

[0845] D6.3. A method of reducing the number and/or viability of, or eliminating, specific cells, comprising administering a composition comprising a compound of any one of embodiments A1-A76, B1-B34, C1-C65 and E1-E23 to a composition containing cells that contain nucleic acid encoding a polypeptide to which the compound binds, wherein the number and/or viability of the cells is reduced.

[0846] D6.4. A method of increasing the number and/or viability of specific cells, comprising administering a composition comprising a compound of any one of embodiments A1- A76, B1-B34, C1-C65 and E1-E23 to a composition containing cells that contain nucleic acid encoding a polypeptide to which the compound binds, wherein the number and/or viability of the cells is increased.

[0847] D6.5. The method of D6.3, wherein the composition containing the cells is a subject.

[0848] D7. The method of any of embodiments D6, D6.1 and D6.2, wherein cells in the subject contain a polynucleotide that encodes a polypeptide comprising a binding domain that binds to a compound of any one of embodiments A1-A76, B1-B34, C1-C65 and E1-E23.

[0849] D8. The method of any of embodiments D6-D7 comprising:

administering the modified cells to a subject; and

[0850] D9. The method of any one of embodiments D1-D8, wherein the polypeptide is a modified polypeptide.

[0851] D10. The method of any one of embodiments D1-D9, wherein the cells contain a first chimeric polypeptide.

[0852] D11. The method of embodiment D10, wherein:

the first chimeric polypeptide comprises a first signaling domain and a first binding domain; and

the first binding domain binds to a compound of any one of embodiments A1-A76, B1- B34, C1-C65 and E1-E23.

[0853] D12. The method of embodiment D1 1 , wherein:

the cells contain a second chimeric polypeptide;

the second chimeric polypeptide comprises a second signaling domain and a second binding domain;

the second binding domain binds to a second multimerizing agent; and the compound binds to the first binding domain with greater affinity than it binds to the second binding domain.

[0854] D13. The method of embodiment D12, wherein the compound binds to the first binding domain with at least 100 times greater affinity than it binds to the second binding domain.

[0855] D14. The method of embodiment D12 or D13, wherein the second multimerizing agent is a homodimerizer agent. [0856] D15. The method of embodiment D14, wherein the homodimerizer agent is rimiducid,

AP20187 or AP1510.

[0857] D15.1 The method of embodiment D14, wherein the homodimerizer agent is a compound of Formula I or Formula II:

wherein:

Z and Z’ are the same or different and each independently is O, NR¹², -N=, S, SO, S0₂ or CH₂;

Y is L, M or Q:

when Y is Q, R³ and R⁴ together with N⁺ may form a heterocyclic or heteroaryl ring optionally substituted with one or more substituents chosen from halogen, hydroxy, alkyl, heteroalkyl, haloalkyl, perhaloalkyl, perhaloalkoxy, alkoxy, haloalkoxy, alkoxyalkyl, acyl, oxo, acyloxy, carboxyl, amido, cyano, amino, alkylamino, alkylaminoalkyl, thiol, alkylthio,

alkylthioalkyl, haloalkylthio, perhaloalkylthio, nitro, aryl, arylalkyl, heterocycle-alkyl, cycloalkylalkyl, cycloalkyl, heterocycloalkyl, heteroaryl, heteroarylalkyl, alkylsulfonyl, sulfonamide, alkylsulfonamido and alkylsilyloxy;

when Y is Q, R¹ and R³ together with -N⁺-RL-N⁺- may form a heterocyclic or heteroaryl ring optionally substituted with one or more substituents chosen from halogen, hydroxy, alkyl, heteroalkyl, haloalkyl, perhaloalkyl, perhaloalkoxy, alkoxy, haloalkoxy, alkoxyalkyl, acyl, oxo, acyloxy, carboxyl, amido, cyano, amino, alkylamino, alkylaminoalkyl, thiol, alkylthio, alkylthioalkyl, haloalkylthio, perhaloalkylthio, nitro, aryl, arylalkyl, heterocycle-alkyl,

when Y is Q, R² and R⁴ together with -N⁺-R_L-N⁺- may form a heterocyclic or heteroaryl ring optionally substituted with one or more substituents chosen from halogen, hydroxy, alkyl, heteroalkyl, haloalkyl, perhaloalkyl, perhaloalkoxy, alkoxy, haloalkoxy, alkoxyalkyl, acyl, oxo, acyloxy, carboxyl, amido, cyano, amino, alkylamino, alkylaminoalkyl, thiol, alkylthio, alkylthioalkyl, haloalkylthio, perhaloalkylthio, nitro, aryl, arylalkyl, heterocycle-alkyl,

Optionally, when Y is Q, one of the groups: R¹ , R², R³ and R⁴ may be nonexistent. If one of the groups: R¹, R², R³ or R⁴ is non-existent, and Y is Q, the compound is a monosalt;

A and A’ are the same or different and each independently are

thiophene, furan, pyrrole, carbonyl, lower dialkyl ether, lower dialkyl thioether, lower dialkylamino, cyclopropylene, alkanylene, cycloalkanylene, alkenylene, cycloalkenylene, lower alkynylene, lower cycloalkynylene, carbamate, sulfanyl, sulfinyl, sulfonyl, thiocarbonyl, imino, or hydroxyimino, in which independently none or one or more carbon atoms are replaced by O, NR¹⁴, S, SO, S0₂, and which is optionally substituted with hydroxyl, alkoxyl, amino, alkylamino, thiol, thioalkyl, or halogen;

R¹⁰ and R¹¹ independently are hydrogen or C1-C2 alkyl;

with the proviso that (i) Y is M or Q when A and A’ are phenyl and Z and Z' are oxygen, or (ii) A and A' are not the same, or one or both of A and A' are not phenyl, or Z and Z' are not the same, or one or both of Z and Z' are not oxygen, when Y is L and R_L is -CH2-CH2-.

[0858] D15.2 The method of embodiment D14, wherein the homodimerizer agent is a compound having the following structure:

[0859] D16. The method of any one of embodiments D1-D15.2, wherein the cells are immune cells.

[0860] D16.2 The method of any one of embodiments D1-D15.2, wherein the cells are chosen from T cells, tumor infiltrating lymphocytes, NK-T cells or NK cells.

[0861] D17. The method of embodiment D16.2, wherein the T-cells are cytotoxic T cells or helper T cells.

[0862] D18. The method of embodiment D16 or D17, wherein the T cells are polyclonal T cells.

[0863] D18.1. The method of any one of embodiments D3-D18, wherein the cells are autologous cells.

[0864] D18.2. The method of any one of embodiments D3-D18, wherein the cells are allogenic cells.

[0865] D19. The method of any one of embodiments D1-D18.2, wherein the cells contain a polynucleotide that encodes a chimeric antigen receptor (CAR) or a T-cell receptor.

[0866] D20. The method of embodiment, D19, wherein the CAR comprises a transmembrane region, a cell activation region, and an antigen recognition region.

[0867] D21. The method of embodiment D20, wherein the cell activation region is a T-cell activation region.

[0868] D22. The method of embodiment D21 , wherein the T-cell activation region is a CD3 zeta-chain region. [0869] D23. The method of any one of embodiments D20-D22, wherein the antigen recognition region specifically binds to a molecule chosen from PSMA, PSCA, Mud CD19, ROR1 , Mesothelin, GD2, CD123, Muc16, CD33, CD38, CD44v6, and Her2/Neu.

[0870] D24. The method of any one of embodiments D10-D23, wherein the first signaling domain comprises a cell activation domain.

[0871] D25. The method of any one of embodiments D10-D23, wherein the first signaling domain comprises a pro-apoptotic signaling domain.

[0872] D26. The method of any one of embodiments D12-D23, wherein the first signaling domain comprises a cell activation domain and the second signaling domain comprises a pro- apoptotic signaling domain.

[0873] D27. The method of any one of embodiments D12-D23, wherein the first signaling domain comprises a pro-apoptotic signaling domain and the second signaling domain comprises a cell activation domain.

[0874] D28. The method of any one of embodiments D24-D27, wherein the cell activation domain comprises a MyD88 polypeptide or a portion thereof.

[0875] D28.1 The method of embodiment D28, wherein the cell activation domain comprises a truncated MyD88 polypeptide lacking the TIR domain.

[0876] D29. The method of embodiment D28 or D28.1 , wherein the MyD88 polypeptide is a human polypeptide.

[0877] D30. The method of embodiment D28 or D28.1 , wherein the MyD88 polypeptide or portion thereof has an amino acid sequence of SEQ ID NO: 101 , SEQ ID NO: 102 or SEQ ID NO: 103 or an amino acid sequence 90% or more identical to SEQ ID NO: 101 , SEQ ID NO:

102 or SEQ ID NO: 103.

[0878] D31. The method of embodiment D30, wherein the MyD88 polypeptide or portion thereof has an amino acid sequence of SEQ ID NO: 101 , SEQ ID NO: 102 or SEQ ID NO: 103 or an amino acid sequence 95% or more identical to SEQ ID NO: 101 , SEQ ID NO: 102 or SEQ ID NO: 103. [0879] D32. The method of embodiment D28.1 , wherein the truncated MyD88 polypeptide has the amino acid sequence of SEQ ID NO: 102 or SEQ ID NO: 103.

[0880] D33. The method of any one of embodiments D24-D32, wherein the cell activation domain comprises a CD40 polypeptide or portion thereof.

[0881] D33.1. The method of embodiment D33, wherein the cell activation domain comprises a CD40 cytoplasmic polypeptide and lacks the CD40 extracellular domain.

[0882] D33.2. The method of embodiment D33 or D33.1 , wherein the CD40 polypeptide is a human polypeptide.

[0883] D33.3 The method of embodiment D33, wherein the CD40 polypeptide has the amino acid sequence of SEQ ID NO: 104 or an amino acid sequence 90% or more identical to SEQ ID NO: 104.

[0884] D34. The method of embodiment D33.1 , wherein the CD40 cytoplasmic polypeptide has the amino acid sequence of SEQ ID NO: 105 or an amino acid sequence 90% or more identical to SEQ ID NO: 105.

[0885] D35. The method of embodiment D34, wherein the CD40 cytoplasmic polypeptide has the amino acid sequence of SEQ ID NO: 105 or an amino acid sequence 95% or more identical to SEQ ID NO: 105.

[0886] D36. The method of embodiment D35, wherein the CD40 cytoplasmic polypeptide has the amino acid sequence of SEQ ID NO: 105.

[0887] D37. The method of any one of embodiments D24-D36, wherein:

the cell activation domain comprises a stimulating polypeptide chosen from CD27,

CD28, ICOS, 4-1 BB, RANK/TRANCE-R and 0X40; and

the stimulating polypeptide includes no extracellular domain or no functional extracellular domain.

[0888] D38. The method of any one of embodiments D24-D37, wherein the pro-apoptotic domain comprises a Caspase-9 polypeptide or portion thereof. [0889] D38.1. The method of embodiment D38, wherein the pro-apoptotic domain comprises a caspase-9 polypeptide lacking one or more caspase recruitment domains (CARD).

[0890] D38.2. The method of embodiment D38 or D38.1 , wherein the caspase-9 polypeptide is a human polypeptide.

[0891] D38.3. The method of embodiment D38 or D38.1 , wherein the caspase-9 polypeptide has the amino acid sequence of SEQ ID NO: 146 or an amino acid sequence 90% or more identical to SEQ ID NO: 146.

[0892] D39. The method of embodiment D38, wherein the Caspase-9 polypeptide has the amino acid sequence of SEQ ID NO: 113 or SEQ ID NO: 1 14 or an amino acid sequence 90% or more identical to SEQ ID NO: 1 13 or SEQ ID NO: 1 14.

[0893] D40. The method of embodiment D39, wherein the Caspase-9 polypeptide has the amino acid sequence of SEQ ID NO: 113 or SEQ ID NO: 1 14 or an amino acid sequence 95% or more identical to SEQ ID NO: 1 13 or SEQ ID NO: 1 14.

[0894] D41. The method of embodiment D40, wherein the Caspase-9 polypeptide has the amino acid sequence of SEQ ID NO: 113 or SEQ ID NO: 1 14.

[0895] D42. The method of any one of embodiments D10-D41 , wherein the first binding domain comprises a FKBP12 region, a FKBP12 variant region, a FKBP12-Rapamycin Binding (FRB) region, a FRB variant region, or combination thereof.

[0896] D43. The method of any one of embodiments D10-D42, wherein the first binding domain comprises a first multimerizing region and a second multimerizing region.

[0897] D44. The method of embodiment D43, wherein the first multimerizing region comprises a FKBP12 region or a FKBP12 variant region, and the second multimerizing region comprises a FKBP12-Rapamycin Binding (FRB) region or a FRB variant region.

[0898] D44.1. The method of embodiment D44, wherein the first multimerizing region comprises a FKBP12 region and the second multimerizing region comprises a FRB region. [0899] D44.2. The method of embodiment D44, wherein the first multimerizing region comprises a FKBP12 region and the second multimerizing region comprises a FRB variant region.

[0900] D44.3. The method of any one of embodiments D43-D44.2, wherein

the cells contain a complex comprising two first chimeric polypeptide molecules;

the compound binds to the first multimerizing region of one of the first chimeric polypeptide molecules in the complex; and

the compound binds to the second multimerizing region of the other first chimeric polypeptide molecule in the complex.

[0901] D44.4. The method of any one of embodiments D43-D44.3, wherein

the compound comprises a first portion and a second portion;

the first portion of the compound binds to the first multimerizing region and the second portion binds to the second multimerizing region.

[0902] D45. The method of any one of embodiments D42-D44.4, wherein the FKBP12 region is from a human protein.

[0903] D45.1 The method of any one of embodiments D42-D44.4, wherein the FKBP12 region has the amino acid sequence of SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 89, or SEQ ID NO: 90 or an amino acid sequence 90% or more identical to SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 89, or SEQ ID NO: 90.

[0904] D46. The method of embodiment D45.1 , wherein the FKBP12 region has the amino acid sequence of SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 89, or SEQ ID NO: 90 or an amino acid sequence 95% or more identical to SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 89, or SEQ ID NO: 90.

[0905] D47. The method of embodiment D46, wherein the FKBP12 region has the amino acid sequence of SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 89, or SEQ ID NO: 90.

[0906] D48. The method of any one of embodiments D42-D47, wherein the FKBP12 variant region has the amino acid sequence of the FKBP12 region of embodiment D45 or D46 with an amino acid substitution at position 36 based on the amino acid numbering of SEQ ID NO: 86 chosen from valine, leucine, isoleuceine and alanine. [0907] D49. The method of embodiment D48, wherein the FKBP12 variant region has the amino acid sequence of SEQ ID NO: 11 1.

[0908] D50. The method of any one of embodiments D42-D49, wherein the FRB region is from a human protein.

[0909] D50.1. The method of any one of embodiments D42-D49, wherein the FRB region has the amino acid sequence of SEQ ID NO: 77 or SEQ ID NO: 78 or an amino acid sequence 90% or more identical to SEQ ID NO: 77 or SEQ ID NO: 78.

[0910] D51. The method of embodiment D50.1 , wherein the FRB region has the amino acid sequence of SEQ ID NO: 77 or SEQ ID NO: 78 or an amino acid sequence 95% or more identical to SEQ ID NO: 77 or SEQ ID NO: 78.

[0911] D52. The method of embodiment D51 , wherein the FRB region has the amino acid sequence of SEQ ID NO: 77 or SEQ ID NO: 78.

[0912] D53. The method of any one of embodiments D50-D52, wherein the FRB variant region has the amino acid sequence of the FRB region of embodiment D50, D50.1 , D51 or D52 with an amino acid substitution at position 2098 relative to SEQ ID NO: 76 chosen from valine, leucine and isoleuceine.

[0913] D54. The method of embodiment D53, wherein the FRB variant region has the amino acid sequence of SEQ ID NO: 79, SEQ ID NO: 80 or SEQ ID NO: 81.

[0914] E1. A compound having a structure of the following Formula A:

Formula A or a pharmaceutically acceptable salt thereof, wherein:

R²⁰ is -R²³ or -R^F-R²³;

R²² is halogen, -N₃, -NR²⁷R²⁸, -R^H-R²⁹, -R^H-R²⁹-R³⁰ or -R^H-R²⁹-R³¹-R³⁰;

n is 1 or 2;

R²³ independently is cycloalkyl, aryl, heterocycloalkyl or heteroaryl, which independently is optionally substituted by one or more substituents chosen from halogen, hydroxy, alkyl, alkenyl, alkynyl, heteroalkyl, haloalkyl, perhaloalkyl, perhaloalkoxy, alkoxy, haloalkoxy, alkoxyalkyl, acyl, oxo, acyloxy, carboxyl, amido, cyano, amino, alkylamino, alkylaminoalkyl, thiol, alkylthio, alkylthioalkyl, haloalkylthio, perhaloalkylthio, nitro, aryl, arylalkyl, cycloalkylalkyl, heterocycle- alkyl, cycloalkyl, heterocycloalkyl, heteroaryl, heteroarylalkyl, alkylsulfonyl, sulfonamide, alkylsulfonamido, alkylsilyloxy, alkylsulfinamido, arylsulfinamido, and arylsulfonamido;

alkylthioalkyl, haloalkylthio, perhaloalkylthio and nitro; and

R²⁷ and R²⁸ each independently is -R³²-R³³, or hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, perhaloalkyl, alkoxy, cycloalkyl, aryl, cycloalkyl, heterocycloalkyl, heterocycle-alkyl or heteroaryl, which independently is optionally substituted with one or more substituents chosen from halogen, hydroxy, alkyl, alkenyl, alkynyl, heteroalkyl, haloalkyl, perhaloalkyl, perhaloalkoxy, alkoxy, haloalkoxy, alkoxyalkyl, acyl, oxo, acyloxy, carboxyl, amido, cyano, amino, alkylamino, alkylaminoalkyl, thiol, alkylthio, alkylthioalkyl, haloalkylthio, perhaloalkylthio, nitro, aryl, arylalkyl, cycloalkylalkyl, heterocycle-alkyl, cycloalkyl, heterocycloalkyl, heteroaryl, heteroarylalkyl, alkylsulfonyl, sulfonamide, alkylsulfonamido and alkylsilyloxy.

[0915] E2. A compound having a structure of the following Formula B:

Formula B or a pharmaceutically acceptable salt thereof, wherein:

R³⁰;

n is 1 or 2;

alkylthioalkyl, haloalkylthio, perhaloalkylthio and nitro; and

[0916] E3. The compound of embodiment E 1 or E2, wherein R²³ is a C5-C7 cycloalkyl, C5- C7 aryl, 5-7 membered heterocycloalkyl or 5-7 membered heteroaryl.

[0917] E4. The compound of embodiment E3, wherein the C5-C7 cycloalkyl, C5-C7 aryl, 5-7 membered heterocycloalkyl or 5-7 membered heteroaryl each independently is optionally substituted by one or more substituents chosen from halogen, hydroxy, C1-C3 alkyl, C1-C3 heteroalkyl, C1-C3 haloalkyl, C1-C3 perhaloalkyl, C1-C3 perhaloalkoxy, C1-C3 alkoxy, C1-C3 haloalkoxy, C1-C3 alkoxy C1-C3 alkyl, C1-C4 acyl, oxo, C1-C4 acyloxy, carboxyl, amido, cyano, amino, C1-C3 alkylamino, C1-C3 alkylamino C1-C3 alkyl, thiol, C1-C3 alkylthio, C1-C3 alkylthio C1-C3 alkyl, C1-C3 haloalkylthio, C1-C3 perhaloalkylthio, nitro, C5-C7 aryl, C5-C7 aryl C1-C3 alkyl, C5-C7 cycloalkyl, C5-C7 cycloalkyl C1-C3 alkyl, 5-7 membered heterocycloalkyl, 5-7 membered heterocycle C1-C3 alkyl, 5-7 membered heteroaryl, 5-7 membered heteroaryl C1-C3 alkyl, C1-C3 alkylsulfonyl, sulfonamide, C1-C3 alkylsulfonamido and C1-C3 alkylsilyloxy.

[0918] E5. The compound of any one of embodiments E1-E4, wherein R²³ is a C5-C7 aryl.

[0919] E6. The compound of embodiment E1 or E2, wherein R²³ has a structure of Formula C-3:

Formula C-3

wherein:

[0920] E7. The compound of embodiment E1 or E2, wherein R²³ has a structure of Formula D-3:

[0921] E8. The compound of embodiment E1 or E2, wherein R²³ has a structure of Formula E- 3:

[0922] E9. The compound of embodiment E8, wherein R⁵¹ and R⁵³ each are methoxy.

[0923] E10. The compound of any one of embodiments E1-E9, wherein R²⁰, R^20A, or R^20B is - R²³.

[0924] E1 1. The compound of embodiment E10, wherein R^20A is hydrogen and R^20B is -R²³, or wherein R^20B is hydrogen and R^20A is -R²³.

[0925] E12. The compound of embodiment E11 , wherein R^20A is hydrogen and R^20B is -R²³.

[0926] E13. The compound of any one of embodiments E1-E9, wherein R²⁰, R^20A, or R^20B is - R^F-R²³. [0927] E14. The compound of embodiment E13, wherein R^20A is hydrogen and R^20B is -R^F- R²³, or wherein R^20B is hydrogen and R^20A is -R^F-R²³.

[0928] E15. The compound of embodiment E14, wherein R^20A is hydrogen and R^20B is -R^F- R²³.

[0929] E16. The compound of any one of embodiments E13-E15, wherein R^F is -O-, -C(O)-, -O-C(O)- or -C(0)-0-.

[0930] E17. The compound of any one of embodiments E13-E15, wherein R^F is -0-.

[0931] E18. The compound of any one of embodiments E13-E15, wherein R^F is -NH-S(O)-.

[0932] E19. The compound of any one of embodiments E1 -E18, wherein R²², R^22A or R^22B is -

NR²⁷R²⁸ or N₃.

[0933] E20. The compound of embodiment E19, wherein R^22A is hydrogen and R^22B is - NR²⁷R²⁸ or N₃, or wherein R^22B is hydrogen and R^22A is -NR²⁷R²⁸ or N₃.

[0934] E21 . The compound of embodiment E19 or E20, wherein R²⁷ and R²⁸ are each hydrogen.

[0935] E22. The compound of embodiment E1 or E2, wherein:

R²⁰ , R^20A or R^20B is -R²³;

R²¹ _R2IA _{or R}2IB _js hydrogen or hydroxy;

R²², _R22A _{o r R}22B jg [^· _{a n d}

R²³ is aryl substituted with alkoxy.

[0936] E23. The compound of embodiment E1 or E2, wherein:

R²⁰, R^20A or R^20B is -R²³;

R²¹ _R2IA _{or R}2IB j_S hydrogen or hydroxy;

R²², R^22A or R^22B is -NR²⁷R²⁸;

R²³ is aryl substituted with alkoxy; and

R²⁷ and R²⁸ are hydrogen. [0937] F. A kit or combination comprising a compound of any one of embodiments A1-A76, B1-B34, C1-C65 and E1-E23.

[0938] F 1. The kit or combination of embodiment F, further comprising nucleic acids encoding one or more chimeric polypeptides.

[0939] F2. The kit or combination of embodiment F, further comprising cells containing nucleic acids encoding one or more chimeric polypeptides.

[0940] F3. The kit or combination of embodiment F, further comprising one or more chimeric polypeptides.

[0941] F4. The kit or combination of any one of embodiments F-F3, wherein one of the one or more chimeric polypeptides comprises one or more polypeptides to which a compound of any one of embodiments A1-A76, B1-B34, C1-C65 and E1-E23 binds.

[0942] F5. The kit or combination of embodiment F3 or F4, wherein one of the one or more chimeric polypeptides comprises an FRB wild type protein, or portion thereof, an FRB variant protein, or portion thereof, an FKBP12 wild type protein, or portion thereof, and/or an FKBP12 variant protein, or portion thereof.

[0943] F6. The kit or combination of embodiment F5, wherein one of the one or more chimeric polypeptides comprises an FRB wild type protein, or portion thereof, or an FRB variant protein, or portion thereof, and an a FKBP12 wild type protein, or portion thereof, or an FKBP12 variant protein, or portion thereof.

[0944] F7. The kit or combination of embodiment F5, wherein one of the one or more chimeric polypeptides comprises an FRB variant protein, or portion thereof, and an a FKBP12 wild type protein, or portion thereof.

[0945] F7.1. The kit or combination of embodiment F5, comprising two or more chimeric polypeptides wherein at least one of the chimeric polypeptides comprises an FRB variant protein, or portion thereof, and an a FKBP12 wild type protein, or portion thereof, and at least one of the chimeric polypeptides comprises an FKBP12 variant protein, or portion thereof. [0946] F8. The kit or combination of any of embodiments F5-F7.1 , wherein a wild type protein, or portion thereof, and/or a variant protein, or portion thereof, of one of the one or more chimeric polypeptides is fused to a cell activation or cell elimination polypeptide.

[0947] F9. The kit or combination of any of embodiments F5-F7.1 , wherein the chimeric polypeptide further comprises a cell activation or cell elimination polypeptide.

[0948] F 10. The kit or combination of embodiment F8 or F9, wherein the cell activation polypeptide comprises a MyD88 polypeptide or a portion thereof.

[0949] F 1 1. The kit or combination of embodiment F10, wherein the cell activation domain comprises a truncated MyD88 polypeptide lacking the TIR domain.

[0950] F 12. The kit or combination of embodiment F10 or F1 1 , wherein the MyD88 polypeptide is a human polypeptide.

[0951] F13. The kit or combination of embodiment F10 or F1 1 , wherein the MyD88 polypeptide or portion thereof has an amino acid sequence of SEQ ID NO: 101 , SEQ ID NO: 102 or SEQ ID NO: 103 or an amino acid sequence 90% or more identical to SEQ ID NO: 101 , SEQ ID NO: 102 or SEQ ID NO: 103.

[0952] F 14. The kit or combination of embodiment F13, wherein the MyD88 polypeptide or portion thereof has an amino acid sequence of SEQ ID NO: 101 , SEQ ID NO: 102 or SEQ ID NO: 103 or an amino acid sequence 95% or more identical to SEQ ID NO: 101 , SEQ ID NO: 102 or SEQ ID NO: 103.

[0953] F 15. The kit or combination of embodiment F1 1 , wherein the truncated MyD88 polypeptide has the amino acid sequence of SEQ ID NO: 102 or SEQ ID NO: 103.

[0954] F 16. The kit or combination of any one of embodiments F9-F 15, wherein the cell activation polypeptide comprises a CD40 polypeptide or portion thereof.

[0955] F 17. The kit or combination of embodiment F16, wherein the cell activation domain comprises a CD40 cytoplasmic polypeptide and lacks the CD40 extracellular domain.

[0956] F 18. The kit or combination of embodiment F16 or F17, wherein the CD40 polypeptide is a human polypeptide. [0957] F 19. The kit or combination of embodiment F16, wherein the CD40 polypeptide has the amino acid sequence of SEQ ID NO: 104 or an amino acid sequence 90% or more identical to SEQ ID NO: 104.

[0958] F20. The kit or combination of embodiment F17, wherein the CD40 cytoplasmic polypeptide has the amino acid sequence of SEQ ID NO: 105 or an amino acid sequence 90% or more identical to SEQ ID NO: 105.

[0959] F21. The kit or combination of embodiment F20, wherein the CD40 cytoplasmic polypeptide has the amino acid sequence of SEQ ID NO: 105 or an amino acid sequence 95% or more identical to SEQ ID NO: 105.

[0960] F22. The kit or combination of embodiment F21 , wherein the CD40 cytoplasmic polypeptide has the amino acid sequence of SEQ ID NO: 105.

[0961] F23. The kit or combination of embodiment F9, wherein the cell elimination polypeptide comprises a pro-apoptotic polypeptide.

[0962] F24. The kit or combination of embodiment F9, wherein the cell elimination polypeptide comprises a caspase-9 polypeptide or portion thereof.

[0963] F25. The kit or combination of embodiment F0, wherein the cell elimination polypeptide comprises a caspase-9 polypeptide lacking one or more caspase recruitment domains (CARD).

[0964] F26. The kit or combination of embodiment F24 or F25, wherein the caspase-9 polypeptide is a human polypeptide.

[0965] F27. The kit or combination of embodiment F24, wherein the caspase-9 polypeptide has the amino acid sequence of SEQ ID NO: 146 or an amino acid sequence 90% or more identical to SEQ ID NO: 146.

[0966] F28. The kit or combination of embodiment F24, wherein the Caspase-9 polypeptide has the amino acid sequence of SEQ ID NO: 1 13 or SEQ ID NO: 1 14 or an amino acid sequence 90% or more identical to SEQ ID NO: 113 or SEQ ID NO: 1 14. [0967] F29. The kit or combination of embodiment F28, wherein the Caspase-9 polypeptide has the amino acid sequence of SEQ ID NO: 1 13 or SEQ ID NO: 1 14 or an amino acid sequence 95% or more identical to SEQ ID NO: 113 or SEQ ID NO: 1 14. [0968] F30. The kit or combination of embodiment F29, wherein the Caspase-9 polypeptide has the amino acid sequence of SEQ ID NO: 1 13 or SEQ ID NO: 1 14.

[0969] F31. The kit or combination of any of embodiments F-F30, comprising a homodimerizer agent that binds to an FKBP12 wild type polypeptide or an FKBP12 variant polypeptide.

[0970] F32. The kit or combination of embodiment F31 , wherein the homodimerizer agent is rimiducid, AP20187 or AP1510.

[0971] F33. The kit or combination of any of embodiments F-F30, further comprising a compound of Formula I or Formula II:

wherein:

Y is L, M or Q:

A and A’ are the same or different and each independently are

R¹⁰ and R¹¹ independently are hydrogen or C1-C2 alkyl;

[0972] F34. The kit or combination of any of embodiments F-F30, further comprising a compound having the following structure:

[0973] Modifications may be made to the foregoing without departing from the basic aspects of the technology. Although the technology has been described in substantial detail with reference to one or more specific embodiments, those of ordinary skill in the art will recognize that changes may be made to the embodiments specifically disclosed in this application, yet these modifications and improvements are within the scope and spirit of the technology.

[0974] The technology illustratively described herein suitably may be practiced in the absence of any element(s) not specifically disclosed herein. Thus, for example, in each instance herein any of the terms“comprising,”“consisting essentially of,” and“consisting of may be replaced with either of the other two terms. Still further, the terms“having”,“including”,“containing” and “comprising” are interchangeable and one of skill in the art is cognizant that these terms are open ended terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and use of such terms and expressions do not exclude any equivalents of the features shown and described or portions thereof, and various modifications are possible within the scope of the technology claimed. The term“a” or“an” can refer to one of or a plurality of the elements it modifies (e.g.,“a reagent” can mean one or more reagents) unless it is contextually clear either one of the elements or more than one of the elements is described. The term“about” as used herein refers to a value within 10% of the underlying parameter (i.e., plus or minus 10%), and use of the term“about” at the beginning of a string of values modifies each of the values (i.e.,“about 1 , 2 and 3” refers to about 1 , about 2 and about 3). For example, a weight of“about 100 grams” can include weights between 90 grams and 1 10 grams. Further, when a listing of values is described herein (e.g., about 50%, 60%, 70%, 80%, 85% or 86%) the listing includes all intermediate and fractional values thereof (e.g., 54%,

85.4%). Thus, it should be understood that although the present technology has been specifically disclosed by representative embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and such modifications and variations are considered within the scope of this technology.

[0975] Certain embodiments of the technology are set forth in the claim(s) that follow(s).

Claims

What is claimed is:

1. A compound having a structure of the following Formula A:

or a pharmaceutically acceptable salt thereof, wherein:

R²⁰ is hydrogen, -R²³ or -R^F-R²³;

n is 1 or 2;

2. The compound of claim 1 , wherein R²² is halogen, -NR²⁷R²⁸ or -R^H-R²⁹-R³¹-R³⁰.

3. A compound having a structure of the following Formula B:

or a pharmaceutically acceptable salt thereof, wherein:

n is 1 or 2;

4. The compound of claim 3, wherein R^22A is hydrogen and R^22B is halogen, -NR²⁷R²⁸ or -R^H- R²⁹— R³ⁱ— R³⁸, or R^22B is hydrogen and R^22A is halogen, -NR²⁷R²⁸ or -R^H-R²⁹-R³¹-R³⁰.

5. The compound of claims 1 -4, wherein R^F, R^G and R^H each independently is - 0-, -C(O)-, - O-C(O)- or -C(0)-0-

6. The compound of claim 1 , wherein R²² is halogen, -R^H-R²⁹-R³⁰ or -R^H-R²⁹-R³¹-R³⁰ and wherein if R²² is -R^H-R²⁹-R³⁰, R²⁹ is substituted with halogen and/or R³⁰ is halogen, or if R²² is - R^H_R²⁹_R³¹_R³⁰ R²⁹ j_S substituted with halogen and/or R³⁰ is halogen and/or R³¹ is substituted with halogen.

7. The compound of claim 6, wherein R²⁰ is hydrogen or -R^F-R²³.

8. The compound of claim 7, wherein R²⁰ is -OCH₃.

9. The compound of claim 3, wherein R^22A is hydrogen and R^22B is halogen, -R^H-R²⁹-R³⁰ or - R^H— R²⁹— R³¹— R³⁰ and wherein if R^22B is -R^H-R²⁹-R³⁰, R²⁹ is substituted with halogen and/or R³⁰ is halogen, or if R^22B is -R^H-R²⁹-R³¹-R³⁰, R²⁹ is substituted with halogen and/or R³⁰ is halogen and/or R³¹ is substituted with halogen.

10. The compound of claim 3, wherein R^22B is hydrogen and R^22A is halogen, -R^H-R²⁹-R³⁰ or - R^H_R²⁹_R³¹_R³⁰ _ancj wherein if R^22A is -R^H-R²⁹-R³⁰, R²⁹ is substituted with halogen and/or R³⁰ is halogen, or if R^22A is -R^H-R²⁹-R³¹-R³⁰, R²⁹ is substituted with halogen and/or R³⁰ is halogen and/or R³¹ is substituted with halogen.

1 1 . The compound of claim 9 or 10, wherein R^20A is hydrogen and R^20B is hydrogen or -R^F-R²³ or wherein R^20B is hydrogen and R^20A is hydrogen or -R^F-R²³.

12. The compound of claim 1 1 , wherein R^20A is hydrogen and R^20B is -OCH₃ or wherein R^20B is hydrogen and R^20A is -OCH₃.

13. The compound of claim 1 , wherein R²² is NR²⁷R²⁸ and R²⁸ is or is substituted with heterocycloalkyl or is substituted with heterocycle-alkyl.

14. The compound of claim 1 , wherein R²² is -R^H-R²⁹-R³⁰ or -R^H-R²⁹-R³¹-R³⁰ and R²⁹ and/or R³⁰ is or is substituted with heterocycloalkyl or is substituted with heterocycle-alkyl.

15. The compound of claim 3, wherein R^22A is hydrogen, R^22B is NR²⁷R²⁸ and R²⁸ is or is substituted with heterocycloalkyl or is substituted with heterocycle-alkyl.

16. The compound of claim 3, wherein R^22B is hydrogen, R^22A is NR²⁷R²⁸ and R²⁸ is or is substituted with heterocycloalkyl or is substituted with heterocycle-alkyl.

17. The compound of claim 3, wherein R^22A is hydrogen, R^22B is -R^H-R²⁹-R³⁰ or -R^H-R²⁹-R³¹- R³⁰ and R²⁹ and/or R³⁰ is or is substituted with heterocycloalkyl or is substituted with

heterocycle-alkyl.

18. The compound of claim 3, wherein R^22B is hydrogen, R^22A is -R^H-R²⁹-R³⁰ or -R^H-R²⁹-R³¹- R³⁰ and R²⁹ and/or R³⁰ is or is substituted with heterocycloalkyl or is substituted with

heterocycle-alkyl.

19. The compound of any one of claims 13-18, wherein the heterocycloalkyl or heterocycle- alkyl comprises one or more nitrogen atoms.

20. The compound of claim 3, wherein R^22A is hydrogen and R^22B is halogen, NR²⁷R²⁸, -R^H-R²⁹- R³⁰ or -R^H-R²⁹-R³¹ -R³⁰.

21 . The compound of claim 4, wherein R^22A is hydrogen and R^22B is halogen, NR²⁷R²⁸, or -R^H-

22. The compound of any one of claims 1 , 3 and 20, wherein R²², R^22A or R^22B is - R^H-R²⁹-R³¹- R³⁰.

23. The compound of any one of claims 1 -22, wherein R^H is -O-C(O)-.

24. The compound of any one of claims 1-12, 14 and 17-23, wherein R²⁹ is C5-C7 cycloalkyl, 5- 7 membered heterocycloalkyl, C5-C7 aryl or 5-7 membered heteroaryl, which independently is optionally substituted by one or more substituents chosen from halogen, hydroxy, C1 -C3 alkyl, C1 -C3 heteroalkyl, C1-C3 haloalkyl, C1 -C3 perhaloalkyl, C1 -C3 perhaloalkoxy, C1 -C3 alkoxy, C1 -C3 haloalkoxy, C1 -C3 alkoxy C1 -C3 alkyl, C1 -C4 acyl, oxo, C1-C4 acyloxy, carboxyl, amido, cyano, amino, C1 -C3 alkylamino, C1 -C3 alkylamino C1 -C3 alkyl, thiol, C1 -C3 alkylthio, C1 -C3 alkylthio C1 -C3 alkyl, C1 -C3 haloalkylthio, C1-C3 perhaloalkylthio, nitro, C5-C7 aryl, C5-C7 aryl C1 -C3 alkyl, C5-C7 cycloalkyl, C5-C7 cycloalkyl C1 -C3 alkyl, 5-7 membered heterocycloalkyl, 5- 7 membered heterocycle C1 -C3 alkyl, 5-7 membered heteroaryl, 5-7 membered heteroaryl C1 - C3 alkyl, C1 -C3 alkylsulfonyl, sulfonamide, C1 -C3 alkylsulfonamido and C1 -C3 alkylsilyloxy.

25. The compound of claim 22, 23 or 24, wherein R²⁹ is a C5-C7 aryl or a 5-7 membered heteroaryl.

26. The compound of claim 25, wherein R²⁹ has a structure of Formula C-1 :

Formula C-1

wherein:

one, two or three of R⁴¹, R⁴², R⁴³, R⁴⁴ and R⁴⁵ independently is R³¹-R³⁰, and each of the remaining R⁴¹, R⁴², R⁴³, R⁴⁴ and R⁴⁵ independently is not present or independently is hydrogen, halogen, hydroxy, C1-C3 alkyl, C1-C3 heteroalkyl, C1-C3 haloalkyl, C1-C3 perhaloalkyl, C1-C3 perhaloalkoxy, C1-C3 alkoxy, C1-C3 haloalkoxy, C1-C3 alkoxy C1-C3 alkyl, C1-C4 acyl, oxo, C1-C4 acyloxy, carboxyl, amido, cyano, amino, C1-C3 alkylamino, C1-C3 alkylamino C1-C3 alkyl, thiol, C1-C3 alkylthio, C1-C3 alkylthio C1-C3 alkyl, C1-C3 haloalkylthio, C1-C3 perhaloalkylthio, nitro, C5-C7 aryl, C5-C7 aryl C1-C3 alkyl, C5-C7 cycloalkyl, C5-C7 cycloalkyl C1-C3 alkyl, 5-7 membered heterocycloalkyl, 5-7 membered heterocycle C1-C3 alkyl, 5-7 membered heteroaryl, 5-7 membered heteroaryl C1 -C3 alkyl, C1-C3 alkylsulfonyl, sulfonamide, C1-C3 alkylsulfonamido or C1-C3 alkylsilyloxy.

27. The compound of claim 26, wherein R²⁹ has a structure of Formula D-1 :

Formula D-1 wherein one, two or three of R⁴¹ , R⁴², R⁴³, R⁴⁴ and R⁴⁵ independently is R³¹-R³⁰, and each of the remaining R⁴¹ , R⁴², R⁴³, R⁴⁴ and R⁴⁵ independently is hydrogen, halogen, hydroxy, C1-C3 alkyl, C1-C3 heteroalkyl, C1-C3 haloalkyl, C1-C3 perhaloalkyl, C1-C3 perhaloalkoxy, C1-C3 alkoxy, C1 -C3 haloalkoxy, C1-C3 alkoxy C1 -C3 alkyl, C1 -C4 acyl, oxo, C1 -C4 acyloxy, carboxyl, amido, cyano, amino, C1 -C3 alkylamino, C1 -C3 alkylamino C1 -C3 alkyl, thiol, C1 -C3 alkylthio, C1 -C3 alkylthio C1 -C3 alkyl, C1-C3 haloalkylthio, C1 -C3 perhaloalkylthio, nitro, C5-C7 aryl, C5-C7 aryl C1 -C3 alkyl, C5-C7 cycloalkyl, C5-C7 cycloalkyl C1 -C3 alkyl, 5-7 membered heterocycloalkyl, 5-7 membered heterocycle C1-C3 alkyl, 5-7 membered heteroaryl, 5-7 membered heteroaryl C1-C3 alkyl, C1-C3 alkylsulfonyl, sulfonamide, C1 -C3 alkylsulfonamido or C1 -C3 alkylsilyloxy.

28. The compound of claim 26, wherein R²⁹ has a structure of Formula E-1 :

Formula E-1 wherein R⁴³ is -R³¹-R³⁰.

29. The compound of any one of claims 1 -28, wherein R³¹ is a C1 -C3 alkyl linker.

30. The compound of claim 29, wherein R³¹ is a methylene linker or ethylene linker.

31 . The compound of any one of claims 1 -30, wherein R³⁰ is a C5-C7 cycloalkyl, 5-7 membered heterocycloalkyl, C5-C7 aryl or 5-7 membered heteroaryl.

32. The compound of any one of claims 1 -31 , wherein R³⁰ is a 5-7 membered heterocycloalkyl or a 5-7 membered heteroaryl.

33. The compound of any one of claims 1 -32, wherein R³⁰ is a 5-7 membered heterocycloalkyl.

34. The compound of claim 33, wherein R³⁰ has a structure of Formula F-1 :

Formula F-1 wherein:

35. The compound of claim 34, wherein R³⁰ has a structure of Formula G-1 :

Formula G-1 wherein:

36. The compound of claim 34, wherein R³⁰ has a structure of Formula H-1 :

Formula H-1 wherein:

37. The compound of claim 35, wherein R³⁰ has a structure of Formula J-1 :

perhaloalkyl.

38. The compound of any one of claims 1 , 3 and 20, wherein R²², R^22A or R^22B is -NR²⁷R²⁸.

39. The compound of claim 38, wherein R²⁷ or R²⁸ is hydrogen.

40. The compound of claim 38, wherein R²⁷ is hydrogen and R²⁸ is C1-C3 alkyl, C1-C3 heteroalkyl, C1-C3 haloalkyl, C1-C3 perhaloalkyl, C1-C4 acyl, amido, C1-C3 alkylamino, C1-C3 alkylamino C1-C3 alkyl, amino, thiol, C1-C3 alkylthio, C1-C3 alkylthio C1-C3 alkyl, C1-C3 haloalkylthio, C1-C3 perhaloalkylthio, C5-C7 aryl, C5-C7 aryl C1-C3 alkyl, C5-C7 cycloalkyl, C5- C7 cycloalkyl C1-C3 alkyl, 5-7 membered heterocycloalkyl, 5-7 membered heterocycle C1-C3 alkyl, 5-7 membered heteroaryl, 5-7 membered heteroaryl C1-C3 alkyl, C1-C3 alkylsulfonyl, sulfonamide, C1-C3 alkylsulfonamido or C1-C3 alkylsilyloxy.

41. The compound of claim 38, wherein R²⁷ is hydrogen and R²⁸ is -R³²-R³³.

42. The compound of claim 41 , wherein R³² is a C1-C4 alkyl linker.

43. The compound of claim 41 or 42, wherein R³² is an unsubstituted C3 alkyl linker.

44. The compound of any one of claims 41-43, wherein R³³ is a C5-C7 cycloalkyl or a 5-7 membered heterocycloalkyl.

45. The compound of any one of claims 41-43, wherein R³³ is a 5-7 membered heteroaryl or a 5-7 membered heterocycloalkyl.

46. The compound of claim 44 or 45, wherein R³³ is a 5-7 membered heterocycloalkyl.

47. The compound of claim 46, wherein R³³ has a structure of Formula F-2:

Formula F-2 wherein:

48. The compound of claim 47, wherein R³³ has a structure of Formula G-2:

Formula G-2 wherein:

49. The compound of claim 47, wherein R³³ has a structure of Formula H-2:

Formula H-2 wherein:

50. The compound of claim 48, wherein R³³ has a structure of Formula J-2:

perhaloalkyl.

51. The compound of any one of claims 1-50, wherein R²⁴ is a C5-C7 cycloalkyl, 5-7 membered heterocycloalkyl, C5-C7 aryl or 5-7 membered heteroaryl, which independently is optionally substituted by one or more substituents chosen from halogen, hydroxy, C1-C3 alkyl, C1-C3 heteroalkyl, C1-C3 haloalkyl, C1-C3 perhaloalkyl, C1-C3 perhaloalkoxy, C1-C3 alkoxy, C1-C3 haloalkoxy, C1-C3 alkoxy C1-C3 alkyl, C1-C4 acyl, oxo, C1-C4 acyloxy, carboxyl, amido, cyano, amino, C1-C3 alkylamino, C1-C3 alkylamino C1-C3 alkyl, thiol, C1-C3 alkylthio, C1-C3 alkylthio C1-C3 alkyl, C1-C3 haloalkylthio, C1-C3 perhaloalkylthio, nitro, C5-C7 aryl, C5-C7 aryl C1-C3 alkyl, C5-C7 cycloalkyl, C5-C7 cycloalkyl C1-C3 alkyl, 5-7 membered heterocycloalkyl, 5-7 membered heterocycle C1-C3 alkyl, 5-7 membered heteroaryl, 5-7 membered heteroaryl C1-C3 alkyl, C1-C3 alkylsulfonyl, sulfonamide, C1-C3 alkylsulfonamido and C1-C3 alkylsilyloxy.

52. The compound of any one of claims 3-51 , wherein R^21B is hydrogen and R^21A is hydroxy, -

R^G-R²⁴, - R^G-R²⁴-R²⁵ or - R^G-R²⁴-R²⁶-R²⁵.

53. The compound of any one of claims 1-51 , wherein R²¹, R^21A or R^21B is - R^G-R²⁴-R²⁶-R²⁵.

54. The compound of any one of claims 1-53, wherein R^G is -O-C(O)-.

55. The compound of claim 53 or 54, wherein R²⁴ is a C5-C7 aryl or a 5-7 membered heteroaryl.

56. The compound of claim 55, wherein R²⁴ has a structure of Formula C-2:

Formula C-2

wherein:

one, two or three of R⁴¹, R⁴², R⁴³, R⁴⁴ and R⁴⁵ independently is R²⁶-R²⁵, and each of the remaining R⁴¹, R⁴², R⁴³, R⁴⁴ and R⁴⁵ independently is not present or independently is hydrogen, halogen, hydroxy, C1-C3 alkyl, C1-C3 heteroalkyl, C1-C3 haloalkyl, C1-C3 perhaloalkyl, C1-C3 perhaloalkoxy, C1-C3 alkoxy, C1-C3 haloalkoxy, C1-C3 alkoxy C1-C3 alkyl, C1-C4 acyl, oxo, C1-C4 acyloxy, carboxyl, amido, cyano, amino, C1-C3 alkylamino, C1-C3 alkylamino C1-C3 alkyl, thiol, C1-C3 alkylthio, C1-C3 alkylthio C1-C3 alkyl, C1-C3 haloalkylthio, C1-C3 perhaloalkylthio, nitro, C5-C7 aryl, C5-C7 aryl C1-C3 alkyl, C5-C7 cycloalkyl, C5-C7 cycloalkyl C1-C3 alkyl, 5-7 membered heterocycloalkyl, 5-7 membered heterocycle C1-C3 alkyl, 5-7 membered heteroaryl, 5-7 membered heteroaryl C1 -C3 alkyl, C1-C3 alkylsulfonyl, sulfonamide, C1-C3 alkylsulfonamido or C1-C3 alkylsilyloxy.

57. The compound of claim 56, wherein R²⁴ has a structure of Formula D-2:

Formula D-2 wherein one, two or three of R⁴¹ , R⁴², R⁴³, R⁴⁴ and R⁴⁵ independently is R²⁶-R²⁵, and each of the remaining R⁴¹ , R⁴², R⁴³, R⁴⁴ and R⁴⁵ independently is hydrogen, halogen, hydroxy, C1-C3 alkyl, C1-C3 heteroalkyl, C1-C3 haloalkyl, C1-C3 perhaloalkyl, C1-C3 perhaloalkoxy, C1-C3 alkoxy, C1-C3 haloalkoxy, C1-C3 alkoxy C1-C3 alkyl, C1-C4 acyl, oxo, C1-C4 acyloxy, carboxyl, amido, cyano, amino, C1-C3 alkylamino, C1-C3 alkylamino C1-C3 alkyl, thiol, C1-C3 alkylthio, C1 -C3 alkylthio C1 -C3 alkyl, C1-C3 haloalkylthio, C1 -C3 perhaloalkylthio, nitro, C5-C7 aryl, C5-C7 aryl C1 -C3 alkyl, C5-C7 cycloalkyl, C5-C7 cycloalkyl C1 -C3 alkyl, 5-7 membered heterocycloalkyl, 5-7 membered heterocycle C1-C3 alkyl, 5-7 membered heteroaryl, 5-7 membered heteroaryl C1-C3 alkyl, C1-C3 alkylsulfonyl, sulfonamide, C1 -C3 alkylsulfonamido or C1 -C3 alkylsilyloxy.

58. The compound of claim 57, wherein R²⁴ has a structure of Formula E-2:

Formula E-2 wherein R⁴³ is -R²⁶-R²⁵.

59. The compound of any one of claims 1-58, wherein R²⁶ is a C1 -C3 alkyl linker.

60. The compound of claim 59, wherein R²⁶ is a methylene linker.

61 . The compound of any one of claims 1 -60, wherein R²⁵ is a 5-7 membered heterocycloalkyl.

62. The compound of claim 61 , wherein R²⁵ has a structure of Formula F-3:

Formula F-3 wherein:

one, two or three of X⁶⁰, X⁶¹ , X⁶², X⁶³, X⁶⁴ and X⁶⁵ are oxygen, nitrogen or sulfur heteroatoms, and the remaining X⁶⁰, X⁶¹ , X⁶², X⁶³, X⁶⁴ and X⁶⁵ are carbon; and R⁶¹, R⁶², R⁶³, R⁶⁴ and R⁶⁵ each represent zero, one or two substituents, each of which substituents independently is hydrogen, halogen, hydroxy, C1-C3 alkyl, C1-C3 heteroalkyl, C1- C3 haloalkyl, C1-C3 perhaloalkyl, C1-C3 perhaloalkoxy, C1-C3 alkoxy, C1-C3 haloalkoxy, C1- C3 alkoxy C1-C3 alkyl, C1-C4 acyl, oxo, C1-C4 acyloxy, carboxyl, amido, cyano, amino, C1-C3 alkylamino, C1-C3 alkylamino C1-C3 alkyl, thiol, C1-C3 alkylthio, C1-C3 alkylthio C1-C3 alkyl, C1-C3 haloalkylthio, C1-C3 perhaloalkylthio, nitro, C5-C7 aryl, C5-C7 aryl C1-C3 alkyl, C5-C7 cycloalkyl, C5-C7 cycloalkyl C1-C3 alkyl, 5-7 membered heterocycloalkyl, 5-7 membered heterocycle C1-C3 alkyl, 5-7 membered heteroaryl, 5-7 membered heteroaryl C1-C3 alkyl, C1- C3 alkylsulfonyl, sulfonamide, C1-C3 alkylsulfonamido or C1-C3 alkylsilyloxy.

63. The compound of claim 62, wherein R²⁵ has a structure of Formula G-3:

Formula G-3 wherein:

64. The compound of claim 63, wherein R²⁵ has a structure of Formula H-3:

Formula H-3 wherein:

65. The compound of claim 63, wherein R²⁵ has a structure of Formula J-3:

perhaloalkyl.

66. The compound of any one of claims 1 , 2, 5, 6, 13, 14, 19 and 22-65, wherein R²⁰ is -R²³.

67. The compound of any one of claims 3, 9, 10 and 15-65, wherein R^20A is hydrogen and R^20B is -R²³, or wherein R^20B is hydrogen and R^20A is -R²³.

68. The compound of claim 67, wherein R^20A is hydrogen and R^20B is -R²³.

69. The compound of any one of claims 1 , 2, 5, 6, 13, 14, 19 and 22-65, wherein R²⁰ is -R^F- R²³.

70. The compound of any one of claims 3, 9, 10 and 15-65, wherein R^20A is hydrogen and R^20B is -R^F-R²³, or wherein R^20B is hydrogen and R^20A is -R^F-R²³.

71. The compound of claim 70, wherein R^20A is hydrogen and R^20B is -R^F-R²³.

72. The compound of any one of claims 1-65, 69, 70 and 71 , wherein R^F is -0-, -C(O)-, -O- C(O)- or -C(0)-0-.

73. The compound of any one of claims 1-65, 69, 70, 71 and 72, wherein R^F is -0-.

74. The compound of any one of claims 1-65, 69, 70 and 71 , wherein R^F is -NH-S(O)-.

75. The compound of any one of claims 1-52 and 54-65, wherein R²¹ is hydroxy; R^21A is hydrogen and R^21B is hydroxy; or R^21A is hydroxy and R^21B is hydrogen.

76. The compound of any one of claims 1-74, wherein R²³ is a C3-C10 alkyl, C5-C7 aryl or 5-7 membered heteroaryl, which independently is optionally substituted by one or more substituents chosen from halogen, hydroxy, C1-C3 alkyl, C1-C3 heteroalkyl, C1-C3 haloalkyl, C1-C3 perhaloalkyl, C1-C3 perhaloalkoxy, C1-C3 alkoxy, C1-C3 haloalkoxy, C1-C3 alkoxy C1-C3 alkyl, C1-C4 acyl, oxo, C1-C4 acyloxy, carboxyl, amido, cyano, amino, C1-C3 alkylamino, C1-C3 alkylamino C1-C3 alkyl, thiol, C1-C3 alkylthio, C1-C3 alkylthio C1-C3 alkyl, C1-C3 haloalkylthio, C1-C3 perhaloalkylthio, nitro, C5-C7 aryl, C5-C7 aryl C1-C3 alkyl, C5-C7 cycloalkyl, C5-C7 cycloalkyl C1-C3 alkyl, 5-7 membered heterocycloalkyl, 5-7 membered heterocycle C1-C3 alkyl, 5-7 membered heteroaryl, 5-7 membered heteroaryl C1 -C3 alkyl, C1-C3 alkylsulfonyl, sulfonamide, C1-C3 alkylsulfonamido and C1-C3 alkylsilyloxy.

77. The compound of any one of claims 1-75, wherein R²³ is a C3-C10 alkyl, C5-C7 aryl, C5-C7 cycloalkyl, or 5-7 membered heteroaryl.

78. The compound of claim 77, wherein R²³ has a structure of Formula C-3:

Formula C-3

wherein:

79. The compound of claim 78, wherein R²³ has a structure of Formula D-3:

80. The compound of claim 78, wherein R²³ has a structure of Formula E-3:

81. The compound of claim 80, wherein R⁵¹ and R⁵³ each are methoxy.

82. The compound of any one of claims 1-77, wherein R²³ is a C3-C10 alkyl.

83. The compound of claim 82, wherein R²³ is a C3 or C4 alkyl.

84. The compound of claim 83, wherein R²³ is isopropyl or isobutyl.

85. The compound of any one of claims 1 , 3 and 20-84, wherein R²³ is a C1-C2 alkyl.

86. The compound of claim 85, wherein R²³ is methyl.

87. The compound of any one of claims 82-86, wherein R^F is -NH-S(O)-.

88. The compound of any one of claims 82-86, wherein R^F is -0-.

89. The compound of any one of claims 82-86, wherein R^F is not present.

90. The compound of any one of claims 1-7, 9-1 1 , 13-77, wherein R²³ is a saturated C5-C7 cycloalkyl or a C5-C7 cycloalkyl comprising one degree of unsaturation.

91. The compound of claim 90, wherein R²³ is a saturated C6 cycloalkyl or a C6 cycloalkyl comprising one degree of unsaturation.

92. The compound of claim 90 or 91 , wherein R²³ comprises one or two C1-C4 alkyl substituents.

93. The compound of any one of claims 90-92, wherein R^F is not present.

94. The compound of any one of claims 90-92, wherein R^F is -NH-S(O)-.

95. The compound of any one of claims 90-92, wherein R^F is -0-.

96. The compound of claim 95, wherein -R^F-R²³ together is p-menth-1-en-3-ol.

97. The compound of claim 95, wherein -R^F-R²³ together is 2-isopropyl-5-methylcyclohexanol.

98. The compound of any one of claims 1-75, wherein R²³ is a 5-7 membered heterocycle.

99. The compound of claim 98, wherein R²³ is a 5 membered heterocycle.

100. The compound of claim 98 or 99, wherein R²³ comprises one, two or three nitrogen ring atoms.

101 . The compound of claim 100, wherein R²³ is imidazole.

102. The compound of any one of claims 98-99, wherein R^F is not present.

103. The compound of any one of claims 1-12, 14, 17-40, 41-102, wherein:

R²², R^22A or R^22B is halogen, -NR²⁷R²⁸ or-R^H-R²⁹-R³¹-R³⁰;

R^H is -O-C(O)-; R²⁷ is hydrogen and R²⁸ is -R³²-R³³;

R²⁹ is C5-C7 aryl or 5-7 membered heteroaryl;

R³¹ is a C1-C3 alkyl linker;

R³⁰ is a 5-7 membered heterocycloalkyl;

R³² is a C1 to C4 alkyl linker; and

R³³ is a 5-7 membered heterocycloalkyl.

104. The compound of claim 103, wherein R²², R^22A or R^22B is halogen.

105. The compound of claim 104, wherein the halogen independently is fluoro, chloro, bromo or iodo.

106. The compound of claim 103, wherein:

R²², R^22A or R^22B is -NR²⁷R²⁸.

107. The compound of claim 103, wherein:

R²², R^22A or R^22B is -R^H-R²⁹-R³¹-R³⁰;

R^H is -O-C(O)-;

R²⁹ is C5-C7 aryl or 5-7 membered heteroaryl;

R³¹ is a C1-C3 alkyl linker; and

R³⁰ is a 5-7 membered heterocycloalkyl.

108. The compound of claim 103 or 107, wherein R³¹ is a methylene linker or ethylene linker.

109 The compound of any one of claims 103, 107 or 108, wherein R²⁹ has a structure of Formula E-1 and R⁴³ is R³¹-R³⁰.

110. The compound of any one of claims 103 and 107-109, wherein:

R³⁰ has a structure of Formula G-1 or Formula H-1 ; and

11 1 . The compound of any one of claims 103 and 107-1 10, wherein:

R³⁰ has a structure of Formula J-1 ; and

R⁶³ is hydrogen or C1-C3 alkyl.

112. The compound of any one of claims 103-1 11 , wherein: R^F is -0-, -NH-S(O)-, or is not present; and

113. The compound of any one of claims 103-1 12, wherein:

R²³ has a structure of Formula D-3; and

114. The compound of any one of claims 103-1 13, wherein:

wherein R²³ has a structure of Formula E-3; and

R⁵¹ and R⁵³ each independently is C1-C3 alkyl or C1-C3 alkoxy.

115. The compound of any one of claims 103-1 14, wherein:

wherein R²³ has a structure of Formula E-3; and

R⁵¹ and R⁵³ each are methoxy.

116. The compound of any one of claims 103-1 12, wherein R²³ is C1-C2 alkyl.

117. The compound of claim 1 16, wherein R²³ is methyl.

118. The compound of any one of claims 103-1 11 , wherein R²¹ is hydroxy; R^21A is hydrogen and R^21B is hydroxy; or R^21A is hydroxy and R^21B is hydrogen.

119. The compound of any one of claims 103-1 12, wherein R²³ is a C3-C10 alkyl.

120. The compound of claim 1 19, wherein R²³ is a C3 or C4 alkyl.

121 . The compound of claim 120, wherein R²³ is isopropyl or isobutyl.

122. The compound of any one of claims 1 19-121 , wherein R^F is -NH-S(O)-.

123. The compound of any one of claims 1 19-121 , wherein R^F is -0-.

124. The compound of any one of claims 1 19-121 , wherein R^F is not present.

125. The compound of any one of claims 103-1 12, wherein R²³ is a saturated C5-C7 cycloalkyl or a C5-C7 cycloalkyl comprising one degree of unsaturation.

126. The compound of claim 125, wherein R²³ is a saturated C6 cycloalkyl or a C6 cycloalkyl comprising one degree of unsaturation.

127. The compound of claim 125 or 126, wherein R²³ comprises one or two C1-C4 alkyl substituents.

128. The compound of any one of claims 125-127, wherein R^F is not present.

129. The compound of any one of claims 125-127, wherein R^F is -NH-S(O)-.

130. The compound of any one of claims 125-127, wherein R^F is -0-.

131 . The compound of claim 130, wherein -R^F-R²³ together is p-menth-1-en-3-ol.

132. The compound of claim 130, wherein -R^F-R²³ together is 2-isopropyl-5- methylcyclohexanol.

133. The compound of any one of claims 103-1 11 , wherein R²³ is a 5-7 membered heterocycle.

134. The compound of claim 133, wherein R²³ is a 5 membered heterocycle.

135. The compound of claim 133 or 134, wherein R²³ comprises one, two or three nitrogen ring atoms.

136. The compound of claim 135, wherein R²³ is imidazole.

137. The compound of any one of claims 133-136, wherein R^F is not present.

138. The compound of claim 37, wherein:

R²², R^22A or R^22B is -R^H-R²⁹-R³¹-R³⁰;

R^H is -O-C(O)-;

R²⁹ is C5-C7 aryl or 5-7 membered heteroaryl; and

R³¹ is a C1-C3 alkyl linker.

139. The compound of claim 138, wherein:

R²¹, R^21A or R^21B is -R^G-R²⁴-R²⁶-R²⁵ ;

R^G is -O-C(O)-;

R²⁴ is C5-C7 aryl or 5-7 membered heteroaryl; and

R²⁶ is a C1-C3 alkyl linker.

140. The compound of claim 139, wherein R²⁵ has a structure of Formula J-3:

Formula J-3 wherein R⁶³ is hydrogen, C1-C3 alkyl, C1-C3 heteroalkyl, C1-C3 haloalkyl or C1-C3 perhaloalkyl.

141 . A method for regulating treatment of a medical condition, comprising:

administering a composition comprising a compound of any one of claims 1-140 to a subject being treated for a medical condition, thereby regulating the treatment thereof.

142. The method of claim 141 , wherein the medical condition is associated with cancer, graft versus host disease, cytokine storms, tumor lysis syndrome, cytokine release syndrome, macrophage activation syndrome and/or an adverse event in connection with therapeutic cell treatment.

143. A method of reducing the number and/or viability of, or eliminating, specific cells, comprising administering a composition comprising a compound of any one of claims 140 to a composition containing cells that contain nucleic acid encoding a polypeptide to which the compound binds, wherein the number and/or viability of the cells is reduced.

144. A method of increasing the number and/or viability of specific cells, comprising

administering a composition comprising a compound of any one of claims 1 to 140 a composition containing cells that contain nucleic acid encoding a polypeptide to which the compound binds, wherein the number and/or viability of the cells is increased.