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WO2020076738A2 - Composés à liaison protéinique - Google Patents

Composés à liaison protéinique Download PDF

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Publication number
WO2020076738A2
WO2020076738A2 PCT/US2019/055067 US2019055067W WO2020076738A2 WO 2020076738 A2 WO2020076738 A2 WO 2020076738A2 US 2019055067 W US2019055067 W US 2019055067W WO 2020076738 A2 WO2020076738 A2 WO 2020076738A2
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Prior art keywords
alkyl
compound
aryl
cycloalkyl
hydrogen
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WO2020076738A3 (fr
Inventor
Joseph Henri Bayle
Slawomir Szymanski
Matthew Robert Collinson-Pautz
Steven Mark TOLER
David Michael Spencer
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Bellicum Pharmaceuticals Inc
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Bellicum Pharmaceuticals Inc
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Publication of WO2020076738A3 publication Critical patent/WO2020076738A3/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D498/12—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains three hetero rings
    • C07D498/18—Bridged systems

Definitions

  • the technology relates in part to compounds that bind to proteins.
  • the compounds can bind to proteins that bind to rapamycin.
  • the compounds can bind to cellular proteins, and/or variant forms of cellular proteins, that bind to rapamycin and/or analogs of rapamycin.
  • 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.
  • 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.
  • mTOR is a protein kinase contained in the protein complex TORC1 involved in activation of protein translation and inhibition of autophagy.
  • 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.
  • 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.
  • rapamycin or a rapalog capable of binding to mTOR
  • rapamycin or a rapalog capable of binding to mTOR
  • 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.
  • 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.
  • 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.
  • 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.
  • FKBP FK506-binding protein
  • a compound provided herein selectively binds to a variant FRB polypeptide.
  • a compound provided herein binds to a variant FRB polypeptide with greater affinity than it has for binding to a wild type FRB polypeptide.
  • the compound may also bind to wild type FKBP12 with similar affinity as rapamycin.
  • a compound provided herein possesses properties (e.g., solubility and/or stability) that are more favorable for pharmaceutical or in vivo use than rapamycin.
  • 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.
  • R 20A is hydrogen and R 20B is -R 23 or -R F -R 23 , or R 20B is hydrogen and R 20A is -R 23 or -R F -R 23 ;
  • R 21A is hydrogen and R 21 B is hydroxy, -R G -R 24 -R G -R 24 -R 25 or -R G -R 24 -R 26 -R 25 , or
  • R 21 B is hydrogen and R 21A is hydroxy, -R G -R 24 -R G -R 24 -R 25 or -R G -R 24 -R 26 -R 25 ;
  • R 22A is hydrogen and R 22B is halogen, -NR 27 R 28 , -R H -R 29 -R 30 or -R H -R 29 -R 31 -R 30 , or R 22B is hydrogen and R 22A is halogen, -NR 27 R 28 , -R H -R 29 -R 30 or -R H -R 29 -R 31 -R 30 ;
  • 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 1 or 2;
  • R 23 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, hetero
  • R 24 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, heteroary
  • R 29 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, alkylsul
  • R 25 , R 30 and R 33 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, heteroarylalky
  • R 26 , R 31 and R 32 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;
  • R 27 and R 28 each independently is -R 32 -R 33 , 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, arylal
  • 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 , R 23 , R 24 , R 25 , R 26 , R 27 , R 28 , R 29 , R 30 , R 31 , R 32 , R 33 and n as set forth above, except that R 22A is hydrogen and R 22B is halogen, -NR 27 R 28 or -R H -R 29 -R 31 - R 30 , or R 22B is hydrogen and R 22A is halogen, -NR 27 R 28 or -R H -R 29 -R 31 -R 30 .
  • compounds provided herein have a structure of Formula B, or a
  • R 20A is hydrogen and R 20B is -R 23 or -R F -R 23 , or R 20B is hydrogen and R 20A is -R 23 or -R F -R 23 ;
  • R 21A is hydrogen and R 21 B is hydrogen, hydroxy or -R G -R 34 , or R 21 B is hydrogen and R 21A is hydrogen, hydroxy or -R G -R 34 ;
  • R 22A is hydrogen and R 22B is -R H -R 35 , or R 22B is hydrogen and R 22A is -R H -R 35 ;
  • 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 1 or 2;
  • R 23 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, heteroary
  • R 34 and R 35 each independently is a C2-C6 alkyl substituted with one or more hydroxy substituents.
  • compounds provided herein have a structure of Formula B, or a pharmaceutically acceptable salt thereof, wherein:
  • R 20A is hydrogen and R 20B is -R 23 , -R F -R 23 or -R'-R 34 , or R 20B is hydrogen and R 20A is -R 23 ,
  • R 21A is hydrogen and R 21 B is hydrogen, hydroxy, -R G -R 24 , -R G -R 24 -R 25 or -R G -R 24 -R 26 -R 25
  • R 21B is hydrogen and R 21A is hydrogen, hydroxy, -R G -R 24 -R G -R 24 -R 25 or -R G -R 24 -R 26 -R 25
  • R 22A is hydrogen and R 22B is hydrogen, hydroxy, halogen, -N 3 , -NR 27 R 28 -R H -R 29 , -R H -R 29 -R 30 or -R H -R 29 -R 31 -R 30
  • R 22B is hydrogen and R 22A is hydrogen, hydroxy, halogen, -N 3 , -NR 27 R 28 -
  • 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 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 1 or 2;
  • R 23 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, al
  • R 24 and R 29 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, sulf
  • R 25 , R 30 and R 33 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, heteroarylalky
  • R 26 , R 31 and R 32 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;
  • R 27 and R 28 each independently is -R 32 -R 33 , 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, arylal
  • R 34 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,
  • 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', R 23 , R 24 , R 25 , R 26 , R 27 , R 28 , R 29 , R 30 , R 31 , R 32 , R 33 and n as set forth above, except that R 22A is hydrogen and R 22B is hydrogen, hydroxy, halogen, N 3 , -NR 27 R 28 or -R H -R 29 -R 31 -R 30 , or R 22B is hydrogen and R 22A is hydrogen, hydroxy, halogen, N 3 , -NR 27 R 28 or -R H -R 29 -R 31 -R 30 .
  • compounds provided herein have a structure of Formula B, or a pharmaceutically acceptable salt thereof, wherein:
  • R 20A is hydrogen and R 20B is -R 23 or -R F -R 23 , or R 20B is hydrogen and R 20A is -R 23 or -R F -R 23 ;
  • R 21A is hydrogen and R 21 B is hydroxy, -R G -R 24 -R G -R 24 -R 25 or -R G -R 24 -R 26 -R 25 , or R 21 B is hydrogen and R 21A is hydroxy, -R G -R 24 -R G -R 24 -R 25 or -R G -R 24 -R 26 -R 25 ;
  • R 22A is hydrogen and R 22B is halogen, -N 3 , -NR 27 R 28 , -R H -R 29 , -R H -R 29 -R 30 or -R H -R 29 -R 31 -R 30 , or R 22B is hydrogen and R 22A is halogen, -N 3 , -NR 27 R 28 , -R H -R 29 , -R H -R 29 -R 30 or -R H -R 29 -R 31 -R 31 -R 30 , or R 22B
  • 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 1 or 2;
  • R 23 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,
  • R 24 and R 29 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, alkylsulf
  • R 25 , R 30 and R 33 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, heteroarylalky
  • R 26 , R 31 and R 32 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
  • R 27 and R 28 each independently is -R 32 -R 33 , 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,
  • 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.
  • kits for using compounds having a structure of a formula provided herein, or compositions containing such compounds are provided.
  • 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.
  • 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.
  • a treatment e.g., a cell-based treatment
  • FIG. 1 depicts the chemical structure of rapamycin.
  • 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.
  • 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 O ⁇ 3z.
  • 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.,
  • 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
  • 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.
  • the activation and elimination components dual switches
  • the cell is referred to as DS-CAR-T.
  • 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.
  • 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 are CMP01 1 , CMP014 and CMP012.
  • 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).
  • 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:
  • FKBP12(F36V) also known as FKBP12v36, Fv36, FKBPv, or F v
  • FKBP12(F36V) also known as FKBP12v36, Fv36, FKBPv, or F v
  • the phenylalanine at amino acid position 36 or 37 if the initial methionine of the protein is counted
  • Acaspase9 the entire fusion protein is termed iC9
  • 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),
  • 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:
  • FKBP12(F36V) also known as FKBP12v36, Fv36, FKBPv, or F v
  • FKBP12(F36V) also known as FKBP12v36, Fv36, FKBPv, or F v
  • the phenylalanine at amino acid position 36 or 37 if the initial methionine of the protein is counted
  • Acaspase9 a portion of human caspase 9 polypeptide
  • 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 O ⁇ 3z polypeptide.
  • FRP5 VH heavy variable region
  • FRP5 VL light chain variable region
  • 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 O ⁇ 3z (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).
  • control T cells circles
  • CAR T cells which were transduced with nucleic acid encoding a chimeric antigen receptor fusion of an
  • 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.
  • 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:
  • FIG. 1 1 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.
  • tumor cells e.g., OE19 cells transduced with nucleic acid encoding firefly luciferase
  • OE19.GFP/uc tumor cells
  • immunodeficient mice e.g., NSG mice.
  • the mice received 5 x10 6 transduced T cells (designated CAR T in FIG. 1 1) via intravenous infusion.
  • compound e.g., CMP001 (i.e.,“Go” drug)
  • 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.
  • FIG. 11 shows a photograph of Renilla luciferase-derived
  • 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.
  • CAR T cells transduced with retrovirus containing plasmids encoding iRMC and iC9 proteins and ONL Rluc Renilla luciferase
  • iRMC-GoCAR-T + iC9 ONL Rluc Renilla luciferase
  • mice designated“iC9: in the figure
  • OE19 tumor cells transduced with nucleic acid encoding GFP FFluc
  • 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.
  • FIG. 12 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.
  • control mice designated“iC9: in the figure
  • the control mice 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.
  • mice imaged were injected with luciferin (firefly luciferase substrate) by an intraperitoneal (i.p.) route in the lower abdomen prior to imaging.
  • luciferin firefly luciferase substrate
  • intraperitoneal i.p.
  • 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.
  • 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).
  • 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.
  • stably label cells e.g., PBMCs
  • RFP nuclear-localized red fluorescent protein
  • 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.
  • Rapamycin also known as sirolimus, is a macrolide lactone natural product having the following structure:
  • 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.
  • rapamycin exists as a mixture of conformational isomers which can be distinguished by NMR spectroscopy.
  • 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.
  • 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).
  • FK506 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.
  • rapamycin 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).
  • FKBP-Rapamycin-Binding (FRB) domain of mTOR mimmalian target of rapamycin; see, e.g., Sabatini et al. (1994) Cell 78:35-43.
  • 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.
  • 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.
  • mTOR also known as FRAP, RAFT, RAPT1 and SEP
  • PI3K cytoplasmic phosphatidylinositol-3-kinase
  • mTORCI cytoplasmic phosphatidylinositol-3-kinase
  • mTORC2 cytoplasmic phosphatidylinositol-3-kinase
  • 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.
  • 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.
  • a rapalog can be a prodrug.
  • a rapalog can be metabolized in vivo to yield rapamycin or another rapalog.
  • a rapalog is not a prodrug.
  • 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.
  • rapalogs 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.
  • Rapamycin and rapalog use as therapeutic agents
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • FKBP12/FRB multimerization-based transcription induction protein localization and protein stabilization
  • 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.
  • 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).
  • 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.
  • 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).
  • rapalog C20- methylallylrapamycin
  • 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).
  • Certain variant FRB proteins are bound by some rapalogs.
  • a mutant human FRB referred to as“PLF” containing three amino acid substitutions, K2095P, T2098L and W2101 F 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).
  • 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
  • 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.
  • 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.
  • T2098L substitution is primarily responsible for the instability of the mutant protein (see, e.g.,
  • T cells also referred to as T 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.
  • NK Natural Killer
  • MHC major histocompatibility
  • 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.
  • 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 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.
  • CAR T cells incorporating an FKBP12/FRB-based multimerization system have a built-in control mechanism that can be regulated 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 repeat et al., PLOS Pathogens 6:1- 13 (2010).
  • 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.
  • T cells are extracted from a patient’s blood and genetically engineered to express chimeric antigen receptors (CARs) on the cell surface.
  • CARs chimeric antigen receptors
  • 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., ⁇ 3z; see SEQ ID NO:
  • 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.
  • another stimulating domain often derived from the cytoplasmic portion of T cell co-stimulating molecules, including CD28, 4-1 BB, 0X40, ICOS and DAP10
  • CD28 co-stimulation provides a clear clinical advantage for the treatment of CD19 + lymphomas.
  • 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 ⁇ 3z cytoplasmic region
  • survival and proliferation are impaired due to lack of co-stimulation.
  • the addition of CD28 or 4-1 BB co-stimulating domain constructs has significantly improved the survival and proliferative capacity of CAR T cells.
  • 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 ⁇ 3z (signal 1) and NF-kB (signal 2) by CD28 or 4-1 BB.32.
  • NFAT nuclear factor of activated T cells
  • Other molecules that similarly activate NF-kB may also be paired with the O ⁇ 3z 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).
  • DC dendritic cell
  • TLR signaling For full activation or licensing of DCs, Toll-like receptor (TLR) signaling is usually involved.
  • 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.
  • IRAK family kinases 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).
  • DD death domains
  • 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.
  • 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
  • TRAFs TNFR-associated factors
  • MC fusion protein
  • O ⁇ 3z which is part of the chimeric antigen receptor (signal 1)
  • NF-kB signal 2
  • CAR T cells expressing MC 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.
  • 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
  • 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.
  • FKBP12 protein that binds a chemical inducer of dimerization
  • FKBP12 and variants bind to homomultimer-inducing agents such as, e.g., FK506 dimer (or a dimeric FK506 analog ligand), AP1903 (rimiducid) or AP20187.
  • 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.
  • heteromultimer-inducing agents such as rapamycin or a rapalog
  • 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).
  • 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.
  • 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.
  • CAR-expressing T cells may be used in various therapies, including cancer therapies.
  • 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.
  • TLS tumor lysis syndrome
  • CRS cytokine release syndrome
  • MAS macrophage activation syndrome
  • the modified cell e.g., a CAR T cell
  • 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.
  • stress conditions e.g., DNA damage
  • pro-apoptotic factors such as cytochrome c
  • Apaf-1 apoptotic protease-activating factor-1
  • 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.
  • caspases that are involved in the downstream steps of the apoptotic cascade 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
  • Activated caspase 9 can, in turn, activate effector caspases leading to a chain of events that ultimately results in cell death.
  • 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).
  • 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.
  • 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-
  • 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 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 V 36 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 FKBP12(F36V) (also known as FKBP12v36, F V 36 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
  • 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.
  • one of the molecular switches can be controlled by a distinct dimerizer ligand, such rapamycin or a rapamycin analog (rapalog).
  • a rapamycin or rapalog-inducible co-stimulatory polypeptide 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.
  • a rapamycin or rapalog-inducible co-stimulatory polypeptide e.g., MyD88/CD40 (iMC)
  • a rimiducid-inducible pro-apoptotic polypeptide e.g., caspase-9
  • a rimiducid-inducible chimeric fusion stimulating polypeptide such as, for example,
  • 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.
  • 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 O ⁇ 3z.
  • scFv extracellular, antibody-derived single chain variable fragment
  • Q Q-bend 10
  • 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).
  • 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.
  • iRMC inducible rapalog MyD88/CD40
  • 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).
  • FKBPv FKBP12 variant FKBP12v36
  • AC9 caspase 9 lacking the CARD domain
  • 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.
  • both the activation and elimination components 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.
  • DS-CAR-T the cell is referred to as DS-CAR-T.
  • 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.
  • proteins e.g., cellular proteins.
  • the compound binds to one or more, or at least two, proteins to which rapamycin binds.
  • 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.
  • protein refers to a molecule having a sequence of amino acids linked by peptide bonds.
  • 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.
  • proteins of intracellular origin e.g., located in the nucleus, cytosol, organelle (e.g., mitochondria or peroxisome) or interstitial space of cells in vivo
  • cell membrane proteins in vivo.
  • variant protein 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.
  • 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.
  • 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.
  • a pro- apoptotic domain or region of a polypeptide refers to a portion(s) thereof that, upon dimerization or multimerization, can participate in the caspase cascade, allowing for, or causing, apoptosis.
  • FRB FKBP12-Rapamycin-Binding
  • 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.
  • rapamycin-interacting residues that have been most analyzed, K2095, T2098, and W2101.
  • 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).
  • 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.
  • 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.
  • 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.
  • the compound, or a pharmaceutically acceptable salt thereof is able to bind to one or more variant FRB proteins.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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,
  • 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,
  • 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.
  • 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.
  • PPIase peptidylprolyl cis/trans isomerase
  • 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.
  • 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.
  • the amino acid substitution can be to a valine, leucine, isoleucine, alanine or other amino acid.
  • 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 ).
  • the compound, or a pharmaceutically acceptable salt thereof is able to bind to one or more variant human FKBP12 proteins or homologs thereof.
  • a compound provided herein binds to a human FKBP12 polypeptide variant that has an amino acid substitution in place of
  • 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.,
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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,
  • 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.
  • a compound described herein, or a pharmaceutically acceptable salt thereof binds to a wild type FKBP12 polypeptide with an IC 5 o, EC 5 o 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.
  • a compound described herein, or a pharmaceutically acceptable salt thereof binds to a wild type FKBP12 polypeptide with an IC 5 o, EC 5 o 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.
  • a compound described herein, or a pharmaceutically acceptable salt thereof binds to a wild type FKBP12 polypeptide with an IC 5 o, EC 5 o 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 FKBP
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • therapeutic applications include, but are not limited to, treatment of conditions which benefit from inhibition of immune cells or immunosuppression.
  • 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.
  • multimeric compounds 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.
  • multimeric compounds provided herein are capable of multimerizing, or heteromerizing, peptides or polypeptides that are different from each other.
  • 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.
  • 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 ).
  • 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.
  • 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.
  • the amino acid substitution is to a leucine.
  • the FRB polypeptide variant is FRBL (or KLW).
  • a rapalog can differ from rapamycin in one or more ways.
  • 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.
  • 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.
  • rapalog 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • compositions 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • CMP001 7-demethoxy-7(S)-o,p-dimethoxyphenylrapamycin
  • 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,
  • rapamycin or a rapalog such as, e.g., 7-demethoxy-7(S)-o,p-dimethoxyphenylrapamycin (CMP001).
  • 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).
  • an intrinsic clearance e.g., as measured in ml/min/mg protein using animal, e.g., human, liver microsome assays or animal model assays
  • an intrinsic clearance e.g.
  • 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
  • compounds provided herein are rapalogs that are epimers of rapamycin.
  • 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.
  • 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.
  • compounds provided herein have a structure of Formula B, or a pharmaceutically acceptable salt thereof, wherein:
  • R 20A is hydrogen and R 20B is -R 23 or -R F -R 23 , or R 20B is hydrogen and R 20A is -R 23 or -R F -R 23 ;
  • R 21A is hydrogen and R 21 B is hydroxy, -R G -R 24 , -R G -R 24 -R 25 or -R G -R 24 -R 26 -R 25 , or
  • R 21 B is hydrogen and R 21A is hydroxy, -R G -R 24 , -R G -R 24 -R 25 or -R G -R 24 -R 26 -R 25 ;
  • R 22A is hydrogen and R 22B is halogen, -NR 27 R 28 , -R H -R 29 -R 30 or -R H -R 29 -R 31 -R 30 , or R 22B is hydrogen and R 22A is halogen, -NR 27 R 28 , -R H -R 29 -R 30 or -R H -R 29 -R 31 -R 30 ;
  • 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 1 or 2;
  • R 23 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, hetero
  • R 24 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, heteroary
  • R 25 , R 30 and R 33 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, heteroarylalky
  • R 26 , R 31 and R 32 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;
  • R 27 and R 28 each independently is -R 32 -R 33 , 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, arylal
  • R 29 has a structure of Formula C-1 :
  • X 40 , X 41 , X 42 , X 43 , X 44 and X 45 together form an aryl or heteroaryl ring; zero, one, two or three of X 40 , X 41 , X 42 , X 43 , X 44 and X 45 are oxygen, nitrogen or sulfur heteroatoms, and the remaining X 40 , X 41 , X 42 , X 43 , X 44 and X 45 are carbon; and
  • R 41 , R 42 , R 43 , R 44 and R 45 independently is R 31 -R 30 , and each of the remaining R 41 , R 42 , R 43 , R 44 and R 45 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
  • 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.
  • R 29 has a structure of Formula C-1
  • R 22A is hydrogen and R 22B is halogen, -NR 27 R 28 or -R H -R 29 -R 31 -R 30
  • R 22B is hydrogen and R 22A is halogen, -NR 27 R 28 or - R H _R 29 _R 31 _R 30
  • n certain embodiments of compounds having a structure of the above- defined Formula B, wherein R 29 has a structure of Formula C-1 , R 22A is hydrogen and R 22B is - R H — R 29 — R 31 — R 30 or R 22B is hydrogen and R 22A is -R H -R 29 -R 31 -R 30 .
  • R H is -O-C(O)-.
  • R 41 , R 42 , R 43 , R 44 and R 45 independently is a Group A moiety chosen from R 31 -R 30 , 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 halo
  • R 41 when X 41 is carbon, R 41 is a Group A moiety; when X 42 is carbon, R 42 is a Group A moiety; when X 43 is carbon, R 43 is a Group A moiety; when X 44 is carbon, R 44 is a Group A moiety; and when X 45 is carbon, R 45 is a Group A moiety.
  • R 22A or R 22B is -R H -R 29 -R 31 -R 30 .
  • R H is -O-C(O)-.
  • R 29 has a structure of Formula C-1
  • R 41 when X 41 is nitrogen, R 41 is a Group B moiety; when X 42 is nitrogen, R 42 is a Group B moiety; when X 43 is nitrogen, R 43 is a Group B moiety; when X 44 is nitrogen, R 44 is a Group B moiety; and when X 45 is nitrogen, R 45 is a Group B moiety.
  • R 22A or R 22B is -R H -R 29 - R3I_R3O I n certain of these embodiments, R H is -O-C(O)-.
  • R 29 has a structure of Formula C-1
  • R 41 , R 42 , R 43 , R 44 and R 45 respectively, independently is not present.
  • R 22A or R 22B is -R H -R 29 -R 31 -R 30 .
  • R H is -O- C(O)-.
  • compounds provided herein have a structure of Formula B, or a pharmaceutically acceptable salt thereof, wherein:
  • R 20A is hydrogen and R 20B is-R 23 or -R F -R 23 , or R 20B is hydrogen and R 20A is -R 23 or -R F -R 23 ;
  • R 21A is hydrogen and R 21 B is hydroxy, -R G -R 24 -R G -R 24 -R 25 or -R G -R 24 -R 26 -R 25 , or
  • R 21 B is hydrogen and R 21A is hydroxy, -R G -R 24 , -R G -R 24 -R 25 or -R G -R 24 -R 26 -R 25 ;
  • R 22A is hydrogen and R 22B is halogen, -NR 27 R 28 , -R H -R 29 -R 30 or -R H -R 29 -R 31 -R 30 , or R 22B is hydrogen and R 22A is halogen, -NR 27 R 28 , -R H -R 29 -R 30 or -R H -R 29 -R 31 -R 30 ;
  • 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 1 or 2;
  • R 23 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, hetero
  • R 29 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, alkylsul
  • R 25 and R 33 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, alky
  • R 26 , R 31 and R 32 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;
  • R 27 and R 28 each independently is -R 32 -R 33 , 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, arylal
  • R 30 has a structure of Formula F-1 : Formula F-1
  • X 60 , X 61 , X 62 , X 63 , X 64 and X 65 together form a heterocycloalkyl ring;
  • X 60 , X 61 , X 62 , X 63 , X 64 and X 65 are oxygen, nitrogen or sulfur heteroatoms, and the remaining X 60 , X 61 , X 62 , X 63 , X 64 and X 65 are carbon; and
  • R 61 , R 62 , R 63 , R 64 and R 65 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 -C
  • R 30 has a structure of Formula F-1
  • R 22A is hydrogen and R 22B is halogen, -NR 27 R 28 or -R H -R 29 -R 31 -R 30
  • R 22B is hydrogen and R 22A is halogen, -NR 27 R 28 or -R H -R 29 -R 31 -R 30
  • R 30 has a structure of Formula F-1
  • R 22A or R 22B is -R H -R 29 -R 31 -R 30
  • R H is -O-C(O)-.
  • R 30 has a structure of Formula F-1 , when X 61 , X 62 , X 63 , X 64 and X 65 independently is carbon, R 61 , R 62 , R 63 , R 64 and R 65 , respectively, independently is two substituents, each of which two substituents
  • R 61 when X 61 is carbon, R 61 is two substituents, each of which two substituents independently is a Group A moiety; when X 62 is carbon, R 62 is two substituents, each of which two substituents independently is a Group A moiety; when X 63 is carbon, R 63 is two substituents, each of which two substituents independently is a Group A moiety; when X 64 is carbon, R 64 is two substituents, each of which two substituents independently is a Group A moiety; and when X 65 is carbon, R 65 is two substituents, each of which two substituents independently is a Group A moiety.
  • R 22A or R 22B is -R H -R 29 -R 31 -R 30 .
  • R H is -O-C(O)-.
  • R 30 has a structure of Formula F-1 , when X 61 , X 62 , X 63 , X 64 and X 65 independently is nitrogen, R 61 , R 62 , R 63 , R 64 and R 65 , 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,
  • R 41 when X 41 is nitrogen, R 41 is a Group B moiety; when X 42 is nitrogen, R 42 is a Group B moiety; when X 43 is nitrogen, R 43 is a Group B moiety; when X 44 is nitrogen, R 44 is a Group B moiety; and when X 45 is nitrogen, R 45 is a Group B moiety.
  • R 22A or R 22B is -R H -R 29 -R 31 -R 30 .
  • R H is -O-C(O)-.
  • compounds provided herein have a structure of Formula B, or a pharmaceutically acceptable salt thereof, wherein:
  • R 20A is hydrogen and R 20B is -R 23 or -R F -R 23 , or R 20B is hydrogen and R 20A is -R 23 or -R F -R 23 ;
  • R 21A is hydrogen and R 21 B is hydroxy, -R G -R 24 -R G -R 24 -R 25 or -R G -R 24 -R 26 -R 25 , or
  • R 21 B is hydrogen and R 21A is hydroxy, -R G -R 24 -R G -R 24 -R 25 or -R G -R 24 -R 26 -R 25 ;
  • R 22A is hydrogen and R 22B is halogen, -NR 27 R 28 , -R H -R 29 -R 30 or -R H -R 29 -R 31 -R 30 , or R 22B is hydrogen and R 22A is halogen, -NR 27 R 28 , -R H -R 29 -R 30 or -R
  • 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 1 or 2;
  • R 23 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, hetero
  • R 29 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, alkylsul
  • R 25 , R 30 and R 33 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, heteroarylalky
  • R 26 , R 31 and R 32 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;
  • R 27 and R 28 each independently is -R 32 -R 33 , 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, arylal
  • R 24 has a structure of Formula C-2:
  • X 40 , X 41 , X 42 , X 43 , X 44 and X 45 together form an aryl or heteroaryl ring;
  • X 40 , X 41 , X 42 , X 43 , X 44 and X 45 are oxygen, nitrogen or sulfur heteroatoms, and the remaining X 40 , X 41 , X 42 , X 43 , X 44 and X 45 are carbon; and
  • R 41 , R 42 , R 43 , R 44 and R 45 independently is R 26 -R 25 , and each of the remaining R 41 , R 42 , R 43 , R 44 and R 45 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
  • R 22A is hydrogen and R 22B is halogen, -NR 27 R 28 or -R H -R 29 -R 31 -R 30
  • R 22B is hydrogen and R 22A is halogen, -NR 27 R 28 or -R H -R 29 -R 31 -R 30
  • R 21A or R 21 B is - R G -R 24 -R 26 -R 25 .
  • R G is -O-C(O)-.
  • R 41 , R 42 , R 43 , R 44 and R 45 independently is carbon
  • R 41 , R 42 , R 43 , R 44 and R 45 respectively, independently is a Group A moiety chosen from R 26 -R 25 , 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,
  • R 41 when X 41 is carbon, R 41 is a Group A moiety; when X 42 is carbon, R 42 is a Group A moiety; when X 43 is carbon, R 43 is a Group A moiety; when X 44 is carbon, R 44 is a Group A moiety; and when X 45 is carbon, R 45 is a Group A moiety.
  • R 21A or R 21 B is - R G -R 24 -R 26 -R 25 .
  • R G is - O-C(O)-.
  • R 24 has a structure of Formula C-2
  • R 41 when X 41 is nitrogen, R 41 is a Group B moiety; when X 42 is nitrogen, R 42 is a Group B moiety; when X 43 is nitrogen, R 43 is a Group B moiety; when X 44 is nitrogen, R 44 is a Group B moiety; and when X 45 is nitrogen, R 45 is a Group B moiety.
  • R 21A or R 21B is - R G -R 24 - R 26 _R25 I n certain of these embodiments, R G is -O-C(O)-.
  • R 24 has a structure of Formula C-2
  • R 41 , R 42 , R 43 , R 44 and R 45 respectively, independently is not present.
  • R 21A or R 21 B is - R G -R 24 - R 26 _R25 I n certain of these embodiments, R G is -O-C(O)-.
  • compounds provided herein have a structure of Formula B, or a pharmaceutically acceptable salt thereof, wherein:
  • R 20A is hydrogen and R 20B is -R 23 , -R F -R 23 or -R'-R 34 , or R 20B is hydrogen and R 20A is -R 23 ,
  • R 21A is hydrogen and R 21 B is hydrogen, hydroxyl, -R G -R 24 -R G -R 24 -R 25 or -R G -R 24 -R 26 -R 25
  • R 21B is hydrogen and R 21A is hydrogen, hydroxyl, -R G -R 24 -R G -R 24 -R 25 or -R G -R 24 -R 26 -R 25
  • R 21A is hydrogen, hydroxyl, -R G -R 24 -R G -R 24 -R 25 or -R G -R 24 -R 26 -R 25 ;
  • R 22A is hydrogen and R 22B is hydrogen, hydroxyl, halogen, -N 3 , -NR 27 R 28 , -R H -R 29 , -R H -R 29 -R 30 or -R H -R 29 -R 31 -R 30 , or R 22B is hydrogen and R 22A is hydrogen, hydroxyl, halogen, -N 3 , - NR 27 R 28 , -R H -R 29 , -R H -R 29 -R 30 or -R H -R 29 -R 31 -R 30 ;
  • 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 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 1 or 2;
  • R 23 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,
  • R 24 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, heteroary
  • R 25 , R 30 and R 33 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, heteroarylalky
  • R 26 , R 31 and R 32 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,
  • R 27 and R 28 each independently is -R 32 -R 33 , 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
  • R 34 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
  • R 29 has a structure of Formula C-1 :
  • X 40 , X 41 , X 42 , X 43 , X 44 and X 45 together form an aryl or heteroaryl ring;
  • X 40 , X 41 , X 42 , X 43 , X 44 and X 45 are oxygen, nitrogen or sulfur heteroatoms, and the remaining X 40 , X 41 , X 42 , X 43 , X 44 and X 45 are carbon; and
  • R 41 , R 42 , R 43 , R 44 and R 45 independently is R 31 -R 30 , and each of the remaining R 41 , R 42 , R 43 , R 44 and R 45 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.
  • each of R 20A , R 20B , R 21A , R 21B , R F , R G , R H , R', R 23 , R 24 , R 25 , R 26 , R 27 , R 28 , R 30 , R 31 , R 32 , R 33 and n are as set forth above, but R 22A is hydrogen and R 22B is hydrogen, hydroxyl, halogen, N 3 , -NR 27 R 28 or -R H -R 29 -R 31 -R 30 , or R 22B is hydrogen and R 22A is hydrogen, hydroxyl, halogen, N 3 , -NR 27 R 28 or -R H -R 29 -R 31 -R 30 .
  • R 29 has a structure of Formula C-1
  • R 22A or R 22B is -R H -R 29 -R 31 -R 30 .
  • R H is -O-C(O)-.
  • R 29 has a structure of Formula C-1
  • R 41 , R 42 , R 43 , R 44 and R 45 independently is carbon
  • R 41 , R 42 , R 43 , R 44 and R 45 respectively, independently is a Group A moiety chosen from R 31 -R 30 , 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 alkylaminoal
  • R 41 when X 41 is carbon, R 41 is a Group A moiety; when X 42 is carbon, R 42 is a Group A moiety; when X 43 is carbon, R 43 is a Group A moiety; when X 44 is carbon, R 44 is a Group A moiety; and when X 45 is carbon, R 45 is a Group A moiety.
  • R 22A or R 22B is -R H -R 29 -R 31 -R 30 .
  • R H is - C(O)-.
  • R 29 has a structure of Formula C-1
  • R 41 when X 41 is nitrogen, R 41 is a Group B moiety; when X 42 is nitrogen, R 42 is a Group B moiety; when X 43 is nitrogen, R 43 is a Group B moiety; when X 44 is nitrogen, R 44 is a Group B moiety; and when X 45 is nitrogen, R 45 is a Group B moiety.
  • R 22A or R 22B is -R H -R 29 - R3I_R3O I n certain of these embodiments, R H is -C(O)-.
  • R 29 has a structure of Formula C-1
  • R 41 , R 42 , R 43 , R 44 and R 45 respectively, independently is not present.
  • R 22A or R 22B is -R H -R 29 - R 3 I_R 3 O I n certain of these embodiments, R H is -C(O)-.
  • compounds provided herein have a structure of Formula B, or a pharmaceutically acceptable salt thereof, wherein:
  • R 20A is hydrogen and R 20B is -R 23 , -R F -R 23 or -R'-R 34 , or R 20B is hydrogen and R 20A is -R 23 ,
  • R 21A is hydrogen and R 21 B is hydrogen, hydroxyl, -R G -R 24 -R G -R 24 -R 25 or -R G -R 24 -R 26 -R 25 , or R 21B is hydrogen and R 21A is hydrogen, hydroxyl, -R G -R 24 -R G -R 24 -R 25 or -R G -R 24 -R 26 -R 25 ;
  • R 22A is hydrogen and R 22B is hydrogen, hydroxy, halogen, -N 3 , -NR 27 R 28 , -R H -R 29 , -R H -R 29 -R 30 or -R H -R 29 -R 31 -R 30 , or R 22B is hydrogen and R 22A is hydrogen, hydroxy, halogen, -N 3 , -NR 27 R 28 , - R H -R 29 , -R H -R 29 -R 30 or -R H -R 29 -R 31 -R 30 ;
  • 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 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 1 or 2;
  • R 23 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,
  • R 24 and R 29 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, sulf
  • R 25 and R 33 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, alky
  • R 26 , R 31 and R 32 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;
  • R 27 and R 28 each independently is -R 32 -R 33 , 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, arylal
  • R 34 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,
  • R 30 has a structure of Formula F-1 :
  • X 60 , X 61 , X 62 , X 63 , X 64 and X 65 together form a heterocycloalkyl ring;
  • X 60 , X 61 , X 62 , X 63 , X 64 and X 65 are oxygen, nitrogen or sulfur heteroatoms, and the remaining X 60 , X 61 , X 62 , X 63 , X 64 and X 65 are carbon; and
  • R 61 , R 62 , R 63 , R 64 and R 65 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 -C
  • each of R 20A , R 20B , R 21A , R 21 B , R F , R G , R H , R 1 , R 23 , R 24 , R 25 , R 26 , R 27 , R 28 , R 29 , R 31 , R 32 , R 33 and n are as set forth above, but R 22A is hydrogen and R 22B is hydrogen, hydroxy, halogen, N 3 , -NR 27 R 28 or -R H -R 29 -R 31 -R 30 , or R 22B is hydrogen and R 22A is hydrogen, hydroxy, halogen, N 3 , -NR 27 R 28 or -R H -R 29 -R 31 -R 30 .
  • R 30 has a structure of Formula F-1
  • R 22A or R 22B is -R H -R 29 -R 31 -R 30 .
  • R H is -O-C(O)-.
  • R 30 has a structure of Formula F-1 , when X 61 , X 62 , X 63 , X 64 and X 65 independently is carbon, R 61 , R 62 , R 63 , R 64 and R 65 , 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 alkyla
  • R 61 when X 61 is carbon, R 61 is two substituents, each of which two substituents independently is a Group A moiety; when X 62 is carbon, R 62 is two substituents, each of which two substituents independently is a Group A moiety; when X 63 is carbon, R 63 is two substituents, each of which two substituents independently is a Group A moiety; when X 64 is carbon, R 64 is two substituents, each of which two substituents independently is a Group A moiety; and when X 65 is carbon, R 65 is two substituents, each of which two substituents independently is a Group A moiety.
  • R 22A or R 22B is -R H -R 29 -R 31 -R 30 .
  • R H is -O-C(O)-.
  • R 30 has a structure of Formula F-1 , when X 61 , X 62 , X 63 , X 64 and X 65 independently is nitrogen, R 61 , R 62 , R 63 , R 64 and R 65 , 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,
  • R 41 when X 41 is nitrogen, R 41 is a Group B moiety; when X 42 is nitrogen, R 42 is a Group B moiety; when X 43 is nitrogen, R 43 is a Group B moiety; when X 44 is nitrogen, R 44 is a Group B moiety; and when X 45 is nitrogen, R 45 is a Group B moiety.
  • R 22A or R 22B is -R H -R 29 -R 31 -R 30 .
  • R H is -O-C(O)-.
  • R 22A or R 22B is -R H -R 29 - R 3 I_R 3 O I n certain of these embodiments, R H is -O-C(O)-.
  • compounds provided herein have a structure of Formula B, or a pharmaceutically acceptable salt thereof, wherein:
  • R 20A is hydrogen and R 20B is -R 23 , -R F -R 23 or -R'-R 34 , or R 20B is hydrogen and R 20A is -R 23 ,
  • R 21A is hydrogen and R 21 B is hydrogen, hydroxy, -R G -R 24 -R G -R 24 -R 25 or -R G -R 24 -R 26 -R 25 , or
  • R 21B is hydrogen and R 21A is hydrogen, hydroxy, -R G -R 24 -R G -R 24 -R 25 or -R G -R 24 -R 26 -R 25 ;
  • R 22A is hydrogen and R 22B is hydrogen, hydroxy, halogen, -N 3 , -NR 27 R 28 , -R H -R 29 , -R H -R 29 -R 30 or
  • R 22B is hydrogen and R 22A is hydrogen, hydroxy, halogen, -N 3 , -NR 27 R 28 , -
  • 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 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 1 or 2;
  • R 23 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,
  • R 29 independently is:
  • alkyl alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl, which
  • acyl 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,
  • R 25 and R 33 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, alky
  • R 26 , R 31 and R 32 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;
  • R 27 and R 28 each independently is -R 32 -R 33 , 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, arylal
  • R 34 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,
  • R 24 has a structure of Formula C-2: Formula C-2
  • X 40 , X 41 , X 42 , X 43 , X 44 and X 45 together form an aryl or heteroaryl ring;
  • X 40 , X 41 , X 42 , X 43 , X 44 and X 45 are oxygen, nitrogen or sulfur heteroatoms, and the remaining X 40 , X 41 , X 42 , X 43 , X 44 and X 45 are carbon; and
  • R 41 , R 42 , R 43 , R 44 and R 45 independently is R 26 -R 25 , and each of the remaining R 41 , R 42 , R 43 , R 44 and R 45 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
  • each of R 20A , R 20B , R 21A , R 21B , R F , R G , R H , R 1 , R 23 , R 24 , R 25 , R 26 , R 27 , R 28 , R 29 , R 30 , R 31 , R 32 , R 33 and n are as set forth above, but R 22A is hydrogen and R 22B is hydrogen, hydroxy, halogen, N 3 , -NR 27 R 28 or -R H -R 29 -R 31 -R 30 , or R 22B is hydrogen and R 22A is hydrogen, hydroxy, halogen, N 3 , -NR 27 R 28 or -R H -R 29 -R 31 -R 30 .
  • R 21A or R 21 B is - R G -R 24 -R 26 -R 25 .
  • R G is -O-C(O)-.
  • R 24 has a structure of Formula C-2
  • R 41 , R 42 , R 43 , R 44 and R 45 independently is carbon
  • R 41 , R 42 , R 43 , R 44 and R 45 respectively, independently is a Group A moiety chosen from R 26 -R 25 , 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 alkylaminoalky
  • R 41 when X 41 is carbon, R 41 is a Group A moiety; when X 42 is carbon, R 42 is a Group A moiety; when X 43 is carbon, R 43 is a Group A moiety; when X 44 is carbon, R 44 is a Group A moiety; and when X 45 is carbon, R 45 is a Group A moiety.
  • R 21A or R 21 B is - R G -R 24 -R 26 -R 25 .
  • R G is - O-C(O)-.
  • R 24 has a structure of Formula C-2
  • R 41 when X 41 is nitrogen, R 41 is a Group B moiety; when X 42 is nitrogen, R 42 is a Group B moiety; when X 43 is nitrogen, R 43 is a Group B moiety; when X 44 is nitrogen, R 44 is a Group B moiety; and when X 45 is nitrogen, R 45 is a Group B moiety.
  • R 21A or R 21B is - R G -R 24 - R 26 _R25 I n certain of these embodiments, R G is -O-C(O)-.
  • R 24 has a structure of Formula C-2
  • R 41 , R 42 , R 43 , R 44 and R 45 respectively, independently is not present.
  • R 21A or R 21 B is - R G -R 24 - R 26 _R25 I n certain of these embodiments, R G is -O-C(O)-.
  • compounds provided herein have a structure of Formula B, or a pharmaceutically acceptable salt thereof, wherein:
  • R 20A is hydrogen and R 20B is -R 23 or -R F -R 23 , or R 20B is hydrogen and R 20A is -R 23 or -R F -R 23 ;
  • R 21A is hydrogen and R 21 B is hydroxy, -R G -R 24 -R G -R 24 -R 25 or -R G -R 24 -R 26 -R 25 , or R 21 B is hydrogen and R 21A is hydroxy, -R G -R 24 -R G -R 24 -R 25 or -R G -R 24 -R 26 -R 25 ;
  • R 22A is hydrogen and R 22B is halogen, -N 3 , -NR 27 R 28 , -R H -R 29 , -R H -R 29 -R 30 or -R H -R 29 -R 31 -R 30 , or R 22B is hydrogen and R 22A is halogen, -N 3 , -NR 27 R 28 , -R H -R 29 , -R H -R 29 -R 30 or -R H -R 29 -R 31 -
  • R 30 ; 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 1 or 2;
  • R 23 has a structure of Formula C-3:
  • X 50 , X 51 , X 52 , X 53 , X 54 and X 55 together form an aryl or heteroaryl ring;
  • X 50 , X 51 , X 52 , X 53 , X 54 and X 55 are oxygen, nitrogen or sulfur heteroatoms, and the remaining X 50 , X 51 , X 52 , X 53 , X 54 and X 55 are carbon; and
  • R 51 , R 52 , R 53 , R 54 and R 55 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 perhaloalkyl
  • R 24 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,
  • R 25 , R 30 and R 33 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, heteroarylalky
  • R 26 , R 31 and R 32 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;
  • R 27 and R 28 each independently is -R 32 -R 33 , 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,
  • R 29 has a structure of Formula C-1 :
  • X 40 , X 41 , X 42 , X 43 , X 44 and X 45 together form an aryl or heteroaryl ring;
  • X 40 , X 41 , X 42 , X 43 , X 44 and X 45 are oxygen, nitrogen or sulfur heteroatoms, and the remaining X 40 , X 41 , X 42 , X 43 , X 44 and X 45 are carbon; and
  • R 41 , R 42 , R 43 , R 44 and R 45 independently is R 31 -R 30 , and each of the remaining R 41 , R 42 , R 43 , R 44 and R 45 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,
  • 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.
  • R 22A or R 22B is -R H -R 29 -R 31 -R 30 .
  • R H is -O-C(O)-.
  • R 41 , R 42 , R 43 , R 44 and R 45 independently is carbon
  • R 41 , R 42 , R 43 , R 44 and R 45 respectively, independently is a Group A moiety chosen from R 31 -R 30 , 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
  • R 41 when X 41 is carbon, R 41 is a Group A moiety; when X 42 is carbon, R 42 is a Group A moiety; when X 43 is carbon, R 43 is a Group A moiety; when X 44 is carbon, R 44 is a Group A moiety; and when X 45 is carbon, R 45 is a Group A moiety.
  • R 22A or R 22B is -R H -R 29 -R 31 -R 30 .
  • R H is -O-C(O)-.
  • R 23 has a structure of Formula C-3 and R 29 has a structure of Formula C-1
  • X 41 , X 42 , X 43 , X 44 and X 45 independently is nitrogen
  • R 41 , R 42 , R 43 , R 44 and R 45 respectively, independently is a Group B moiety chosen from hydrogen, C1-C3 alkyl or C1 -C3 alkyl substituted with hydroxyl, halogen or NR 27 R 28 .
  • R 41 when X 41 is nitrogen, R 41 is a Group B moiety; when X 42 is nitrogen, R 42 is a Group B moiety; when X 43 is nitrogen, R 43 is a Group B moiety; when X 44 is nitrogen, R 44 is a Group B moiety; and when X 45 is nitrogen, R 45 is a Group B moiety.
  • R 22A or R 22B is -R H -R 29 -R 31 -R 30 .
  • R H is -O-C(O)-.
  • R 22A or R 22B is -R H -R 29 -R 31 -R 30 .
  • R H is -O-C(O)-.
  • compounds provided herein have a structure of Formula B, or a pharmaceutically acceptable salt thereof, wherein:
  • R 20A is hydrogen and R 20B is -R 23 or -R F -R 23 , or R 20B is hydrogen and R 20A is -R 23 or -R F -R 23 ;
  • R 21A is hydrogen and R 21 B is hydroxy, -R G -R 24 , -R G -R 24 -R 25 or -R G -R 24 -R 26 -R 25 , or R 21 B is hydrogen and R 21A is hydroxy, -R G -R 24 -R G -R 24 -R 25 or -R G -R 24 -R 26 -R 25 ;
  • R 22A is hydrogen and R 22B is halogen, -N 3 , -NR 27 R 28 , -R H -R 29 , -R H -R 29 -R 30 or -R H -R 29 -R 31 -R 30 , or R 22B is hydrogen and R 22A is halogen, -N 3 , -NR 27 R 28 , -R H -R 29 , -R H -R 29 -R 30 or -R H -R 29 -R 31 -
  • 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 1 or 2;
  • R 23 has a structure of Formula C-3:
  • X 50 , X 51 , X 52 , X 53 , X 54 and X 55 together form an aryl or heteroaryl ring;
  • X 50 , X 51 , X 52 , X 53 , X 54 and X 55 are oxygen, nitrogen or sulfur heteroatoms, and the remaining X 50 , X 51 , X 52 , X 53 , X 54 and X 55 are carbon; and
  • R 51 , R 52 , R 53 , R 54 and R 55 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,
  • R 24 and R 29 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, alkylsulf
  • R 25 and R 33 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, alky
  • R 26 , R 31 and R 32 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;
  • R 27 and R 28 each independently is -R 32 -R 33 , 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,
  • R 30 has a structure of Formula F-1 : Formula F- 1
  • X 60 , X 61 , X 62 , X 63 , X 64 and X 65 together form a heterocycloalkyl ring;
  • X 60 , X 61 , X 62 , X 63 , X 64 and X 65 are oxygen, nitrogen or sulfur heteroatoms, and the remaining X 60 , X 61 , X 62 , X 63 , X 64 and X 65 are carbon; and
  • R 61 , R 62 , R 63 , R 64 and R 65 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
  • R 22A or R 22B is -R H -R 29 -R 31 -R 30 .
  • R H is -O-C(O)-.
  • R 23 has a structure of Formula C-3 and R 30 has a structure of Formula F-1
  • R 61 , R 62 , R 63 , R 64 and X 65 independently is carbon
  • R 61 , R 62 , R 63 , R 64 and R 65 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
  • R 22A or R 22B is -R H -R 29 -R 31 -R 30 .
  • R H is - O-C(O)-.
  • R 23 has a structure of Formula C-3 and R 30 has a structure of Formula F-1
  • R 61 , R 62 , R 63 , R 64 and R 65 independently is nitrogen
  • R 61 , R 62 , R 63 , R 64 and R 65 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
  • R 41 when X 41 is nitrogen, R 41 is a Group B moiety; when X 42 is nitrogen, R 42 is a Group B moiety; when X 43 is nitrogen, R 43 is a Group B moiety; when X 44 is nitrogen, R 44 is a Group B moiety; and when X 45 is nitrogen, R 45 is a Group B moiety.
  • R 22A or R 22B is -R H -R 29 -R 31 -R 30 .
  • R H is -O-C(O)-.
  • R 22A or R 22B is -R H -R 29 -R 31 -R 30 . In certain of these embodiments,
  • R H is -O-C(O)-.
  • compounds provided herein have a structure of Formula B, or a pharmaceutically acceptable salt thereof, wherein:
  • R 20A is hydrogen and R 20B is -R 23 or -R F -R 23 , or R 20B is hydrogen and R 20A is -R 23 or -R F -R 23 ;
  • R 21A is hydrogen and R 21 B is hydroxy, -R G -R 24 -R G -R 24 -R 25 or -R G -R 24 -R 26 -R 25 , or
  • R 21 B is hydrogen and R 21A is hydroxy, -R G -R 24 -R G -R 24 -R 25 or -R G -R 24 -R 26 -R 25 ;
  • R 22A is hydrogen and R 22B is halogen, -N 3 , -NR 27 R 28 , -R H -R 29 , -R H -R 29 -R 30 or -R H -R 29 -R 31 -R 30 , or R 22B is hydrogen and R 22A is halogen, -N 3 , -NR 27 R 28 , -R H -R 29 , -R H -R 29 -R 30 or -R H -R 29 -R 31 -
  • 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 1 or 2;
  • R 23 has a structure of Formula C-3:
  • X 50 , X 51 , X 52 , X 53 , X 54 and X 55 together form an aryl or heteroaryl ring;
  • X 50 , X 51 , X 52 , X 53 , X 54 and X 55 are oxygen, nitrogen or sulfur heteroatoms, and the remaining X 50 , X 51 , X 52 , X 53 , X 54 and X 55 are carbon; and
  • R 51 , R 52 , R 53 , R 54 and R 55 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 haloalky
  • R 29 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,
  • R 25 , R 30 and R 33 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, heteroarylalky
  • R 26 , R 31 and R 32 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;
  • R 27 and R 28 each independently is -R 32 -R 33 , 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,
  • R 24 has a structure of Formula C-2:
  • X 40 , X 41 , X 42 , X 43 , X 44 and X 45 together form an aryl or heteroaryl ring;
  • X 40 , X 41 , X 42 , X 43 , X 44 and X 45 are oxygen, nitrogen or sulfur heteroatoms, and the remaining X 40 , X 41 , X 42 , X 43 , X 44 and X 45 are carbon; and
  • R 41 , R 42 , R 43 , R 44 and R 45 independently is R 26 -R 25 , and each of the remaining R 41 , R 42 , R 43 , R 44 and R 45 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 alky
  • 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.
  • R 21A or R 21B is - R G -R 24 -R 26 -R 25 .
  • R G is -O-C(O)-.
  • R 41 , R 42 , R 43 , R 44 and R 45 independently is a Group A moiety chosen from R 26 -R 25 , 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 perhalo
  • R 41 when X 41 is carbon, R 41 is a Group A moiety; when X 42 is carbon, R 42 is a Group A moiety; when X 43 is carbon, R 43 is a Group A moiety; when X 44 is carbon, R 44 is a Group A moiety; and when X 45 is carbon, R 45 is a Group A moiety.
  • R 21A or R 21B is - R G -R 24 -R 26 -R 25 .
  • R G is -O-C(O)-.
  • R 41 , R 42 , R 43 , R 44 and R 45 independently is a Group B moiety chosen from hydrogen, C1-C3 alkyl or C1-C3 alkyl substituted with hydroxyl, halogen or NR 27 R 28 .
  • R 41 , R 42 , R 43 , R 44 and R 45 independently is a Group B moiety chosen from hydrogen, C1-C3 alkyl or C1-C3 alkyl substituted with hydroxyl, halogen or NR 27 R 28 .
  • R 21A or R 21 B is - R G -R 24 -R 26 -R 25 .
  • R G is -O-C(O)-.
  • 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:
  • R 20A , R 20B , R 22A and R 22B are as listed and R 21A is hydrogen and R 21 B is hydroxy
  • R 20A , R 20B , R 22A and R 22B are as listed and R 21A is hydroxy and R 21B is hydrogen,
  • 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
  • R 20A , R 20B , R 22A and R 22B are as listed and R 21A is p-bromomethylbenzoyl and R 21B is hydrogen,
  • 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
  • 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 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:
  • R 20A , R 20B , R 22A and R 22B are as listed and R 21A is hydrogen and R 21B is hydroxy
  • R 21A is hydroxy and R 21B is hydrogen
  • R 21A is hydrogen and R 21B is p-bromomethylbenzoyl
  • R 20A , R 20B , R 22A and R 22B are as listed and R 21A is p-bromomethylbenzoyl and R 21B is hydrogen,
  • 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
  • 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 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,
  • R 20A , R 20B , R 22A and R 22B are as listed and R 21A is hydroxy and R 21B is hydrogen,
  • R 20A , R 20B , R 22A and R 22B are as listed and R 21A is hydrogen and R 21B is p-bromomethylbenzoyl, and
  • R 20A , R 20B , R 22A and R 22B are as listed and R 21A is p-bromomethylbenzoyl and R 21B is hydrogen,
  • 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
  • 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 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:
  • R 21A is hydrogen and R 21B is hydroxy
  • R 21B is hydrogen
  • R 20A , R 20B , R 22A and R 22B are as listed and R 21A is hydrogen and R 21B is p-bromomethylbenzoyl, and
  • R 20A , R 20B , R 22A and R 22B are as listed and R 21A is p-bromomethylbenzoyl and R 21B is hydrogen,
  • 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
  • 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.
  • 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.
  • the counter ion is phosphate.
  • the counter ion is hydrochloride.
  • a number or letter normally designated as a superscript for example, the“1” in R 1 , or the“L” in R L
  • a subscript for example, Ri or R L
  • any modification of script such as, for example, R1 or RL.
  • any definition herein may be used in combination with any other definition to describe a composite structural group.
  • the trailing element of any such definition is that which attaches to a parent moiety.
  • the composite group alkylamido would represent an alkyl group attached to a parent molecule through an amido group
  • the term alkoxyalkyl would represent an alkoxy group attached to a parent molecule through an alkyl group, for example.
  • a group is defined to be“null,” the group is absent.
  • the term“optionally substituted” means the anteceding group may be substituted or unsubstituted.
  • substituted 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,
  • 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., -CH 2 CH 3 ), fully substituted (e.g., - CF 2 CF 3 ), monosubstituted (e.g., -CH 2 CH 2 F) or substituted at a level anywhere in-between fully substituted and monosubstituted (e.g., -CH 2 CF 3 ).
  • 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.
  • 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.
  • acyl 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.
  • An“acetyl” group refers to a -C(0)CH 3 group.
  • aliphatic 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.
  • 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.
  • 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.
  • alkenyl 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.
  • alkenyl radicals include ethenyl, propenyl, 2-methylpropenyl, 1 ,4-butadienyl and the like.
  • the term“alkenyl” may include“alkenylene” groups.
  • alkoxy refers to an alkyl ether radical, where the term alkyl is as defined below.
  • alkyl ether radicals include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, iso-butoxy, sec-butoxy, tert-butoxy, and the like.
  • alkyl 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.
  • 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.
  • alkylene refers to a saturated aliphatic group derived from a straight or branched chain saturated hydrocarbon attached at two or more positions, such as methylene (-CH 2 -). Unless otherwise specified, the term“alkyl” may include“alkylene” groups.
  • alkylamino 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.
  • alkylidene 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.
  • alkylthio 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.
  • alkyl thioether radicals include methylthio, ethylthio, n-propylthio, isopropylthio, n-butylthio, iso-butylthio, sec-butylthio, tert-butylthio, methanesulfonyl, ethanesulfinyl, and the like.
  • alkynyl 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.
  • alkynylene refers to a carbon-carbon triple bond attached at two positions such as ethynylene (-C:::C-, -CoC-).
  • 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.
  • the term“alkynyl” may include“alkynylene” groups.
  • 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 (CH 3 C(0)NH-).
  • amino 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.
  • aryl 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).
  • 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.
  • the polycyclic ring system is a bicyclic aryl group, where the bicyclic aryl group 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.
  • aryl ring systems include phenyl (monocyclic, C6), naphthyl (bicyclic, C10), anthracenyl (tricyclic, C14) and phenanthryl (tricyclic, C14).
  • 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.
  • 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.
  • 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.
  • 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.
  • aryloxy refers to an aryl group attached to a parent molecular moiety through an oxy.
  • carbamate 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.
  • N-carbamyl refers to a
  • 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.
  • cycloalkyl 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”).
  • each cyclic moiety contains from 3 to 12 carbon ring member atoms which may be optionally substituted as defined herein.
  • a cycloalkyl group contains 3 to 10 carbon ring member atoms.
  • a cycloalkyl includes 5 to 7 carbon atoms.
  • 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.
  • 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.
  • the polycyclic ring system is a tricyclic cycloalkyl group, where the tricyclic cycloalkyl group is C1 1-C18, or, for example, C12-C16.
  • cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, octahydronaphthalene,
  • 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.
  • attachment of the polycycle to the indicated point of attachment on the parent molecule may be through any ring atom of the polycycle rings.
  • the polycycle is attached to the indicated point of attachment through a ring member atom of a cycloalkyl ring.
  • 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.
  • 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.
  • esters refers to a carboxy group bridging two moieties linked at carbon atoms.
  • ether refers to an oxy group bridging two moieties linked at carbon atoms.
  • halo refers to fluorine, chlorine, bromine, or iodine.
  • haloalkoxy refers to a haloalkyl group attached to a parent molecular moiety through an oxygen atom.
  • haloalkyl 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
  • heteroaliphatic 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.
  • a heteroaliphatic group contains from one to three heteroatoms chosen from O,
  • 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.
  • heteroalkyl 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.
  • 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.
  • the heteroatom(s) may be placed at any interior position of the heteroalkyl group.
  • 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.
  • heteroalkenyl 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.
  • 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.
  • the heteroatom(s) may be placed at any interior position of the heteroalkenyl group. In some embodiments, up to two heteroatoms may be consecutive.
  • a heteroalkenyl includes 2 to 20 carbon atoms, 2 to 10 carbon atoms, 2 to 8 carbon atoms or 2 to 6 carbon atoms.
  • heteroalkynyl 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.
  • 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.
  • the heteroatom(s) may be placed at any interior position of the heteroalkynyl group. In some embodiments, up to two heteroatoms may be consecutive.
  • a heteroalkynyl includes 2 to 20 carbon atoms, 2 to 10 carbon atoms, 2 to 8 carbon atoms or 2 to 6 carbon atoms.
  • heteroaryl 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the polycycle is attached to the indicated point of attachment through a ring member atom of a heteroaryl ring.
  • the monocyle or polycycle is attached to the indicated point of attachment through a ring member heteroatom of a heteroaryl ring.
  • 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, NR n , S, SO, S0 2 , where “n” denotes any positive integer.
  • heteroarylalkyl refers to an unsubstituted or substituted heteroaryl group attached to a parent molecular moiety through an alkyl group.
  • heterocycle-alkyl refers to a substituted or unsubstituted heterocycle group attached to a parent molecular moiety through an alkyl group.
  • the one or more heteroatoms that can be in the ring include, for example, nitrogen, oxygen, sulfur, phosphorous and/or silicon.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the polycycle is attached to the indicated point of attachment through a ring member atom of a heterocycloalkyl ring.
  • the monocyle or polycycle is attached to the indicated point of attachment through a ring member heteroatom of a heterocycloalkyl ring.
  • 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.
  • 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,
  • 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, NR n , S, SO, S0 2 , where n denotes any positive integer.
  • hydrazinyl refers to two amino groups joined by a single bond, i.e., -HN-NH-.
  • hydroxyalkyl refers to a hydroxy group attached to a parent molecular moiety through an alkyl group.
  • 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.
  • isocyanato refers to a -NCO group.
  • the phrase“linear chain of atoms” refers to the longest straight chain of atoms independently selected from carbon, nitrogen, oxygen and sulfur.
  • lower aryl 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.
  • lower heteroaryl 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.
  • lower cycloalkyl 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.
  • lower heterocycloalkyl 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.
  • lower amino 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.
  • 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.
  • mercaptyl or“mercaptan” as used herein, alone or in combination refers to an RS- group, where R is as defined herein.
  • menthol 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.
  • 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 iso
  • menthyl 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.
  • 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.
  • perhaloalkoxy refers to an alkoxy group where all of the hydrogen atoms are replaced by halogen atoms.
  • ring member atoms refers to all of the atoms that form the covalent structure of a cyclic ring structure.
  • 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.
  • thiocarbonyl when alone includes thioformyl -C(S)H and in combination is a -C(S)- group.
  • N-thiocarbamyl refers to an ROC(S)NR’- group, with R and R’ as defined herein.
  • O-thiocarbamyl refers to a -OC(S)NRR’, group with R and R’ as defined herein.
  • trihalomethanesulfonamido refers to a X 3 CS(0) 2 NR- group with X is a halogen and R as defined herein.
  • trihalomethanesulfonyl refers to a X 3 CS(0) 2 - group where X is a halogen.
  • trihalomethoxy refers to a X 3 CO- group where X is a halogen.
  • trisubstituted silyl 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.
  • ureido refers to the univalent radical NH 2 CONH- derived from urea.
  • Non-limiting examples include ureidoproprionate and ureidosuccinate.
  • 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.
  • the methoxy group bonded to the carbon at position 7 of rapamycin is substituted with another moiety.
  • some of the compounds in these embodiments can be synthesized from rapamycin through hydrolysis of the 7-methylether linkage followed by nucleophilic substitution.
  • 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.
  • the hydroxyl group at carbon number 40 of rapamycin is substituted with a halogen.
  • 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.
  • rapamycin or an analog thereof, such as, e.g., 7(S)-dimethoxyphenyl-rapamycin
  • trifluoromethanesulfonic anhydride followed by reaction with a tetrabutylammonium halide to yield site-directed halogenation at the C40 position.
  • the hydroxyl group at carbon number 40 of rapamycin is substituted with a benzoic acid ester-containing moiety.
  • 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.,
  • DCC dicyclohexylcarbodiimide
  • DMAP nucleophilic acylation catalyst
  • 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.
  • esterification refers to chemical reaction conditions used to create an ester.
  • reaction refers to a chemical redox reaction in which the oxygen atom of a carboxylic group is substituted by two hydrogen atoms, reducing C0 2 H to CH 2 OH.
  • activation of primary OH 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.
  • N-alkylation conditions refers to chemical reaction conditions used to form an N-R bond.
  • Coupled conditions refers to chemical reaction conditions used to create a chemical bond between N and CO as in a peptide bond.
  • 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.
  • Compounds provided herein can be evaluated for the ability to bind proteins using methods described herein or known in the art.
  • 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.
  • 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
  • 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.
  • the number of cells undergoing activation or apoptosis is less than 10, 5, 4, 3, 2 or 1 %.
  • 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.
  • polypeptides such as, for example, rapamycin-binding polypeptides.
  • 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).
  • binding affinity 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.
  • 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.
  • Some functional assays incorporate secreted alkaline phosphatase (SEAP) as a readily detectable reporter molecule.
  • SEAP secreted alkaline phosphatase
  • 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 IC 5 o 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.
  • SeAP assay 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
  • 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).
  • 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
  • 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.
  • a transcription activator e.g., the herpes simplex virus (HSV) VP16 protein
  • HSV herpes simplex virus
  • 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.
  • the reporter plasmid can contain five GAL4-specific DNA recognition sites proximal to the transcriptional start site.
  • a compound such as rapamycin or a rapalog
  • reporter gene e.g., SeAP-encoding DNA
  • 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.
  • 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).
  • 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
  • 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.;
  • 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
  • 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.
  • 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.
  • 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
  • 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).
  • 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.
  • 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).
  • 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.
  • 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
  • CID chemically induced dimerization
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • nucleic acid vectors for expression of fusion proteins 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
  • 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
  • compositions 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.
  • nucleic acid vectors for expression of fusion proteins 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).
  • 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).
  • transcription factors and splicing regulator proteins e.g., SR proteins or RS domains thereof; see, e.g., Graveley (2005) RNA 1 1 :355-358.
  • 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).
  • 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.
  • 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 e.g., containing transcriptional activator and/or repressor proteins
  • monitoring cells for multimerization of fusion proteins and effects thereof e.g., activation and/or repression of transcription
  • fusion proteins e.g., containing transcriptional activator and/or repressor proteins
  • monitoring cells for multimerization of fusion proteins and effects thereof e.g., activation and/or repression of transcription
  • 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).
  • an mRNA splicing regulator protein e.g., an SR protein
  • RS domain thereof e.g., an FKBP12 protein
  • 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
  • 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.
  • nucleic acid vectors for expression of fusion proteins 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).
  • 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.
  • 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.
  • cell elimination 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 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.
  • 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.
  • Ligand-controlled cell elimination methods 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • the cell is an immune cell, e.g., a T cell.
  • the cell co-expresses a chimeric antigen receptor or a recombinant T cell receptor that recognizes an antigen expressed by a target cell.
  • 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.
  • a co stimulatory polypeptide such as, for example, a CD40 and/or MyD88 polypeptide, or portions thereof.
  • 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.
  • 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.
  • 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.
  • 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 .
  • the expression construct encodes a truncated caspase-9 polypeptide encoded by the caspase-9 nucleotide sequences of pM006 or pM009.
  • “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.
  • “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.
  • 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.
  • 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.
  • 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.
  • 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.
  • activation 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.
  • cell activation is the activation of immune cell function.
  • 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.
  • 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.
  • immune cells e.g., T cells
  • CAR-expressing immune cells e.g., T cells
  • 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.
  • 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.
  • NFAT NFAT
  • NF-kB NF-kB
  • JNK p38 mitogen-activated protein kinase
  • AP1 activator protein 1
  • ERK ERK
  • AKT ERK
  • 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.
  • Ligand-controlled cell activation methods 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.
  • 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).
  • 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.
  • 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.
  • the cell is a T cell.
  • the cell co-expresses a chimeric antigen receptor or a recombinant T cell receptor that recognizes an antigen expressed by a target cell.
  • 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.
  • 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.
  • 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).
  • truncated is meant that the protein is not full length and may lack, for example, a domain.
  • cytoplasmic CD40 or“CD40 lacking the CD40 extracellular domain” is meant a CD40 polypeptide that lacks the CD40 extracellular domain.
  • 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.
  • an expression construct encodes a CD40 polypeptide containing the intracellular domain of the CD40 protein.
  • 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.
  • 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).
  • 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.
  • 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.
  • the method may also be used, or the construct designed to refer to a truncated CD40 polypeptide.
  • an expression construct encodes an MyD88 polypeptide
  • the polypeptide may be a portion of the full-length MyD88 polypeptide.
  • 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.
  • an expression construct encodes a portion of the MyD88 polypeptide lacking the TIR domain.
  • the expression construct encodes a portion of the MyD88 polypeptide containing the DD (death domain) or the DD and intermediary domains.
  • truncated is meant that the protein is not full length and may lack, for example, a domain.
  • 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.
  • 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).
  • 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.
  • 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.
  • the method may also be used, or the construct designed to refer to a truncated MyD88 polypeptide.
  • 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.
  • the MyD88 polypeptide (or portion thereof) is located upstream of the CD40 polypeptide (or portion thereof).
  • 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.
  • 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.
  • 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).
  • chimeric proteins designed to provide for inducible cell activation and chimeric proteins designed to provide for inducible cell death bind to different multimerizing agents.
  • 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.
  • 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.
  • chimeric proteins containing co-stimulatory polypeptides e.g., CD40 and/or MyD88
  • chimeric proteins containing a pro-apoptotic polypeptide e.g., caspase-9
  • 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.
  • 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).
  • 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):
  • Y is L, M or Q:
  • R ⁇ R 2 , R 3 , and R 4 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;
  • R 1 and R 2 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,
  • R 3 and R 4 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;
  • R 1 and R 3 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,
  • cycloalkylalkyl cycloalkyl, heterocycloalkyl, heteroaryl, heteroarylalkyl, alkylsulfonyl, sulfonamide, alkylsulfonamido and alkylsilyloxy;
  • R 2 and R 4 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,
  • cycloalkylalkyl cycloalkyl, heterocycloalkyl, heteroaryl, heteroarylalkyl, alkylsulfonyl, sulfonamide, alkylsulfonamido and alkylsilyloxy;
  • one of the groups: R 1 , R 2 , R 3 and R 4 may be nonexistent. If one of the groups: R 1 , R 2 , R 3 or R 4 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, NR 13 , S, SO, S0 2 , 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, NR 14 , S, SO, S0 2 , and which is optionally substituted with hydroxyl, alkoxyl, amino, alkylamino, thiol, thioalkyl, or halogen;
  • R 12 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, cycloalkyl
  • R 13 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, cycloalkyl
  • R 14 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, cycloalkylal
  • X 1 , X 2 , X 3 , X 4 , X 5 and X 6 independently are carbon or nitrogen with the proviso that none, one, two or three of X 1 , X 2 , X 3 , X 4 , X 5 and X 6 are nitrogen;
  • R 5 , R 6 , R 7 , R 8 or R 9 independently is hydrogen, hydroxyl, halogen, C1-C2 alkyl or C1-C2 alkyl substituted with hydroxyl, halogen or NR 10 R 11 ;
  • R 10 and R 11 independently are hydrogen or C1-C2 alkyl
  • the method involves use of chimeric fusion proteins and/or nucleic acids encoding such proteins, such as, for example, those described herein.
  • the method involves use of expression vectors or constructs, such as, for example, those described herein, containing nucleic acids encoding such proteins.
  • 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.
  • the method involves use of cells, such as, for example, cells described herein.
  • 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.
  • 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.
  • 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.
  • the chimeric polypeptides expressed from such expression constructs may be contacted by a multimeric compound described herein, or a pharmaceutically acceptable salt thereof.
  • 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.
  • 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
  • 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).
  • 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 FRB L polypeptide (see, e.g., SEQ ID NO: 79 for an example of an amino acid sequence containing an FRB L polypeptide).
  • SEQ ID NO: 79 for an example of an amino acid sequence containing an FRB L polypeptide.
  • 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.
  • 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.
  • 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.
  • 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)).
  • an FKBP12 polypeptide variant, or FKBP12 mutant binds to a ligand, such as rimiducid or a compound described in U.S. patent application no.
  • FKBP12 polypeptide variants include, for example, those discussed in Kopytek et al. ((2000) Chem & Biol 7:313-321), Gestwicki et al.
  • 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.
  • 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).
  • 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).
  • inducible chimeric polypeptides contain an F v F Vis sequence, which comprises two FKBP12v36 polypeptides.
  • 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.
  • 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.
  • An expression construct may or may not encode a membrane-targeting sequence.
  • the chimeric polypeptide may contain a membrane targeting region.
  • 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.
  • 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.
  • 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).
  • multimerization region “ligand binding region” and“multimeric ligand binding region” are interchangeable.
  • 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.
  • a heterologous protein e.g., heterologous to an apoptosis-inducing polypeptide or costimulating polypeptide
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 2A-like 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.
  • 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.
  • a 2A linker includes the amino acid sequence of SEQ ID NO: 108.
  • 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.
  • 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.
  • 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.
  • a nucleic acid construct is contained within a viral vector.
  • the viral vector is a retroviral vector.
  • 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.
  • 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.
  • 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.
  • 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.
  • expression includes both transcription of nucleic acid and translation of mRNA into a product.
  • 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.
  • 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.
  • treatment refers to prophylaxis and/or therapy.
  • 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.
  • cDNA is intended to refer to DNA prepared using messenger RNA (mRNA) as template.
  • mRNA messenger RNA
  • polynucleotide is defined as a chain of nucleotides.
  • nucleic acids are polymers of nucleotides.
  • nucleic acids and nucleic acids are polymers of nucleotides.
  • 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 PCRTM, and the like, and by synthetic means.
  • recombinant means i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCRTM, and the like, and by synthetic means.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • polypeptides derived from antibodies such as, for example, single-chain variable fragments (scFv), Fab, Fab’, F(ab’)2, and Fv fragments
  • T Cell receptors such as, for example, TCR variable domains
  • any ligand or receptor fragment that binds to the extracellular cognate protein such as, for example, TCR variable domains.
  • 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.
  • 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.
  • Immune cells 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.
  • peripheral blood 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.
  • red blood cells e.g., red blood cells, white blood cells and platelets
  • platelets e.g., red blood cells, white blood cells and platelets
  • PBMCs Peripheral blood mononuclear cells
  • PMBCs are blood cells with round nuclei and include lymphocytes, monocytes and macrophages.
  • PMBCs are obtained by density gradient centrifugation of anticoagulated peripheral venous blood using a hydrophilic colloid.
  • 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 can be achieved by any one of a number of means including electroporation, microinjection, gene gun delivery, retroviral infection, lipofection, superfection and the like.
  • developmentally differentiated cells 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
  • 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.
  • 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.
  • kits for treating, preventing and/or delaying the onset of a disease, disorder or condition are also provided herein.
  • methods of regulating a treatment e.g., a cell-based treatment
  • the methods include contacting a cell with a compound, or pharmaceutically acceptable salt thereof, provided herein.
  • 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.
  • 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.
  • 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.
  • the cell contacted with a compound provided herein is a cell contained within a subject that had not been removed from the subject.
  • a cell is contacted ex vivo or in vitro with a compound, or
  • 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.
  • binding of the compound to the chimeric polypeptide results in multimerization of the chimeric polypeptide.
  • 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.
  • 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.
  • 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.
  • 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.
  • the chimeric polypeptide may contain a polypeptide that activates cell activity, for example, immune activity, such as, for example, a co-stimulating polypeptide.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • GvHD graft versus host disease
  • GvHD can be divided into an acute form and a chronic form.
  • Acute GVHD 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
  • Acute GvHD of the skin can result in a diffuse maculopapular rash, sometimes in a lacy pattern.
  • 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 10 6 , greater than 10 7 , greater than 10 8 , or greater than 10 9 cells.
  • the number of T cells/kg subject body weight that may be administered may be, for example, from about 1 x 10 4 T cells/kg subject body weight to about 9 x 10 7 T cells/kg subject body weight, for example about 1 , 2, 3, 4, 5, 6, 7, 8, or 9 x 10 4 ; 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 10 6 ; or about 1 , 2, 3, 4, 5, 6, 7, 8, or 9 x 10 7 T cells/kg subject body weight.
  • 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 10 4 therapeutic cells/kg body weight to about 9 x 10 7 therapeutic cells/kg body weight, for example about 1 , 2, 3, 4, 5, 6, 7, 8, or 9 x 10 4 ; about 1 , 2,
  • unit dose 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.
  • an "effective amount" of a compound or pharmaceutical composition 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.
  • 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.”
  • 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.”
  • 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.”
  • 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.
  • the term may refer to the size of a tumor, or the number of tumors present in an organ or tissue.
  • 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.
  • 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.
  • 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.
  • 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.
  • “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.
  • 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.
  • 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.
  • one or more agents may be administered within from
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a compound provided herein, or a pharmaceutically acceptable salt thereof may be administered to the patient.
  • 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.
  • 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.
  • 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.
  • a symptom or condition associated with the presence of the target cells may be administered, 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • cell therapy is terminated before the appearance of negative symptoms.
  • the therapy may be terminated after the transplant has made progress toward engraftment, but before clinically observable GvHD, or other negative symptoms, can occur.
  • 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 containing nucleic acids, cells, proteins and/or compounds are also provided herein.
  • 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.
  • CID chemically induced multimerization
  • kits or combination provided herein contains a compound provided herein. .
  • a kit or combination provided herein contains a compound of Formula B as described herein.
  • 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
  • the instructions include instructions for administering the compound to a cell and/or a subject.
  • 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.
  • 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.
  • the compound provided herein e.g., a compound of Formula B
  • a compound described in U.S. Patent application no. 62/608,552 e.g.,
  • kits 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.
  • CID chemically inducing multimerization
  • the instructions include instructions for administering the compounds to a cell and/or a subject.
  • 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.
  • 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).
  • cytokine storms e.g., tumor lysis syndrome, cytokine release syndrome, macrophage activation syndrome
  • serious adverse events associated with therapeutic cell (e.g., CAR T cell) treatment 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
  • kits 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.
  • the one or more chimeric polypeptides includes one or more of any of the chimeric polypeptides described herein and/or in references incorporated herein.
  • one of the one or more chimeric polypeptides includes one or more polypeptides that bind to a compound provided herein.
  • 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.
  • 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.
  • 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).
  • 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).
  • 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).
  • kits 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.
  • at least one of the two or more chimeric polypeptides includes one or more polypeptides that bind to a compound provided herein.
  • 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
  • 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).
  • 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.
  • the polypeptide to which an FRB and/or FKBP12 is fused is a cell elimination polypeptide
  • the polypeptide to which an FKBP12v36 is fused is a cell activation polypeptide.
  • the polypeptide to which an FRB and/or FKBP12 is fused is a cell activation polypeptide
  • the polypeptide to which an FKBP12v36 is fused is a cell elimination polypeptide.
  • 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).
  • 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).
  • 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).
  • kits 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 includes a compound provided herein.
  • the compound is a compound of Formula B as described herein.
  • 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.
  • kits 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.
  • instructions e.g., written material
  • CID chemically inducing multimerization
  • a compound provided herein can be prepared as a pharmaceutically acceptable salt.
  • 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.
  • 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.
  • conventional non-toxic salts include those derived from bases, such as potassium hydroxide, sodium hydroxide, ammonium hydroxide, caffeine, various amines, and the like.
  • a pharmaceutically acceptable salt can be prepared from a parent compound, which contains a basic or acidic moiety, by conventional chemical methods.
  • 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.
  • nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile.
  • pharmaceutically acceptable 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.
  • preparations may meet sterility, pyrogenicity, and general safety and purity standards as required by FDA Office of Biologies standards.
  • a compound provided herein often is a stable compound and often has a stable structure in a composition provided.
  • 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.
  • pharmaceutical compositions comprising a compound described herein, or a 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.
  • solvents, and diluents include water, saline, dextrose, ethanol, glycerol, oil, and the like.
  • 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.
  • 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.
  • 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.
  • a pharmaceutical composition is a liquid composition.
  • a pharmaceutical composition is provided as a dry powder composition.
  • a pharmaceutical composition is in a liposomal composition, sometimes as a micro-emulsion.
  • a pharmaceutical composition is a spray dried composition.
  • a pharmaceutical composition comprises a pharmaceutically acceptable co-polymer.
  • 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.
  • 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.
  • 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.
  • 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.
  • Water or saline used for preparing a pharmaceutical composition may be buffered or not buffered.
  • 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)).
  • D5NS normal saline
  • D10NS 5% dextrose in normal saline
  • D10NS 5% dextrose in normal saline
  • D5HNS 5% dextrose in half-normal saline
  • D10HNS 10% dextrose in half-normal saline
  • 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).
  • PBS phosphate buffered saline
  • HBSS Hank's balanced salt solution
  • EBSS Earle's balanced salt solution
  • SSC standard saline citrate
  • HBS HEPES- buffered saline
  • GBSS Gey's balanced salt solution
  • 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.
  • a liquid pharmaceutical composition has an 80%
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Nitrogen And Oxygen Or Sulfur-Condensed Heterocyclic Ring Systems (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

La technologie concerne en partie des composés qui se lient à des protéines. Dans certains exemples, les composés peuvent se lier à des protéines de liaison à la rapamycine. Dans certains exemples, les composés peuvent se lier à des protéines cellulaires et/ou des formes de variants de protéines cellulaires de liaison à la rapamycine et/ou à des analogues de la rapamycine. Dans certains exemples, les composés se lient à des protéines multimériques de liaison à la rapamycine et/ou à des analogues de la rapamycine, par exemple une protéine FKBP12 (et/ou des variants de celle-ci) et un polypeptide mTOR et/ou un domaine tel que FRB, et/ou des variants de celui-ci. L'invention concerne des composés comprenant ceux ayant une structure de formule A, dans laquelle les fractions R20, R21 et R22 sont décrites ici.
PCT/US2019/055067 2018-10-12 2019-10-07 Composés à liaison protéinique Ceased WO2020076738A2 (fr)

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Cited By (7)

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US11021492B2 (en) 2019-01-22 2021-06-01 Aeovian Pharmaceuticals, Inc. mTORC modulators and uses thereof
WO2022020522A2 (fr) 2020-07-21 2022-01-27 Aeovian Pharmaceuticals, Inc. Modulateurs de mtorc1 et leurs utilisations
CN114539288A (zh) * 2020-11-24 2022-05-27 鲁南制药集团股份有限公司 一种高效的依维莫司制备方法
US11603377B2 (en) 2020-03-27 2023-03-14 Aeovian Pharmaceuticals, Inc. MTORC1 modulators and uses thereof
US11819476B2 (en) 2019-12-05 2023-11-21 Janssen Pharmaceutica Nv Rapamycin analogs and uses thereof
US11944605B2 (en) 2018-06-15 2024-04-02 Janssen Pharmaceutica Nv Rapamycin analogs and uses thereof
EP4281463A4 (fr) * 2021-01-22 2025-07-23 Janssen Pharmaceutica Nv Analogues de la rapamycine et leurs utilisations

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US4894366A (en) * 1984-12-03 1990-01-16 Fujisawa Pharmaceutical Company, Ltd. Tricyclo compounds, a process for their production and a pharmaceutical composition containing the same
GB0417852D0 (en) * 2004-08-11 2004-09-15 Biotica Tech Ltd Production of polyketides and other natural products
SG158143A1 (en) * 2004-12-20 2010-01-29 Wyeth Corp Rapamycin analogues and the uses thereof in the treatment of neurological, proliferative, and inflammatory disorders
GB201312318D0 (en) * 2013-07-09 2013-08-21 Isomerase Therapeutics Ltd Novel methods and compounds

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11944605B2 (en) 2018-06-15 2024-04-02 Janssen Pharmaceutica Nv Rapamycin analogs and uses thereof
US11021492B2 (en) 2019-01-22 2021-06-01 Aeovian Pharmaceuticals, Inc. mTORC modulators and uses thereof
US11230557B2 (en) 2019-01-22 2022-01-25 Aeovian Pharmaceuticals, Inc. mTORC modulators and uses thereof
US11702429B2 (en) 2019-01-22 2023-07-18 Aeovian Pharmaceuticals, Inc. mTORC modulators and uses thereof
EP4069223A4 (fr) * 2019-12-05 2023-12-20 Janssen Pharmaceutica NV Analogues de la rapamycine et leurs utilisations
US11819476B2 (en) 2019-12-05 2023-11-21 Janssen Pharmaceutica Nv Rapamycin analogs and uses thereof
US11634432B2 (en) 2020-03-27 2023-04-25 Aeovian Pharmaceuticals, Inc. mTORC1 modulators and uses thereof
US11603377B2 (en) 2020-03-27 2023-03-14 Aeovian Pharmaceuticals, Inc. MTORC1 modulators and uses thereof
CN116322677A (zh) * 2020-07-21 2023-06-23 艾奥维安制药公司 Mtorc1调节剂及其用途
WO2022020522A3 (fr) * 2020-07-21 2022-03-03 Aeovian Pharmaceuticals, Inc. Modulateurs de mtorc1 et leurs utilisations
WO2022020522A2 (fr) 2020-07-21 2022-01-27 Aeovian Pharmaceuticals, Inc. Modulateurs de mtorc1 et leurs utilisations
CN116322677B (zh) * 2020-07-21 2025-09-23 艾奥维安制药公司 Mtorc1调节剂及其用途
CN114539288A (zh) * 2020-11-24 2022-05-27 鲁南制药集团股份有限公司 一种高效的依维莫司制备方法
CN114539288B (zh) * 2020-11-24 2024-01-30 鲁南制药集团股份有限公司 一种依维莫司的制备方法
EP4281463A4 (fr) * 2021-01-22 2025-07-23 Janssen Pharmaceutica Nv Analogues de la rapamycine et leurs utilisations

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