WO2023107597A2 - Bicyclic heteroaromatic amide compounds and uses thereof - Google Patents

Bicyclic heteroaromatic amide compounds and uses thereof Download PDF

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WO2023107597A2
WO2023107597A2 PCT/US2022/052216 US2022052216W WO2023107597A2 WO 2023107597 A2 WO2023107597 A2 WO 2023107597A2 US 2022052216 W US2022052216 W US 2022052216W WO 2023107597 A2 WO2023107597 A2 WO 2023107597A2
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optionally substituted
compound
weeks
phenyl
alkyl
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PCT/US2022/052216
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WO2023107597A3 (en
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Gnanasambandam Kumaravel
Madeline MACDONNELL
Hairuo Peng
Iwona WRONA
Kerem OZBOYA
Bertrand Le Bourdonnec
Vanessa KURIA
Matthew Lucas
Byron Delabarre
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Kineta, Inc.
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Publication of WO2023107597A2 publication Critical patent/WO2023107597A2/en
Publication of WO2023107597A3 publication Critical patent/WO2023107597A3/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
    • C07D209/04—Indoles; Hydrogenated indoles
    • C07D209/30—Indoles; Hydrogenated indoles with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to carbon atoms of the hetero ring
    • C07D209/42—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
    • C07D277/02—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings
    • C07D277/20—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D277/32—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D277/56—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
    • C07D295/16—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms acylated on ring nitrogen atoms
    • C07D295/18—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms acylated on ring nitrogen atoms by radicals derived from carboxylic acids, or sulfur or nitrogen analogues thereof
    • C07D295/182—Radicals derived from carboxylic acids
    • C07D295/185—Radicals derived from carboxylic acids from aliphatic carboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D333/00—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom
    • C07D333/02—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings
    • C07D333/04—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings not substituted on the ring sulphur atom
    • C07D333/26—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings not substituted on the ring sulphur atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D333/38—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
    • C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
    • C07D491/10—Spiro-condensed systems
    • C07D491/107—Spiro-condensed systems with only one oxygen atom as ring hetero atom in the oxygen-containing ring
    • 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
    • C07D498/02—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 two hetero rings
    • C07D498/04—Ortho-condensed systems
    • 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
    • C07D498/02—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 two hetero rings
    • C07D498/08—Bridged systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00

Definitions

  • TDP­43 is a nuclear DNA/RNA binding protein involved in RNA splicing. Under pathological cell stress, TDP­43 translocates to the cytoplasm and aggregates into stress granules. These phenotypes are hallmarks of degenerating motor neurons and are found in 97% of all ALS cases. The highly penetrant nature of this pathology indicates that TDP­43 is broadly involved in both familial and sporadic ALS. Additionally, TDP­43 mutations that promote aggregation are linked to higher risk of developing ALS, suggesting protein misfolding and aggregation act as drivers of toxicity. TDP­43 toxicity can be recapitulated in yeast models, where the protein induces a viability deficit and localizes to stress granules.
  • the present inventors have discovered that the CYP51A1 inhibitors described herein are capable of reversing TDP­43 induced toxicity. Accordingly, the present invention describes such CYP51A1 compounds and methods of using these compounds for the treatment of disorders related to TDP­43 toxicity such as ALS.
  • the invention features a compound, or a pharmaceutically acceptable salt thereof, having the structure: Formula I wherein m is 0, 1, 2, 3, or 4; X 1 is CH, S, or N; X 2 and X 3 are, independently, N, CH, or CR 1 ; X 4 is NH or S; each R 1 is, independently, halo, optionally substituted C1­C6 alkyl, or optionally substituted C1­C6 alkoxy; Ar is optionally substituted C6­C10 aryl or optionally substituted C2­C9 heteroaryl; L 1 is ­CONR­ or­NRCO­; R is hydrogen or optionally substituted C1­C6 alkyl; L 2 is optionally substituted C1­C6 alkylene or optionally substituted C3­C8 cycloalkylene; R A is ­CH2CONHR 2 , ­CONHR 2 , or ­COR 2 ; R 2 is optionally substituted aryl, optionally substituted heteroaryl, optional
  • R is hydrogen. In some embodiments, R is optionally substituted C1­C6 alkyl (e.g., methyl). In some embodiments, X 1 is CH. In some embodiments, X 1 is S. In some embodiments, X 1 is N. In some embodiments, X 2 is CR 1 and X 3 is N. In some embodiments, the compound has the structure: or a pharmaceutically acceptable salt thereof. In some embodiments, X 2 is N and X 3 is CR 1 . In some embodiments, the compound has the structure: . Formula 1b or a pharmaceutically acceptable salt thereof. In some embodiments, X 2 and X 3 are CR 1 .
  • the compound has the structure: , Formula 1c or a pharmaceutically acceptable salt thereof, where each R 1A is independently H or R 1 .
  • at least one R 1A is halo (e.g., fluoro, chloro, or bromo).
  • at least one R 1A is optionally substituted C1­C6 alkyl (e.g., methyl, ethyl, iso­propyl).
  • at least one R 1A is optionally substituted C1­C6 alkoxy (e.g., methoxy, ethoxy, or iso­propoxy).
  • At least one R 1 is halo (e.g., fluoro, chloro, or bromo). In some embodiments, at least one R 1 is optionally substituted C1­C6 alkyl (e.g., methyl, ethyl, iso­propyl). In some embodiments, at least one R 1 is optionally substituted C1­C6 alkoxy (e.g., methoxy, ethoxy, or iso­propoxy).
  • Ar is optionally substituted C6­C10 aryl (e.g., phenyl, 2­chloro­phenyl, 3­chloro­ phenyl, 4­chloro­phenyl, 2­fluoro­phenyl, 3­fluoro­phenyl, 4­fluoro­phenyl, 2­methyl­phenyl, 4­benzoxy­ phenyl, 2­methoxy­phenyl, 3­methoxy­phenyl, 4­methoxy­phenyl, 2­cyano­phenyl, 3­cyano­phenyl, 4­cyano­ phenyl, 2­chloro­4­fluoro­phenyl, 2,4­fluoro­phenyl, 2­chloro­3­fluoro­phenyl, 2­chloro­4­cyano­phenyl, 2­cyano­4­phenyl, 2­cyano­4­phenyl, 2­cyano­4­phenyl, 2
  • R 2 has the structure of Formula II. In some embodiments, R 2 has the structure of Formula III. In some embodiments, R 2 has the structure:
  • R 10 is optionally substituted C1­C6 alkyl; and each R 8 is, independently, halo, hydroxy, optionally substituted C1­C6 alkyl, or optionally substituted C1­C6 alkoxy.
  • the compound has the structure: Formula 1e or a pharmaceutically acceptable salt thereof, wherein R 13 is optionally substituted pyridin­4­yl or optionally substituted phenyl; and R 14 is optionally substituted piperidin­4­yl, optionally substituted 2­oxa­6­azaspiro[3.3]heptan­6­yl, 2­ azaspiro[3.3]heptan­2­yl substituted with hydroxy.
  • R 13 is optionally substituted phenyl.
  • the optionally substituted phenyl is 2,4­difluorphenyl.
  • R 13 is optionally substituted pyridin­4­yl.
  • the compound has the structure: or a pharmaceutically acceptable salt thereof, wherein R 15 is optionally substituted 4­azaspiro[2.4]heptan­4­yl.
  • the compound has the structure: Formula 1g or a pharmaceutically acceptable salt thereof, wherein R 16 is optionally substituted 2­oxa­5­azabicyclo[2.2.1]heptan­5­yl, or optionally substituted 3­ azabicyclo[3.1.0]hexan­3­yl.
  • the compound has the structure: Formula 1h or a pharmaceutically acceptable salt thereof.
  • R 17 and R 18 are each, independently, H or F; and R 19 is 2­oxa­5­azabicyclo[2.2.1]heptan­5­yl, or 3­azabicyclo[3.1.0]hexan­3­yl.
  • the compound has the structure: Formula 1i or a pharmaceutically acceptable salt thereof, wherein R 20 is optionally substituted 2­oxa­6­azaspiro[3.3]heptan­6­yl.
  • the compound has the structure: Formula 1j or a pharmaceutically acceptable salt thereof, wherein: R 21 is optionally substituted pyridinyl; and R 22 is piperidin­1­yl optionally substituted with methoxy; azetidin­1­yl optionally substituted with methyl, methoxy, or fluoro; 2­oxa­5­azabicyclo[2.2.1]heptan­5­yl, or optionally substituted morpholin­4­yl.
  • the compound has the structure or a pharmaceutically acceptable salt thereof, wherein In some embodiments, the compound has the structure: Formula 1l or a pharmaceutically acceptable salt thereof, wherein In some embodiments, the compound has the structure: Formula 1m or a pharmaceutically acceptable salt thereof, wherein X 14 is N or CH; and , In some embodiments, X 14 is N. In some embodiments, X 14 is CH.
  • the invention features a compound, or pharmaceutically acceptable salt thereof, having the structure: Formula 2 or a pharmaceutically acceptable salt thereof, wherein m is 0, 1, 2, 3, or 4; X 6 is CH, or N; X 7 is NH or S; Ar 1 is optionally substituted C6­C10 aryl; R 23 is hydrogen, halo, or optionally substituted C1­C6 alkoxy; L 3 is ­NR 24 CO­ or ­CONR 24 ­, R 24 is H or optionally substituted C1­C6 alkyl; R B is ­CH2CONHR 25 , or ­COR 25 or NR 25 ; R 25 is optionally substituted heteroaryl, optionally substituted cyclohexyl, or has the structure Formula II Formula III wherein R 26 is optionally substituted C1­C3 alkyl; R 27 is optionally substituted C1­C6 alkyl, or optionally substituted C1­C6 heteroalkyl;
  • X 6 is CH. In some embodiments, X 6 is N. In some embodiments, X 7 is N. In some embodiments, X 7 is S. In some embodiments, the compound has the structure: or a pharmaceutically acceptable salt thereof, In some embodiments, the compound has the structure: or a pharmaceutically acceptable salt thereof, In some embodiments, the compound has the structure: Formula 2c or a pharmaceutically acceptable salt thereof, In some embodiments, the compound has the structure: or a pharmaceutically acceptable salt thereof, In some embodiments, the compound has the structure: Formula 2e or a pharmaceutically acceptable salt thereof.
  • Ar 1 is phenyl, 2­chloro­phenyl, 4­chloro­phenyl, 2­fluoro­phenyl, 3­fluoro­ phenyl, 4­fluoro­phenyl, 2­methyl­phenyl, 2­methoxy­phenyl, 3­methoxy­phenyl, 4­methoxy­phenyl, 2­chloro­ 4­fluoro­phenyl, 2,4­fluoro­phenyl, 2­chloro­3­fluoro­phenyl, 2­cyano­4­fluoro­phenyl, 2­cyano­4­chloro­ phenyl, 2,3­difluoro­phenyl, 2­chloro­6­fluoro­phenyl, or 2­fluoro­4­chloro­phenyl.
  • R 22 has the structure , , , some embodiments, R 22 has the structure: In some embodiments, the compound has the structure: Formula 2f or a pharmaceutically acceptable salt thereof, wherein R 57 is halo. In some embodiments, R 57 is chloro. a pharmaceutically acceptable salt thereof. In some embodiments, the compound has the structure: a pharmaceutically acceptable salt thereof.
  • the invention features a compound, or pharmaceutically acceptable salt thereof, having the structure: Formula 3 or a pharmaceutically acceptable salt thereof, wherein n is 0, 1, 2, 3, or 4; L 4 is optionally substituted C1­C6 alkylene or optionally substituted C3­C8 cycloalkylene; X 9 is N and X 10 is CH, or X 9 is CH and X 10 is N; X 11 is N or CH; each R 30 is, independently, halo, optionally substituted C1­C6 alkyl, or optionally substituted C1­C6 alkoxy; Ar C is optionally disubstituted C6­C10 aryl, or C6­C10 aryl optionally monosubstituted with chloro, optionally substituted C 1 ­C 6 heteroalkyl, cyano, meta­fluoro, or ortho­fluoro; R C is COR 31 , or ­R 31 ; R 31 is optionally substituted C2­C5 heteroaryl, or has the
  • X 12 is NR 34 , CR 34 R 35 , O, or SR 34 R 35 ;
  • R 34 is absent, halo, oxo, hydrogen, hydroxy, optionally substituted amino, cyano, optionally substituted C1­C6 alkyl, optionally substituted C1­C6 heteroalkyl, ­SO2­optionally substituted C1­C6 alkyl, optionally substituted C2­C9 heterocyclyl, optionally substituted C6­C10 aryl, or optionally substituted C2­C9 heteroaryl, or R 33 and R 34 combine with the atoms to which they are attached to form an optionally substituted C2­C9 heteroaryl;
  • R 35 is absent, halo, oxo, hydroxy, optionally substituted amino, cyano, optionally substituted C1­C6 alkyl,
  • X 9 is N and X 10 is CH. In some embodiments, X 9 is CH and X 10 is N. In some embodiments, X 11 is N. In some embodiments, X 11 is CH. In some embodiments, the compound has the structure: Formula 3a or a pharmaceutically acceptable salt thereof. In some embodiments, the compound has the structure Formula 3b or a pharmaceutically acceptable salt thereof. In some embodiments at least one R 30 is halo. In some embodiments, at least one R 30 is chloro. In some embodiments, at least one R 30 is optionally substituted C1­C6 alkyl. In some embodiments, at least one R 30 is methyl. In some embodiments, at least one R 30 is optionally substituted C1­C6 alkoxy.
  • At least one R 30 is ethoxy.
  • Ar C is 2­chloro­phenyl, 3­chloro­phenyl, 4­chloro­phenyl, 2­fluoro­phenyl, 3­ fluoro­phenyl, 4­benzoxy­phenyl, 2­cyano­phenyl4­cyano­phenyl, 2­chloro­4­fluoro­phenyl, 2,4­difluoro­ phenyl, 2­chloro­3­fluoro­phenyl, 2­chloro­4­cyano­phenyl, 2­cyano­4­phenyl, 2­cyano­4­chloro­phenyl, 2,3­difluoro­phenyl, 2­fluoro­4­cyano­phenyl, 2­chloro­6­fluoro­phenyl, 2­fluoro­4­chloro­phenyl, 2,6­d
  • R 31 has the structure .
  • R 31 has the structure: wherein p is 0, 1, 2, 3, 4, or 5; q is 0, 1, 2, 3, or 4; and each R 36 is, independently, halo, hydroxy, optionally substituted C1­C6 alkyl, or optionally substituted C1­C6 alkoxy.
  • the compound has the structure: or a pharmaceutically acceptable salt thereof, wherein R 37 is cyano and R 38 is fluoro, or R 37 is fluoro and R 38 is cyano; and R 39 is or 3,3­difluoro­azetidin­1­yl.
  • the compound has the structure: or a pharmaceutically acceptable salt thereof, wherein l is 0 or 1; L 4 is is optionally substituted C1­C6 alkylene or optionally substituted C3­C8 cycloalkylene; and R 40 is 4­hydroxy­piperidin­1­yl, 3­methoxy­piperidin­1­yl, optionally substituted diazapen­1­yl, triazolopiperazinyl substituted with methyl, 4,4­difluoro­piperidin­1­yl, 1,1­dioxothiomorpholin­4­yl, 2­ (methoxymethyl)­pyrroloin­1­yl, tetrahydro­1,3­oxazin­3­yl, 4­isopropyl­piperazin­1­yl, 4­(2­oxazolidin­3­yl)­ piperidin­1­yl, optionally substituted 1,2,4 oxadizol
  • l is 1.
  • the compound has the structure Formula 3e or a pharmaceutically acceptable salt thereof, wherein R 41 is piperidin­1­yl substituted with optionally substituted dialkylamino.
  • the compound has the structure: Formula 3f or a pharmaceutically acceptable salt thereof, wherein R 43 is F or CN; and R 42 is optionally substituted azetidin­1­yl.
  • optionally substituted azetidine­1­yl is 3,3­difluoro­azetidin­1­yl.
  • the compound has the structure Formula 3g or a pharmaceutically acceptable salt thereof, wherein R 44 is optionally substituted azetidin­1­yl. In some embodiments, wherein optionally substituted azetidine­1­yl is 3,3­difluoro­azetidin­1­yl. In some embodiments, the compound has the structure:
  • the invention features a compound, or pharmaceutically acceptable salt thereof, having the structure: or a pharmaceutically acceptable salt thereof, wherein n is 0, 1, 2, 3, or 4; R 45 is halo; and R 46 is optionally substituted azetidinyl.
  • the compound has the structure: or a pharmaceutically acceptable salt thereof.
  • the compound is: , or a pharmaceutically acceptable salt thereof.
  • the invention features a compound, or pharmaceutically acceptable salt thereof, having the structure: Formula 5 or a pharmaceutically acceptable salt thereof, wherein p is 0 or 1; X 13 is a single bond or O; R 48 is optionally substituted C6­C10 aryl, optionally substituted C2­C5 heteroaryl, or trifluoromethyl; R 49 is H or optionally substituted C1­C6 alkyl; R 50 is optionally substituted C 6 ­C 10 aryl or optionally substituted C 2 ­C 5 hetetoaryl; and R 51 is optionally substituted C2­C5 heterocyclyl.
  • p is 0.
  • p is 1.
  • X 13 is O.
  • R 48 is optionally substituted C6­C10 aryl. In some embodiments, R 48 is , , . In some embodiments, R 48 is optionally substituted C2­C5 heteroaryl. In some embodiments, R 48 is In some embodiments, R 49 is H. In some embodiments, R 49 is C1­C6 alkyl. In some embodiments, R 49 is methyl. In some embodiments, R 50 is optionally substituted C6­C10 aryl.
  • R 50 is e
  • R 51 is morpholin­4­yl, 2­oxa­6­azaspiro[3.3]heptan­6­yl, or 4­hydroxy­ piperidin­4­yl.
  • the invention features a compound having the structure of any one of compounds 1­ 198, 356, 373, 386, 419, 435, or 436 in Table 1, or pharmaceutically acceptable salt thereof.
  • the invention features a compound having the structure of any one of compounds 199- 355, 357-372, 374-385, 387-418, 420-434, or 437-453 in Table 1 , or pharmaceutically acceptable salt thereof.
  • the invention features a pharmaceutical composition comprising any of the foregoing compounds, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.
  • the invention features a method of treating a neurological disorder (e.g,, frontotemporal dementia (FTLD-TDP), chronic traumatic encephalopathy, ALS, Alzheimer's disease, limbic-predominant age-related TDP-42 encephalopathy (LATE), or frontotemporal lobar degeneration) in a subject in need thereof.
  • a neurological disorder e.g, frontotemporal dementia (FTLD-TDP), chronic traumatic encephalopathy, ALS, Alzheimer's disease, limbic-predominant age-related TDP-42 encephalopathy (LATE), or frontotemporal lobar degeneration
  • This method includes administering an effective amount of any of the foregoing compounds or pharmaceutical compositions.
  • the invention features a method of inhibiting toxicity in a cell (e.g., mammalian neural cell) related to a protein (e.g., TDP-43).
  • This method includes administering an effective amount of any of the foregoing compounds or pharmaceutical compositions.
  • the invention features a method of treating a CYP51A1 -associated disorder (e.g., FTLD-TDP, chronic traumatic encephalopathy, ALS, Alzheimer’s disease, LATE, or frontotemporal lobar degeneration) in a subject in need thereof.
  • a CYP51A1 -associated disorder e.g., FTLD-TDP, chronic traumatic encephalopathy, ALS, Alzheimer’s disease, LATE, or frontotemporal lobar degeneration
  • This method includes administering an effective amount of any of the foregoing compounds pharmaceutical compositions.
  • the invention features a method of inhibiting CYP51 A1 . This method includes contacting a cell with an effective amount of any of the foregoing compounds or pharmaceutical compositions.
  • the invention features a method of treating a neurological disorder in a patient, such as a human patient, identified as likely to benefit from treatment with a CYP51 A1 inhibitor on the basis of TDP-43 aggregation.
  • the method may include (I) determining that the patient exhibits, or is prone to develop, TDP-43 aggregation, and (ii) providing to the patient a therapeutically effective amount of a CYP51A1 inhibitor.
  • the patient has previously been determined to exhibit, or to be prone to developing, TDP-43 aggregation, and the method includes providing to the patient a therapeutically effective amount of a CYP51A1 inhibitor.
  • the susceptibility of the patient to developing TDP-43 aggregation may be determined, e.g., by determining whether the patient expresses a mutant isoform of TDP-43 containing a mutation that is associated with TDP-43 aggregation and toxicity, such as a mutation selected from Q331 K, M337V, Q343R, N345K, R361 S, and N390D. This may be performed, for example, by determining the amino acid sequence of a TDP-43 isoform isolated from a sample obtained from the patient or by determining the nucleic acid sequence of a TDP-43 gene isolated from a sample obtained from the patient. In some embodiments, the method includes the step of obtaining the sample from the patient.
  • the invention features a method of treating a neurological disorder in a patient, such as a human patient, identified as likely to benefit from treatment with a CYP51 A1 inhibitor on the basis of TDP-43 expression.
  • the method includes (i) determining that the patient expresses a mutant form of TDP-43 having a mutation associated with TDP-43 aggregation (e.g., a mutation selected from Q331 K, M337V, Q343R, N345K, R361 S, and N390D), and (ii) providing to the patient a therapeutically effective amount of a CYP51 A1 inhibitor.
  • a mutant form of TDP-43 having a mutation associated with TDP-43 aggregation e.g., a mutation selected from Q331 K, M337V, Q343R, N345K, R361 S, and N390D
  • the patient has previously been determined to express a mutant form of TDP-43 having a mutation associated with TDP-43 aggregation, such as a Q331 K, M337V, Q343R, N345K, R361S, or N390D mutation, and the method includes providing to the patient a therapeutically effective amount of a CYP51 A1 inhibitor.
  • a mutation associated with TDP-43 aggregation such as a Q331 K, M337V, Q343R, N345K, R361S, or N390D mutation
  • the invention features a method of determining whether a patient (e.g., a human patient) having a neurological disorder is likely to benefit from treatment with a CYP51 A1 inhibitor by (I) determining whether the patient exhibits, or is prone to develop, TDP-43 aggregation and (ii) identifying the patient as likely to benefit from treatment with a CYP51 A1 inhibitor if the patient exhibits, or is prone to develop, TDP-43 aggregation.
  • the method further includes the step of (ill) informing the patient whether he or she is likely to benefit from treatment with a CYP51A1 inhibitor.
  • the susceptibility of the patient to developing TDP-43 aggregation may be determined, e.g., by determining whether the patient expresses a mutant isoform of TDP-43 containing a mutation that is associated with TDP-43 aggregation and toxicity, such as a mutation selected from Q331 K, M337V, Q343R, N345K, R361 S, and N390D. This may be performed, for example, by determining the amino acid sequence of a TDP-43 isoform isolated from a sample obtained from the patient or by determining the nucleic acid sequence of a TDP-43 gene isolated from a sample obtained from the patient. In some embodiments, the method includes the step of obtaining the sample from the patient.
  • the invention features a method of determining whether a patient (e.g., a human patient) having a neurological disorder is likely to benefit from treatment with a CYP51 A1 inhibitor by (i) determining whether the patient expresses a TDP-43 mutant having a mutation associated with TDP-43 aggregation (e.g., a mutation selected from Q331 K, M337V, Q343R, N345K, R361S, and N390D) and (ii) identifying the patient as likely to benefit from treatment with a CYP51A1 inhibitor if the patient expresses a TDP-43 mutant.
  • a mutation associated with TDP-43 aggregation e.g., a mutation selected from Q331 K, M337V, Q343R, N345K, R361S, and N390D
  • the method further includes the step of (iii) informing the patient whether he or she is likely to benefit from treatment with a CYP51 A1 inhibitor.
  • the TDP-43 isoform expressed by the patient may be assessed, for example, by isolated TDP-43 protein from a sample obtained from the patient and sequencing the protein using molecular biology techniques described herein or known in the art.
  • the TDP-43 isoform expressed by the patient is determined by analyzing the patient’s genotype at the TDP-43 locus, for example, by sequencing the TDP-43 gene in a sample obtained from the patient.
  • the method includes the step of obtaining the sample from the patient.
  • the CYP51A1 inhibitor is provided to the patient by administration of the CYP51A1 inhibitor to the patient. In some embodiments, the CYP51A1 inhibitor is provided to the patient by administration of a prodrug that is converted in vivo to the CYP51A1 inhibitor.
  • the neurological disorder is a neuromuscular disorder, such as a neuromuscular disorder selected from amyotrophic lateral sclerosis, congenital myasthenic syndrome, congenital myopathy, cramp fasciculation syndrome, Duchenne muscular dystrophy, glycogen storage disease type II, hereditary spastic paraplegia, inclusion body myositis, Isaac’s Syndrome, Kearns-Sayre syndrome, Lambert-Eaton myasthenic syndrome, mitochondrial myopathy, muscular dystrophy, myasthenia gravis, myotonic dystrophy, peripheral neuropathy, spinal and bulbar muscular atrophy, spinal muscular atrophy, Stiff person syndrome, Troyer syndrome, and Guillain-Barre syndrome.
  • the neurological disorder is amyotrophic lateral sclerosis.
  • the neurological disorder is selected from frontotemporal degeneration (also referred to as frontotemporal lobar degeneration and frontotemporal dementia), Alzheimer’s disease, Parkinson’s disease, dementia with Lewy Bodies, corticobasal degeneration, progressive supranuclear palsy, dementia parkinsonism ALS complex of Guam, Huntington's disease, Inclusion body myopathy with early-onset Paget disease and frontotemporal dementia (IBMPFD), sporadic inclusion body myositis, myofibrillar myopathy, dementia pugilistica, chronic traumatic encephalopathy, Alexander disease, and hereditary inclusion body myopathy.
  • frontotemporal degeneration also referred to as frontotemporal lobar degeneration and frontotemporal dementia
  • Alzheimer’s disease Parkinson’s disease
  • dementia with Lewy Bodies corticobasal degeneration
  • progressive supranuclear palsy progressive supranuclear palsy
  • dementia parkinsonism ALS complex of Guam Huntington's disease
  • the neurological disorder is amyotrophic lateral scierosis
  • the CYP51A1 inhibitor following administration of the CYP51A1 inhibitor to the patient, the patient exhibits one or more, or ail, of the following responses:
  • an increase in slow vital capacity such as an increase in the patient’s slow vital capacity within one or more days, weeks, or months following administration of the CYP51A.1 inhibitor
  • an increase in the patient’s slow vital capacity within from about 1 day to about 48 weeks (e.g., within from about 2 days to about 36 weeks, from about 4 weeks to about 24 weeks, from about 8 weeks to about 20 weeks, or from about 12 weeks to about 16 weeks), or more, following the initial administration of the CYP51A1 inhibitor to the patient, such as within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34
  • (ill) a reduction in decremental responses exhibited by the patient upon repetitive nerve stimulation, such as a reduction that is observed within one or more days, weeks, or months following administration of the CYP51 A1 inhibitor (e.g., a reduction that is observed within from about 1 day to about 48 weeks (e.g., within from about 2 days to about 36 weeks, from about 4 weeks to about 24 weeks, from about 8 weeks to about 20 weeks, or from about 12 weeks io about 16 weeks), or more, foilowing the initial administration of the CYP51A1 inhibitor to the patient, such as within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks
  • Council muscle testing scale (as described, e.g., in Jagtap et al., Ann. Indian. Acad. Neurol. 17:336-339 (2014), the disclosure of which is incorporated herein by reference as it pertains to measuring patient response to neurological disease treatment), such as an improvement that is observed within one or more days, weeks, or months following administration of the CYP51A1 inhibitor (e.g., an improvement that is observed within from about 1 day to about 48 weeks (e.g., within from about 2 days to about 36 weeks, from about 4 weeks to about 24 weeks, from about 8 weeks to about 20 weeks, or from about 12 weeks to about 16 weeks), or more, following the initial administration of the CYP51A1 inhibitor to the patient, such as within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19
  • an improvement in quality of life as assessed, for example, using the amyotrophic lateral sclerosis-specific quality of life (ALS-specific QOL) questionnaire, such as an improvement in the patient’s quality of life that is observed within one or more days, weeks, or months following administration of the CYP51 A1 inhibitor (e.g., an improvement in the subject’s quality of life that is observed within from about 1 day to about 48 weeks (e.g., within from about 2 days to about 36 weeks, from about 4 weeks to about 24 weeks, from about 8 weeks to about 20 weeks, or from about 12 weeks to about 16 weeks), or more, following the initial administration of the CYP51A1 inhibitor to the patient, such as within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22
  • a decrease in the frequency and/or severity of muscle cramps such as a decrease in cramp frequency and/or severity within one or more days, weeks, or months following administration of the CYP51 A1 inhibitor (e.g., a decrease in cramp frequency and/or severity within from about 1 day to about 48 weeks (e.g., within from about 2 days to about 36 weeks, from about 4 weeks to about 24 weeks, from about 8 weeks to about 20 weeks, or from about 12 weeks to about 16 weeks), or more, following the initial administration of the CYP51A1 inhibitor to the patient, such as within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32
  • a decrease in TDP-43 aggregation such as a decrease in TDP-43 aggregation within one or more days, weeks, or months following administration of the CYP51A1 inhibitor (e.g., a decrease in TDP-43 aggregation within from about 1 day to about 48 weeks (e.g., within from about 2 days to about 36 weeks, from about 4 weeks to about 24 weeks, from about 8 weeks to about 20 weeks, or from about 12 weeks to about 16 weeks), or more, following the initial administration of the CYP51A1 inhibitor to the patient, such as within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks
  • one or more compounds depicted herein may exist in different tautomeric forms.
  • references to such compounds encompass all such tautomeric forms
  • tautomeric forms result from the swapping of a single bond with an adjacent double bond and the concomitant migration of a proton.
  • a tautomeric form may be a prototropic tautomer, which is an isomeric protonation states having the same empirical formula and total charge as a reference form.
  • moieties with prototropic tautomeric forms are ketone - enol pairs, amide - imidic acid pairs, lactam - lactim pairs, amide - imidic acid pairs, enamine ⁇ imine pairs, and annular forms where a proton can occupy two or more positions of a heterocyclic system, such as, 1 H- and 3H-imidazole, 1H-, 2H- and 4H- 1 ,2,4-triazole, 1 H- and 2H- isoindoie, and 1 H- and 2H-pyrazole.
  • tautomeric forms can be in equilibrium or sterically locked into one form by appropriate substitution.
  • tautomeric forms result from acetal interconversion, e.g., the interconversion illustrated in the scheme below:
  • isotopes of compounds described herein may be prepared and/or utilized in accordance with the present invention.
  • “Isotopes” refers to atoms having the same atomic number but different mass numbers resulting from a different number of neutrons in the nuclei.
  • isotopes of hydrogen include tritium and deuterium.
  • an isotopic substitution e.g., substitution of hydrogen with deuterium ⁇ may alterthe physiciochemical properties of the molecules, such as metabolism and/or the rate of racemization of a chiral center.
  • compounds described and/or depicted herein may be provided and/or utilized in salt form.
  • compounds described and/or depicted herein may be provided and/or utilized in hydrate or solvate form.
  • substituents of compounds of the present disclosure are disclosed in groups or in ranges. It is specifically intended that the present disclosure include each and every individual subcombination of the members of such groups and ranges.
  • C1-C5 alkyl is specifically intended to individually disclose methyl, ethyl, Cs alkyl, C4 alkyl, C5 alkyl, and Co alkyl.
  • the present disclosure is intended to cover individual compounds and groups of compounds (e.g., genera and subgenera) containing each and every individual subcombination of members at each position.
  • optionally substituted X e.g., optionally substituted alkyl
  • X is optionally substituted
  • alkyl wherein said alkyl is optionally substituted
  • acyl represents a hydrogen or an alkyl group, as defined herein that is attached to a parent molecular group through a carbonyl group, as defined herein, and is exemplified by formyl (i.e., a carboxyaldehyde group), acetyl, trifluoroacetyl, propionyl, and butanoyl.
  • exemplary unsubstituted acyl groups include from 1 to 6, from 1 to 11 , or from 1 to 21 carbons.
  • alkyl refers to a branched or straight-chain monovalent saturated aliphatic hydrocarbon radical of 1 to 20 carbon atoms (e.g,, 1 to 16 carbon atoms, 1 to 10 carbon atoms, or 1 to 6 carbon atoms).
  • An alkylene is a divalent alkyl group.
  • alkenyl refers to a straight-chain or branched hydrocarbon residue having a carbon-carbon double bond and having 2 io 20 carbon atoms (e.g., 2 to 16 carbon atoms, 2 to 10 carbon atoms, 2 to 6, or 2 carbon atoms).
  • alkynyl refers to a straight-chain or branched hydrocarbon residue having a carbon-carbon triple bond and having 2 to 20 carbon atoms (e.g., 2 to 16 carbon atoms, 2 to 10 carbon atoms, 2 io 6, or 2 carbon atoms).
  • amino represents -N(R N1 )2, wherein each R N1 is, independently, H, OH, NO2, N(R N2 )2, SO2OR N2 , SOZR N2 , SOR N2 , an ⁇ /-protecting group, alkyl, alkoxy, aryl, arylalkyl, cycloalkyl, acyl (e.g., acetyl, trifluoroacetyl, or others described herein), wherein each of these recited R N1 groups can be optionally substituted; or two R N1 combine to form an alkylene or heteroalkylene, and wherein each R N2 is, independently, H, alkyl, or aryl.
  • the amino groups of the invention can be an unsubstituted amino (i.e., -NH2) or a substituted amino (i.e., -N(R N1 )2>.
  • aryl refers to an aromatic mono- or polycarbocyclic radical of 6 to 12 carbon atoms having at least one aromatic ring.
  • groups include, but are not limited to, phenyl, naphthyl, 1 ,2,3,4-tetrahydronaphthyl, 1 ,2-dihydronaphthyl, indanyl, and 7/7-indenyl.
  • arylalkyl represents an alkyl group substituted with an aryl group.
  • exemplary unsubstituted arylalkyl groups are from 7 to 30 carbons (e.g., from 7 to 16 or from 7 to 20 carbons, such as CB-IO aryl Ci-Cs alkyl, Cs-10 aryl C1-C10 alkyl, or Ca-10 aryl C1-C20 alkyl), such as, benzyl and phenethyl.
  • the akyl and the aryl each can be further substituted with 1 , 2, 3, or 4 substituent groups as defined herein for the respective groups.
  • cyano represents a CN group.
  • Carbocyclyl refer to a non-aromatic C3-C12 monocyclic, bicyclic, or tricyclic structure in which the rings are formed by carbon atoms.
  • Carbocyclyl structures include cycloalkyl groups and unsaturated carbocyclyl radicals.
  • cycloalkyl refers to a saturated, non-aromatic, monovalent mono- or polycarbocyclic radical of three to ten, preferably three to six carbon atoms. This term is further exemplified by radicals such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, norbornyl, and adamantyl.
  • a cycloalkylene is a divalent alkyl group.
  • halo means a fluorine (fluoro), chlorine (chloro), bromine (bromo), or iodine (iodo) radical.
  • heteroalkyl refers to an alkyl group, as defined herein, in which one or more of the constituent carbon atoms have been replaced by nitrogen, oxygen, or sulfur.
  • the heteroalkyl group can be further substituted with 1 , 2, 3, or 4 substituent groups as described herein for alkyl groups.
  • heteroalkyl groups include an “alkoxy” which, as used herein, refers alkyl-O- (e.g., methoxy and ethoxy).
  • a heteroalkylene is a divalent heteroalkyl group.
  • heteroaikenyl refers to an alkenyl group, as defined herein, in which one or more of the constituent carbon atoms have been replaced by nitrogen, oxygen, or sulfur.
  • the heteroalkenyl group can be further substituted with 1 , 2, 3, or 4 substituent groups as described herein for alkenyl groups.
  • heteroaikenyl groups include an “alkenoxy” which, as used herein, refers alkenyl-O-.
  • a heteroalkenylene is a divalent heteroaikenyl group.
  • heteroalkynyl refers to an alkynyl group, as defined herein, in which one or more of the constituent carbon atoms have been replaced by nitrogen, oxygen, or sulfur.
  • the heteroalkynyl group can be further substituted with 1 , 2, 3, or 4 substituent groups as described herein tor alkynyl groups.
  • heteroalkynyl groups include an “alkynoxy” which, as used herein, refers alkynyl-O-.
  • a heteroalkynylene is a divalent heteroalkynyl group.
  • heteroaryl refers to an aromatic mono- or polycyclic radical of 5 to 12 atoms having at least one aromatic ring and containing one, two, or three ring heteroatoms selected from N, O, and S, with the remaining ring atoms being C. One or two ring carbon atoms of the heteroaryl group may be replaced with a carbonyl group.
  • heteroaryl groups include pyridyl, pyrazoyl, benzooxazolyl, benzoimidazolyl, benzothiazolyl, imidazolyl, oxaxolyl, and thiazolyl.
  • heteroarylalkyl represents an alkyl group substituted with a heteroaryl group.
  • exemplary unsubstituted heteroarylalkyl groups are from 7 to 30 carbons (e.g., from 7 to 16 or from 7 to 20 carbons, such as C2-C9 heteroaryl Ci-Ce alkyl, C2-C9 heteroaryl Ci-Cw alkyl, or C2-C9 heteroaryl C1-C20 alkyl).
  • the akyl and the heteroaryl each can be further substituted with 1 , 2, 3, or 4 substituent groups as defined herein for the respective groups.
  • heterocyclyl denotes a mono- or polycyclic radical having 3 to 12 atoms having at least one ring containing one, two, three, or four ring heteroatoms selected from N, O or S, wherein no ring is aromatic.
  • heterocyclyl groups include, but are not limited to, morpholinyl, thiomorpholinyl, furyl, piperazinyl, piperidinyl, pyranyl, pyrrolidinyl, tetrahydropyranyl, tetrahydrofuranyl, and 1 ,3-dioxanyl.
  • heterocyclyciylalkyl represents an alkyl group substituted with a heterocyclyl group.
  • exemplary unsubstituted heterocyciylalkyl groups are from 7 to 30 carbons (e.g., from 7 to 16 or from 7 to 20 carbons, such as C2-C9 heterocyclyl Ci-Cs alkyl, C2-C9 heterocyclyl C1-C10 alkyl, or C2-C9 heterocyclyl C1-C20 alkyl), in some embodiments, the akyl and the heterocyclyl each can be further substituted with 1 , 2, 3, or 4 substituent groups as defined herein for the respective groups.
  • hydroxyl represents an -OH group.
  • A/-protecting group represents those groups intended to protect an amino group against undesirable reactions during synthetic procedures. Commonly used /V-protecting groups are disclosed in Greene, “Protective Groups in Organic Synthesis,” 3 rd Edition (John Wiley & Sons, New York, 1999).
  • A/-protecting groups include acyl, aryloyl, or carbamyl groups such as formyl, acetyl, propionyl, pivaloyl, t-butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoroac-etyl, trichloroacetyl, phthalyl, o-nitrophenoxyacetyl, a-chlorobuiyryl, benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl, and chiral auxiliaries such as protected or unprotected D, L or D, L-amino acids such as alanine, leucine, and phenylalanine; sulfonyl-coniaining groups such as benzenesulfonyl, and p-toluenesulfonyl; carbamate forming groups such as benzyl
  • Preferred /V-protecting groups are alloc, formyl, acetyl, benzoyl, pivaloyl, t-butylacetyl, alanyl, phenylsulfonyl, benzyl, t-butyloxycarbonyl (Boc), and benzyloxycarbonyl (Cbz).
  • nitro represents an NO2 group.
  • thiol represents an -SH group.
  • alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl (e.g., cycloalkyl), aryl, heteroaryl, and heterocyclyl groups may be substituted or unsubstituted. When substituted, there will generally be 1 to 4 substituents present, unless otherwise specified.
  • Substituents include, for example, aryl (e.g., substituted and unsubstituted phenyl), carbocyclyl (e.g., substituted and unsubstituted cycloalkyl), halo (e.g., fluoro), hydroxyl, oxo, heteroalkyl (e.g., substituted and unsubstituted methoxy, ethoxy, or thioalkoxy), heteroaryl, heterocyclyl, amino (e.g., NHz or mono- or dialkyl amino), azido, cyano, nitro, or thiol.
  • aryl e.g., substituted and unsubstituted phenyl
  • carbocyclyl e.g., substituted and unsubstituted cycloalkyl
  • halo e.g., fluoro
  • hydroxyl oxo
  • heteroalkyl e.g., substituted and un
  • Aryl, carbocyclyl (e.g., cycloalkyl), heteroaryl, and heterocyclyl groups may also be substituted with alkyl (unsubstituted and substituted such as arylalkyl (e.g., substituted and unsubstituted benzyl)).
  • Compounds of the invention can have one or more asymmetric carbon atoms and can exist in the form of optically pure enantiomers, mixtures of enantiomers such as, for example, racemates, optically pure diastereoisomers, mixtures of diastereoisomers, diastereoisomeric racemates or mixtures of diastereoisomeric racemates.
  • the optically active forms can be obtained, for example, by resolution of the racemates, by asymmetric synthesis or asymmetric chromatography (chromatography with a chiral adsorbent or eluant). That is, certain of the disclosed compounds may exist in various stereoisomeric forms.
  • Stereoisomers are compounds that differ only in their spatial arrangement.
  • Enantiomers are pairs of stereoisomers whose mirror images are not superimposable, most commonly because they contain an asymmetrically substituted carbon atom that acts as a chiral center. "Enantiomer” means one of a pair of molecules that are mirror images of each other and are not superimposable. Diastereomers are stereoisomers that are not related as mirror images, most commonly because they contain two or more asymmetrically substituted carbon atoms and represent the configuration of substituents around one or more chiral carbon atoms. Enantiomers of a compound can be prepared, for example, by separating an enantiomer from a racemate using one or more well-known techniques and methods, such as, for example, chiral chromatography and separation methods based thereon.
  • Racemate or “racemic mixture” means a compound containing two enantiomers, wherein such mixtures exhibit no opticai activity; i.e., they do not rotate the plane of polarized light.
  • Geometric- isomer means isomers that differ in the orientation of substituent atoms in relationship to a carbon-carbon double bond, to a cycloalkyl ring, or to a bridged bicyclic system.
  • Atoms (other than H) on each side of a carbon- carbon double bond may be in an E (substituents are on opposite sides of the carbon- carbon double bond) or Z (substituents are oriented on the same side) configuration.
  • E substituted amino acid
  • Z substituted amino acid
  • R* ,” “E,” “Z,” “cis,” and “trans,” indicate configurations relative to the core molecule.
  • Certain of the disclosed compounds may exist in atropisomeric forms.
  • Atropisomers are stereoisomers resulting from hindered rotation about single bonds where the steric strain barrier to rotation is high enough to allow for the isolation of the conformers.
  • the compounds of the invention may be prepared as individual isomers by either isomer-specific synthesis or resolved from an isomeric mixture.
  • Conventional resolution techniques include forming the salt of a free base of each isomer of an isomeric pair using an optically active acid (followed by fractional crystallization and regeneration of the free base), forming the salt of the acid form of each isomer of an isomeric pair using an optically active amine (followed by fractional crystallization and regeneration of the free acid), forming an ester or amide of each of the isomers of an isomeric pair using an optically pure acid, amine or alcohol (followed by chromatographic separation and removal of the chiral auxiliary), or resolving an isomeric mixture of either a starting material or a final product using various well known chromatographic methods.
  • the stereochemistry of a disclosed compound is named or depicted by structure
  • the named or depicted stereoisomer is at least 60%, 70%, 80%, 90%, 99% or 99.9%) by weight relative to the other stereoisomers.
  • the depicted or named enantiomer is at least 60%, 70%, 80%, 90%, 99% or 99.9% by weight optically pure.
  • the depicted or named diastereomer is at least 60%, 70%, 80%, 90%, 99% or 99.9% by weight pure.
  • Percent optical purity is the ratio of the weight of the enantiomer or over the weight of the enantiomer plus the weight of its optical isomer. Diastereomeric purity by weight is the ratio of the weight of one diastereomer or over the weight of all the diastereomers.
  • the stereochemistry of a disclosed compound is named or depicted by structure, the named or depicted stereoisomer is at least 60%, 70%, 80%, 90%, 99% or 99.9% by mole fraction pure relative to the other stereoisomers.
  • the depicted or named enantiomer is at least 60%, 70%, 80%, 90%, 99% or 99.9% by mole fraction pure.
  • diastereomer When a single diastereomer is named or depicted by structure, the depicted or named diastereomer is at least 60%, 70%, 80%, 90%, 99% or 99.9% by mole fraction pure. Percent purity by mole fraction is the ratio of the moles of the enantiomer or over the moles of the enantiomer plus the moles of its optical isomer. Similarly, percent purity by moles fraction is the ratio of the moles of the diastereomer or over the moles of the diastereomer plus the moles of its isomer.
  • the term “a” may be understood to mean “at least one”; (ii) the term “or” may be understood to mean “and/or”; (ill) the terms “comprising” and “including” may be understood to encompass itemized components or steps whether presented by themselves or together with one or more additional components or steps; and (iv) the terms “about” and “approximately” may be understood to permit standard variation as would be understood by those of ordinary skill in the art; and (v) where ranges are provided, endpoints are included.
  • the term “administration” refers to the administration of a composition (e.g., a compound, a complex or a preparation that includes a compound or complex as described herein) to a subject or system.
  • Administration to an animal subject may be by any appropriate route.
  • administration may be bronchial (including by bronchial instillation), buccal, enteral, interdermal, intra-arterial, intradermal, intragastric, intra med u I la ry, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (including by intratracheal instillation), transdermal, vaginal and vitreal.
  • bronchial including by bronchial instillation
  • the term “animal” refers to any member of the animal kingdom. In some embodiments, “animal” refers to humans, at any stage of development. In some embodiments, “animal” refers to non-human animals, at any stage of development. In some embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, and/or worms. In some embodiments, an animal may be a transgenic animal, genetically-engineered animal, and/or a clone.
  • the terms “approximately” and “about” are each intended to encompass normal statistical variation as would be understood by those of ordinary skill in the art as appropriate to the relevant context.
  • the terms “approximately” or “about” each refer to a range of values that fail within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11 %, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of a stated value, unless otherwise stated or otherwise evident from the context (e.g., where such number would exceed 100% of a possible value).
  • Two events or entities are “associated” with one another, as that term is used herein, if the presence, level and/or form of one is correlated with that of the other.
  • a particular entity e.g., polypeptide
  • a particular disease, disorder, or condition if its presence, level and/or form correlates with incidence of and/or susceptibility of the disease, disorder, or condition (e.g., across a relevant population).
  • a subject such as a human subject undergoing therapy for the treatment of a neurological disorder, for example, amyotrophic lateral sclerosis, frontotemporal degeneration (also referred to as frontotemporal lobar degeneration and frontotemporal dementia), Alzheimer's disease, Parkinson’s disease, dementia with Lewy Bodies, corticobasal degeneration, progressive supranuclear palsy, dementia parkinsonism ALS complex of Guam, Huntington’s disease, Inclusion body myopathy with early-onset Paget disease and frontotemporal dementia (IBMPFD), sporadic inclusion body myositis, myofibrillar myopathy, dementia pugilistica, chronic traumatic encephalopathy, Alexander disease, and hereditary inclusion body myopathy.
  • a neurological disorder for example, amyotrophic lateral sclerosis, frontotemporal degeneration (also referred to as frontotemporal lobar degeneration and frontotemporal dementia), Alzheimer's disease, Parkinson’s disease, dementia with Lewy Bodies, cor
  • a subject undergoing treatment for a neurological disorder using the compositions and methods described herein e.g., in the context of a human subject undergoing treatment for a neurological disorder described herein, such as amyotrophic lateral sclerosis, with a cytochrome P450 isoform 51 A1 (CYP51A1) inhibitor described herein, such as an inhibitory small molecule, antibody, antigen-binding fragment thereof, or interfering RNA molecule
  • CYP51A1 cytochrome P450 isoform 51 A1
  • an inhibitory small molecule, antibody, antigen-binding fragment thereof, or interfering RNA molecule include the slowing and halting of disease progression, as well as suppression of one or more symptoms associated with the disease.
  • examples of clinical “benefits” and “responses” are (i) an improvement in the subject’s condition as assessed using the amyotrophic lateral sclerosis functional rating scale (ALSFRS) or the revised ALSFRS (ALSFRS-R) following administration of the CYP51A1 inhibitor, such as an improvement in the subject's ALSFRS or ALSFRS-R score within one or more days, weeks, or months following administration of the CYP51A1 inhibitor (e.g., an improvement in the subject’s ALSFRS or ALSFRS-R score within from about 1 day to about 48 weeks (e.g., within from about 2 days to about 36 weeks, from about 4 weeks to about 24 weeks, from about 8 weeks io about 20 weeks, or from about 12 weeks to about 16 weeks), or more, following the initial administration of the CYP51A1 inhibitor
  • an “effective amount” of any one of the compounds of the invention or a combination of any of the compounds of the invention or a pharmaceutically acceptable salt thereof is administered via any of the usual and acceptable methods known in the art, either singly or in combination.
  • cytochrome P450 isoform 51 A1 As used herein, the terms “cytochrome P450 isoform 51 A1 ,” “CYP51A1 ,” and “lanosterol 14-alpha demethylase” are used interchangeably and refer to the enzyme that catalyzes the conversion of lanosterol to 4,4-dimethylcholesta-8(9),14,24-trien-3p-ol, for example, in human subjects.
  • the terms “cytochrome P450 isoform 51A1 ,” “CYP51A1 ,” and “lanosterol 14-alpha demethylase” refer not only to wild-type forms of CYP51A1 , but also to variants of wild-type CYP51A1 proteins and nucleic acids encoding the same.
  • amino acid sequence and corresponding mRNA sequence of a wild-type form of human CYP51A1 are provided herein as SEQ ID NOs: 1 and 2, which correspond to GenBank Accession No. AAC50951.1 and NCBI Reference Sequence NO. NM_000786.3, respectively. These sequences are shown in Table 2, below.
  • cytochrome P450 isoform 51A1 “CYP51A1 ,” and “lanosterol 14-alpha demethyiase” as used herein include, for example, forms of the human CYP51A1 protein that have an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 1 (e.g., 85%, 86%, 87%, 88%, 89%,
  • cytochrome P450 isoform 51A1 includes, for example, forms of the human CYP51A1 gene that encode an mRNA transcript having a nucleic acid sequence that is at least 85% identical to the nucleic acid sequence of SEQ ID NO: 2 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical io the amino acid sequence of SEQ ID NO: 2).
  • cytochrome P450 isoform 51A1 inhibitor As used herein, the terms “cytochrome P450 isoform 51A1 inhibitor,” “CYP51A1 inhibitor,” and “lanosterol 14-alpha demethylase inhibitor” are used interchangeably and refer to substances, such as compounds of Formula I.
  • Inhibitors of this type may, for example, competitively inhibit CYP51A1 activity by specifically binding the CYP51A1 enzyme (e.g., by virtue of the affinity of the inhibitor for the CYP51A1 active site), thereby precluding, hindering, or halting the entry of one or more endogenous substrates of CYP51A1 into the enzyme’s active site.
  • cytochrome P45Q isoform 51A1 inhibitor refers to substances that reduce the concentration and/or stability of CYP51 A1 mRNA transcripts in vivo, as well as those that suppress the translation of functional CYP51 A1 enzyme.
  • CYP51A1-associated disorder refers to an undesired physiological condition, disorder, or disease that is associated with and/or mediated at least in pari by CYP51 A1 .
  • CYP51A1 -associated disorders are associated with excess CYP51A1 levels and/or activity.
  • Exemplary CYP51A1 -associated disorders include CYP51A1 -associated disorders include but are noi limited to central nervous system (CNS) disorders, dementia, Alzheimer's Disease, chronic traumatic encephalopathy, FTLD-TDP, LATE, or frontotemporal lobar degeneration.
  • the term “combination therapy” refers to those situations in which a subject is simultaneously exposed to two or more therapeutic agents.
  • two or more compounds may be administered simultaneously; in some embodiments, such compounds may be administered sequentially; in some embodiments, such compounds are administered in overlapping dosing regimens.
  • the term “dosage form” refers to a physically discrete unit of an active compound (e.g., a therapeutic or diagnostic agent) for administration to a subject.
  • Each unit contains a predetermined quantity of active agent.
  • such quantity is a unit dosage amount (or a whole fraction thereof) appropriate for administration in accordance with a dosing regimen that has been determined to correlate with a desired or beneficial outcome when administered to a relevant population (i.e., with a therapeutic dosing regimen).
  • a dosage amount or a whole fraction thereof
  • a dosing regimen refers to a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time.
  • a given therapeutic compound has a recommended dosing regimen, which may involve one or more doses.
  • a dosing regimen comprises a plurality of doses each of which are separated from one another by a time period of the same length; in some embodiments, a dosing regimen comprises a plurality of doses and at least two different time periods separating individual doses.
  • all doses within a dosing regimen are of the same unit dose amount. In some embodiments, different doses within a dosing regimen are of different amounts.
  • a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount different from the first dose amount. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount same as the first dose amount. In some embodiments, a dosing regimen is correlated with a desired or beneficial outcome when administered across a relevant population (i.e., is a therapeutic dosing regimen).
  • neuromuscular disorder refers to a disease impairing the ability of one or more neurons to control the activity of an associated muscle.
  • Examples of neuromuscular disorders are amyotrophic lateral sclerosis, congenital myasthenic syndrome, congenital myopathy, cramp fasciculation syndrome, Duchenne muscular dystrophy, glycogen storage disease type II, hereditary spastic paraplegia, inclusion body myositis, Isaac's Syndrome, Kearns-Sayre syndrome, Lambert-Eaton myasthenic syndrome, mitochondrial myopathy, muscular dystrophy, myasthenia gravis, myotonic dystrophy, peripheral neuropathy, spinal and bulbar muscular atrophy, spinal muscular atrophy, Stiff person syndrome, Troyer syndrome, and Guillain-Barre syndrome, among others.
  • composition represents a composition containing a compound described herein formulated with a pharmaceutically acceptable excipient, and manufactured or sold with the approval of a governmental regulatory agency as part of a therapeutic regimen for the treatment of disease in a mammal.
  • Pharmaceutical compositions can be formulated, for example, for oral administration in unit dosage form (e.g., a tablet, capsule, caplet, gelcap, or syrup); for topical administration (e.g., as a cream, gel, lotion, or ointment); for intravenous administration (e.g., as a sterile solution free of particulate emboli and in a solvent system suitable for intravenous use); or in any other pharmaceutically acceptable formulation.
  • pharmaceutically acceptable excipient refers any ingredient other than the compounds described herein (e.g., a vehicle capable of suspending or dissolving the active compound) and having the properties of being substantially nontoxic and non-inflammatory in a patient.
  • Excipients may include, for example, antiadherents, antioxidants, binders, coatings, compression aids, dis integrants, dyes (colors), emollients, emulsifiers, fillers (diluents), film formers or coatings, flavors, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, sorbents, suspensing or dispersing agents, sweeteners, and waters of hydration.
  • antiadherents antioxidants, binders, coatings, compression aids, dis integrants, dyes (colors), emollients, emulsifiers, fillers (diluents), film formers or coatings, flavors, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, sorbents, suspensing or dispersing agents, sweeteners, and waters of hydration.
  • the term “pharmaceutically acceptable salt” means any pharmaceutically acceptable salt of the compound of formula (I).
  • pharmaceutically acceptable salts of any of the compounds described herein include those that are within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without undue toxicity, irritation, allergic response and are commensurate with a reasonable benefit/risk ratio.
  • Pharmaceutically acceptable salts are well known in the art. For example, pharmaceutically acceptable salts are described in: Berge et al., J. Pharmaceutical Sciences 66:1-19, 1977 and in Pharmaceutical Salts: Properties, Selection, and Use, (Eds. P.H. Stahl and C.G. Wermuth), Wiley-VCH, 2008.
  • the salts can be prepared in situ during the final isolation and purification of the compounds described herein or separately by reacting a free base group with a suitable organic acid.
  • the compounds of the invention may have ionizable groups so as to be capable of preparation as pharmaceutically acceptable salts.
  • These salts may be acid addition salts involving inorganic or organic acids or the salts may, in the case of acidic forms of the compounds of the invention be prepared from inorganic or organic bases.
  • the compounds are prepared or used as pharmaceutically acceptable salts prepared as addition products of pharmaceutically acceptable acids or bases.
  • Suitable pharmaceutically acceptable acids and bases and methods for preparation of the appropriate salts are well-known in the art. Salts may be prepared from pharmaceutically acceptable non-toxic acids and bases including inorganic and organic acids and bases.
  • pure means substantially pure or free of unwanted components (e.g., other compounds and/or other components of a cell lysate), material defilement, admixture or imperfection.
  • a variety of clinical indicators can be used to identify a patient as “at risk” of developing a particular neurological disease.
  • patients e.g., human patients
  • that are “at risk” of developing a neurological disease such as amyotrophic lateral sclerosis, frontotemporal degeneration, Alzheimer’s disease, Parkinson’s disease, dementia with Lewy Bodies, corticobasal degeneration, progressive supranuclear palsy, dementia parkinsonism ALS complex of Guam, Huntington’s disease, Inclusion body myopathy with early-onset Paget disease and frontotemporal dementia (IBMPFD), sporadic inclusion body myositis, myofibrillar myopathy, dementia pugilistica, chronic traumatic encephalopathy, Alexander disease, and hereditary inclusion body myopathy, include (i) subjects exhibiting or prone to exhibit aggregation of TAR-DNA binding protein (TDP)-43, and (ii) subjects expressing a mutant form of TDP-43 containing a mutation associated with TDP-
  • TAR-DNA binding protein-43 and “TDP-43” are used interchangeably and refer to the transcription repressor protein involved in modulating HIV-1 transcription and alternative splicing of the cystic fibrosis transmembrane conductance regulator (CFTR) pre-mRNA transcript, for example, in human subjects.
  • the terms “TAR-DNA binding protein-43” and “TDP-43” refer not only to wildtype forms of TDP-43, but also to variants of wild-type TDP-43 proteins and nucleic acids encoding the same.
  • the amino acid sequence and corresponding mRNA sequence of a wild-type form of human TDP-43 are provided herein as SEQ ID NOs: 3 and 4, which correspond to NCBI Reference Sequence NOs. NMJD07375.3 and NP_Q31401.1 , respectively. These sequences are shown in Table 3, below.
  • TAR-DNA binding protein-43 and “TDP-43” as used herein include, for example, forms of the human TDP-43 protein that have an amino acid sequence that is at least 85% identical io the amino acid sequence of SEQ ID NO: 3 (e.g consecutive 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
  • patients that may be treated for a neurological disorder as described herein include amyotrophic lateral sclerosis, frontotemporal degeneration, Alzheimer’s disease, Parkinson’s disease, dementia with Lewy Bodies, corticobasal degeneration, progressive supranuclear palsy, dementia parkinsonism ALS complex of Guam, Huntington’s disease, inclusion body myopathy with early-onset Paget disease and frontotemporal dementia (IBMPFD), sporadic inclusion body myositis, myofibrillar myopathy, dementia pugilistica, chronic traumatic encephalopathy, Alexander disease, and hereditary inclusion body myopathy, include human patients that express a form of TDP-43 having a mutation associated with elevated TDP-43 aggregation and toxicity, such as a mutation selected from Q331 K, M337V, Q343R, N345K, R361 S, and N39QD.
  • a neurological disorder as described herein such as amyotrophic lateral sclerosis, frontotempo
  • TAR-DNA binding protein-43 and “TDP-43” as used herein include, for example, forms of the human TDP-43 gene that encode an mRNA transcript having a nucleic acid sequence that is at least 85% identical to the nucleic acid sequence of SEQ ID NO: 4 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical to the amino acid sequence of SEQ ID NO: 4).
  • the term “subject” refers to any organism to which a composition in accordance with the invention may be administered, e.g., for experimental, diagnostic, prophylactic, and/or therapeutic purposes. Typical subjects include any animal (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans). A subject may seek or be in need of treatment, require treatment, be receiving treatment, be receiving treatment in the future, or be a human or animal who is under care by a trained professional for a particular disease or condition.
  • animal e.g., mammals such as mice, rats, rabbits, non-human primates, and humans.
  • a subject may seek or be in need of treatment, require treatment, be receiving treatment, be receiving treatment in the future, or be a human or animal who is under care by a trained professional for a particular disease or condition.
  • treat means both therapeutic treatment and prophylactic or preventative measures wherein the object is to prevent or slow down (lessen) an undesired physiological condition, disorder, or disease, or obtain beneficial or desired clinical results.
  • Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; dimmishment of the extent of a condition, disorder, or disease; stabilized (i.e., not worsening) state of condition, disorder, or disease; delay in onset or slowing of condition, disorder, or disease progression: amelioration of the condition, disorder, or disease state or remission (whether partial or total), whether detectable or undetectable; an amelioration of at least one measurable physical parameter, not necessarily discernible by the patient; or enhancement or improvement of condition, disorder, or disease.
  • Treatment includes eliciting a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival as compared to expected survival if not receiving treatment.
  • a “therapeutic regimen” refers to a dosing regimen whose administration across a relevant population is correlated with a desired or beneficial therapeutic outcome.
  • terapéuticaally effective amount means an amount that is sufficient, when administered to a population suffering from or susceptible to a disease, disorder, and/or condition in accordance with a therapeutic dosing regimen, to treat the disease, disorder, and/or condition.
  • a therapeutically effective amount is one that reduces the incidence and/or severity of, and/or delays onset of, one or more symptoms of the disease, disorder, and/or condition.
  • therapeutically effective amount does not in fact require successful treatment be achieved in a particular individual. Rather, a therapeutically effective amount may be that amount that provides a particular desired pharmacological response in a significant number of subjects when administered to patients in need of such treatment.
  • a refractory subject may have a low bioavailability such that clinical efficacy is not obtainable.
  • reference to a therapeutically effective amount may be a reference to an amount as measured in one or more specific tissues (e.g., a tissue affected by the disease, disorder or condition) or fluids (e.g., blood, saliva, serum, sweat, tears, urine, etc).
  • tissue e.g., a tissue affected by the disease, disorder or condition
  • fluids e.g., blood, saliva, serum, sweat, tears, urine, etc.
  • a therapeutically effective amount may be formulated and/or administered in a single dose.
  • a therapeutically effective amount may be formulated and/or administered in a plurality of doses, for example, as part of a dosing regimen.
  • FiGS. 1 A - 1 C demonstrate that the viability of a yeast TDP-43 model is restored by the Erg11 inhibitor, fluconazole.
  • FIG. 1A Structure of the Erg11 inhibitor and anti-fungal, fluconazole.
  • FIG. 1 B Fluconazole rescues viability of TDP-43-expressing yeast using a resazurin-reduction endpoint. A 2-fold serial dilution of fluconazole was applied to TDP-43-expressing yeast for 24 hours prior to analysis.
  • FIG. 1C Wild-type yeast cultures were treated with fluconazole for eight hours prior to HPLC analysis for lanosterol and ergosterol. Data are expressed as the area under the curve (AUG) normalized to cell mass based on optical density of cultures at 600 nm. Fluconazole treatment reduces ergosterol, while simultaneously leading to an increase in the Erg11 substrate, lanosterol.
  • F!G. 2 shows the structures of compounds used in primary rat cortical neuron TDP-43 wild type and Q331 K mutant survival studies.
  • FiGS. 3A and 3B demonstrate that compound A promotes survival in primary rat cortical neurons transfected with wild-type TDP-43.
  • Rat primary cortical neurons were co-transfected with a red fluorescent protein (RFP) as a morphological marker and either control (empty vector) or wild-type TDP-43 expression plasmids and treated with vehicle (DMSO) or a titration of compound A.
  • RFP red fluorescent protein
  • FIG. 3A Risk of neuron death plots. The lifetime of each neuron was determined by either loss of RFP signal or morphological indicators of death such as loss of neurites and cell blobbing and used to generate cumulative hazard plots of risk of death over time (hrs) post-transfection.
  • FIG. 3B Forest plots.
  • Hazard ratios for each treatment group were determined by cox regression analysis and used to generate forest plots.
  • Hazard ratios (HR) ⁇ 1 in which the confidence interval (Ci) does not encompass 1 represent treatments that significantly reduce probability of neuron death relative to the TDP-43 DMSO control.
  • P p-value.
  • FiGS. 4A and 4B demonstrate that compound A promotes survival in primary rat cortical neurons transfected with Q331 K Mutant TDP-43.
  • Rat primary cortical neurons were co-transfected with a red fluorescent protein (RFP) as a morphological marker and either control (empty vector) or Q331 K mutant TDP- 43 expression plasmids and treated with vehicle (DMSO) or a titration of compound A.
  • RFP red fluorescent protein
  • FIG. 4A Risk of neuron death plots. The lifetime of each neuron was determined by either loss of RFP signal or morphological indicators of death such as loss of neurites and cell blebbing and used to generate cumulative hazard plots of risk of death over time (hrs) post-transfection.
  • Hazard ratios for each treatment group were determined by cox regression analysis and used to generate forest plots.
  • Hazard ratios (HR) ⁇ 1 in which the confidence interval (Cl) does not encompass 1 represent treatments that significantly reduce probability of neuron death relative to the TDP-43 DMSO control.
  • P p-value.
  • FIGS. SA and SB demonstrate that compound B promotes survival in primary rat cortical neurons transfected with wild-type TDP-43.
  • Rat primary cortical neurons were co-transfected with a red fluorescent protein (RFP) as a morphological marker and either control (empty vector) or wild type TDP-43 expression plasmids and treated with vehicle (DMSO) or a titration of compound B.
  • RFP red fluorescent protein
  • FIG. 5A Risk of neuron death plots. The lifetime of each neuron was determined by either loss of RFP signal or morphological indicators of death such as loss of neurites and cell biebbing and used to generate cumulative hazard plots of risk of death over time (hrs) post-transfection.
  • FIG. 5B Forest plots.
  • Hazard ratios for each treatment group were determined by cox regression analysis and used to generate forest plots.
  • Hazard ratios (HR) ⁇ 1 in which the confidence interval (Cl) does not encompass 1 represent treatments that significantly reduce probability of neuron death relative to the TDP-43 DMSO control.
  • P p-value.
  • the present invention features compositions and methods for treating neurological disorders, such as amyotrophic lateral sclerosis and other neuromuscular disorders, as well as frontotemporal degeneration, Alzheimer’s disease, Parkinson’s disease, dementia with Lewy Bodies, corticobasal degeneration, progressive supranuclear palsy, dementia parkinsonism ALS complex of Guam, Huntington’s disease, Inclusion body myopathy with early-onset Paget disease and frontotemporal dementia (IBMPFD), sporadic inclusion body myositis, myofibrillar myopathy, dementia pugilistica, chronic traumatic encephalopathy, Alexander disease, and hereditary inclusion body myopathy among others.
  • neurological disorders such as amyotrophic lateral sclerosis and other neuromuscular disorders, as well as frontotemporal degeneration, Alzheimer’s disease, Parkinson’s disease, dementia with Lewy Bodies, corticobasal degeneration, progressive supranuclear palsy, dementia parkinsonism ALS complex of Guam, Huntington’
  • the invention provides inhibitors of cytochrome P450 isoform 51A1 (CYP51A1), also referred to herein as lanosterol 14- alpha demethylase, that may be administered to a patient (e.g., a human patient) so as to treat or prevent a neurological disorder, such as one or more of the foregoing conditions.
  • a patient e.g., a human patient
  • the CYP51 A1 inhibitor may be administered to the patient to alleviate one or more symptoms of the disorder and/or to remedy an underlying molecular pathology associated with the disease, such as to suppress or prevent aggregation of TAR-DNA binding protein (TDP)-43.
  • TDP TAR-DNA binding protein
  • TDP-43 aggregation modulates TDP-43 aggregation in vivo. Suppression of TDP-43 aggregation exerts beneficial effects in patients suffering from a neurological disorder.
  • Many pathological conditions have been correlated with TDP-43-promoted aggregation and toxicity, such as amyotrophic lateral sclerosis, frontotemporal degeneration, Alzheimer’s disease, Parkinson’s disease, dementia with Lewy Bodies, corticobasal degeneration, progressive supranuclear palsy, dementia parkinsonism ALS complex of Guam, Huntington's disease, IBMPFD, sporadic inclusion body myositis, myofibrillar myopathy, dementia pugilistica, chronic traumatic encephalopathy, Alexander disease, and hereditary inclusion body myopathy.
  • patients suffering from diseases associated with TDP­43 aggregation and toxicity may be treated, for example, due to the suppression of TDP­43 aggregation induced by the CYP51A1 inhibitor.
  • Patients that are likely to respond to CYP51A1 inhibition as described herein include those that have or are at risk of developing TDP­43 aggregation, such as those that express a mutant form of TDP­43 associated with TDP­43 aggregation and toxicity in vivo.
  • compositions and methods described herein thus provide the additional clinical benefit of enabling the identification of patients that are likely to respond to CYP51A1 inhibitor therapy, as well as processes for treating these patients accordingly.
  • the sections that follow provide a description of exemplary CYP51A1 inhibitors that may be used in conjunction with the compositions and methods disclosed herein.
  • the sections below additionally provide a description of various exemplary routes of administration and pharmaceutical compositions that may be used for delivery of these substances for the treatment of a neurological disorder.
  • CYP51A1 inhibitors described herein include compounds, or a pharmaceutically acceptable salts thereof, having the structure: Formula I wherein m is 0, 1, 2, 3, or 4; X 1 is CH, S, or N; X 2 and X 3 are, independently, N, CH, or CR 1 ; X 4 is NH or S; each R 1 is, independently, halo, optionally substituted C1­C6 alkyl, or optionally substituted C1­C6 alkoxy; Ar is optionally substituted C6­C10 aryl or optionally substituted C2­C9 heteroaryl; L 1 is ­CONR­ or­NRCO­; R is hydrogen or optionally substituted C 1 ­C 6 alkyl; L 2 is optionally substituted C1­C6 alkylene or optionally substituted C3­C8 cycloalkylene; R A is ­CH2CONHR 2 , ­CONHR 2 , or ­COR 2 ; R 2 is optionally substituted
  • Additional exemplary CYP51A1 inhibitors described herein include compounds, or a pharmaceutically acceptable salts thereof, having the structure: Formula 2 wherein m is 0, 1, 2, 3, or 4; X 6 is CH, or N; X 7 is NH or S; Ar 1 is optionally substituted C6­C10 aryl; R 23 is hydrogen, halo, or optionally substituted C1­C6 alkoxy; L 3 is ­NR 24 CO­ or ­CONR 24 ­, R 24 is H or optionally substituted C1­C6 alkyl; R B is ­CH2CONHR 25 , or ­COR 25 or NR 25 ; R 25 is optionally substituted heteroaryl, optionally substituted cyclohexyl, or has the structure Formula II Formula III wherein R 26 is optionally substituted C1­C3 alkyl; R 27 is optionally substituted C1­C6 alkyl, or optionally substituted C1­C6 heteroalkyl; n is 0 or 1; o is
  • CYP51A1 inhibitors described herein include compounds having the structure: and pharmaceutically acceptable salts thereof.
  • CYP51A1 inhibitors described herein include compounds having the structure: and pharmaceutically acceptable salts thereof.
  • CYP51A1 inhibitors described herein include compounds having the structure: Formula 1c wherein each R 1A is independently H or R 1 , and pharmaceutically acceptable salts thereof.
  • CYP51A1 inhibitors described herein include compounds having the structure: and pharmaceutically acceptable salts thereof.
  • CYP51A1 inhibitors described herein include compounds having the structure: R 12 is optionally substituted 2­oxa­6­azaspiro[3.3]heptan­6­yl, and pharmaceutically acceptable salts thereof.
  • CYP51A1 inhibitors described herein include compounds having the structure: Formula 1e wherein R 13 is optionally substituted pyridin­4­yl or optionally substituted phenyl; and R 14 is optionally substituted piperidin­4­yl, optionally substituted 2­oxa­6­azaspiro[3.3]heptan­6­yl, 2­ azaspiro[3.3]heptan­2­yl substituted with hydroxy, and pharmaceutically acceptable salts thereof.
  • CYP51A1 inhibitors described herein include compounds having the structure: wherein R 15 is optionally substituted 4­azaspiro[2.4]heptan­4­yl, and pharmaceutically acceptable salts thereof.
  • CYP51A1 inhibitors described herein include compounds having the structure: Formula 1g wherein R 16 is optionally substituted 2­oxa­5­azabicyclo[2.2.1]heptan­5­yl, or optionally substituted 3­ azabicyclo[3.1.0]hexan­3­yl, and pharmaceutically acceptable salts thereof.
  • CYP51A1 inhibitors described herein include compounds having the structure: , wherein R 17 and R 18 are each, independently, H or F; and R 19 is 2­oxa­5­azabicyclo[2.2.1]heptan­5­yl, or 3­azabicyclo[3.1.0]hexan­3­yl, and pharmaceutically acceptable salts thereof.
  • CYP51A1 inhibitors described herein include compounds having the structure: Formula 1i wherein R 20 is optionally substituted 2­oxa­6­azaspiro[3.3]heptan­6­yl, and pharmaceutically acceptable salts thereof.
  • CYP51A1 inhibitors described herein include compounds having the structure: Formula 1j wherein R 21 is optionally substituted pyridinyl; and R 22 is piperidin­1­yl optionally substituted with methoxy; azetidin­1­yl optionally substituted with methyl, methoxy, or fluoro; 2­oxa­5­azabicyclo[2.2.1]heptan­5­yl; or optionally substituted morpholin­4­yl, and pharmaceutically acceptable salts thereof.
  • CYP51A1 inhibitors described herein include compounds having the structure: Formula 2 wherein m is 0, 1, 2, 3, or 4; X 6 is CH, or N; X 7 is NH or S; Ar 1 is optionally substituted C6­C10 aryl; R 23 is hydrogen, halo, or optionally substituted C1­C6 alkoxy; L 3 is ­NR 24 CO­ or ­CONR 24 ­, R 24 is H or optionally substituted C1­C6 alkyl; R B is ­CH2CONHR 25 , or ­COR 25 or NR 25 ; R 25 is optionally substituted heteroaryl, optionally substituted cyclohexyl, or has the structure Formula II Formula III wherein R 26 is optionally substituted C1­C3 alkyl; R 27 is optionally substituted C1­C6 alkyl, or optionally substituted C1­C6 heteroalkyl; n is 0 or 1; o is 0 or 1 or 2; X 8 is NR 28
  • CYP51A1 inhibitors described herein include compounds having the structure: Formula 2a and pharmaceutically acceptable salts thereof.
  • CYP51A1 inhibitors described herein include compounds having the structure: Formula 2b and pharmaceutically acceptable salts thereof.
  • CYP51A1 inhibitors described herein include compounds having the structure: Formula 2c and pharmaceutically acceptable salts thereof.
  • CYP51A1 inhibitors described herein include compounds having the structure: Formula 2d and pharmaceutically acceptable salts thereof.
  • CYP51A1 inhibitors described herein include compounds having the structure: Formula 2e and pharmaceutically acceptable salts thereof.
  • CYP51A1 inhibitors described herein include compounds having the structure: Formula 3 and pharmaceutically acceptable salts thereof, Wherein n is 0, 1, 2, 3, or 4; L 4 is optionally substituted C1­C6 alkylene or optionally substituted C3­C8 cycloalkylene; X 9 is N and X 10 is CH, or X 9 is CH and X 10 is N; X 11 is N or CH; each R 30 is, independently, halo, optionally substituted C1­C6 alkyl, or optionally substituted C1­C6 alkoxy; Ar C is optionally disubstituted C6­C10 aryl, or C6­C10 aryl optionally monosubstituted with chloro, optionally substituted C 1 ­C 6 heteroalkyl, cyano, meta­fluoro, or ortho­fluoro; R C is COR 31 , or ­R 31 ; R 31 is optionally substituted C2­C5 heteroaryl, or has the structure: Formula II Formula
  • X 12 is NR 34 , CR 34 R 35 , O, or SR 34 R 35 ;
  • R 34 is absent, halo, oxo, hydrogen, hydroxy, optionally substituted amino, cyano, optionally substituted C1­C6 alkyl, optionally substituted C1­C6 heteroalkyl, ­SO2­optionally substituted C1­C6 alkyl, optionally substituted C 2 ­C 9 heterocyclyl, optionally substituted C 6 ­C 10 aryl, or optionally substituted C 2 ­C 9 heteroaryl, or R 33 and R 34 combine with the atoms to which they are attached to form an optionally substituted C2­C9 heteroaryl;
  • R 35 is absent, halo, oxo, hydroxy, optionally substituted amino, cyano, optionally substituted C1­C6 al
  • CYP51A1 inhibitors described herein include compounds having the structure: Formula 3a and pharmaceutically acceptable salts thereof.
  • CYP51A1 inhibitors described herein include compounds having the structure: Formula 3b and pharmaceutically acceptable salts thereof.
  • CYP51A1 inhibitors described herein include compounds having the structure: Formula 3c Wherein R 37 is cyano and R 38 is fluoro, or R 37 is fluoro and R 38 is cyano; and R 39 is or 3,3­difluoro­azetidin­1­yl, and pharmaceutically acceptable salts thereof.
  • CYP51A1 inhibitors described herein include compounds having the structure: wherein l is 0 or 1; L 4 is is optionally substituted C1­C6 alkylene or optionally substituted C3­C8 cycloalkylene; and R 40 is 4­hydroxy­piperidin­1­yl, 3­methoxy­piperidin­1­yl, optionally substituted diazapen­1­yl, triazolopiperazinyl substituted with methyl, 4,4­difluoro­piperidin­1­yl, 1,1­dioxothiomorpholin­4­yl, 2­ (methoxymethyl)­pyrroloin­1­yl, tetrahydro­1,3­oxazin­3­yl, 4­isopropyl­piperazin­1­yl, 4­(2­oxazolidin­3­yl)­ piperidin­1­yl, optionally substituted 1,2,4 oxadizol­5
  • CYP51A1 inhibitors described herein include compounds having the structure: and pharmaceutically acceptable salts thereof.
  • CYP51A1 inhibitors described herein include compounds having the structure: Formula 3f wherein R 43 is F or CN; and R 42 is optionally substituted azetidin­1­yl, and pharmaceutically acceptable salts thereof.
  • CYP51A1 inhibitors described herein include compounds having the structure: Formula 3g wherein R 44 is optionally substituted azetidin­1­yl, and pharmaceutically acceptable salts thereof.
  • CYP51A1 inhibitors described herein include compounds having the structure: Formula 4 wherein n is 0, 1, 2, 3, or 4; R 45 is halo; and R 46 is optionally substituted azetidinyl, and pharmaceutically acceptable salts thereof.
  • CYP51A1 inhibitors described herein include compounds having the structure: Formula 4a and pharmaceutically acceptable salts thereof.
  • CYP51A1 inhibitors described herein include compounds having the structure: Formula 5 and pharmaceutically acceptable salts thereof, wherein p is 0 or 1; X 13 is a single bond or O; R 48 is optionally substituted C 6 ­C 10 aryl, optionally substituted C 2 ­C 5 heteroaryl, or trifluoromethyl; R 49 is H or optionally substituted C1­C6 alkyl; R 50 is optionally substituted C6­C10 aryl or optionally substituted C2­C5 hetetoaryl; and R 51 is optionally substituted C2­C5 heterocyclyl.
  • exemplary CYP51A1 inhibitors described herein include compounds, or pharmaceutically acceptable salts thereof, having the structure: Formula 1k and pharmaceutically acceptable salts thereof, wherein CYP51A1 inhibitors described herein include compounds having the structure: Formula 1l and pharmaceutically acceptable salts thereof, wherein CYP51A1 inhibitors described herein include compounds having the structure: Formula 1m and pharmaceutically acceptable salts thereof, CYP51A1 inhibitors described herein include compounds having the structure: Formula 2f and pharmaceutically acceptable salts thereof, wherein R 57 is halo. CYP51A1 inhibitors described herein include compounds having the structure: Formula 3h and pharmaceutically acceptable salts thereof, wherein R 57 and R 58 are each halo. Exemplary CYP51A1 inhibitors described herein include any one of the compounds in Table 1, or pharmaceutically acceptable salts thereof. Table 1. Compounds of the Invention
  • the compound has the structure of any one of compounds 1­453 in Table 1.
  • Other embodiments, as well as exemplary methods for the synthesis or production of these compounds, are described herein. Methods of Treatment
  • a patient suffering from a neurological disorder may be administered a CYP51A1 inhibitor, such as a small molecule, antibody, antigen-binding fragment thereof, or interfering RNA molecule described herein, so as to treat the disorder and/or to suppress one or more symptoms associated with the disorder.
  • a CYP51A1 inhibitor such as a small molecule, antibody, antigen-binding fragment thereof, or interfering RNA molecule described herein, so as to treat the disorder and/or to suppress one or more symptoms associated with the disorder.
  • Exemplary neurological disorders that may be treated using the compositions and methods described herein are, without limitation, amyotrophic lateral sclerosis, frontotemporal degeneration, Alzheimer’s disease, Parkinson's disease, dementia with Lewy Bodies, corticobasai degeneration, progressive supranuclear palsy, dementia parkinsonism ALS complex of Guam, Huntington’s disease, IBMPFD, sporadic inclusion body myositis, myofibrillar myopathy, dementia pugilistica, chronic traumatic encephalopathy, Alexander disease, and hereditary inclusion body myopathy, as well as neuromuscular diseases such as congenital myasthenic syndrome, congenital myopathy, cramp fasciculation syndrome, Duchenne muscular dystrophy, glycogen storage disease type II, hereditary spastic paraplegia, inclusion body myositis, Isaac's Syndrome, Kearns-Sayre syndrome, Lambert-Eaton myasthenic syndrome, mitochondrial myopathy, muscular dystrophy, myasth
  • the present disclosure is based, in part, on the discovery that CYP51A1 inhibitors, such as the agents described herein, are capable of attenuating TDP-43 aggregation in vivo. TDP-43-promoted aggregation and toxicity have been associated with various neurological diseases.
  • the discovery that CYP51A1 inhibitors modulate TDP-43 aggregation provides an important therapeutic benefit.
  • a CYP51A1 inhibitor such as a CYP51A1 inhibitor described herein
  • a patient suffering from a neurological disorder or at risk of developing such a condition may be treated in a manner that remedies an underlying molecular etiology of the disease.
  • the compositions and methods described herein can be used to treat or prevent such neurological conditions, for example, by suppressing the TDP-43 aggregation that promotes pathology.
  • compositions and methods described herein provide the beneficial feature of enabling the identification and treatment of patients that are likely to respond to CYP51A1 inhibitor therapy.
  • a patient e.g., a human patient suffering from or at risk of developing a neurological disease described herein, such as amyotrophic lateral sclerosis
  • a CYP51A1 inhibitor if the patient is identified as likely to respond to this form of treatment.
  • Patients may be identified as such on the basis, for example, of susceptibility to TDP-43 aggregation.
  • the patient is identified is likely to respond to CYP51A1 inhibitor treatment based on the isoform of TDP-43 expressed by the patient.
  • TDP-43 isoforms having a mutation selected from Q331 K, M337V, Q343R, N345K, R361S, and N390D, among others are more likely to develop TDP-43-promoted aggregation and toxicity relative to patients that do not express such isoforms of TDP-43.
  • a patient may be identified as likely to respond to CYP51A1 inhibitor therapy on the basis of expressing such an isoform of TDP-43, and may subsequently be administered a CYP51 A1 inhibitor so as to treat or prevent one or more neurological disorders, such as one or more of the neurological disorders described herein.
  • a patient having a neurological disorder e.g,, a patient at risk of developing TDP-43 aggregation, such as a patient expressing a mutant form of TDP-43 having a mutation associated with elevated TDP-43 aggregation and toxicity, for example, a mutation selected from Q331 K, M337V, Q343R, N345K, R361 S, and N390D
  • a patient having a neurological disorder e.g, a patient at risk of developing TDP-43 aggregation, such as a patient expressing a mutant form of TDP-43 having a mutation associated with elevated TDP-43 aggregation and toxicity, for example, a mutation selected from Q331 K, M337V, Q343R, N345K, R361 S, and N390D
  • successful treatment of a patient having a neurological disease such as amyotrophic lateral sclerosis, with a CYP51A1 inhibitor described herein may be signaled by:
  • an improvement in condition as assessed using the amyotrophic lateral sclerosis functional rating scale (ALSFRS) or the revised ALSFRS (ALSFRS-R), such as an improvement in the patient’s ALSFRS or ALSFRS-R score within one or more days, weeks, or months following administration of the CYP51A1 inhibitor e.g,, an improvement in the patient’s ALSFRS or ALSFRS-R score within from about 1 day to about 48 weeks (e.g., within from about 2 days to about 36 weeks, from about 4 weeks to about 24 weeks, from about 8 weeks to about 20 weeks, or from about 12 weeks to about 16 weeks), or more, following the initial administration of the CYP51A1 inhibitor to the patient, such as within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks,
  • an increase in slow vital capacity such as an increase in the patient’s slow vital capacity within one or more days, weeks, or months following administration of the CYP51A1 inhibitor (e.g., an increase in the patient's slow vital capacity within from about 1 day to about 48 weeks (e.g., within from about 2 days to about 36 weeks, from about 4 weeks to about 24 weeks, from about 8 weeks to about 20 weeks, or from about 12 weeks to about 16 weeks), or more, following the initial administration of the CYP51A1 inhibitor to the patient, such as within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks
  • a reduction in decremental responses exhibited by the patient upon repetitive nerve stimulation such as a reduction that is observed within one or more days, weeks, or months following administration of the CYP51 A1 inhibitor (e.g., a reduction that is observed within from about 1 day to about 48 weeks (e.g., within from about 2 days to about 36 weeks, from about 4 weeks to about 24 weeks, from about 8 weeks to about 20 weeks, or from about 12 weeks to about 16 weeks), or more, foilowing the initial administration of the CYP51A1 inhibitor to the patient, such as within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks,
  • Council muscle testing scale (as described, e.g., in Jagtap et al., Ann. Indian. Acad. Neurol. 17:336-339 (2014), the disclosure of which is incorporated herein by reference as it pertains to measuring patient response to neurological disease treatment), such as an improvement that is observed within one or more days, weeks, or months following administration of the CYP51A1 inhibitor (e.g., an improvement that is observed within from about 1 day to about 48 weeks (e.g., within from about 2 days to about 36 weeks, from about 4 weeks to about 24 weeks, from about 8 weeks to about 20 weeks, or from about 12 weeks to about 16 weeks), or more, following the initial administration of the CYP51A1 inhibitor to the patient, such as within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19
  • an improvement in quality of life as assessed, for example, using the amyotrophic lateral sclerosis-specific quality of life (ALS-specific QOL) questionnaire, such as an improvement in the patient’s quality of life that is observed within one or more days, weeks, or months following administration of the CYP51 A1 inhibitor (e.g., an improvement in the subject’s quality of life that is observed within from about 1 day to about 48 weeks (e.g., within from about 2 days to about 36 weeks, from about 4 weeks to about 24 weeks, from about 8 weeks to about 20 weeks, or from about 12 weeks to about 16 weeks), or more, following the initial administration of the CYP51A1 inhibitor to the patient, such as within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22
  • a decrease in the frequency and/or severity of muscle cramps such as a decrease in cramp frequency and/or severity within one or more days, weeks, or months following administration of the CYP51 A1 inhibitor (e.g., a decrease in cramp frequency and/or severity within from about 1 day to about 48 weeks (e.g., within from about 2 days to about 36 weeks, from about 4 weeks to about 24 weeks, from about 8 weeks to about 20 weeks, or from about 12 weeks to about 16 weeks), or more, following the initial administration of the CYP51A1 inhibitor to the patient, such as within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32
  • the compounds of the invention can be combined with one or more therapeutic agents.
  • the therapeutic agent can be one that treats or prophylactically treats any neurological disorder described herein.
  • Combination Therapies A compound of the invention can be used alone or in combination with other agents that treat neurological disorders or symptoms associated therewith, or in combination with other types of treatment to treat, prevent, and/or reduce the risk of any neurological disorders.
  • the dosages of one or more of the therapeutic compounds may be reduced from standard dosages when administered alone. For example, doses may be determined empirically from drug combinations and permutations or may be deduced by isobolographic analysis (e.g., Black et al., Neurology 65:S3­S6, 2005).
  • compositions for administration to human subjects in a biologically compatible form suitable for administration in vivo.
  • the present invention provides a pharmaceutical composition comprising a compound of the invention in admixture with a suitable diluent, carrier, or excipient.
  • the compounds of the invention may be used in the form of the free base, in the form of salts, solvates, and as prodrugs. All forms are within the scope of the invention.
  • the described compounds or salts, solvates, or prodrugs thereof may be administered to a patient in a variety of forms depending on the selected route of administration, as will be understood by those skilled in the art.
  • the compounds of the invention may be administered, for example, by oral, parenteral, buccal, sublingual, nasal, rectal, patch, pump, or transdermal administration and the pharmaceutical compositions formulated accordingly.
  • Parenteral administration includes intravenous, intraperitoneal, subcutaneous, intramuscular, transepithelial, nasal, intrapulmonary, intrathecal, rectal, and topical modes of administration. Parenteral administration may be by continuous infusion over a selected period of time.
  • A. compound of the invention may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsules, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet.
  • a compound of the invention may be incorporated with an excipient and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, and wafers.
  • a compound of the invention may also be administered parenterally.
  • Solutions of a compound of the invention can be prepared in water suitably mixed with a surfactant.
  • Dispersions can also be prepared in glycerol, liquid polyethylene glycols, DMSO and mixtures thereof with or without alcohol, and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.
  • Conventional procedures and ingredients for the selection and preparation of suitable formulations are described, for example, in Remington's Pharmaceutical Sciences (2003, 20 th ed.) and in The United States Pharmacopeia: The National Formulary (USP 24 NF19), published in 1999.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that may be easily administered via syringe.
  • compositions for nasal administration may conveniently be formulated as aerosols, drops, gels, and powders.
  • Aerosol formulations typically include a solution or fine suspension of the active substance in a physiologically acceptable aqueous or non-aqueous solvent and are usually presented in single or multidose quantities in sterile form in a sealed container, which can take the form of a cartridge or refill for use with an atomizing device.
  • the sealed container may be a unitary dispensing device, such as a single dose nasal inhaler or an aerosol dispenser fitted with a metering valve which is intended for disposal after use.
  • the dosage form comprises an aerosol dispenser
  • a propellant which can be a compressed gas, such as compressed air or an organic propellant, such as fluorochlorohydrocarbon.
  • the aerosol dosage forms can also take the form of a pump-atomizer.
  • compositions suitable for buccal or sublingual administration include tablets, lozenges, and pastilles, where the active ingredient is formulated with a carrier, such as sugar, acacia, tragacanth, gelatin, and glycerine.
  • Compositions for rectal administration are conveniently in the form of suppositories containing a conventional suppository base, such as cocoa butter.
  • the compounds of the invention may be administered to an animal, e.g., a human, alone or in combination with pharmaceutically acceptable carriers, as noted herein, the proportion of which is determined by the solubility and chemical nature of the compound, chosen route of administration, and standard pharmaceutical practice.
  • the dosage of the compounds of the invention, and/or compositions comprising a compound of the invention can vary depending on many factors, such as the pharmacodynamic properties of the compound; the mode of administration; the age, health, and weight of the recipient; the nature and extent of the symptoms; the frequency of the treatment, and the type of concurrent treatment, if any; and the clearance rate of the compound in the animal to be treated.
  • One of skill in the art can determine the appropriate dosage based on the above factors.
  • the compounds of the invention may be administered initially in a suitable dosage that may be adjusted as required, depending on the clinical response. In general, satisfactory results may be obtained when the compounds of the invention are administered to a human at a daily dosage of, for example, between 0.05 mg and 3000 mg (measured as the solid form).
  • Dose ranges include, for example, between 10­1000 mg.
  • the dosage amount can be calculated using the body weight of the patient.
  • the dose of a compound, or pharmaceutical composition thereof, administered to a patient may range from 0.1­50 mg/kg.
  • the acid­labile (e.g., tert­butyloxycarbonyl) or base­labile (e.g., fluorenylmethyloxycarbonyl) protecting group of appropriately substituted amine I is removed under acidic or basic conditions to yield deprotected amine II.
  • Amine II is reacted with appropriately substituted carboxylic acid III under a variety of coupling conditions (e.g., HATU) to afford appropriately substituted amide IV.
  • the acid­ or base­labile protecting group of amine IV is removed under acid or basic conditions to yield deprotected amine V.
  • Amine V is reacted with appropriately substituted carboxylic acid VI under a variety of coupling conditions (e.g., HATU) to yield desired amide VII.
  • Carboxylic acid VI is coupled with appropriately substituted amine VII under a variety of coupling conditions (e.g., HATU) to afford amide VIII.
  • the enantiomers of VIII are separated via chiral­HPLC to afford desired compounds IX and X.
  • An appropriately substituted amino acid I is reacted with (Z)­N'­hydroxypropionimidamide II under a variety of coupling conditions (e.g., EDCI) to afford appropriately substituted oxime ester III.
  • Oxime ester III is intramolecularly cyclized under basic conditions (e.g., potassium hydroxide) to afford appropriately substituted oxadiazole IV, which is subsequently deprotected under acidic conditions (e.g., trifluoroacetic acid) to form appropriately substituted amine V.
  • Amine V is reacted with appropriately substituted carboxylic acid VI under a variety of coupling conditions (e.g., HATU) to form appropriately substituted amide VII.
  • the enantiomers of VII are separated via chiral­HPLC to afford desired compounds VIII and IX.
  • Step 2 ethyl 5-methyl-1H-pyrrolo[3,2-b]pyridine-2-carboxylate.
  • ethyl 3­(6­methyl­3­nitropyridin­2­yl)­2­oxopropanoate 2.9 g, 11.5 mmol
  • ethanol 50 mL
  • aqueous saturated ammonium chloride solution 8 mL
  • iron powder 3.8 g, 69.9 mmol
  • Step 1a (S)-tert-butyl 3-(2-chlorophenyl)-1-(4-hydroxypiperidin-1-yl)-1-oxopropan-2-ylcarbamate.
  • Step 4 (S)-N-(3-(2-chlorophenyl)-1-(4-hydroxypiperidin-1-yl)-1-oxopropan-2-yl)-5-methyl-1H- pyrrolo[3,2-b]pyridine-2-carboxamide.
  • the combined organic phase was concentrated and purified by silica gel column chromatography (10% methanol in ethyl acetate) to afford 300 mg of a yellow oil, which was further purified by prep­HPLC (ammonium bicarbonate as buffer) to afford the target compound (90.0 mg, 0.21 mmol, yield:21%) as a white solid.
  • Step 2 Preparation of tert-butyl (S)-(3-(2,4-difluorophenyl)-1-(6-hydroxy-2-azaspiro[3.3]heptan-2-yl)-1- oxopropan-2-yl)carbamate.
  • Step 4 Preparation of (S)-N-(3-(2,4-difluorophenyl)-1-(6-hydroxy-2-azaspiro[3.3]heptan-2-yl)-1- oxopropan-2-yl)-5-methyl-1H-pyrrolo[2,3-b]pyridine-2-carboxamide.
  • Enantiomer 1 (Compound 146) was obtained as white solid.
  • Enantiomer 2 (Compound 145) was obtained as white solid.
  • Step 1 Preparation of 2-(bromomethyi)-5 ⁇ chiorobenzonitriie.
  • Step 3 Preparation of 2-amino-3-(4-chioro ⁇ 2-cyanophenyi)propanoic acid.
  • Step 4 Preparation of 2-(5-chtoro-1 H ⁇ pyrroio[2,3-b]pyridine ⁇ 2-carboxamido)-3 ⁇ (4-chloro-2- cyanophenyi)propanoic acid.
  • Step 5 Preparation of 5-chloro-N-(3-(4-chloro-2-cyanopheny!)-1-(6-methoxy-2-azaspiro[3.3]heptan-2- yi) ⁇ 1 -oxopropan-2 ⁇ yi)-1 H-pyrroio[2,3-b]pyridine-2 -carboxamide.
  • the elution system used was a gradient of 5%-95% over 1 .5 min at 2ml/min and the solvent was acetonitrile/0.01% aqueous NH4HCO3.) to afford the title compound (100 mg, 0.195 mmol, 52.8 % yield) as white solid. It was separated by Chiral-HPLC using the conditions described above to obtain two isomers.
  • Step 1 Preparation of 5-chloro-1H-pyrro!o[2,3-b]pyridme-2-carboxy!ic acid.
  • Step 2 Preparation of 2,5-daoxopyrrobdm-l-yl S-chSoro-IH-pyrrolo ⁇ -bJpyndme ⁇ -carboxylateTM
  • Step 3 Preparation of 2-(5-chtoro-1H-pyrroio[2,3-b]pyndine-2 ⁇ carboxamido) ⁇ 3-(4- cyanopheny!propanoic acid.
  • Step 4 Synthesis of enantiomer 1 (Compound 196) and enantiomer 2 (compound 197) of 5-ch toro-N- [3-(4-cyanophenyi)-1 -(3,3 ⁇ difiuoroazetidin-1 -yi)-1 -oxopropan ⁇ 2-yi] ⁇ 1 H-pyrroto[2,3 ⁇ b]pyndine-2- carboxamide.
  • Enantiomer 1 (Compound 259) and enantiomer s (Compound 260) of 5-chtoro-N-(3-(2-cyanophenyl) ⁇ 1-
  • Step 1 2-(bro:nomethyl)benzomtriie.
  • Step 2 tert-buty! 3-(2-cyanophenyi) ⁇ 2-(diphenyimethyleneamino)propanoate.
  • Step 5 Synthesis of enantiomer 1 and enantiomer 2 of tert-butyl 3-(2-cyanophenyl)-1-(3,3- difluoroazetidin-1-yl)-1-oxopropan-2-ylcarbamate.
  • a mixture of 2­(tert­butoxycarbonylamino)­3­(2­cyanophenyl)propanoic acid 500 mg, 1.7 mmol
  • 3,3­ difluoroazetidine hydrochloride 220 mg, 1.7 mmol
  • HATU 950 mg, 2.5 mmol
  • DIPEA 658 mg, 5.1 mmol
  • DMF 8 mL
  • Step 6 2-(2-amino-3-(3,3-difluoroazetidin-1-yl)-3-oxopropyl)benzonitrile (from enantiomer 2 of step-5).
  • enantiomer 2 70 mg, 0.2 mmol
  • dichloromethane 4 mL
  • trifluoroacetic acid 1.5 mL
  • the mixture was then concentrated to afford the target compound (95 mg, crude) as a colorless oil, which was used in the next step without further purification.
  • Step 7 5-chloro-N-(3-(2-cyanophenyl)-1-(3,3-difluoroazetidin-1-yl)-1-oxopropan-2-yl)-1H-pyrrolo[2,3- b]pyridine-2-carboxamide (Compound 259, from enantiomer 2 of step-5).
  • Step 2 Preparation of enantiomer 1 and enantiomer 2 of 2-(5-chloro-1H-pyrrolo[2,3-b]pyridine-2- carboxamido)-3-(4-cyano-2-fluorophenyl)propanoic acid.
  • Step 3 Synthesis of Compound 261: (from enantiomer 1 of step-2) A mixture of enantiomer 1 (70 mg, 0.18 mmol) of 2­(5­chloro­1H­pyrrolo[2,3­b]pyridine­2­ carboxamido)­3­(4­cyano­2­fluorophenyl)propanoic acid from step­2, 2­oxa­6­azaspiro[3.3]heptane (17.9 mg, 0.18 mmol), PyAOP (141.7 mg, 0.27 mmol) and DIPEA (70.2 mg, 0.54 mmol) in DMF (2 mL) was stirred at 0 o C for 1 h.
  • the resultant mixture was subjected to prep­HPLC (BOSTON pHlex ODS 10um 21.2 ⁇ 250mm120A.
  • the mobile phase was acetonitrile/0.1% Ammonium bicarbonate) to obtain the desired product as white solid (34 mg, 0.073 mmol, 40% yield).
  • Step 4 Synthesis of Compound 262 (from enantiomer 2 of step-2).
  • a mixture of enantiomer 2 of 2­(5­chloro­1H­pyrrolo[2,3­b]pyridine­2­carboxamido)­3­(4­cyano­2­ fluorophenyl)propanoic acid (60 mg, 0.16 mmol) from step­2, 2­oxa­6­azaspiro[3.3]heptane (15.4 mg, 0.16 mmol), PyAOP (121.5 mg, 0.23 mmol) and DIPEA (60.2 mg, 0.47 mmol) in DMF (2 mL) was stirred at 0 o C for 1 h.
  • the mixture was subjected to prep­HPLC (BOSTON pHlex ODS 10um 21.2 ⁇ 250mm120A.
  • the mobile phase was acetonitrile/0.1% Ammonium bicarbonate) to get the desired product as white solid (53.1 mg, 0.114 mmol, 73.1% yield).
  • the compound 265 and 266 were synthesized according to the protocol described above. However, the enantiomers were separated at the last step.
  • Step 3 Preparation of tert-butyl 3-(5-chloropyridin-2-yl)-2-(diphenylmethyleneamino)propanoate.
  • Step 6 Synthesis of enantiomer 1 (Compound 275) and enantiomer 2 (Compound 276) of 5-chioro-N- (3-(5-chloropyridin-2-yi)-1 -(3,3-difiuoroazetidin-1 -y $)-1 -oxopropan-2-yi)-1 H-pyrroio[2,3-b]pyridine-2- carboxamide
  • Enantiomer 1 (Compound 275) was obtained (5.8 mg, 0.05 mmol) as white solid.
  • Enantiomer 2 (Compound 276) was obtained (5 mg, 0.04 mmol) as white solid.
  • Step 3 tert-butyl 3-(5-chloropyridin-2-yl)-2-(diphenylmethyleneamino)propanoate.
  • 5­chloro­2­(chloromethyl)pyridine hydrochloride (2.25 g, 11.25 mmol) TBAB (200 mg) in dichloromethane (100 mL) were added potassium hydroxide (50%) (3.78 g, 67.5 mmol) and tert­ butyl 2­(diphenylmethyleneamino) acetate (4 g, 13.5 mmol) at ­10 o C and the resultant mixture was stirred for 2h between ­10 ⁇ 25 o C.
  • Step 4 Preparation of tert-butyl 3-(4-ch!oroth!azol-2-yl)-2-(dipheny!methyieneamino)propanoate.
  • Step 5 Preparation of 2-amino-3-(4-chiorothsazoi ⁇ 2-y!propanoic acid.
  • Step 4 Preparation of tert-butyl 3-(5-ch!oroth!azoi-2-yl)-2-(dipheny!methyleneamino)propanoate.
  • Step 5 Preparation of 2-ammo-3-(5-chlorothiazol ⁇ 2-y!propanoic acid.
  • Step 2 2-(chloromethyl)-5-methylthiophene.
  • dichloromethane 200 mL
  • brine 100 mL
  • DMF 100 uL
  • Step 3 tert-butyl 2-(diphenylmethyleneamino)-3-(5-methylthiophen-2-yl)propanoate.
  • Step 1 Preparation of 4-(chioromethyl)-5-metiiyi-1 H-imidazoie.
  • Step 2 Preparation of diethyl 2-acetamido-2-((5-methyl-1H-imidazol-4-yl)methyl)malonate.
  • diethyl 2­acetamidomalonate 3.52 g, 16.2 mmol
  • sodium hydride 1.35 g, 33.8 mmol
  • the mixture was stirred at 25 o C for 1h.
  • 4­ (chloromethyl)­5­methyl­1H­imidazole (2.26 g, 13.5 mmol) was added and the mixture was stirred at 25 o C for 16 h.
  • the resultant crude product was purified by Prep­HPLC (Boston C1821*250mm 10 ⁇ m column.
  • the mobile phase was acetonitrile/0.01% aqueous trifluoroacetic acid.) to give the desired 2­(5­chloro­1H­indole­2­carboxamido)­3­(5­methyl­1H­imidazol­4­yl)propanoic acid (170 mg, 0.49 mmol, yield: 30 %) as a white solid.
  • Step 6 Preparation of 5-chloro-N-(1-(3,3-difluoropyrrolidin-1-yl)-3-(5-methyl-1H-imidazol-4-yl)-1- oxopropan-2-yl)-1H-indole-2-carboxamide.
  • the mobile phase was acetonitrile/0.01% aqueous trifluoroacetic acid.) to give the desired product 5- chtoro-N-(1-(3,3-difluoropyrrolidin-1-yl)-3-(5-methyl-1 H-imidazol-4-yl)-1-oxopropan-2-yl)-1H-indote-2- carboxamide (135 mg, 0.31 mmol, yield: 77 %) as a white solid.
  • Step 7 Separation of enantiomer 1 (Compound 301) and enantiomer 2 (Compound 302) of S-ch!oro-N- (1 -(3,3-difiuoropyrroiidin-l -yi) ⁇ 3-(5-methyi-1 H-imidazoi-4-yl) ⁇ 1 -oxopropan-2-yi)-1 H-indoie-2- carboxamide.
  • Enantiomer 1 was obtained (30.8 mg, 0.07 mmol) as white solid.
  • Enantiomer 2 was obtained (41.7 mg, 0.10 mmol) as white solid.
  • Step 2 1-(2,4-dif!uorophenyi)cyctopropanecarba!dehyde.
  • Step 4 2-amino-2-(1-(2,4-difluorophenyl)cyclopropyl)acetic acid.
  • a mixture of 5­(1­(2,4­difluorophenyl)cyclopropyl)imidazolidine­2,4­dione (900 mg, 3.6 mmol) in aqueous sodium hydroxide (6.0N, 6 mL) and ethane­1,2­diol (20 mL) was stirred at 130 ⁇ for 6 hours.
  • the mixture was acidified to pH 1 ⁇ 2 with 36% hydrochloric acid.
  • the resultant precipitate was filtered off.
  • Step 8 Synthesis of compounds 321 and 322: A mixture of 5­chloro­1H­pyrrolo[2,3­b]pyridine­2­carboxylic acid (63 mg, 0.32 mmol), 2­amino­2­(1­ (2,4­difluorophenyl)cyclopropyl)­1­(4­hydroxypiperidin­1­yl)ethanone (100 mg, 0.32 mmol), PyAOP (203 mg, 0.39 mmol), DIPEA (124 mg, 0.96 mmol) and DMF (4 mL) was stirred at 15 ⁇ for 1 hour.
  • Step 3 Preparation of methyl 3-amino-4-(2-chlorophenyl)butanoate.
  • ethyl 4­(2­chlorophenyl)­3­oxobutanoate (2 g, 8.33 mmol) in methanol (40 mL) were added ammonium acetate (6.4 g, 83.3 mmol), magnesium sulfate (3 g, 25 mmol) and sodium cyanoborohydride (1.05 g, 16.67 mmol). Then the mixture was stirred at 70°C (reflux) for 16h.
  • Step 5 Preparation of 3-(5-chloro-1H-indole-2-carboxamido)-4-(2-chlorophenyl)butanoic acid.
  • methyl 3­(5­chloro­1H­indole­2­carboxamido)­4­(2­chlorophenyl)butanoate 0.7 g, 1.73 mmol
  • lithium hydroxide monohydrate 87 mg, 2.08 mmol
  • Step 6 Synthesis of enantiomer 1 and enantiomer 2 of 5-chloro-N-(1-(2-chlorophenyl)-4-(4- hydroxypiperidin-1-yl)-4-oxobutan-2-yl)-1H-indole-2-carboxamide: To a solution of 3­(5­chloro­1H­indole­2­carboxamido)­4­(2­chlorophenyl)butanoic acid (0.2 g, 0.51 mmol), piperidin­4­ol (52 mg, 0.51 mmol) and HATU (0.29 g, 0.77 mmol) in DMF (20 mL) was added DIPEA (0.2 g, 1.54 mmol) drop­wise under nitrogen.
  • reaction was stirred at 20°C for 2 hour.
  • the reaction mixture was then diluted with ethyl acetate/water (30 mL/30 mL) and extracted with ethyl acetate (2 X 30 mL). The combined organic layers were washed with brine (40 mL), and concentrated.
  • stereoisomer 1 (Compound 325), stereoisomer 2 (Compound 326), stereoisomer 3 (Compound 327) and stereoisomer 4 (Compound 328) of (S)-N-(5-chioro-1 H ⁇ indoi-2-yi) ⁇ 3-(2- ch!orophenyi) ⁇ 2-((T*,4*)-4-hydroxycyciohexyiamino)propenamide.
  • Step 1 tert-butyi 5-chtoro-1 H ⁇ indoi-2-yicarbamate.
  • Step 2 5-chloro-1 H-indo!-2-amine.2,2,2-tnfluoroacetate.
  • Step 3 (S)-fert-bufyl 1 -(5-chioro-1 H-indoi-2-ylammo)-3-(2 ⁇ chtorophenyi)-1 -oxopropan-2 ⁇ y!carbamate.
  • the crude product was purified by Prep-HPLC (BOSTON pHlex ODS 10um 21 ,2x250mm120A.
  • the mobile phase was acetonitrile/0.1 % trifluoroacetate) to get (S)-tert- butyl 1-(5-chloro-1H-indol-2-ylamino)-3-(2-chloropheny!-1-oxopropan-2-ylcarbamate as white solid (200 mg, 0.356 mmol, 25%).
  • Step 4 (S)-2-amino-N-(5-chioro-1 H-indoi-2 ⁇ yi)-3 ⁇ (2-chtorophenyl)propenamide
  • Step 5 synthesis of diastereomer 1 and diastereomer 2 of (S)-N-(5-chloro-1 H ⁇ indoi-2-yl) ⁇ 3-(2- ch!orophenyi)-2-((T*,4*)-4-hydroxycyclohexylamino)propenamide.
  • Step 1 Preparation of (S.Z)-tert-buty! 1-(1-aminopropySideneaminooxy)-3-(2,4-difiuorophenyi)-1- oxopropan-2-yicarbamate.
  • Step 2 Preparation of (S)-tert-butyl 2-(2,4-difiuoropheny!)-1-(3 ⁇ ethy!-1,2,4-oxadiazol-5- yi)ethyicarbamate.
  • Step 3 Preparation of (S)-2-(2,4-difluorophenyl)-1-(3-ethyl-1 ,2,4-oxadiazoL5-yi)ethanamine.
  • Step 4 Preparation of enantiomer 1 and enantiomer 2 of 5 ⁇ ctsioro ⁇ N-(2-(2,4 ⁇ difkiorophenyi ⁇ -1 ⁇ (3-ettsyl- 1 ,2,4-oxadiazoi-5-yl)ethyi)-1 H-pyrroio[2,3-b]pyridine-2-carboxamide,
  • Step 3 (S)-N-(1-(4-acetylpiperazin-1-yl)-3-(3-chlorophenyl)-1-oxopropan-2-yl)-5-chloro-1H-indole-2- carboxamide.
  • Step 2 Preparation of 1-(2,6-diazaspiro[3.3]heptan-2-yl)ethan-1-one.
  • Step 3 Preparation of tert-butyl (S)-(1-(6-acetyi-2,6-diazaspiro[3.3]beptan-2-yi)-3-(2-cblorophenyi)-1- oxopropan-2-yl)carbamate.
  • Step 4 Preparation of (S)-1-(6 ⁇ acestyl-2,6-diazaspiro[3.3]hepfan-2-yl)-2-amino-3-(2- chlorophenyi)propan-1 -one.
  • Step S Preparation of (S)-M-(1-(6-acetyl-2,8-diazaspiro[3.3]beptan ⁇ 2-yl) ⁇ 3-(2-chioropheriyl) ⁇ 1- oxopropan-2-yl)-5-chioro-1H-indoie-2-carboxamide.
  • Step 1 Preparation of (S)-methy! 2-(tert-butoxycarbonylamino)-3-(2 ⁇ chlorophenyi)propanoate.
  • Step 2 Preparation of (S)-tert-butyl 1 -(2-cb!orophei"syi)-3-hydroxypropai"s-2-yicarbamate.
  • Step 3 Preparation of (S)-tert-butyl 1 -(2-ch!orophenyl)-3-oxopropan ⁇ 2-yicarbamate.
  • Step 5 Preparation of (2S)-2-amino-3-(2-chtorophenyi)-1-(2-methoxypyridin-4-yl)propan-1-oi hydrochloride.
  • Step 6 Preparation of 5-chioro-N-((2S)-3 ⁇ (2-chlorophenyl)-1 -hydroxy-1 -(2-methoxypyndsn-4-yi)propan- 2-yl)-1 H-indoie-2 -carboxamide.
  • Step 7 Preparation of 5-chioro-N-((2S)-3 ⁇ (2-chtorophenyl)-1-hydroxy ⁇ 1-(2-oxo-1,2-dihydropyridsn-4 ⁇ yi)propafi-2-yi)-1 H-sndoie-2 -carboxamide.
  • Step 8 Preparation of (S)-5 ⁇ chioro ⁇ N-(3-(2-chioropheriyl) ⁇ 1-oxo-1-(2-oxo-1 ,2-dibydropyridm-4- y!propan-2-yl)-1 H-ii"sdole-2-carboxainide.
  • Step 1 (S)-tert-butyi 3 ⁇ (2-chlorophenyl)-1-(methoxy(methyi)amino)-1-oxopropan-2-ylcarbamate.
  • Step 2 (S)-tert-butyl 3-(2-cb iorophenyl)-1 -(6-methoxypyndm ⁇ 3-yi) ⁇ 1 -oxopropan-2 ⁇ yicarbamate,
  • 5-bromo-2-methoxypyridine (733 mg, 3.9 mmol) was slowly added to a suspension of sodium hydride (156 mg, 3.9 mmol) in tetrahydrofuran (16 mL) at 0°C. After the mixture was stirred for 10 minutes, n- butyllithium (1.56 mL 2.5 M in tetrahydrofuran) was added drop wise over a period of 15 min at -78°C.
  • Step 3 (S)-2-x3mino-3-(2-chioropheny!)-1-(6-methaxypyndin-3-yl)propan-1-one hydrochioride and (S)- 5-(2-amino-3-(2-chiorophenyi)propanoyi)pyridin-2(1 H) ⁇ one hydrochioride.
  • Step 4 5-ch toro-N-(3 ⁇ (2-ch torophenyl)-1 -(6 ⁇ methoxypyridm-3 ⁇ yi)-1 -oxopropan ⁇ 2-yi) ⁇ 1 H-indoie-2- carboxamide (S isomer enriched) and 5-cbtoro-N-(3-(2-chtorophenyi)-1-oxo-1-(6-oxo-1 ,6- dshydropyridin-3-y!propan-2 ⁇ yi)-1 H-indo!e-2-carboxamide (S isomer enriched).
  • the crude product thus obtained was purified by Prep- HPLC (BOSTON pHlex ODS 10um 21 2x250mm120A.
  • the mobile phase was acetonitrile/0.1 % Ammonium bicarbonate) to get two white solids, 5-chloro-N-(3-(2-chlorophenyl)-1-(6-methoxypyridin-3-yl)-1-oxopropan-2- yl)-1 H-indole-2-carboxamide (S isomer enriched) (180 mg, 0.384 mmol, 36.6%), LCMS (ESI) m/z:
  • Step 5 (S)-5-chioro-N-(3-(2-chioropheny!)-1 -(6-methoxypyndin-3-yi)-1 -oxapropan-2-yl)-1 H-indofe-2- carboxamide (Compound 391 ) and (R) ⁇ 5-chloro-N ⁇ (3-(2-chiorophenyi)-1 ⁇ (6-methoxypyridin-3-yl)-t- oxopropan-2-yl)-1 H-mdo!e-2-cart3oxamide (Compound 392).
  • Step 6 (R)-5 ⁇ ctiioro ⁇ N-(3-(2 ⁇ ctiiorophenyl) ⁇ 1 -oxo-1 -(6 ⁇ oxo-1 ,6-dihydropyridin-3 ⁇ yi)propan-2-yl)-1 H ⁇ indole-2 -carboxamide (Compound 394) and (S)-5-chloro-N-(3-(2-chlorophenyi)-1-oxo-1-(8-oxo-1,6- dihydropyridin ⁇ 3-yi)propan-2 ⁇ yi)-1 H-indoie-2-carboxamide (Compound 393).
  • Step 1 (S)-2-ximino-3-(2,4-difluorophenyl)-1 -(2-methoxypyndin-4-yl)propan-1 -one hydrochloride.
  • Step 2 5-chioro-N-(3-(2,4-difluorophenyi)-1 -(2 ⁇ methoxypyridin-4-y!-1 -oxopropan-2-yi) ⁇ 1 H-indo!e-2- carboxamide (Compound 395).
  • the mixture with CP- 0022561-166 was purified by Prep-HPLC (BOSTON pHlex ODS 10um 21.2x250mm120A.
  • the mobile phase was acetonitrile/0.1% Formic acid) to get 5-chtoro-N-(3-(2,4-difluorophenyl)-1-oxo-1-(2-oxo-1 ,2- dihydropyridin-4-yl)propan-2-yl)-1 H-indole-2-carboxamide (25 mg, 0.055 mmol, 64.5%) as white solid.
  • Step 1 l-tert-butyi 2 -methyl 4-bromo-1 H-pyiToie-1 ,2-dicarboxylate.
  • Step 3 4-cydopropyM H ⁇ pyrroie ⁇ 2-carboxyiic acid.
  • Step 4 (S)-N-(3-(2 ⁇ chlorophenyi) ⁇ 1 -(4-hydroxypiperidin-1 -yi)-1 -oxopropan ⁇ 2-y!) ⁇ 4-cyclopropyl-1 H- pyrroie-2 -carboxamide.
  • Step 1 Preparation of (tert-butoxycarbonyi)-L-histidine.
  • Step 2 Preparation of tert-butyl (S)-(3-(1 H-imidazoM-yl)-1-morphoiino-1 -oxopropan-2-yl)carbamate.
  • Step 3 Preparation of (S)-(3-(1 H-imidazoi-4 ⁇ yl)-1 ⁇ morphoiino-1 ⁇ oxopropan-2-yi)carbamate,
  • Step 1 (S)-terf-butyl 3-(2,4-difluorophenyl)-1 -oxo-1 -(2-propionyihydraziny i)propan ⁇ 2-yicarbamate.
  • Step 2 (S)-tert-butyl 2-(2,4-difiuorophenyl)-1-(5-ethy -1 ,3,4-oxadiazol-2-yl)ethyicarbamate.
  • the Burgess reagent (Methoxycarbonylsulfamoyl)triethylammonium hydroxide) (428 mg, 1.8 mmol) was added to a solution of (S)-tert-butyl 3-(2,4-difluorophenyj)-1-oxo-1-(2-propionylhydrazinyi)propan-2- ylcarbamate (450 mg, 1 .2 mmol) in tetra hydrofuran (20 mL) and the resultant mixture was stirred at 70°C for 2 h. The reaction was then quenched with water and concentrated to remove the organics and ethyl acetate (100 mL) was added.
  • Step 3 (S)-2-(2,4-dif!tiorophenyi)-1 -(5-ethyM ,3,4-oxadiazo!-2-yi)ef hanamine.
  • Step 4 (S)-5 ⁇ chloro-N-(2 ⁇ (2,4-difluorophenyl)-1 -(5-ethyM ,3,4-oxad!azoi-2-yl)ethyi)-1 H-pyrroio[2,3- bJpyndine-2 -carboxamide.
  • the mobile phase was acetonitrile/0.1% Ammonium bicarbonate) to get (S)-5-chloro-N- (2-(2,4-difluorophenyl)-1-(5-ethyl-1 ,3,4-oxadiazol-2-yl)ethyl)-1 H-pyrrolo[2,3-b]pyridine-2-carboxamide (16.5 mg, 0.038 mmol, 10.7%) as a white solid.
  • Step 2 Preparation of (S)-3 ⁇ (2,4-difluoropbenyl)-2-(methyiamino)propanoic acid bydrochioride.
  • Step 3 Preparation of methyi (S)-3 ⁇ (2,4-difiuorophenyl) ⁇ 2-(methylamino)propanoate.
  • Step 5 Preparation of (S)-2-(5-chtoro-N-methyMH-pyrroio[2,3-b]pyiidine-2-carboxamido)-3-(2,4- difiuorophenyl)propanoic acid hydrochloride.
  • Step 6 Preparation of (S) ⁇ 5-cbioro-M ⁇ (3 ⁇ (2,4-difiuorophenyi) ⁇ 1-oxo-1 -(2 ⁇ oxa-6 ⁇ azaspiro[3.3]heptan ⁇ 6- yi)propan-2-yi)-N-methyi ⁇ 1 H-pyrroio[2,3-b]pyr!dine-2 -carboxamide.
  • Step 1 (S)-2-(tert-biitoxycarbonyi(methyi)amirio) ⁇ 3-(2-chlorophenyi)propanoic acid.
  • Step 3 (S)-3-(2-ch!orophei"syi)-1 -(4-hydroxypiperidm-l -y!-2-(methy!amino)propan-1 -one hydrochloride.
  • Step 4 (S)-5-chloro-N-(3-(2-chtorophenyl)-1 -(4-hydroxypiperidin-l -yl)-1 -oxopropan-2-yl)-N-methyM H- indole-2 -carboxamide.
  • the combined organic phase was concentrated and purified by silica gel column chromatography (10% methanol in ethyl acetate) to afford 800 mg of a yellow oil, which was further purified by prep-HPLC(ammonium bicarbonate as buffer) to afford the target compound (40.3 mg, 0.085 mmol, yield: 2.8%) as a white solid.
  • Step 1 Preparation of (S) ⁇ 2-(5-chioro-1 H ⁇ indoie-2 ⁇ carboxamido) ⁇ 3-(pyndin-4-yl)propanoic acid.
  • Step 2 Preparation of (S)-5-chiaro-N-(1-moipholino-1-oxo-3-(pyndin-4-yl)propan-2-y!-1 H-indate-2- carboxamide.
  • Step 1 methyl (2S)-2-(tert-butoxycarbonylamino)-3-(3-chioro-4-pyridyi)propanoate.
  • Step 2 (2S)-2 ⁇ (tert-butoxycarbonylamino)-3 ⁇ (3-chtoro-4 ⁇ pyr!dyl)propanoic acid.
  • Step 4 (2S) ⁇ 2-ammo ⁇ 3-(3-chioro ⁇ 4-pyiidyi)-1 ⁇ (2 ⁇ oxa-6 ⁇ azaspiro[3.3]heptan-6-yi)propan-1 -one.
  • Step 5 2-(2-tert-butylpynmsdin ⁇ 5-yi) ⁇ N-[(1S)-1 -[(3-chtoro-4-pyridyl)methyl]-2-(2 ⁇ oxa-6- azaspiro[3.3]heptan-6-yl)-2-oxo-ethyi]acetamide.
  • reaction mixture was concentrated and the residue was purified by prep-HPLC (Waters Xbridge Prep OBD C18 150*40mm*10um column; 15-35% acetonitrile in an a 10mM ammonium bicarbonate solution in water and in an a 0.05% ammonia solution in water, 8 min gradient) to obtain 2-(2-tert- butylpyrimidin-5-yl)-N-[(1S)-1-[(3-chloro-4-pyridyi)methyip2-(2-oxa-6-azaspiro[3.3]heptan-6-yi)-2-oxo- ethyljacetamide (38 mg, 83 umol, 55%) as a white solid.
  • SFC (Rt 2.366) method:AD EtOH IPAm 5 50 34 35 3min.
  • Step 1 methyl (2R)-2-(tert ⁇ butoxycarbonyiamino) ⁇ 3-(4-methoxy-3-pyndyi)propanoate.
  • Step 2 (2R)-2-(tert-butoxycarbonylam!no)-3-(4-methoxy ⁇ 3-pyridyl)propanoic acid.
  • Step 3 tert-butyl N ⁇ [(1 R)-2-(3 ⁇ methoxy-3-methyl-azet!din-1 -y S)-1 ⁇ [(4 ⁇ methoxy-3 ⁇ pyr!dyl)methyl] ⁇ 2-oxo ⁇ ethyijcarbamate.
  • Step 4 (2R)-2-amino-1 ⁇ (3 ⁇ methoxy-3 ⁇ methyl ⁇ azetidin-1 -yl)-3-(4-methoxy ⁇ 3-pyi'idyl)propan-1 ⁇ one.
  • the filtrate was purified by prep-HPLC (Phenomenex Gemini-NX C18 75*30mm*3um column; 20-50 % acetonitrile in an a 0.05% ammonium hydroxide and 10mM ammonium bicarbonate solution in water, 8 min gradient) to obtain 5-chloro-N-[(1R)-2- (3-methoxy-3-methyl-azetidin-1-yl)-1-[(4-methoxy-3-pyridyl)methyl]-2-oxo-ethyl]-1 H-pyrrolo[2,3-b]pyridine-2- carboxamide (40 mg, 88 umol, 27%) as pale yellow solid.
  • Step 1 methyl (2R)-2 ⁇ (tert-butoxycarbonyiamino)-3 ⁇ (3-methoxy-4-pyndy!propanoate.
  • the crude product was purified by flash column (ISCO 12 g silica, 0-100 % ethyl acetate in petroleum ether, gradient over 20 min) to obtain methyl (2R)-2-(tert-butoxycarbonylamino)-3-(3-methoxy-4- pyridyl)propanoate (250 mg, 483 umol, 15%) as yellow oil.
  • Step 2 (2R)-2-(tert-butoxycarbonyiamino)-3-(3 ⁇ methoxy-4 ⁇ pyndyi)propanoic acid.
  • Step 3 tert-butyl N-[(1 R)-2-(3-methoxy-3-methyi-azetidin ⁇ 1 -yl) ⁇ 1 -[(3-methoxy ⁇ 4-pyridyi)methyi]-2 ⁇ oxo- ethyljcarbamate.
  • Step 4 (2R)-2-arnmo-1 -(3-methoxy-3-rnethyl-azetidm-1 -yl)-3-(3-rnethoxy-4-pyiidyl)propan-1-one.
  • Step 5 5-cbioro ⁇ N-[(1 R)-2 ⁇ (3-methoxy-3-methyl-azetsdifi-1 ⁇ yl)-1 -[(3 ⁇ methoxy-4-pyridy!metby!]-2-oxo- ethyi]-1 H-pyrrolo[2, 3-b]pyridme-2 -carboxamide.
  • Step 3 During step 3, the conditions used ted the complete racemization of the R-enantiomer that was used as a starting material.
  • Step 1 methyl (2R)-2-(tert ⁇ butoxycarbonyiammo) ⁇ 3-(3-chioro-4-pyridlyi)propa8TOate.
  • Step 2 methyl (2R)-2 ⁇ amino-3-(3-chioro-4 ⁇ pyridyl)propanoate.
  • Step 3 methyl 3-(3-chtoro-4-pyridyl)-2-[(2,4-dimefhoxyphenyi)methyl-methyl-amino]propanoate.
  • Step 4 3-(3-chioro-4-pyridyi)-2-[(2,4-dimethoxypheny!)methy!-methy!-amino]propanoic acid.
  • Step 6 3-(3-cbioro-4 ⁇ pyridyi) ⁇ 1-(4-tiydroxy ⁇ 4-methyl-1 -pipendyi)-2 ⁇ (mettiylamino)propan-1 ⁇ one.
  • Step 7 5-chtoro ⁇ N-[1-[(3-chioro-4 ⁇ pyndyl)methyl] ⁇ 2-(4-hydroxy ⁇ 4-methyM ⁇ piperidyi)-2 ⁇ oxo-ethyl]-N- methyl-1 H-pyrrolo[2,3-b]pyridirie-2-carboxamide.
  • the reaction mixture was concentrated and purified first by prep-HPLC (Waters Xbridge Prep OBD C18 150*40mm*10um column; 20-50% acetonitrile in an a 10mM ammonium bicarbonate solution in water and in an a 0.05% ammonia solution in water, 8 min gradient) and then by prep-HPLC (Waters Xbridge Prep OBD C18 150*40mm*10um column; 20-50% acetonitrile in an a 10mM ammonium bicarbonate solution in water and in an a 0.05% ammonia solution in water, 8 min gradient) to obtain 5-chloro-N-[1-[(3-chloro-4-pyridyl)methyl]-2-(4-hydroxy-4-methyl-1-piperidyl)-2-oxo-ethyl]- N-methy!-1 H-pyrrolo[2,3-b]pyridine-2-carboxamide (62 mg, 127 umol, 18%) as a white solid.
  • Step 1 Preparation of methyl 2-methyM H-imidazoie-5-carboxylate,
  • Step 2 Preparation of (2-methyi-1 H-im!dazoi-5-yi)methanoi.
  • Step 3 Preparation of 5-(chtoromethyl)-2-methyM H ⁇ imidazoie hydrochloride.
  • Step 5 Preparation of 2-amino-3 ⁇ (2-methyi-1 H-imidazol-4-yl)propanoic acid hydrochloride.
  • Step 6 Preparation of methyl 2 ⁇ amino-3-(2-methyM H-lmidazoi-4 ⁇ yl)propanoate hydrochloride.
  • Step 7 Preparation of 2 ⁇ (5-chloro-1 H-indole ⁇ 2-carboxamido)-3 ⁇ (2-methyl-1 H-imidazoi-4-yl)propanoic acid.
  • the mobile phase was acetonitrile/0.01 % aqueous trifluoroacetic acid.) to give the desired 2-(5-chloro-1 H-indole-2-carboxamido)-3-(2-methyl-1 H-imidazoi-4-yl)propanoic acid (275 mg, 0.8 mmol, yield: 26 %) as a off-white solid.
  • Step 8 Preparation of 5-chioro-N ⁇ (1 -(3,3-diflnoropyrroiidin-l -yl)-3 ⁇ (2-methyi-1 H-imidazoi-4 ⁇ yl)-1 - oxopropan-2-yl)-1 H-indole-2-carboxamide.
  • the mobile phase was acetonitrile/0.01 % aqueous trifluoroacetic acid.).
  • the product 5-chloro-N-(1- (3,3-difluoropyrrolidin-1-yl)-3-(2-methyl-1 H-imidazol-4-yl)-1-oxopropan-2-yl)-1 H-indole-2-carboxamide (58.7 mg, 0.13 mmol, yield: 34 %) was obtained as a white solid.
  • Recombinant human CYP51A1 (lanosterol-14a-demethylase) enzyme was co-expressed with CYP reductase in bacterial membranes and the fluorescent substrate BOMCC (a non-natural substrate that causes increases in fluorescence upon CYP51A1 -dependent demethylation) was used to obtain 8-point dose concentration-response curves for each compound.
  • Example 7 inhibition of CYP51A1 moduiates TDP-43 aggregation
  • ALS Amyotrophic lateral sclerosis
  • Lou Gehrig's disease is an aggressive, debilitating disease in which affected patients succumb within two to five years after diagnosis.
  • ALS presents with heterogeneous clinical features but has a common underlying pathology of motor neuron loss that limits the central nervous system’s ability to effectively regulate voluntary and involuntary muscle activity. Additionally, without neuronal trophic support muscles being to atrophy, further exacerbating motor deterioration.
  • Cellular and tissue degeneration results in motor impairment such as fasciculations and weakening in the arms, legs and neck, difficulty swallowing, slurred speech and ultimately failure of the diaphragm muscles that control breathing.
  • TDP-43 is a DNA/RNA binding protein involved in RNA splicing and is typically localized to the nucleus but can be translocated to the cytoplasm under conditions of cell stress. Nuclear clearing and cytoplasmic accumulation of misfolded and aggregated TDP-43 are hallmarks of degenerating motor neurons in ALS, but it remains unclear if mechanism of toxicity is due to aggregation-dependent loss of TDP-43 function or if the aggregates acquire toxic gain of function.
  • TDP-43 Aggregates of TDP-43 accumulate in discrete cellular domains known as stress granules, which are also enriched with translationally inactive mRNAs. Stress granules are observed in multiple cellular types and are thought to be directly related to TDP-43-dependent toxicity in ALS and FTD. Dysfunction in DNA/RNA binding protein activity plays a crucial role in susceptible motor neurons in ALS, as familial cases have also been traced to mutations in the protein Fused in Sarcoma (FUS), a DNA/RNA binding protein that recently has been shown to be involved in gene silencing. Preclinical studies suggest that FUS mutations promote a toxic gain of function that may be causative in motor neuron degeneration.
  • FUS protein Fused in Sarcoma
  • TDP-43 gene Mutations in the TDP-43 gene (TARDBP) have also been causally linked to familial forms of ALS.
  • a common TDP-43 mutation is known as Q331 K, in which glutamine (Q) 331 has been mutated to a lysine (K). This mutation results in a TDP-43 protein that is more aggregation prone and exhibits enhanced toxicity.
  • Q331 K mutation can confer a toxic gain of function in a TDP-43 knock-in mouse, which exhibits cognitive deficits and histological abnormalities similar to that which occurs in frontotemporal dementia (FTD).
  • FTD refers to a group of degenerative disorders that are characterized by atrophy in the frontal and temporal cortices due to progressive neuron loss.
  • FTD neurodegenerative disease
  • C9orf72 progranulin
  • GNN progranulin
  • MART multifactorial involving mutations in genes such as C9orf72, progranulin (GRN) and MART, but intracellular inclusions of aggregated TDP-43, FUS and tau have been observed.
  • ALS and FTD may have different genetic and molecular triggers and occur in different cell types, similar protein misfolding and degenerative mechanisms may operate in multiple diseases.
  • TDP-43 The toxic gain of function features of TDP-43 can be faithfully recapitulated in the simple model organism, budding yeast, where the protein also localizes to stress granules.
  • Human disease mutations in TDP-43 enhance toxicity and yeast genetic screens have revealed key connections that are conserved io humans.
  • the yeast model thus provides a robust cell­based screening platform for small molecules capable of ameliorating toxicity.
  • To validate compounds from such phenotypic screens it is imperative to test compounds in a mammalian neuronal context. In an effort to develop TDP­43­related mammalian models of neuron loss that occurs in ALS and FTD, primary cultures of rat cortical neurons were transfected with human wild type or Q331K mutant TDP­43.
  • Erg11 reduces ergosterol synthesis (yeast equivalent of cholesterol), while increasing lanosterol levels, the substrate of Erg11 (FIG.1C).
  • the human homolog of Erg11 is Cyp51A1, a member of the cytochrome P450 superfamily of enzymes but does not appear to have a role in detoxification of xenobiotics.
  • Cyp51A1 has also been known as lanosterol 14­alpha demethylase, which describes its function in removing the 14­alpha­methyl group from lanosterol to generate 4,4­dimethylcholesta­8(9),14,24­trien­3 ⁇ ­ol, which is a critical step in the cholesterol biosynthetic pathway.

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Abstract

The present invention features compounds useful in the treatment of neurological disorders. The compounds of the invention, alone or in combination with other pharmaceutically active agents, can be used for treating or preventing neurological disorders.

Description

BICYCLIC HETEROAROMATIC AMIDE COMPOUNDS AND USES THEREOF Background An incomplete understanding of the molecular perturbations that cause disease, as well as a limited arsenal of robust model systems, has contributed to a failure to generate successful disease­modifying therapies against common and progressive neurological disorders, such as ALS and FTD. Progress is being made on many fronts to find agents that can arrest the progress of these disorders. However, the present therapies for most, if not all, of these diseases provide very little relief. Accordingly, a need exists to develop therapies that can alter the course of neurodegenerative diseases. More generally, a need exists for better methods and compositions for the treatment of neurodegenerative diseases in order to improve the quality of the lives of those afflicted by such diseases. Summary TDP­43 is a nuclear DNA/RNA binding protein involved in RNA splicing. Under pathological cell stress, TDP­43 translocates to the cytoplasm and aggregates into stress granules. These phenotypes are hallmarks of degenerating motor neurons and are found in 97% of all ALS cases. The highly penetrant nature of this pathology indicates that TDP­43 is broadly involved in both familial and sporadic ALS. Additionally, TDP­43 mutations that promote aggregation are linked to higher risk of developing ALS, suggesting protein misfolding and aggregation act as drivers of toxicity. TDP­43 toxicity can be recapitulated in yeast models, where the protein induces a viability deficit and localizes to stress granules. The present inventors have discovered that the CYP51A1 inhibitors described herein are capable of reversing TDP­43 induced toxicity. Accordingly, the present invention describes such CYP51A1 compounds and methods of using these compounds for the treatment of disorders related to TDP­43 toxicity such as ALS. In an aspect, the invention features a compound, or a pharmaceutically acceptable salt thereof, having the structure:
Figure imgf000002_0001
Formula I wherein m is 0, 1, 2, 3, or 4; X1 is CH, S, or N; X2 and X3 are, independently, N, CH, or CR1; X4 is NH or S; each R1 is, independently, halo, optionally substituted C1­C6 alkyl, or optionally substituted C1­C6 alkoxy; Ar is optionally substituted C6­C10 aryl or optionally substituted C2­C9 heteroaryl; L1 is ­CONR­ or­NRCO­; R is hydrogen or optionally substituted C1­C6 alkyl; L2 is optionally substituted C1­C6 alkylene or optionally substituted C3­C8 cycloalkylene; RA is ­CH2CONHR2, ­CONHR2, or ­COR2; R2 is optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cyclohexyl, or has the structure:
Figure imgf000003_0001
Formula II Formula III wherein R3 is optionally substituted C1­C3 alkyl; R4 is optionally substituted C2­C6 alkyl, optionally substituted C2­C6 heteroalkyl, optionally substituted C3­C8 cycloalkyl, optionally substituted C2­C9 heterocyclyl, or optionally substituted C6­C10 aryl C1­C6 alkyl; n is 0 or 1; o is 0 or 1 or 2; p is 0, 1, 2, 3, 4, 5, 6, 7, or 8; X5 is NR5, CR5R6, O, or SR5R6; R5 is absent, halo, oxo, hydrogen, hydroxy, optionally substituted amino, cyano, optionally substituted C1­C6 alkyl, optionally substituted C1­C6 alkoxy, ­SO2­optionally substituted C1­C6 alkyl, optionally substituted C2­C9 heterocyclyl, or optionally substituted C2­C9 heteroaryl; and R6 is absent, halo, oxo, hydroxy, optionally substituted amino, cyano, optionally substituted C1­C6 alkyl, optionally substituted C1­C6 alkoxy, ­SO2­optionally substituted C1­C6 alkyl, optionally substituted C2­C9 heterocyclyl, or optionally substituted C2­C9 heteroaryl; or R5 and R6 combine with the atoms to which they are attached to form an optionally substituted C2­C9 heterocyclyl, or optionally substituted C2­C9 heteroaryl; and each R7 is, independently, halo, optionally substituted C1­C6 heteroalkyl, or optionally substituted C1­C6 alkyl, and/or two R7, together with the atoms to which they are attached, combine to form an optionally substituted C2­C9 heterocyclyl; or R5 is absent, halo, oxo, hydrogen, hydroxy, optionally substituted amino, cyano, optionally substituted C1­C6 alkyl, optionally substituted C1­C6 alkoxy, ­SO2­optionally substituted C1­C6 alkyl, optionally substituted C2­C9 heterocyclyl, or optionally substituted C2­C9 heteroaryl; R6 and one R7, together with the atoms to which they are attached, combine to form an optionally substituted C2­C9 heterocyclyl or optionally substituted C2­C9 heteroaryl; and the remaining R7 groups, if present, are independently halo, optionally substituted C1­C6 heteroalkyl, or optionally substituted C1­C6 alkyl; or R6 is absent, halo, oxo, hydroxy, optionally substituted amino, cyano, optionally substituted C1­C6 alkyl, optionally substituted C1­C6 alkoxy, ­SO2­optionally substituted C1­C6 alkyl, optionally substituted C2­C9 heterocyclyl, or optionally substituted C2­C9 heteroaryl; R5 and one R7, together with the atoms to which they are attached, combine to form an optionally substituted C2­C9 heterocyclyl or optionally substituted C2­C9 heteroaryl; and the remaining R7 groups, if present, are independently halo, optionally substituted C1­C6 heteroalkyl, or optionally substituted C1­C6 alkyl; wherein if X5 is S, then each of R5 and R6 is, independently, absent or oxo; wherein if Ar is optionally substituted C6­C10 aryl, one of n and o is 1 and the other of n and o is 0 or 1, then X5 is NR5, wherein R5 is hydrogen, hydroxy, optionally substituted amino, cyano, optionally substituted C1­C6 alkoxy, ­SO2­optionally substituted C1­C6 alkyl, optionally substituted C2­C9 heterocyclyl, or optionally substituted C2­C9 heteroaryl; each R7 is, independently, halo, optionally substituted C1­C6 heteroalkyl, or optionally substituted C1­C6 alkyl, and/or two R7, together with the atoms to which they are attached, combine to form an optionally substituted C2­C9 heterocyclyl; or R5 and one R7, together with the atoms to which they are attached, combine to form an optionally substituted C2­C9 heterocyclyl or optionally substituted C2­C9 heteroaryl; X5 is CR5R6, wherein R5 is halo, hydrogen, hydroxy, optionally substituted amino, cyano, optionally substituted C1­C6 alkyl, optionally substituted C1­C6 alkoxy, ­SO2­optionally substituted C1­ C6 alkyl, optionally substituted C2­C9 heterocyclyl, or optionally substituted C2­C9 heteroaryl, and R6 is halo, optionally substituted amino, cyano, optionally substituted C1­C6 alkyl, optionally substituted C1­ C6 alkoxy, ­SO2­optionally substituted C1­C6 alkyl, optionally substituted C2­C9 heterocyclyl, or optionally substituted C2­C9 heteroaryl, and each R7 is, independently, halo, optionally substituted C1­ C6 heteroalkyl, or optionally substituted C1­C6 alkyl, and/or two R7, together with the atoms to which they are attached, combine to form an optionally substituted C2­C9 heterocyclyl; or R5 is halo, hydrogen, hydroxy, optionally substituted amino, cyano, optionally substituted C1­C6 alkyl, optionally substituted C1­C6 alkoxy, ­SO2­optionally substituted C1­C6 alkyl, optionally substituted C2­C9 heterocyclyl, or optionally substituted C2­C9 heteroaryl; R6 and one R7, together with the atoms to which they are attached, combine to form an optionally substituted C2­C9 heterocyclyl or optionally substituted C2­C9 heteroaryl; and the remaining R7 groups, if present, are independently halo, optionally substituted C1­C6 heteroalkyl, or optionally substituted C1­C6 alkyl; or R6 is halo, hydroxy, optionally substituted amino, cyano, optionally substituted C1­C6 alkyl, optionally substituted C1­C6 alkoxy, ­SO2­optionally substituted C1­C6 alkyl, optionally substituted C2­C9 heterocyclyl, or optionally substituted C2­C9 heteroaryl; R5 and one R7, together with the atoms to which they are attached, combine to form an optionally substituted C2­C9 heterocyclyl or optionally substituted C2­C9 heteroaryl; and the remaining R7 groups, if present, are independently halo, optionally substituted C1­ C6 alkyl, or optionally substituted C1­C6 alkyl; X is O, wherein p is 1, 2, 3, 4, 5, 6, 7, or 8; or X is SR5R6, wherein each of R5 and R6 is oxo. In some embodiments, R is hydrogen. In some embodiments, R is optionally substituted C1­C6 alkyl (e.g., methyl). In some embodiments, X1 is CH. In some embodiments, X1 is S. In some embodiments, X1 is N. In some embodiments, X2 is CR1 and X3 is N. In some embodiments, the compound has the structure:
Figure imgf000005_0001
or a pharmaceutically acceptable salt thereof. In some embodiments, X2 is N and X3 is CR1. In some embodiments, the compound has the structure:
Figure imgf000005_0002
. Formula 1b or a pharmaceutically acceptable salt thereof. In some embodiments, X2 and X3 are CR1. In some embodiments, the compound has the structure:
Figure imgf000005_0003
, Formula 1c or a pharmaceutically acceptable salt thereof, where each R1A is independently H or R1. In some embodiments, at least one R1A is halo (e.g., fluoro, chloro, or bromo). In some embodiments, at least one R1A is optionally substituted C1­C6 alkyl (e.g., methyl, ethyl, iso­propyl). In some embodiments, at least one R1A is optionally substituted C1­C6 alkoxy (e.g., methoxy, ethoxy, or iso­propoxy). In some embodiments, at least one R1 is halo (e.g., fluoro, chloro, or bromo). In some embodiments, at least one R1 is optionally substituted C1­C6 alkyl (e.g., methyl, ethyl, iso­propyl). In some embodiments, at least one R1 is optionally substituted C1­C6 alkoxy (e.g., methoxy, ethoxy, or iso­propoxy). In some embodiments, Ar is optionally substituted C6­C10 aryl (e.g., phenyl, 2­chloro­phenyl, 3­chloro­ phenyl, 4­chloro­phenyl, 2­fluoro­phenyl, 3­fluoro­phenyl, 4­fluoro­phenyl, 2­methyl­phenyl, 4­benzoxy­ phenyl, 2­methoxy­phenyl, 3­methoxy­phenyl, 4­methoxy­phenyl, 2­cyano­phenyl, 3­cyano­phenyl, 4­cyano­ phenyl, 2­chloro­4­fluoro­phenyl, 2,4­fluoro­phenyl, 2­chloro­3­fluoro­phenyl, 2­chloro­4­cyano­phenyl, 2­ cyano­4­fluoro­phenyl, 2­cyano­4­chloro­phenyl, 2,3­fluoro­phenyl, 2­fluoro­4­cyano­phenyl, 2­chloro­6­fluoro­ phenyl, 2­fluoro­4­chloro­phenyl, 2,6­fluoro­phenyl, 2,5­fluoro­phenyl, or 2­fluoro­4­methoxy­phenyl.
Figure imgf000006_0001
Formula 1d or a pharmaceutically acceptable salt thereof. In some embodiments, R2 has the structure of Formula II. In some embodiments, R2 has the structure of Formula III. In some embodiments, R2 has the structure:
Figure imgf000006_0002
Figure imgf000007_0001
wherein q is 0, 1, 2, 3, 4, 5, 6, 7, or 8; R10 is optionally substituted C1­C6 alkyl; and each R8 is, independently, halo, hydroxy, optionally substituted C1­C6 alkyl, or optionally substituted C1­C6 alkoxy. In some embodiments, the compound has the structure:
Figure imgf000008_0001
Formula 1e or a pharmaceutically acceptable salt thereof, wherein R13 is optionally substituted pyridin­4­yl or optionally substituted phenyl; and R14 is optionally substituted piperidin­4­yl, optionally substituted 2­oxa­6­azaspiro[3.3]heptan­6­yl, 2­ azaspiro[3.3]heptan­2­yl substituted with hydroxy. In some embodiments, R13 is optionally substituted phenyl. In some embodiments, the optionally substituted phenyl is 2,4­difluorphenyl. In some embodiments, R13 is optionally substituted pyridin­4­yl. In some embodiments, the optionally substituted pyridin­
Figure imgf000008_0002
In some embodiments, the compound has the structure:
Figure imgf000008_0003
or a pharmaceutically acceptable salt thereof, wherein R15 is optionally substituted 4­azaspiro[2.4]heptan­4­yl. In some embodiments, the compound has the the structure:
Figure imgf000008_0004
Formula 1g or a pharmaceutically acceptable salt thereof, wherein R16 is optionally substituted 2­oxa­5­azabicyclo[2.2.1]heptan­5­yl, or optionally substituted 3­ azabicyclo[3.1.0]hexan­3­yl. In some embodiments, the compound has the structure:
Figure imgf000009_0001
Formula 1h or a pharmaceutically acceptable salt thereof. wherein R17 and R18 are each, independently, H or F; and R19 is 2­oxa­5­azabicyclo[2.2.1]heptan­5­yl, or 3­azabicyclo[3.1.0]hexan­3­yl. In some embodiments, the compound has the structure:
Figure imgf000009_0002
Formula 1i or a pharmaceutically acceptable salt thereof, wherein R20 is optionally substituted 2­oxa­6­azaspiro[3.3]heptan­6­yl. In some embodiments, the compound has the structure:
Figure imgf000009_0003
Formula 1j or a pharmaceutically acceptable salt thereof, wherein: R21 is optionally substituted pyridinyl; and R22 is piperidin­1­yl optionally substituted with methoxy; azetidin­1­yl optionally substituted with methyl, methoxy, or fluoro; 2­oxa­5­azabicyclo[2.2.1]heptan­5­yl, or optionally substituted morpholin­4­yl. In some embodiments,
Figure imgf000009_0004
In some embodiments, the compound has the structure
Figure imgf000010_0001
or a pharmaceutically acceptable salt thereof, wherein
Figure imgf000010_0002
In some embodiments, the compound has the structure:
Figure imgf000010_0003
Formula 1l or a pharmaceutically acceptable salt thereof, wherein
Figure imgf000010_0004
In some embodiments, the compound has the structure:
Figure imgf000010_0005
Formula 1m or a pharmaceutically acceptable salt thereof, wherein X14 is N or CH; and
Figure imgf000011_0001
, In some embodiments, X14 is N. In some embodiments, X14 is CH.
Figure imgf000011_0002
Figure imgf000012_0001
pharmaceutically acceptable salt thereof.
Figure imgf000013_0001
Figure imgf000013_0002
, p ,
Figure imgf000013_0003
In an aspect, the invention features a compound, or pharmaceutically acceptable salt thereof, having the structure:
Figure imgf000014_0001
Formula 2 or a pharmaceutically acceptable salt thereof, wherein m is 0, 1, 2, 3, or 4; X6 is CH, or N; X7 is NH or S; Ar1 is optionally substituted C6­C10 aryl; R23 is hydrogen, halo, or optionally substituted C1­C6 alkoxy; L3 is ­NR24CO­ or ­CONR24­, R24 is H or optionally substituted C1­C6 alkyl; RB is ­CH2CONHR25, or ­COR25 or NR25; R25 is optionally substituted heteroaryl, optionally substituted cyclohexyl, or has the structure
Figure imgf000014_0002
Formula II Formula III wherein R26 is optionally substituted C1­C3 alkyl; R27 is optionally substituted C1­C6 alkyl, or optionally substituted C1­C6 heteroalkyl; n is 0 or 1; o is 0 or 1 or 2; X8 is NR28, CR28R29, or O; R28 is absent, hydrogen, hydroxy, optionally substituted amino, optionally substituted C1­C6 alkyl, OR optionally substituted C1­C6 alkoxy; R29 is absent, hydrogen, hydroxy, optionally substituted amino, optionally substituted C1­C6 alkyl, OR optionally substituted C1­C6 alkoxy; each R25 is, independently, halo or optionally substituted C1­C6 alkyl. In some embodiments, X6 is CH. In some embodiments, X6 is N. In some embodiments, X7 is N. In some embodiments, X7 is S. In some embodiments, the compound has the structure:
Figure imgf000014_0003
or a pharmaceutically acceptable salt thereof, In some embodiments, the compound has the structure:
Figure imgf000015_0001
or a pharmaceutically acceptable salt thereof, In some embodiments, the compound has the structure:
Figure imgf000015_0002
Formula 2c or a pharmaceutically acceptable salt thereof, In some embodiments, the compound has the structure:
Figure imgf000015_0003
or a pharmaceutically acceptable salt thereof, In some embodiments, the compound has the structure:
Figure imgf000015_0004
Formula 2e or a pharmaceutically acceptable salt thereof. In some embodiments, Ar1 is phenyl, 2­chloro­phenyl, 4­chloro­phenyl, 2­fluoro­phenyl, 3­fluoro­ phenyl, 4­fluoro­phenyl, 2­methyl­phenyl, 2­methoxy­phenyl, 3­methoxy­phenyl, 4­methoxy­phenyl, 2­chloro­ 4­fluoro­phenyl, 2,4­fluoro­phenyl, 2­chloro­3­fluoro­phenyl, 2­cyano­4­fluoro­phenyl, 2­cyano­4­chloro­ phenyl, 2,3­difluoro­phenyl, 2­chloro­6­fluoro­phenyl, or 2­fluoro­4­chloro­phenyl. In some embodiments, R22 has the structure
Figure imgf000016_0001
, , ,
Figure imgf000016_0002
some embodiments, R22 has the structure:
Figure imgf000016_0003
In some embodiments, the compound has the structure:
Figure imgf000016_0004
Formula 2f or a pharmaceutically acceptable salt thereof, wherein R57 is halo. In some embodiments, R57 is chloro.
Figure imgf000016_0005
a pharmaceutically acceptable salt thereof. In some embodiments, the compound has the structure:
Figure imgf000016_0006
a pharmaceutically acceptable salt thereof. In an aspect, the invention features a compound, or pharmaceutically acceptable salt thereof, having the structure:
Figure imgf000017_0001
Formula 3 or a pharmaceutically acceptable salt thereof, wherein n is 0, 1, 2, 3, or 4; L4 is optionally substituted C1­C6 alkylene or optionally substituted C3­C8 cycloalkylene; X9 is N and X10 is CH, or X9 is CH and X10 is N; X11 is N or CH; each R30 is, independently, halo, optionally substituted C1­C6 alkyl, or optionally substituted C1­C6 alkoxy; ArC is optionally disubstituted C6­C10 aryl, or C6­C10 aryl optionally monosubstituted with chloro, optionally substituted C1­C6 heteroalkyl, cyano, meta­fluoro, or ortho­fluoro; RC is COR31, or ­R31; R31 is optionally substituted C2­C5 heteroaryl, or has the structure:
Figure imgf000017_0002
Formula II Formula III wherein R32 is optionally substituted C1­C3 alkyl; R33 is optionally substituted C1­C6 alkyl C6 aryl, optionally substituted C1­C6 alkyl C2­C5 heteroaryl, optionally substituted C5 cycloalkyl, optionally substituted C3­C6 alkyl, or optionally substituted C3 heteroalkyl. n is 0 or 1; o is 0 or 1 or 2; p is 0, 1, 2, 3, 4, 5, 6, 7, or 8; X12 is NR34, CR34R35, O, or SR34R35; R34 is absent, halo, oxo, hydrogen, hydroxy, optionally substituted amino, cyano, optionally substituted C1­C6 alkyl, optionally substituted C1­C6 heteroalkyl, ­SO2­optionally substituted C1­C6 alkyl, optionally substituted C2­C9 heterocyclyl, optionally substituted C6­C10 aryl, or optionally substituted C2­C9 heteroaryl, or R33 and R34 combine with the atoms to which they are attached to form an optionally substituted C2­C9 heteroaryl; R35 is absent, halo, oxo, hydroxy, optionally substituted amino, cyano, optionally substituted C1­C6 alkyl, optionally substituted C1­C6 heteroalkyl, ­SO2­optionally substituted C1­C6 alkyl, optionally substituted C2­C9 heterocyclyl, optionally substituted C6­C10 aryl or optionally substituted C2­C9 heteroaryl, or R33 and R35 combine with the atoms to which they are attached to form an optionally substituted C2­C9 heteroaryl; and each R33 is, independently, halo, oxo, or optionally substituted C1­C6 alkyl, or two R33 combine with the atoms to which they are attached to form an optionally substituted C4 cycloalkyl. In some embodiments, X9 is N and X10 is CH. In some embodiments, X9 is CH and X10 is N. In some embodiments, X11 is N. In some embodiments, X11 is CH. In some embodiments, the compound has the structure:
Figure imgf000018_0001
Formula 3a or a pharmaceutically acceptable salt thereof. In some embodiments, the compound has the structure
Figure imgf000018_0002
Formula 3b or a pharmaceutically acceptable salt thereof. In some embodiments at least one R30 is halo. In some embodiments, at least one R30 is chloro. In some embodiments, at least one R30 is optionally substituted C1­C6 alkyl. In some embodiments, at least one R30 is methyl. In some embodiments, at least one R30 is optionally substituted C1­C6 alkoxy. In some embodiments, at least one R30 is ethoxy. In some embodiments, ArC is 2­chloro­phenyl, 3­chloro­phenyl, 4­chloro­phenyl, 2­fluoro­phenyl, 3­ fluoro­phenyl, 4­benzoxy­phenyl, 2­cyano­phenyl4­cyano­phenyl, 2­chloro­4­fluoro­phenyl, 2,4­difluoro­ phenyl, 2­chloro­3­fluoro­phenyl, 2­chloro­4­cyano­phenyl, 2­cyano­4­fluoro­phenyl, 2­cyano­4­chloro­phenyl, 2,3­difluoro­phenyl, 2­fluoro­4­cyano­phenyl, 2­chloro­6­fluoro­phenyl, 2­fluoro­4­chloro­phenyl, 2,6­difluoro­ phenyl, 2,5­difluoro­phenyl, 2­chloro­3­fluoro­phenyl, 3,4­difluoro­phenyl, 2,3­difluoro­phenyl, or 2­fluoro­4­ methoxy­phenyl.
In some embodiments, R31 has the structure
Figure imgf000019_0001
. In some embodiments, R31 has the structure:
Figure imgf000019_0002
wherein p is 0, 1, 2, 3, 4, or 5; q is 0, 1, 2, 3, or 4; and each R36 is, independently, halo, hydroxy, optionally substituted C1­C6 alkyl, or optionally substituted C1­C6 alkoxy. In some embodiments, the compound has the structure:
Figure imgf000019_0003
or a pharmaceutically acceptable salt thereof, wherein R37 is cyano and R38 is fluoro, or R37 is fluoro and R38 is cyano; and R39 is or 3,3­difluoro­azetidin­1­yl. In some embodiments, the compound has the structure:
Figure imgf000019_0004
or a pharmaceutically acceptable salt thereof, wherein l is 0 or 1; L4 is is optionally substituted C1­C6 alkylene or optionally substituted C3­C8 cycloalkylene; and R40 is 4­hydroxy­piperidin­1­yl, 3­methoxy­piperidin­1­yl, optionally substituted diazapen­1­yl, triazolopiperazinyl substituted with methyl, 4,4­difluoro­piperidin­1­yl, 1,1­dioxothiomorpholin­4­yl, 2­ (methoxymethyl)­pyrroloin­1­yl, tetrahydro­1,3­oxazin­3­yl, 4­isopropyl­piperazin­1­yl, 4­(2­oxazolidin­3­yl)­ piperidin­1­yl, optionally substituted 1,2,4 oxadizol­5­yl, or optionally substituted 1,3,4 oxadizol­2­yl. In some embodiments, l is 1. In some embodiments, the compound has the structure
Figure imgf000020_0001
Formula 3e or a pharmaceutically acceptable salt thereof, wherein R41 is piperidin­1­yl substituted with optionally substituted dialkylamino. In some embodiments, the compound has the structure:
Figure imgf000020_0002
Formula 3f or a pharmaceutically acceptable salt thereof, wherein R43 is F or CN; and R42 is optionally substituted azetidin­1­yl. In some embodiments, optionally substituted azetidine­1­yl is 3,3­difluoro­azetidin­1­yl. In some embodiments, the compound has the structure
Figure imgf000020_0003
Formula 3g or a pharmaceutically acceptable salt thereof, wherein R44 is optionally substituted azetidin­1­yl. In some embodiments, wherein optionally substituted azetidine­1­yl is 3,3­difluoro­azetidin­1­yl. In some embodiments, the compound has the structure:
Figure imgf000021_0001
or a pharmaceutically acceptable salt thereof, wherein R57 and R58 are each halo. In some embodiments, R57 and R58 are each fluoro. In an aspect, the invention features a compound, or pharmaceutically acceptable salt thereof, having the structure:
Figure imgf000021_0002
or a pharmaceutically acceptable salt thereof, wherein n is 0, 1, 2, 3, or 4; R45 is halo; and R46 is optionally substituted azetidinyl. In some embodiments, the compound has the structure:
Figure imgf000021_0003
or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is:
Figure imgf000021_0004
, or a pharmaceutically acceptable salt thereof. In an aspect, the invention features a compound, or pharmaceutically acceptable salt thereof, having the structure:
Figure imgf000022_0001
Formula 5 or a pharmaceutically acceptable salt thereof, wherein p is 0 or 1; X13 is a single bond or O; R48 is optionally substituted C6­C10 aryl, optionally substituted C2­C5 heteroaryl, or trifluoromethyl; R49 is H or optionally substituted C1­C6 alkyl; R50 is optionally substituted C6­C10 aryl or optionally substituted C2­C5 hetetoaryl; and R51 is optionally substituted C2­C5 heterocyclyl. In some embodiments, p is 0. In some embodiments, p is 1. In some embodiments, X13 is O. In some embodiments, R48 is optionally substituted C6­C10 aryl. In some embodiments, R48 is
Figure imgf000022_0002
,
Figure imgf000022_0003
, . In some embodiments, R48 is optionally substituted C2­C5 heteroaryl. In some embodiments, R48 is
Figure imgf000022_0004
In some embodiments, R49 is H. In some embodiments, R49 is C1­C6 alkyl. In some embodiments, R49 is methyl. In some embodiments, In some embodiments, R50 is optionally substituted C6­C10 aryl. In some embodiments, R50 is e
Figure imgf000022_0005
, In some embodiments, R51 is morpholin­4­yl, 2­oxa­6­azaspiro[3.3]heptan­6­yl, or 4­hydroxy­ piperidin­4­yl. In an aspect, the invention features a compound having the structure of any one of compounds 1­ 198, 356, 373, 386, 419, 435, or 436 in Table 1, or pharmaceutically acceptable salt thereof. In an aspect, the invention features a compound having the structure of any one of compounds 199- 355, 357-372, 374-385, 387-418, 420-434, or 437-453 in Table 1 , or pharmaceutically acceptable salt thereof. in an aspect, the invention features a pharmaceutical composition comprising any of the foregoing compounds, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.
In an aspect, the invention features a method of treating a neurological disorder (e.g,, frontotemporal dementia (FTLD-TDP), chronic traumatic encephalopathy, ALS, Alzheimer's disease, limbic-predominant age-related TDP-42 encephalopathy (LATE), or frontotemporal lobar degeneration) in a subject in need thereof. This method includes administering an effective amount of any of the foregoing compounds or pharmaceutical compositions.
In an aspect, the invention features a method of inhibiting toxicity in a cell (e.g., mammalian neural cell) related to a protein (e.g., TDP-43). This method includes administering an effective amount of any of the foregoing compounds or pharmaceutical compositions.
In an aspect, the invention features a method of treating a CYP51A1 -associated disorder (e.g., FTLD-TDP, chronic traumatic encephalopathy, ALS, Alzheimer’s disease, LATE, or frontotemporal lobar degeneration) in a subject in need thereof. This method includes administering an effective amount of any of the foregoing compounds pharmaceutical compositions. in an aspect, the invention features a method of inhibiting CYP51 A1 . This method includes contacting a cell with an effective amount of any of the foregoing compounds or pharmaceutical compositions.
In another aspect, the invention features a method of treating a neurological disorder in a patient, such as a human patient, identified as likely to benefit from treatment with a CYP51 A1 inhibitor on the basis of TDP-43 aggregation. In this aspect, the method may include (I) determining that the patient exhibits, or is prone to develop, TDP-43 aggregation, and (ii) providing to the patient a therapeutically effective amount of a CYP51A1 inhibitor. In some embodiments, the patient has previously been determined to exhibit, or to be prone to developing, TDP-43 aggregation, and the method includes providing to the patient a therapeutically effective amount of a CYP51A1 inhibitor. The susceptibility of the patient to developing TDP-43 aggregation may be determined, e.g., by determining whether the patient expresses a mutant isoform of TDP-43 containing a mutation that is associated with TDP-43 aggregation and toxicity, such as a mutation selected from Q331 K, M337V, Q343R, N345K, R361 S, and N390D. This may be performed, for example, by determining the amino acid sequence of a TDP-43 isoform isolated from a sample obtained from the patient or by determining the nucleic acid sequence of a TDP-43 gene isolated from a sample obtained from the patient. In some embodiments, the method includes the step of obtaining the sample from the patient. in an additional aspect, the invention features a method of treating a neurological disorder in a patient, such as a human patient, identified as likely to benefit from treatment with a CYP51 A1 inhibitor on the basis of TDP-43 expression. In this aspect, the method includes (i) determining that the patient expresses a mutant form of TDP-43 having a mutation associated with TDP-43 aggregation (e.g., a mutation selected from Q331 K, M337V, Q343R, N345K, R361 S, and N390D), and (ii) providing to the patient a therapeutically effective amount of a CYP51 A1 inhibitor. In some embodiments, the patient has previously been determined to express a mutant form of TDP-43 having a mutation associated with TDP-43 aggregation, such as a Q331 K, M337V, Q343R, N345K, R361S, or N390D mutation, and the method includes providing to the patient a therapeutically effective amount of a CYP51 A1 inhibitor.
In another aspect, the invention features a method of determining whether a patient (e.g., a human patient) having a neurological disorder is likely to benefit from treatment with a CYP51 A1 inhibitor by (I) determining whether the patient exhibits, or is prone to develop, TDP-43 aggregation and (ii) identifying the patient as likely to benefit from treatment with a CYP51 A1 inhibitor if the patient exhibits, or is prone to develop, TDP-43 aggregation. In some embodiments, the method further includes the step of (ill) informing the patient whether he or she is likely to benefit from treatment with a CYP51A1 inhibitor. The susceptibility of the patient to developing TDP-43 aggregation may be determined, e.g., by determining whether the patient expresses a mutant isoform of TDP-43 containing a mutation that is associated with TDP-43 aggregation and toxicity, such as a mutation selected from Q331 K, M337V, Q343R, N345K, R361 S, and N390D. This may be performed, for example, by determining the amino acid sequence of a TDP-43 isoform isolated from a sample obtained from the patient or by determining the nucleic acid sequence of a TDP-43 gene isolated from a sample obtained from the patient. In some embodiments, the method includes the step of obtaining the sample from the patient.
In another aspect, the invention features a method of determining whether a patient (e.g., a human patient) having a neurological disorder is likely to benefit from treatment with a CYP51 A1 inhibitor by (i) determining whether the patient expresses a TDP-43 mutant having a mutation associated with TDP-43 aggregation (e.g., a mutation selected from Q331 K, M337V, Q343R, N345K, R361S, and N390D) and (ii) identifying the patient as likely to benefit from treatment with a CYP51A1 inhibitor if the patient expresses a TDP-43 mutant. In some embodiments, the method further includes the step of (iii) informing the patient whether he or she is likely to benefit from treatment with a CYP51 A1 inhibitor. The TDP-43 isoform expressed by the patient may be assessed, for example, by isolated TDP-43 protein from a sample obtained from the patient and sequencing the protein using molecular biology techniques described herein or known in the art. In some embodiments, the TDP-43 isoform expressed by the patient is determined by analyzing the patient’s genotype at the TDP-43 locus, for example, by sequencing the TDP-43 gene in a sample obtained from the patient. In some embodiments, the method includes the step of obtaining the sample from the patient.
In some embodiments of any of the above aspects, the CYP51A1 inhibitor is provided to the patient by administration of the CYP51A1 inhibitor to the patient. In some embodiments, the CYP51A1 inhibitor is provided to the patient by administration of a prodrug that is converted in vivo to the CYP51A1 inhibitor.
In some embodiments of any of the above aspects, the neurological disorder is a neuromuscular disorder, such as a neuromuscular disorder selected from amyotrophic lateral sclerosis, congenital myasthenic syndrome, congenital myopathy, cramp fasciculation syndrome, Duchenne muscular dystrophy, glycogen storage disease type II, hereditary spastic paraplegia, inclusion body myositis, Isaac’s Syndrome, Kearns-Sayre syndrome, Lambert-Eaton myasthenic syndrome, mitochondrial myopathy, muscular dystrophy, myasthenia gravis, myotonic dystrophy, peripheral neuropathy, spinal and bulbar muscular atrophy, spinal muscular atrophy, Stiff person syndrome, Troyer syndrome, and Guillain-Barre syndrome. In some embodiments, the neurological disorder is amyotrophic lateral sclerosis.
In some embodiments of any of the above aspects, the neurological disorder is selected from frontotemporal degeneration (also referred to as frontotemporal lobar degeneration and frontotemporal dementia), Alzheimer’s disease, Parkinson’s disease, dementia with Lewy Bodies, corticobasal degeneration, progressive supranuclear palsy, dementia parkinsonism ALS complex of Guam, Huntington's disease, Inclusion body myopathy with early-onset Paget disease and frontotemporal dementia (IBMPFD), sporadic inclusion body myositis, myofibrillar myopathy, dementia pugilistica, chronic traumatic encephalopathy, Alexander disease, and hereditary inclusion body myopathy.
In some embodiments, the neurological disorder is amyotrophic lateral scierosis, and following administration of the CYP51A1 inhibitor to the patient, the patient exhibits one or more, or ail, of the following responses:
(i) an improvement in condition as assessed using the amyotrophic lateral sclerosis functional rating scaie (ALSFRS) or the revised ALSFRS (ALSFRS-R), such as an improvement in the patient’s ALSFRS or ALSFRS-R score within one or more days, weeks, or months foilowing administration of the CYP51A1 inhibitor (e.g., an improvement in the patient's ALSFRS or ALSFRS-R score within from about 1 day to about 48 weeks (e.g., within from about 2 days to about 36 weeks, from about 4 weeks to about 24 weeks, from about 8 weeks to about 20 weeks, or from about 12 weeks to about 16 weeks), or more, following the initial administration of the CYP51A1 inhibitor to the patient, such as within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 weeks, or more, following the initial administration of the CYP51A1 inhibitor to the patient);
(ii) an increase in slow vital capacity, such as an increase in the patient’s slow vital capacity within one or more days, weeks, or months following administration of the CYP51A.1 inhibitor (e.g., an increase in the patient’s slow vital capacity within from about 1 day to about 48 weeks (e.g., within from about 2 days to about 36 weeks, from about 4 weeks to about 24 weeks, from about 8 weeks to about 20 weeks, or from about 12 weeks to about 16 weeks), or more, following the initial administration of the CYP51A1 inhibitor to the patient, such as within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 weeks, or more, following the initial administration of the CYP51A1 inhibitor to the patient);
(ill) a reduction in decremental responses exhibited by the patient upon repetitive nerve stimulation, such as a reduction that is observed within one or more days, weeks, or months following administration of the CYP51 A1 inhibitor (e.g., a reduction that is observed within from about 1 day to about 48 weeks (e.g., within from about 2 days to about 36 weeks, from about 4 weeks to about 24 weeks, from about 8 weeks to about 20 weeks, or from about 12 weeks io about 16 weeks), or more, foilowing the initial administration of the CYP51A1 inhibitor to the patient, such as within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 weeks, or more, following the initial administration of the CYP51A1 inhibitor to the patient);
(iv) an improvement in muscle strength, as assessed, for example, by way of the Medical Research
Council muscle testing scale (as described, e.g., in Jagtap et al., Ann. Indian. Acad. Neurol. 17:336-339 (2014), the disclosure of which is incorporated herein by reference as it pertains to measuring patient response to neurological disease treatment), such as an improvement that is observed within one or more days, weeks, or months following administration of the CYP51A1 inhibitor (e.g., an improvement that is observed within from about 1 day to about 48 weeks (e.g., within from about 2 days to about 36 weeks, from about 4 weeks to about 24 weeks, from about 8 weeks to about 20 weeks, or from about 12 weeks to about 16 weeks), or more, following the initial administration of the CYP51A1 inhibitor to the patient, such as within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 weeks, or more, following the initial administration of the CYP51 A1 inhibitor to the patient);
(v) an improvement in quality of life, as assessed, for example, using the amyotrophic lateral sclerosis-specific quality of life (ALS-specific QOL) questionnaire, such as an improvement in the patient’s quality of life that is observed within one or more days, weeks, or months following administration of the CYP51 A1 inhibitor (e.g., an improvement in the subject’s quality of life that is observed within from about 1 day to about 48 weeks (e.g., within from about 2 days to about 36 weeks, from about 4 weeks to about 24 weeks, from about 8 weeks to about 20 weeks, or from about 12 weeks to about 16 weeks), or more, following the initial administration of the CYP51A1 inhibitor to the patient, such as within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 weeks, or more, following the initial administration of the CYP51A1 inhibitor to the patient);
(vi) a decrease in the frequency and/or severity of muscle cramps, such as a decrease in cramp frequency and/or severity within one or more days, weeks, or months following administration of the CYP51 A1 inhibitor (e.g., a decrease in cramp frequency and/or severity within from about 1 day to about 48 weeks (e.g., within from about 2 days to about 36 weeks, from about 4 weeks to about 24 weeks, from about 8 weeks to about 20 weeks, or from about 12 weeks to about 16 weeks), or more, following the initial administration of the CYP51A1 inhibitor to the patient, such as within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 weeks, or more, following the initial administration of the CYP51A1 inhibitor to the patient); and/or
(vii) a decrease in TDP-43 aggregation, such as a decrease in TDP-43 aggregation within one or more days, weeks, or months following administration of the CYP51A1 inhibitor (e.g., a decrease in TDP-43 aggregation within from about 1 day to about 48 weeks (e.g., within from about 2 days to about 36 weeks, from about 4 weeks to about 24 weeks, from about 8 weeks to about 20 weeks, or from about 12 weeks to about 16 weeks), or more, following the initial administration of the CYP51A1 inhibitor to the patient, such as within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 weeks, or more, following the initial administration of the CYP51A1 inhibitor to the patient.
Chemical Terms
It is to be understood that the terminology employed herein is for the purpose of describing particular embodiments and is not intended to be limiting.
Those skilled in the art will appreciate that certain compounds described herein can exist in one or more different isomeric (e.g., stereoisomers, geometric isomers, tautomers) and/or isotopic (e.g., in which one or more atoms has been substituted with a different isotope of the atom, such as hydrogen substituted for deuterium) forms. Unless otherwise indicated or clear from context, a depicted structure can be understood to represent any such isomeric or isotopic form, individually or in combination.
In some embodiments, one or more compounds depicted herein may exist in different tautomeric forms. As will be clear from context, unless explicitly excluded, references to such compounds encompass all such tautomeric forms, in some embodiments, tautomeric forms result from the swapping of a single bond with an adjacent double bond and the concomitant migration of a proton. In certain embodiments, a tautomeric form may be a prototropic tautomer, which is an isomeric protonation states having the same empirical formula and total charge as a reference form. Examples of moieties with prototropic tautomeric forms are ketone - enol pairs, amide - imidic acid pairs, lactam - lactim pairs, amide - imidic acid pairs, enamine ~ imine pairs, and annular forms where a proton can occupy two or more positions of a heterocyclic system, such as, 1 H- and 3H-imidazole, 1H-, 2H- and 4H- 1 ,2,4-triazole, 1 H- and 2H- isoindoie, and 1 H- and 2H-pyrazole. In some embodiments, tautomeric forms can be in equilibrium or sterically locked into one form by appropriate substitution. In certain embodiments, tautomeric forms result from acetal interconversion, e.g., the interconversion illustrated in the scheme below:
Figure imgf000028_0001
Those skilled in the art will appreciate that, in some embodiments, isotopes of compounds described herein may be prepared and/or utilized in accordance with the present invention. “Isotopes” refers to atoms having the same atomic number but different mass numbers resulting from a different number of neutrons in the nuclei. For example, isotopes of hydrogen include tritium and deuterium. In some embodiments, an isotopic substitution (e.g., substitution of hydrogen with deuterium} may alterthe physiciochemical properties of the molecules, such as metabolism and/or the rate of racemization of a chiral center.
As is known in the art, many chemical entities (in particular many organic molecules and/or many small molecules) can adopt a variety of different solid forms such as, for example, amorphous forms and/or crystalline forms (e.g., polymorphs, hydrates, solvates, etc). In some embodiments, such entities may be utilized in any form, including in any solid form. In some embodiments, such entities are utilized in a particular form, e.g., in a particular solid form.
In some embodiments, compounds described and/or depicted herein may be provided and/or utilized in salt form.
In certain embodiments, compounds described and/or depicted herein may be provided and/or utilized in hydrate or solvate form.
At various places in the present specification, substituents of compounds of the present disclosure are disclosed in groups or in ranges. It is specifically intended that the present disclosure include each and every individual subcombination of the members of such groups and ranges. For example, the term “C1-C5 alkyl” is specifically intended to individually disclose methyl, ethyl, Cs alkyl, C4 alkyl, C5 alkyl, and Co alkyl. Furthermore, where a compound includes a plurality of positions at which substitutes are disclosed in groups or in ranges, unless otherwise indicated, the present disclosure is intended to cover individual compounds and groups of compounds (e.g., genera and subgenera) containing each and every individual subcombination of members at each position.
Herein a phrase of the form “optionally substituted X” (e.g., optionally substituted alkyl) is intended to be equivalent to “X, wherein X is optionally substituted" (e.g., “alkyl, wherein said alkyl is optionally substituted”). It is not intended to mean that the feature “X” (e.g., alkyl) per se is optional.
The term “acyl,” as used herein, represents a hydrogen or an alkyl group, as defined herein that is attached to a parent molecular group through a carbonyl group, as defined herein, and is exemplified by formyl (i.e., a carboxyaldehyde group), acetyl, trifluoroacetyl, propionyl, and butanoyl. Exemplary unsubstituted acyl groups include from 1 to 6, from 1 to 11 , or from 1 to 21 carbons. The term “alkyl," as used herein, refers to a branched or straight-chain monovalent saturated aliphatic hydrocarbon radical of 1 to 20 carbon atoms (e.g,, 1 to 16 carbon atoms, 1 to 10 carbon atoms, or 1 to 6 carbon atoms). An alkylene is a divalent alkyl group.
The term “alkenyl," as used herein, alone or in combination with other groups, refers to a straight-chain or branched hydrocarbon residue having a carbon-carbon double bond and having 2 io 20 carbon atoms (e.g., 2 to 16 carbon atoms, 2 to 10 carbon atoms, 2 to 6, or 2 carbon atoms).
The term “alkynyl,” as used herein, alone or in combination with other groups, refers to a straight-chain or branched hydrocarbon residue having a carbon-carbon triple bond and having 2 to 20 carbon atoms (e.g., 2 to 16 carbon atoms, 2 to 10 carbon atoms, 2 io 6, or 2 carbon atoms).
The term “amino,” as used herein, represents -N(RN1)2, wherein each RN1 is, independently, H, OH, NO2, N(RN2)2, SO2ORN2, SOZRN2, SORN2, an ^/-protecting group, alkyl, alkoxy, aryl, arylalkyl, cycloalkyl, acyl (e.g., acetyl, trifluoroacetyl, or others described herein), wherein each of these recited RN1 groups can be optionally substituted; or two RN1 combine to form an alkylene or heteroalkylene, and wherein each RN2 is, independently, H, alkyl, or aryl. The amino groups of the invention can be an unsubstituted amino (i.e., -NH2) or a substituted amino (i.e., -N(RN1)2>.
The term “aryl,” as used herein, refers to an aromatic mono- or polycarbocyclic radical of 6 to 12 carbon atoms having at least one aromatic ring. Examples of such groups include, but are not limited to, phenyl, naphthyl, 1 ,2,3,4-tetrahydronaphthyl, 1 ,2-dihydronaphthyl, indanyl, and 7/7-indenyl.
The term “arylalkyl,” as used herein, represents an alkyl group substituted with an aryl group. Exemplary unsubstituted arylalkyl groups are from 7 to 30 carbons (e.g., from 7 to 16 or from 7 to 20 carbons, such as CB-IO aryl Ci-Cs alkyl, Cs-10 aryl C1-C10 alkyl, or Ca-10 aryl C1-C20 alkyl), such as, benzyl and phenethyl. In some embodiments, the akyl and the aryl each can be further substituted with 1 , 2, 3, or 4 substituent groups as defined herein for the respective groups.
The term “azido,” as used herein, represents a -N3 group.
The term “cyano,” as used herein, represents a CN group.
The terms “carbocyclyl,” as used herein, refer to a non-aromatic C3-C12 monocyclic, bicyclic, or tricyclic structure in which the rings are formed by carbon atoms. Carbocyclyl structures include cycloalkyl groups and unsaturated carbocyclyl radicals.
The term “cycloalkyl,” as used herein, refers to a saturated, non-aromatic, monovalent mono- or polycarbocyclic radical of three to ten, preferably three to six carbon atoms. This term is further exemplified by radicals such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, norbornyl, and adamantyl. A cycloalkylene is a divalent alkyl group.
The term “halo,” as used herein, means a fluorine (fluoro), chlorine (chloro), bromine (bromo), or iodine (iodo) radical.
The term “heteroalkyl,” as used herein, refers to an alkyl group, as defined herein, in which one or more of the constituent carbon atoms have been replaced by nitrogen, oxygen, or sulfur. In some embodiments, the heteroalkyl group can be further substituted with 1 , 2, 3, or 4 substituent groups as described herein for alkyl groups. Examples of heteroalkyl groups include an “alkoxy” which, as used herein, refers alkyl-O- (e.g., methoxy and ethoxy). A heteroalkylene is a divalent heteroalkyl group. The term “heteroaikenyl,” as used herein, refers to an alkenyl group, as defined herein, in which one or more of the constituent carbon atoms have been replaced by nitrogen, oxygen, or sulfur. In some embodiments, the heteroalkenyl group can be further substituted with 1 , 2, 3, or 4 substituent groups as described herein for alkenyl groups. Examples of heteroaikenyl groups include an “alkenoxy” which, as used herein, refers alkenyl-O-. A heteroalkenylene is a divalent heteroaikenyl group.
The term “heteroalkynyl,” as used herein, refers to an alkynyl group, as defined herein, in which one or more of the constituent carbon atoms have been replaced by nitrogen, oxygen, or sulfur. In some embodiments, the heteroalkynyl group can be further substituted with 1 , 2, 3, or 4 substituent groups as described herein tor alkynyl groups. Examples of heteroalkynyl groups include an “alkynoxy” which, as used herein, refers alkynyl-O-. A heteroalkynylene is a divalent heteroalkynyl group.
The term “heteroaryl,” as used herein, refers to an aromatic mono- or polycyclic radical of 5 to 12 atoms having at least one aromatic ring and containing one, two, or three ring heteroatoms selected from N, O, and S, with the remaining ring atoms being C. One or two ring carbon atoms of the heteroaryl group may be replaced with a carbonyl group. Examples of heteroaryl groups include pyridyl, pyrazoyl, benzooxazolyl, benzoimidazolyl, benzothiazolyl, imidazolyl, oxaxolyl, and thiazolyl.
The term “heteroarylalkyl,” as used herein, represents an alkyl group substituted with a heteroaryl group. Exemplary unsubstituted heteroarylalkyl groups are from 7 to 30 carbons (e.g., from 7 to 16 or from 7 to 20 carbons, such as C2-C9 heteroaryl Ci-Ce alkyl, C2-C9 heteroaryl Ci-Cw alkyl, or C2-C9 heteroaryl C1-C20 alkyl). In some embodiments, the akyl and the heteroaryl each can be further substituted with 1 , 2, 3, or 4 substituent groups as defined herein for the respective groups.
The term “heterocyclyl,” as used herein, denotes a mono- or polycyclic radical having 3 to 12 atoms having at least one ring containing one, two, three, or four ring heteroatoms selected from N, O or S, wherein no ring is aromatic. Examples of heterocyclyl groups include, but are not limited to, morpholinyl, thiomorpholinyl, furyl, piperazinyl, piperidinyl, pyranyl, pyrrolidinyl, tetrahydropyranyl, tetrahydrofuranyl, and 1 ,3-dioxanyl.
The term “heterocyciylalkyl,” as used herein, represents an alkyl group substituted with a heterocyclyl group. Exemplary unsubstituted heterocyciylalkyl groups are from 7 to 30 carbons (e.g., from 7 to 16 or from 7 to 20 carbons, such as C2-C9 heterocyclyl Ci-Cs alkyl, C2-C9 heterocyclyl C1-C10 alkyl, or C2-C9 heterocyclyl C1-C20 alkyl), in some embodiments, the akyl and the heterocyclyl each can be further substituted with 1 , 2, 3, or 4 substituent groups as defined herein for the respective groups.
The term “hydroxyl,” as used herein, represents an -OH group.
The term “A/-protecting group,” as used herein, represents those groups intended to protect an amino group against undesirable reactions during synthetic procedures. Commonly used /V-protecting groups are disclosed in Greene, “Protective Groups in Organic Synthesis,” 3rd Edition (John Wiley & Sons, New York, 1999). A/-protecting groups include acyl, aryloyl, or carbamyl groups such as formyl, acetyl, propionyl, pivaloyl, t-butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoroac-etyl, trichloroacetyl, phthalyl, o-nitrophenoxyacetyl, a-chlorobuiyryl, benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl, and chiral auxiliaries such as protected or unprotected D, L or D, L-amino acids such as alanine, leucine, and phenylalanine; sulfonyl-coniaining groups such as benzenesulfonyl, and p-toluenesulfonyl; carbamate forming groups such as benzyloxycarbonyl, p-chtorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl, 3,5-dimethoxybenzyloxycarbonyl, 2,4-dimethoxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxycarbonyl, 3,4,5-trimethoxybenzyloxycarbonyl, 1-(p-biphenylyl)-1 -methylethoxycarbonyl, a,a-dimethyl-3,5-dimethoxybenzyloxycarbonyL benzhydryloxy carbonyl, t-butyloxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl, 2,2,2,-trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxy carbonyl, fIuorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl, adamantyloxycarbonyl, cyclohexyloxycarbonyl, and phenytthiocarbonyl, arylalkyl groups such as benzyl, triphenylmethyl, and benzyloxymethyl, and silyl groups, such as trimethylsilyl. Preferred /V-protecting groups are alloc, formyl, acetyl, benzoyl, pivaloyl, t-butylacetyl, alanyl, phenylsulfonyl, benzyl, t-butyloxycarbonyl (Boc), and benzyloxycarbonyl (Cbz).
The term “nitro,” as used herein, represents an NO2 group.
The term “thiol,” as used herein, represents an -SH group.
The alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl (e.g., cycloalkyl), aryl, heteroaryl, and heterocyclyl groups may be substituted or unsubstituted. When substituted, there will generally be 1 to 4 substituents present, unless otherwise specified. Substituents include, for example, aryl (e.g., substituted and unsubstituted phenyl), carbocyclyl (e.g., substituted and unsubstituted cycloalkyl), halo (e.g., fluoro), hydroxyl, oxo, heteroalkyl (e.g., substituted and unsubstituted methoxy, ethoxy, or thioalkoxy), heteroaryl, heterocyclyl, amino (e.g., NHz or mono- or dialkyl amino), azido, cyano, nitro, or thiol. Aryl, carbocyclyl (e.g., cycloalkyl), heteroaryl, and heterocyclyl groups may also be substituted with alkyl (unsubstituted and substituted such as arylalkyl (e.g., substituted and unsubstituted benzyl)).
Compounds of the invention can have one or more asymmetric carbon atoms and can exist in the form of optically pure enantiomers, mixtures of enantiomers such as, for example, racemates, optically pure diastereoisomers, mixtures of diastereoisomers, diastereoisomeric racemates or mixtures of diastereoisomeric racemates. The optically active forms can be obtained, for example, by resolution of the racemates, by asymmetric synthesis or asymmetric chromatography (chromatography with a chiral adsorbent or eluant). That is, certain of the disclosed compounds may exist in various stereoisomeric forms. Stereoisomers are compounds that differ only in their spatial arrangement. Enantiomers are pairs of stereoisomers whose mirror images are not superimposable, most commonly because they contain an asymmetrically substituted carbon atom that acts as a chiral center. "Enantiomer" means one of a pair of molecules that are mirror images of each other and are not superimposable. Diastereomers are stereoisomers that are not related as mirror images, most commonly because they contain two or more asymmetrically substituted carbon atoms and represent the configuration of substituents around one or more chiral carbon atoms. Enantiomers of a compound can be prepared, for example, by separating an enantiomer from a racemate using one or more well-known techniques and methods, such as, for example, chiral chromatography and separation methods based thereon. The appropriate technique and/or method for separating an enantiomer of a compound described herein from a racemic mixture can be readily determined by those of skill in the art. "Racemate” or "racemic mixture” means a compound containing two enantiomers, wherein such mixtures exhibit no opticai activity; i.e., they do not rotate the plane of polarized light. “Geometric- isomer" means isomers that differ in the orientation of substituent atoms in relationship to a carbon-carbon double bond, to a cycloalkyl ring, or to a bridged bicyclic system. Atoms (other than H) on each side of a carbon- carbon double bond may be in an E (substituents are on opposite sides of the carbon- carbon double bond) or Z (substituents are oriented on the same side) configuration. "R," "S,'
"R* ," "E," "Z," "cis," and "trans," indicate configurations relative to the core molecule. Certain of the disclosed compounds may exist in atropisomeric forms. Atropisomers are stereoisomers resulting from hindered rotation about single bonds where the steric strain barrier to rotation is high enough to allow for the isolation of the conformers. The compounds of the invention may be prepared as individual isomers by either isomer-specific synthesis or resolved from an isomeric mixture. Conventional resolution techniques include forming the salt of a free base of each isomer of an isomeric pair using an optically active acid (followed by fractional crystallization and regeneration of the free base), forming the salt of the acid form of each isomer of an isomeric pair using an optically active amine (followed by fractional crystallization and regeneration of the free acid), forming an ester or amide of each of the isomers of an isomeric pair using an optically pure acid, amine or alcohol (followed by chromatographic separation and removal of the chiral auxiliary), or resolving an isomeric mixture of either a starting material or a final product using various well known chromatographic methods. When the stereochemistry of a disclosed compound is named or depicted by structure, the named or depicted stereoisomer is at least 60%, 70%, 80%, 90%, 99% or 99.9%) by weight relative to the other stereoisomers. When a single enantiomer is named or depicted by structure, the depicted or named enantiomer is at least 60%, 70%, 80%, 90%, 99% or 99.9% by weight optically pure. When a single diastereomer is named or depicted by structure, the depicted or named diastereomer is at least 60%, 70%, 80%, 90%, 99% or 99.9% by weight pure. Percent optical purity is the ratio of the weight of the enantiomer or over the weight of the enantiomer plus the weight of its optical isomer. Diastereomeric purity by weight is the ratio of the weight of one diastereomer or over the weight of all the diastereomers. When the stereochemistry of a disclosed compound is named or depicted by structure, the named or depicted stereoisomer is at least 60%, 70%, 80%, 90%, 99% or 99.9% by mole fraction pure relative to the other stereoisomers. When a single enantiomer is named or depicted by structure, the depicted or named enantiomer is at least 60%, 70%, 80%, 90%, 99% or 99.9% by mole fraction pure. When a single diastereomer is named or depicted by structure, the depicted or named diastereomer is at least 60%, 70%, 80%, 90%, 99% or 99.9% by mole fraction pure. Percent purity by mole fraction is the ratio of the moles of the enantiomer or over the moles of the enantiomer plus the moles of its optical isomer. Similarly, percent purity by moles fraction is the ratio of the moles of the diastereomer or over the moles of the diastereomer plus the moles of its isomer. When a disclosed compound is named or depicted by structure without indicating the stereochemistry, and the compound has at least one chiral center, it is to be understood that the name or structure encompasses either enantiomer of the compound free from the corresponding optical isomer, a racemic mixture of the compound or mixtures enriched in one enantiomer relative to its corresponding optical isomer. When a disclosed compound is named or depicted by structure without indicating the stereochemistry and has two or more chiral centers, it is to be understood that the name or structure encompasses a diastereomer free of other diastereomers, a number of diastereomers free from other diastereomeric pairs, mixtures of diastereomers, mixtures of diastereomeric pairs, mixtures of diastereomers in which one diastereomer is enriched relative to the other diastereomer(s) or mixtures of diastereomers in which one or more diastereomer is enriched relative to the other diastereomers. The invention embraces all of these forms.
Definitions
In this application, unless otherwise clear from context, (I) the term “a” may be understood to mean “at least one”; (ii) the term “or” may be understood to mean “and/or"; (ill) the terms “comprising" and “including" may be understood to encompass itemized components or steps whether presented by themselves or together with one or more additional components or steps; and (iv) the terms “about” and “approximately” may be understood to permit standard variation as would be understood by those of ordinary skill in the art; and (v) where ranges are provided, endpoints are included.
As used herein, the term “administration” refers to the administration of a composition (e.g., a compound, a complex or a preparation that includes a compound or complex as described herein) to a subject or system. Administration to an animal subject (e.g., to a human) may be by any appropriate route. For example, in some embodiments, administration may be bronchial (including by bronchial instillation), buccal, enteral, interdermal, intra-arterial, intradermal, intragastric, intra med u I la ry, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (including by intratracheal instillation), transdermal, vaginal and vitreal.
As used herein, the term “animal” refers to any member of the animal kingdom. In some embodiments, “animal" refers to humans, at any stage of development. In some embodiments, “animal” refers to non-human animals, at any stage of development. In some embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, and/or worms. In some embodiments, an animal may be a transgenic animal, genetically-engineered animal, and/or a clone.
As used herein, the terms “approximately” and “about” are each intended to encompass normal statistical variation as would be understood by those of ordinary skill in the art as appropriate to the relevant context. In certain embodiments, the terms “approximately” or “about” each refer to a range of values that fail within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11 %, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of a stated value, unless otherwise stated or otherwise evident from the context (e.g., where such number would exceed 100% of a possible value).
Two events or entities are “associated” with one another, as that term is used herein, if the presence, level and/or form of one is correlated with that of the other. For example, a particular entity (e.g., polypeptide) is considered to be associated with a particular disease, disorder, or condition, if its presence, level and/or form correlates with incidence of and/or susceptibility of the disease, disorder, or condition (e.g., across a relevant population).
As used herein, the terms “benefit" and “response” are used interchangeably in the context of a subject, such as a human subject undergoing therapy for the treatment of a neurological disorder, for example, amyotrophic lateral sclerosis, frontotemporal degeneration (also referred to as frontotemporal lobar degeneration and frontotemporal dementia), Alzheimer's disease, Parkinson’s disease, dementia with Lewy Bodies, corticobasal degeneration, progressive supranuclear palsy, dementia parkinsonism ALS complex of Guam, Huntington’s disease, Inclusion body myopathy with early-onset Paget disease and frontotemporal dementia (IBMPFD), sporadic inclusion body myositis, myofibrillar myopathy, dementia pugilistica, chronic traumatic encephalopathy, Alexander disease, and hereditary inclusion body myopathy. The terms “benefit" and “response” refer to any clinical improvement in the subject’s condition. Exemplary benefits in the context, of a subject undergoing treatment for a neurological disorder using the compositions and methods described herein (e.g., in the context of a human subject undergoing treatment for a neurological disorder described herein, such as amyotrophic lateral sclerosis, with a cytochrome P450 isoform 51 A1 (CYP51A1) inhibitor described herein, such as an inhibitory small molecule, antibody, antigen-binding fragment thereof, or interfering RNA molecule) include the slowing and halting of disease progression, as well as suppression of one or more symptoms associated with the disease. Particularly, in the context of a patient (e.g., a human patient) undergoing treatment for amyotrophic lateral sclerosis with a CYP51 A1 inhibitor described herein, examples of clinical “benefits” and “responses” are (i) an improvement in the subject’s condition as assessed using the amyotrophic lateral sclerosis functional rating scale (ALSFRS) or the revised ALSFRS (ALSFRS-R) following administration of the CYP51A1 inhibitor, such as an improvement in the subject's ALSFRS or ALSFRS-R score within one or more days, weeks, or months following administration of the CYP51A1 inhibitor (e.g., an improvement in the subject’s ALSFRS or ALSFRS-R score within from about 1 day to about 48 weeks (e.g., within from about 2 days to about 36 weeks, from about 4 weeks to about 24 weeks, from about 8 weeks io about 20 weeks, or from about 12 weeks to about 16 weeks), or more, following the initial administration of the CYP51A1 inhibitor to the subject, such as within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 weeks, or more, following the initial administration of the CYP51A1 inhibitor to the subject); (ii) an increase in the subject's slow vital capacity following administration of the CYP51A1 inhibitor, such as an increase in the subject’s slow vital capacity within one or more days, weeks, or months following administration of the CYP51A1 inhibitor (e.g., an increase in the subject’s slow vital capacity within from about 1 day to about 48 weeks (e.g., within from about 2 days to about 36 weeks, from about 4 weeks to about 24 weeks, from about 8 weeks to about 20 weeks, or from about 12 weeks to about 16 weeks), or more, following the initial administration of the CYP51 A1 inhibitor to the subject, such as within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 weeks, or more, following the initial administration of the CYP51A1 inhibitor to the subject); (iii) a reduction in decremental responses exhibited by the subject upon repetitive nerve stimulation, such as a reduction that is observed within one or more days, weeks, or months following administration of the CYP51A1 inhibitor (e.g., a reduction that is observed within from about 1 day to about 48 weeks (e.g., within from about 2 days to about 36 weeks, from about 4 weeks to about 24 weeks, from about 8 weeks to about 20 weeks, or from about 12 weeks to about 16 weeks), or more, following the initial administration of the CYP51A1 inhibitor to the subject, such as within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 weeks, or more, following the initial administration of the CYP51A1 inhibitor to the subject); (iv) an improvement in the subject’s muscle strength, as assessed, for example, by way of the Medical Research Council muscle testing scale (as described, e.g., in Jagtap et al., Ann. Indian. Acad. Neurol. 17:336-339 (2014), the disclosure of which is incorporated herein by reference as it pertains to measuring patient response to neurological disease treatment), such as an improvement that is observed within one or more days, weeks, or months following administration of the CYP51A1 inhibitor (e.g., an improvement that is observed within from about 1 day to about 48 weeks (e.g., within from about 2 days to about 36 weeks, from about 4 weeks to about 24 weeks, from about 8 weeks to about 20 weeks, or from about 12 weeks to about 16 weeks), or more, following the initial administration of the CYP51A1 inhibitor to the subject, such as within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 weeks, or more, following the initial administration of the CYP51A1 inhibitor to the subject); (v) an improvement in the subject’s quality of life, as assessed, for example, using the amyotrophic lateral sclerosis-specific quality of life (ALS-specific QOL) questionnaire, such as an improvement in the subject’s quality of life that is observed within one or more days, weeks, or months following administration of the CYP51A1 inhibitor (e.g., an improvement in the subject’s quality of life that is observed within from about 1 day to about 48 weeks (e.g., within from about 2 days to about 36 weeks, from about 4 weeks to about 24 weeks, from about 8 weeks to about 20 weeks, or from about 12 weeks to about 16 weeks), or more, following the initial administration of the CYP51A1 inhibitor to the subject, such as within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 weeks, or more, following the initial administration of the CYP51 A1 inhibitor to the subject); and (vi) a decrease in the frequency and/or severity of muscle cramps exhibited by the subject, such as a decrease in cramp frequency and/or severity within one or more days, weeks, or months foltowing administration of the CYP51A1 inhibitor (e.g., a decrease in cramp frequency and/or severity within from about 1 day to about 48 weeks (e.g,, within from about 2 days to about 36 weeks, from about 4 weeks io about 24 weeks, from about 8 weeks to about 20 weeks, or from about 12 weeks to about 16 weeks), or more, following the initial administration of the CYP51A1 inhibitor to the subject, such as within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 weeks, or more, following the initial administration of the CYP51A1 inhibitor to the subject).
In the practice of the methods of the present invention, an “effective amount” of any one of the compounds of the invention or a combination of any of the compounds of the invention or a pharmaceutically acceptable salt thereof, is administered via any of the usual and acceptable methods known in the art, either singly or in combination.
As used herein, the terms “cytochrome P450 isoform 51 A1 ,” “CYP51A1 ,” and “lanosterol 14-alpha demethylase” are used interchangeably and refer to the enzyme that catalyzes the conversion of lanosterol to 4,4-dimethylcholesta-8(9),14,24-trien-3p-ol, for example, in human subjects. The terms “cytochrome P450 isoform 51A1 ,” “CYP51A1 ,” and “lanosterol 14-alpha demethylase” refer not only to wild-type forms of CYP51A1 , but also to variants of wild-type CYP51A1 proteins and nucleic acids encoding the same. The amino acid sequence and corresponding mRNA sequence of a wild-type form of human CYP51A1 are provided herein as SEQ ID NOs: 1 and 2, which correspond to GenBank Accession No. AAC50951.1 and NCBI Reference Sequence NO. NM_000786.3, respectively. These sequences are shown in Table 2, below.
Table 2. Amino acid and nucleic acid sequences of wild-type human CYP5A1
Figure imgf000036_0001
Figure imgf000037_0001
The terms “cytochrome P450 isoform 51A1 ,” “CYP51A1 ,” and “lanosterol 14-alpha demethyiase” as used herein include, for example, forms of the human CYP51A1 protein that have an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 1 (e.g., 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical to the amino acid sequence of SEQ ID NO: 1) and/or forms of the human CYP51 A1 protein that contain one or more substitutions, insertions, and/or deletions (e.g., one or more conservative and/or nonconservative amino acid substitutions, such as up to 5, 10, 15, 20, 25, or more, conservative or nonconservative amino acid substitutions) relative to a wild-type CYP51 A1 protein. Similarly, the terms “cytochrome P450 isoform 51A1 ,” “CYP51A1 and “lanosterol 14-alpha demethylase” as used herein include, for example, forms of the human CYP51A1 gene that encode an mRNA transcript having a nucleic acid sequence that is at least 85% identical to the nucleic acid sequence of SEQ ID NO: 2 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical io the amino acid sequence of SEQ ID NO: 2).
As used herein, the terms “cytochrome P450 isoform 51A1 inhibitor,” “CYP51A1 inhibitor,” and “lanosterol 14-alpha demethylase inhibitor” are used interchangeably and refer to substances, such as compounds of Formula I. Inhibitors of this type may, for example, competitively inhibit CYP51A1 activity by specifically binding the CYP51A1 enzyme (e.g., by virtue of the affinity of the inhibitor for the CYP51A1 active site), thereby precluding, hindering, or halting the entry of one or more endogenous substrates of CYP51A1 into the enzyme’s active site. Additional examples of CYP51A1 inhibitors that suppress the activity of the CYP51 A1 enzyme include substances that may bind CYP51A1 at a site distal from the active site and attenuate the binding of endogenous substrates to the CYP51 Al active site by way of a change in the enzyme’s spatial conformation upon binding of the inhibitor. In addition to encompassing substances that modulate CYP51A1 activity, the terms “cytochrome P45Q isoform 51A1 inhibitor,” “CYP51A1 inhibitor,” and “lanosterol 14-alpha demethylase inhibitor” refer to substances that reduce the concentration and/or stability of CYP51 A1 mRNA transcripts in vivo, as well as those that suppress the translation of functional CYP51 A1 enzyme.
As used herein, the term “CYP51A1-associated disorder” refers to an undesired physiological condition, disorder, or disease that is associated with and/or mediated at least in pari by CYP51 A1 . In some instances, CYP51A1 -associated disorders are associated with excess CYP51A1 levels and/or activity. Exemplary CYP51A1 -associated disorders include CYP51A1 -associated disorders include but are noi limited to central nervous system (CNS) disorders, dementia, Alzheimer's Disease, chronic traumatic encephalopathy, FTLD-TDP, LATE, or frontotemporal lobar degeneration.
As used herein, the term “combination therapy” refers to those situations in which a subject is simultaneously exposed to two or more therapeutic agents. In some embodiments, two or more compounds may be administered simultaneously; in some embodiments, such compounds may be administered sequentially; in some embodiments, such compounds are administered in overlapping dosing regimens.
As used herein, the term “dosage form” refers to a physically discrete unit of an active compound (e.g., a therapeutic or diagnostic agent) for administration to a subject. Each unit contains a predetermined quantity of active agent. In some embodiments, such quantity is a unit dosage amount (or a whole fraction thereof) appropriate for administration in accordance with a dosing regimen that has been determined to correlate with a desired or beneficial outcome when administered to a relevant population (i.e., with a therapeutic dosing regimen). Those of ordinary skill in the art appreciate that the total amount of a therapeutic composition or compound administered to a particular subject is determined by one or more attending physicians and may involve administration of multiple dosage forms.
As used herein, the term “dosing regimen” refers to a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time. In some embodiments, a given therapeutic compound has a recommended dosing regimen, which may involve one or more doses. In some embodiments, a dosing regimen comprises a plurality of doses each of which are separated from one another by a time period of the same length; in some embodiments, a dosing regimen comprises a plurality of doses and at least two different time periods separating individual doses. In some embodiments, all doses within a dosing regimen are of the same unit dose amount. In some embodiments, different doses within a dosing regimen are of different amounts. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount different from the first dose amount. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount same as the first dose amount. In some embodiments, a dosing regimen is correlated with a desired or beneficial outcome when administered across a relevant population (i.e., is a therapeutic dosing regimen).
As used herein, the term “neuromuscular disorder” refers to a disease impairing the ability of one or more neurons to control the activity of an associated muscle. Examples of neuromuscular disorders are amyotrophic lateral sclerosis, congenital myasthenic syndrome, congenital myopathy, cramp fasciculation syndrome, Duchenne muscular dystrophy, glycogen storage disease type II, hereditary spastic paraplegia, inclusion body myositis, Isaac's Syndrome, Kearns-Sayre syndrome, Lambert-Eaton myasthenic syndrome, mitochondrial myopathy, muscular dystrophy, myasthenia gravis, myotonic dystrophy, peripheral neuropathy, spinal and bulbar muscular atrophy, spinal muscular atrophy, Stiff person syndrome, Troyer syndrome, and Guillain-Barre syndrome, among others.
The term “pharmaceutical composition," as used herein, represents a composition containing a compound described herein formulated with a pharmaceutically acceptable excipient, and manufactured or sold with the approval of a governmental regulatory agency as part of a therapeutic regimen for the treatment of disease in a mammal. Pharmaceutical compositions can be formulated, for example, for oral administration in unit dosage form (e.g., a tablet, capsule, caplet, gelcap, or syrup); for topical administration (e.g., as a cream, gel, lotion, or ointment); for intravenous administration (e.g., as a sterile solution free of particulate emboli and in a solvent system suitable for intravenous use); or in any other pharmaceutically acceptable formulation.
A. “pharmaceutically acceptable excipient,” as used herein, refers any ingredient other than the compounds described herein (e.g., a vehicle capable of suspending or dissolving the active compound) and having the properties of being substantially nontoxic and non-inflammatory in a patient. Excipients may include, for example, antiadherents, antioxidants, binders, coatings, compression aids, dis integrants, dyes (colors), emollients, emulsifiers, fillers (diluents), film formers or coatings, flavors, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, sorbents, suspensing or dispersing agents, sweeteners, and waters of hydration.
As used herein, the term “pharmaceutically acceptable salt” means any pharmaceutically acceptable salt of the compound of formula (I). For example, pharmaceutically acceptable salts of any of the compounds described herein include those that are within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without undue toxicity, irritation, allergic response and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, pharmaceutically acceptable salts are described in: Berge et al., J. Pharmaceutical Sciences 66:1-19, 1977 and in Pharmaceutical Salts: Properties, Selection, and Use, (Eds. P.H. Stahl and C.G. Wermuth), Wiley-VCH, 2008. The salts can be prepared in situ during the final isolation and purification of the compounds described herein or separately by reacting a free base group with a suitable organic acid.
The compounds of the invention may have ionizable groups so as to be capable of preparation as pharmaceutically acceptable salts. These salts may be acid addition salts involving inorganic or organic acids or the salts may, in the case of acidic forms of the compounds of the invention be prepared from inorganic or organic bases. Frequently, the compounds are prepared or used as pharmaceutically acceptable salts prepared as addition products of pharmaceutically acceptable acids or bases. Suitable pharmaceutically acceptable acids and bases and methods for preparation of the appropriate salts are well-known in the art. Salts may be prepared from pharmaceutically acceptable non-toxic acids and bases including inorganic and organic acids and bases.
The term “pure” means substantially pure or free of unwanted components (e.g., other compounds and/or other components of a cell lysate), material defilement, admixture or imperfection.
A variety of clinical indicators can be used to identify a patient as “at risk” of developing a particular neurological disease. Examples of patients (e.g., human patients) that are “at risk” of developing a neurological disease, such as amyotrophic lateral sclerosis, frontotemporal degeneration, Alzheimer’s disease, Parkinson’s disease, dementia with Lewy Bodies, corticobasal degeneration, progressive supranuclear palsy, dementia parkinsonism ALS complex of Guam, Huntington’s disease, Inclusion body myopathy with early-onset Paget disease and frontotemporal dementia (IBMPFD), sporadic inclusion body myositis, myofibrillar myopathy, dementia pugilistica, chronic traumatic encephalopathy, Alexander disease, and hereditary inclusion body myopathy, include (i) subjects exhibiting or prone to exhibit aggregation of TAR-DNA binding protein (TDP)-43, and (ii) subjects expressing a mutant form of TDP-43 containing a mutation associated with TDP-43 aggregation and toxicity, such as a mutation selected from Q331K, M337V, Q343R, N345K, R361S, and N390D. Subjects that are “at risk" of developing amyotrophic lateral sclerosis may exhibit one or both of these characteristics, for example, prior to the first administration of a CYP51A.1 inhibitor in accordance with the compositions and methods described herein.
As used herein, the terms “TAR-DNA binding protein-43” and “TDP-43” are used interchangeably and refer to the transcription repressor protein involved in modulating HIV-1 transcription and alternative splicing of the cystic fibrosis transmembrane conductance regulator (CFTR) pre-mRNA transcript, for example, in human subjects. The terms “TAR-DNA binding protein-43” and “TDP-43” refer not only to wildtype forms of TDP-43, but also to variants of wild-type TDP-43 proteins and nucleic acids encoding the same. The amino acid sequence and corresponding mRNA sequence of a wild-type form of human TDP-43 are provided herein as SEQ ID NOs: 3 and 4, which correspond to NCBI Reference Sequence NOs. NMJD07375.3 and NP_Q31401.1 , respectively. These sequences are shown in Table 3, below.
Table 3. Amino acid and nucleic acid sequences of wild-type human TDP-43
Figure imgf000041_0001
The terms “TAR-DNA binding protein-43” and “TDP-43” as used herein include, for example, forms of the human TDP-43 protein that have an amino acid sequence that is at least 85% identical io the amino acid sequence of SEQ ID NO: 3 (e.g„ 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99%, 99.9%, or 100% identical to the amino acid sequence of SEQ ID NO: 3) and/or forms of the human TDP-43 protein that contain one or more substitutions, insertions, and/or deletions (e.g., one or more conservative and/or nonconservative amino acid substitutions, such as up to 5, 10, 15, 20, 25, or more, conservative or nonconservative amino acid substitutions) relative to a wild-type TDP-43 protein. For instance, patients that may be treated for a neurological disorder as described herein, such as amyotrophic lateral sclerosis, frontotemporal degeneration, Alzheimer’s disease, Parkinson’s disease, dementia with Lewy Bodies, corticobasal degeneration, progressive supranuclear palsy, dementia parkinsonism ALS complex of Guam, Huntington’s disease, inclusion body myopathy with early-onset Paget disease and frontotemporal dementia (IBMPFD), sporadic inclusion body myositis, myofibrillar myopathy, dementia pugilistica, chronic traumatic encephalopathy, Alexander disease, and hereditary inclusion body myopathy, include human patients that express a form of TDP-43 having a mutation associated with elevated TDP-43 aggregation and toxicity, such as a mutation selected from Q331 K, M337V, Q343R, N345K, R361 S, and N39QD. Similarly, the terms “TAR-DNA binding protein-43” and “TDP-43” as used herein include, for example, forms of the human TDP-43 gene that encode an mRNA transcript having a nucleic acid sequence that is at least 85% identical to the nucleic acid sequence of SEQ ID NO: 4 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical to the amino acid sequence of SEQ ID NO: 4).
As used herein, the term “subject” refers to any organism to which a composition in accordance with the invention may be administered, e.g., for experimental, diagnostic, prophylactic, and/or therapeutic purposes. Typical subjects include any animal (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans). A subject may seek or be in need of treatment, require treatment, be receiving treatment, be receiving treatment in the future, or be a human or animal who is under care by a trained professional for a particular disease or condition.
As used herein, the terms "treat," "treated," or "treating" mean both therapeutic treatment and prophylactic or preventative measures wherein the object is to prevent or slow down (lessen) an undesired physiological condition, disorder, or disease, or obtain beneficial or desired clinical results. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; dimmishment of the extent of a condition, disorder, or disease; stabilized (i.e., not worsening) state of condition, disorder, or disease; delay in onset or slowing of condition, disorder, or disease progression: amelioration of the condition, disorder, or disease state or remission (whether partial or total), whether detectable or undetectable; an amelioration of at least one measurable physical parameter, not necessarily discernible by the patient; or enhancement or improvement of condition, disorder, or disease. Treatment includes eliciting a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival as compared to expected survival if not receiving treatment.
A “therapeutic regimen” refers to a dosing regimen whose administration across a relevant population is correlated with a desired or beneficial therapeutic outcome.
The term “therapeutically effective amount” means an amount that is sufficient, when administered to a population suffering from or susceptible to a disease, disorder, and/or condition in accordance with a therapeutic dosing regimen, to treat the disease, disorder, and/or condition. In some embodiments, a therapeutically effective amount is one that reduces the incidence and/or severity of, and/or delays onset of, one or more symptoms of the disease, disorder, and/or condition. Those of ordinary skill in the art will appreciate that the term “therapeutically effective amount” does not in fact require successful treatment be achieved in a particular individual. Rather, a therapeutically effective amount may be that amount that provides a particular desired pharmacological response in a significant number of subjects when administered to patients in need of such treatment. It is specifically understood that particular subjects may, in fact, be “refractory” to a “therapeutically effective amount.” To give but one example, a refractory subject may have a low bioavailability such that clinical efficacy is not obtainable. In some embodiments, reference to a therapeutically effective amount may be a reference to an amount as measured in one or more specific tissues (e.g., a tissue affected by the disease, disorder or condition) or fluids (e.g., blood, saliva, serum, sweat, tears, urine, etc). Those of ordinary skill in the art will appreciate that, in some embodiments, a therapeutically effective amount may be formulated and/or administered in a single dose. In some embodiments, a therapeutically effective amount may be formulated and/or administered in a plurality of doses, for example, as part of a dosing regimen.
Brief Description of the Drawings
FiGS. 1 A - 1 C demonstrate that the viability of a yeast TDP-43 model is restored by the Erg11 inhibitor, fluconazole. (FIG. 1A) Structure of the Erg11 inhibitor and anti-fungal, fluconazole. (FIG. 1 B) Fluconazole rescues viability of TDP-43-expressing yeast using a resazurin-reduction endpoint. A 2-fold serial dilution of fluconazole was applied to TDP-43-expressing yeast for 24 hours prior to analysis. (FIG. 1C) Wild-type yeast cultures were treated with fluconazole for eight hours prior to HPLC analysis for lanosterol and ergosterol. Data are expressed as the area under the curve (AUG) normalized to cell mass based on optical density of cultures at 600 nm. Fluconazole treatment reduces ergosterol, while simultaneously leading to an increase in the Erg11 substrate, lanosterol.
F!G. 2 shows the structures of compounds used in primary rat cortical neuron TDP-43 wild type and Q331 K mutant survival studies.
FiGS. 3A and 3B demonstrate that compound A promotes survival in primary rat cortical neurons transfected with wild-type TDP-43. Rat primary cortical neurons were co-transfected with a red fluorescent protein (RFP) as a morphological marker and either control (empty vector) or wild-type TDP-43 expression plasmids and treated with vehicle (DMSO) or a titration of compound A. (FIG. 3A) Risk of neuron death plots. The lifetime of each neuron was determined by either loss of RFP signal or morphological indicators of death such as loss of neurites and cell blobbing and used to generate cumulative hazard plots of risk of death over time (hrs) post-transfection. (FIG. 3B) Forest plots. Hazard ratios for each treatment group (relative to TDP- 43 DMSO group) were determined by cox regression analysis and used to generate forest plots. Hazard ratios (HR) < 1 in which the confidence interval (Ci) does not encompass 1 represent treatments that significantly reduce probability of neuron death relative to the TDP-43 DMSO control. P, p-value.
FiGS. 4A and 4B demonstrate that compound A promotes survival in primary rat cortical neurons transfected with Q331 K Mutant TDP-43. Rat primary cortical neurons were co-transfected with a red fluorescent protein (RFP) as a morphological marker and either control (empty vector) or Q331 K mutant TDP- 43 expression plasmids and treated with vehicle (DMSO) or a titration of compound A. (FIG. 4A) Risk of neuron death plots. The lifetime of each neuron was determined by either loss of RFP signal or morphological indicators of death such as loss of neurites and cell blebbing and used to generate cumulative hazard plots of risk of death over time (hrs) post-transfection. (FIG. 4B) Forest plots. Hazard ratios for each treatment group (relative to TDP-43 DMSO group) were determined by cox regression analysis and used to generate forest plots. Hazard ratios (HR) < 1 in which the confidence interval (Cl) does not encompass 1 represent treatments that significantly reduce probability of neuron death relative to the TDP-43 DMSO control. P, p-value.
FIGS. SA and SB demonstrate that compound B promotes survival in primary rat cortical neurons transfected with wild-type TDP-43. Rat primary cortical neurons were co-transfected with a red fluorescent protein (RFP) as a morphological marker and either control (empty vector) or wild type TDP-43 expression plasmids and treated with vehicle (DMSO) or a titration of compound B. (FIG. 5A) Risk of neuron death plots. The lifetime of each neuron was determined by either loss of RFP signal or morphological indicators of death such as loss of neurites and cell biebbing and used to generate cumulative hazard plots of risk of death over time (hrs) post-transfection. (FIG. 5B) Forest plots. Hazard ratios for each treatment group (relative to TDP- 43 DMSO group) were determined by cox regression analysis and used to generate forest plots. Hazard ratios (HR) < 1 in which the confidence interval (Cl) does not encompass 1 represent treatments that significantly reduce probability of neuron death relative to the TDP-43 DMSO control. P, p-value.
Detailed Description
The present invention features compositions and methods for treating neurological disorders, such as amyotrophic lateral sclerosis and other neuromuscular disorders, as well as frontotemporal degeneration, Alzheimer’s disease, Parkinson’s disease, dementia with Lewy Bodies, corticobasal degeneration, progressive supranuclear palsy, dementia parkinsonism ALS complex of Guam, Huntington’s disease, Inclusion body myopathy with early-onset Paget disease and frontotemporal dementia (IBMPFD), sporadic inclusion body myositis, myofibrillar myopathy, dementia pugilistica, chronic traumatic encephalopathy, Alexander disease, and hereditary inclusion body myopathy among others. Particularly, the invention provides inhibitors of cytochrome P450 isoform 51A1 (CYP51A1), also referred to herein as lanosterol 14- alpha demethylase, that may be administered to a patient (e.g., a human patient) so as to treat or prevent a neurological disorder, such as one or more of the foregoing conditions. In the context of therapeutic treatment, the CYP51 A1 inhibitor may be administered to the patient to alleviate one or more symptoms of the disorder and/or to remedy an underlying molecular pathology associated with the disease, such as to suppress or prevent aggregation of TAR-DNA binding protein (TDP)-43.
The disclosure herein is based, in part, on the discovery that CYP51A1 inhibition modulates TDP-43 aggregation in vivo. Suppression of TDP-43 aggregation exerts beneficial effects in patients suffering from a neurological disorder. Many pathological conditions have been correlated with TDP-43-promoted aggregation and toxicity, such as amyotrophic lateral sclerosis, frontotemporal degeneration, Alzheimer’s disease, Parkinson’s disease, dementia with Lewy Bodies, corticobasal degeneration, progressive supranuclear palsy, dementia parkinsonism ALS complex of Guam, Huntington's disease, IBMPFD, sporadic inclusion body myositis, myofibrillar myopathy, dementia pugilistica, chronic traumatic encephalopathy, Alexander disease, and hereditary inclusion body myopathy. Without being limited by mechanism, by administering an inhibitor of CYP51A1, patients suffering from diseases associated with TDP­43 aggregation and toxicity may be treated, for example, due to the suppression of TDP­43 aggregation induced by the CYP51A1 inhibitor. Patients that are likely to respond to CYP51A1 inhibition as described herein include those that have or are at risk of developing TDP­43 aggregation, such as those that express a mutant form of TDP­43 associated with TDP­43 aggregation and toxicity in vivo. Examples of such mutations in TDP­43 that have been correlated with elevated TDP­43 aggregation and toxicity include Q331K, M337V, Q343R, N345K, R361S, and N390D, among others. The compositions and methods described herein thus provide the additional clinical benefit of enabling the identification of patients that are likely to respond to CYP51A1 inhibitor therapy, as well as processes for treating these patients accordingly. The sections that follow provide a description of exemplary CYP51A1 inhibitors that may be used in conjunction with the compositions and methods disclosed herein. The sections below additionally provide a description of various exemplary routes of administration and pharmaceutical compositions that may be used for delivery of these substances for the treatment of a neurological disorder. CYP51A1 Inhibitors Exemplary CYP51A1 inhibitors described herein include compounds, or a pharmaceutically acceptable salts thereof, having the structure:
Figure imgf000045_0001
Formula I wherein m is 0, 1, 2, 3, or 4; X1 is CH, S, or N; X2 and X3 are, independently, N, CH, or CR1; X4 is NH or S; each R1 is, independently, halo, optionally substituted C1­C6 alkyl, or optionally substituted C1­C6 alkoxy; Ar is optionally substituted C6­C10 aryl or optionally substituted C2­C9 heteroaryl; L1 is ­CONR­ or­NRCO­; R is hydrogen or optionally substituted C1­C6 alkyl; L2 is optionally substituted C1­C6 alkylene or optionally substituted C3­C8 cycloalkylene; RA is ­CH2CONHR2, ­CONHR2, or ­COR2; R2 is optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cyclohexyl, or has the structure:
Figure imgf000046_0001
Formula II Formula III wherein R3 is optionally substituted C1­C3 alkyl; R4 is optionally substituted C2­C6 alkyl, optionally substituted C2­C6 heteroalkyl, optionally substituted C3­C8 cycloalkyl, optionally substituted C2­C9 heterocyclyl, or optionally substituted C6­C10 aryl C1­C6 alkyl; n is 0 or 1; o is 0 or 1 or 2; p is 0, 1, 2, 3, 4, 5, 6, 7, or 8; X5 is NR5, CR5R6, O, or SR5R6; R5 is absent, halo, oxo, hydrogen, hydroxy, optionally substituted amino, cyano, optionally substituted C1­C6 alkyl, optionally substituted C1­C6 alkoxy, ­SO2­optionally substituted C1­C6 alkyl, optionally substituted C2­C9 heterocyclyl, or optionally substituted C2­C9 heteroaryl; and R6 is absent, halo, oxo, hydroxy, optionally substituted amino, cyano, optionally substituted C1­C6 alkyl, optionally substituted C1­C6 alkoxy, ­SO2­ optionally substituted C1­C6 alkyl, optionally substituted C2­C9 heterocyclyl, or optionally substituted C2­C9 heteroaryl; or R5 and R6 combine with the atoms to which they are attached to form an optionally substituted C2­C9 heterocyclyl, or optionally substituted C2­C9 heteroaryl; and each R7 is, independently, halo, optionally substituted C1­C6 heteroalkyl, or optionally substituted C1­C6 alkyl, and/or two R7, together with the atoms to which they are attached, combine to form an optionally substituted C2­C9 heterocyclyl; or R5 is absent, halo, oxo, hydrogen, hydroxy, optionally substituted amino, cyano, optionally substituted C1­C6 alkyl, optionally substituted C1­C6 alkoxy, ­SO2­optionally substituted C1­C6 alkyl, optionally substituted C2­C9 heterocyclyl, or optionally substituted C2­C9 heteroaryl; R6 and one R7, together with the atoms to which they are attached, combine to form an optionally substituted C2­C9 heterocyclyl or optionally substituted C2­C9 heteroaryl; and the remaining R7 groups, if present, are independently halo, optionally substituted C1­C6 heteroalkyl, or optionally substituted C1­C6 alkyl; or R6 is absent, halo, oxo, hydroxy, optionally substituted amino, cyano, optionally substituted C1­C6 alkyl, optionally substituted C1­C6 alkoxy, ­SO2­optionally substituted C1­C6 alkyl, optionally substituted C2­C9 heterocyclyl, or optionally substituted C2­C9 heteroaryl; R5 and one R7, together with the atoms to which they are attached, combine to form an optionally substituted C2­C9 heterocyclyl or optionally substituted C2­C9 heteroaryl; and the remaining R7 groups, if present, are independently halo, optionally substituted C1­C6 heteroalkyl, or optionally substituted C1­C6 alkyl; wherein if X5 is S, then each of R5 and R6 is, independently, absent or oxo; wherein if Ar is optionally substituted C6­C10 aryl, one of n and o is 1 and the other of n and o is 0 or 1, then X5 is NR5, wherein R5 is hydrogen, hydroxy, optionally substituted amino, cyano, optionally substituted C1­C6 alkoxy, ­SO2­optionally substituted C1­C6 alkyl, optionally substituted C2­C9 heterocyclyl, or optionally substituted C2­C9 heteroaryl; each R7 is, independently, halo, optionally substituted C1­C6 heteroalkyl, or optionally substituted C1­C6 alkyl, and/or two R7, together with the atoms to which they are attached, combine to form an optionally substituted C2­C9 heterocyclyl; or R5 and one R7, together with the atoms to which they are attached, combine to form an optionally substituted C2­C9 heterocyclyl or optionally substituted C2­C9 heteroaryl; X5 is CR5R6, wherein R5 is halo, hydrogen, hydroxy, optionally substituted amino, cyano, optionally substituted C1­C6 alkyl, optionally substituted C1­C6 alkoxy, ­SO2­optionally substituted C1­C6 alkyl, optionally substituted C2­C9 heterocyclyl, or optionally substituted C2­C9 heteroaryl, and R6 is halo, optionally substituted amino, cyano, optionally substituted C1­C6 alkyl, optionally substituted C1­C6 alkoxy, ­SO2­optionally substituted C1­C6 alkyl, optionally substituted C2­C9 heterocyclyl, or optionally substituted C2­C9 heteroaryl, and each R7 is, independently, halo, optionally substituted C1­C6 heteroalkyl, or optionally substituted C1­C6 alkyl, and/or two R7, together with the atoms to which they are attached, combine to form an optionally substituted C2­C9 heterocyclyl; or R5 is halo, hydrogen, hydroxy, optionally substituted amino, cyano, optionally substituted C1­C6 alkyl, optionally substituted C1­C6 alkoxy, ­SO2­optionally substituted C1­C6 alkyl, optionally substituted C2­C9 heterocyclyl, or optionally substituted C2­C9 heteroaryl; R6 and one R7, together with the atoms to which they are attached, combine to form an optionally substituted C2­C9 heterocyclyl or optionally substituted C2­C9 heteroaryl; and the remaining R7 groups, if present, are independently halo, optionally substituted C1­C6 heteroalkyl, or optionally substituted C1­C6 alkyl; or R6 is halo, hydroxy, optionally substituted amino, cyano, optionally substituted C1­C6 alkyl, optionally substituted C1­C6 alkoxy, ­SO2­ optionally substituted C1­C6 alkyl, optionally substituted C2­C9 heterocyclyl, or optionally substituted C2­C9 heteroaryl; R5 and one R7, together with the atoms to which they are attached, combine to form an optionally substituted C2­C9 heterocyclyl or optionally substituted C2­C9 heteroaryl; and the remaining R7 groups, if present, are independently halo, optionally substituted C1­C6 alkyl, or optionally substituted C1­C6 alkyl; X is O, wherein p is 1, 2, 3, 4, 5, 6, 7, or 8; or X is SR5R6, wherein each of R5 and R6 is oxo. Additional exemplary CYP51A1 inhibitors described herein include compounds, or a pharmaceutically acceptable salts thereof, having the structure:
Figure imgf000047_0001
Formula 2 wherein m is 0, 1, 2, 3, or 4; X6 is CH, or N; X7 is NH or S; Ar1 is optionally substituted C6­C10 aryl; R23 is hydrogen, halo, or optionally substituted C1­C6 alkoxy; L3 is ­NR24CO­ or ­CONR24­, R24 is H or optionally substituted C1­C6 alkyl; RB is ­CH2CONHR25, or ­COR25 or NR25; R25 is optionally substituted heteroaryl, optionally substituted cyclohexyl, or has the structure
Figure imgf000048_0001
Formula II Formula III wherein R26 is optionally substituted C1­C3 alkyl; R27 is optionally substituted C1­C6 alkyl, or optionally substituted C1­C6 heteroalkyl; n is 0 or 1; o is 0 or 1 or 2; X8 is NR28, CR28R29, or O; R28 is absent, hydrogen, hydroxy, optionally substituted amino, optionally substituted C1­C6 alkyl, OR optionally substituted C1­C6 alkoxy; R29 is absent, hydrogen, hydroxy, optionally substituted amino, optionally substituted C1­C6 alkyl, OR optionally substituted C1­C6 alkoxy; each R25 is, independently, halo or optionally substituted C1­C6 alkyl. CYP51A1 inhibitors described herein include compounds having the structure:
Figure imgf000048_0002
and pharmaceutically acceptable salts thereof. CYP51A1 inhibitors described herein include compounds having the structure:
Figure imgf000048_0003
and pharmaceutically acceptable salts thereof. CYP51A1 inhibitors described herein include compounds having the structure:
Figure imgf000048_0004
Formula 1c wherein each R1A is independently H or R1, and pharmaceutically acceptable salts thereof. CYP51A1 inhibitors described herein include compounds having the structure:
Figure imgf000049_0001
and pharmaceutically acceptable salts thereof. CYP51A1 inhibitors described herein include compounds having the structure:
Figure imgf000049_0002
R12 is optionally substituted 2­oxa­6­azaspiro[3.3]heptan­6­yl, and pharmaceutically acceptable salts thereof. CYP51A1 inhibitors described herein include compounds having the structure:
Figure imgf000049_0003
Formula 1e wherein R13 is optionally substituted pyridin­4­yl or optionally substituted phenyl; and R14 is optionally substituted piperidin­4­yl, optionally substituted 2­oxa­6­azaspiro[3.3]heptan­6­yl, 2­ azaspiro[3.3]heptan­2­yl substituted with hydroxy, and pharmaceutically acceptable salts thereof. CYP51A1 inhibitors described herein include compounds having the structure:
Figure imgf000049_0004
wherein R15 is optionally substituted 4­azaspiro[2.4]heptan­4­yl, and pharmaceutically acceptable salts thereof. CYP51A1 inhibitors described herein include compounds having the structure:
Figure imgf000050_0001
Formula 1g wherein R16 is optionally substituted 2­oxa­5­azabicyclo[2.2.1]heptan­5­yl, or optionally substituted 3­ azabicyclo[3.1.0]hexan­3­yl, and pharmaceutically acceptable salts thereof. CYP51A1 inhibitors described herein include compounds having the structure: ,
Figure imgf000050_0002
wherein R17 and R18 are each, independently, H or F; and R19 is 2­oxa­5­azabicyclo[2.2.1]heptan­5­yl, or 3­azabicyclo[3.1.0]hexan­3­yl, and pharmaceutically acceptable salts thereof. CYP51A1 inhibitors described herein include compounds having the structure:
Figure imgf000050_0003
Formula 1i wherein R20 is optionally substituted 2­oxa­6­azaspiro[3.3]heptan­6­yl, and pharmaceutically acceptable salts thereof. CYP51A1 inhibitors described herein include compounds having the structure:
Figure imgf000050_0004
Formula 1j wherein R21 is optionally substituted pyridinyl; and R22 is piperidin­1­yl optionally substituted with methoxy; azetidin­1­yl optionally substituted with methyl, methoxy, or fluoro; 2­oxa­5­azabicyclo[2.2.1]heptan­5­yl; or optionally substituted morpholin­4­yl, and pharmaceutically acceptable salts thereof. CYP51A1 inhibitors described herein include compounds having the structure:
Figure imgf000051_0001
Formula 2 wherein m is 0, 1, 2, 3, or 4; X6 is CH, or N; X7 is NH or S; Ar1 is optionally substituted C6­C10 aryl; R23 is hydrogen, halo, or optionally substituted C1­C6 alkoxy; L3 is ­NR24CO­ or ­CONR24­, R24 is H or optionally substituted C1­C6 alkyl; RB is ­CH2CONHR25, or ­COR25 or NR25; R25 is optionally substituted heteroaryl, optionally substituted cyclohexyl, or has the structure
Figure imgf000051_0002
Formula II Formula III wherein R26 is optionally substituted C1­C3 alkyl; R27 is optionally substituted C1­C6 alkyl, or optionally substituted C1­C6 heteroalkyl; n is 0 or 1; o is 0 or 1 or 2; X8 is NR28, CR28R29, or O; R28 is absent, hydrogen, hydroxy, optionally substituted amino, optionally substituted C1­C6 alkyl, OR optionally substituted C1­C6 alkoxy; R29 is absent, hydrogen, hydroxy, optionally substituted amino, optionally substituted C1­C6 alkyl, OR optionally substituted C1­C6 alkoxy; each R25 is, independently, halo or optionally substituted C1­C6 alkyl, and pharmaceutically acceptable salts thereof. CYP51A1 inhibitors described herein include compounds having the structure:
Figure imgf000051_0003
Formula 2a and pharmaceutically acceptable salts thereof. CYP51A1 inhibitors described herein include compounds having the structure:
Figure imgf000052_0001
Formula 2b and pharmaceutically acceptable salts thereof. CYP51A1 inhibitors described herein include compounds having the structure:
Figure imgf000052_0002
Formula 2c and pharmaceutically acceptable salts thereof. CYP51A1 inhibitors described herein include compounds having the structure:
Figure imgf000052_0003
Formula 2d and pharmaceutically acceptable salts thereof. CYP51A1 inhibitors described herein include compounds having the structure:
Figure imgf000052_0004
Formula 2e and pharmaceutically acceptable salts thereof. CYP51A1 inhibitors described herein include compounds having the structure:
Figure imgf000052_0005
Formula 3 and pharmaceutically acceptable salts thereof, Wherein n is 0, 1, 2, 3, or 4; L4 is optionally substituted C1­C6 alkylene or optionally substituted C3­C8 cycloalkylene; X9 is N and X10 is CH, or X9 is CH and X10 is N; X11 is N or CH; each R30 is, independently, halo, optionally substituted C1­C6 alkyl, or optionally substituted C1­C6 alkoxy; ArC is optionally disubstituted C6­C10 aryl, or C6­C10 aryl optionally monosubstituted with chloro, optionally substituted C1­C6 heteroalkyl, cyano, meta­fluoro, or ortho­fluoro; RC is COR31, or ­R31; R31 is optionally substituted C2­C5 heteroaryl, or has the structure:
Figure imgf000053_0001
Formula II Formula III wherein R32 is optionally substituted C1­C3 alkyl; R33 is optionally substituted C1­C6 alkyl C6 aryl, optionally substituted C1­C6 alkyl C2­C5 heteroaryl, optionally substituted C5 cycloalkyl, optionally substituted C3­C6 alkyl, or optionally substituted C3 heteroalkyl. n is 0 or 1; o is 0 or 1 or 2; p is 0, 1, 2, 3, 4, 5, 6, 7, or 8; X12 is NR34, CR34R35, O, or SR34R35; R34 is absent, halo, oxo, hydrogen, hydroxy, optionally substituted amino, cyano, optionally substituted C1­C6 alkyl, optionally substituted C1­C6 heteroalkyl, ­SO2­optionally substituted C1­C6 alkyl, optionally substituted C2­C9 heterocyclyl, optionally substituted C6­C10 aryl, or optionally substituted C2­C9 heteroaryl, or R33 and R34 combine with the atoms to which they are attached to form an optionally substituted C2­C9 heteroaryl; R35 is absent, halo, oxo, hydroxy, optionally substituted amino, cyano, optionally substituted C1­C6 alkyl, optionally substituted C1­C6 heteroalkyl, ­SO2­optionally substituted C1­C6 alkyl, optionally substituted C2­C9 heterocyclyl, optionally substituted C6­C10 aryl or optionally substituted C2­C9 heteroaryl, or R33 and R35 combine with the atoms to which they are attached to form an optionally substituted C2­C9 heteroaryl; and each R33 is, independently, halo, oxo, or optionally substituted C1­C6 alkyl, or two R33 combine with the atoms to which they are attached to form an optionally substituted C4 cycloalkyl. CYP51A1 inhibitors described herein include compounds having the structure:
Figure imgf000053_0002
Formula 3a and pharmaceutically acceptable salts thereof. CYP51A1 inhibitors described herein include compounds having the structure:
Figure imgf000054_0001
Formula 3b and pharmaceutically acceptable salts thereof. CYP51A1 inhibitors described herein include compounds having the structure:
Figure imgf000054_0002
Formula 3c Wherein R37 is cyano and R38 is fluoro, or R37 is fluoro and R38 is cyano; and R39 is or 3,3­difluoro­azetidin­1­yl, and pharmaceutically acceptable salts thereof. CYP51A1 inhibitors described herein include compounds having the structure:
Figure imgf000054_0003
wherein l is 0 or 1; L4 is is optionally substituted C1­C6 alkylene or optionally substituted C3­C8 cycloalkylene; and R40 is 4­hydroxy­piperidin­1­yl, 3­methoxy­piperidin­1­yl, optionally substituted diazapen­1­yl, triazolopiperazinyl substituted with methyl, 4,4­difluoro­piperidin­1­yl, 1,1­dioxothiomorpholin­4­yl, 2­ (methoxymethyl)­pyrroloin­1­yl, tetrahydro­1,3­oxazin­3­yl, 4­isopropyl­piperazin­1­yl, 4­(2­oxazolidin­3­yl)­ piperidin­1­yl, optionally substituted 1,2,4 oxadizol­5­yl, or optionally substituted 1,3,4 oxadizol­2­yl. and pharmaceutically acceptable salts thereof. CYP51A1 inhibitors described herein include compounds having the structure:
Figure imgf000054_0004
and pharmaceutically acceptable salts thereof. CYP51A1 inhibitors described herein include compounds having the structure:
Figure imgf000055_0001
Formula 3f wherein R43 is F or CN; and R42 is optionally substituted azetidin­1­yl, and pharmaceutically acceptable salts thereof. CYP51A1 inhibitors described herein include compounds having the structure:
Figure imgf000055_0002
Formula 3g wherein R44 is optionally substituted azetidin­1­yl, and pharmaceutically acceptable salts thereof. CYP51A1 inhibitors described herein include compounds having the structure:
Figure imgf000055_0003
Formula 4 wherein n is 0, 1, 2, 3, or 4; R45 is halo; and R46 is optionally substituted azetidinyl, and pharmaceutically acceptable salts thereof. CYP51A1 inhibitors described herein include compounds having the structure:
Figure imgf000055_0004
Formula 4a and pharmaceutically acceptable salts thereof. CYP51A1 inhibitors described herein include compounds having the structure:
Figure imgf000056_0001
Formula 5 and pharmaceutically acceptable salts thereof, wherein p is 0 or 1; X13 is a single bond or O; R48 is optionally substituted C6­C10 aryl, optionally substituted C2­C5 heteroaryl, or trifluoromethyl; R49 is H or optionally substituted C1­C6 alkyl; R50 is optionally substituted C6­C10 aryl or optionally substituted C2­C5 hetetoaryl; and R51 is optionally substituted C2­C5 heterocyclyl. Additional exemplary CYP51A1 inhibitors described herein include compounds, or pharmaceutically acceptable salts thereof, having the structure:
Figure imgf000056_0002
Formula 1k and pharmaceutically acceptable salts thereof, wherein
Figure imgf000056_0003
CYP51A1 inhibitors described herein include compounds having the structure:
Figure imgf000056_0004
Formula 1l and pharmaceutically acceptable salts thereof, wherein
Figure imgf000057_0001
CYP51A1 inhibitors described herein include compounds having the structure:
Figure imgf000057_0002
Formula 1m and pharmaceutically acceptable salts thereof,
Figure imgf000057_0003
CYP51A1 inhibitors described herein include compounds having the structure:
Figure imgf000057_0004
Formula 2f and pharmaceutically acceptable salts thereof, wherein R57 is halo. CYP51A1 inhibitors described herein include compounds having the structure:
Figure imgf000057_0005
Formula 3h and pharmaceutically acceptable salts thereof, wherein R57 and R58 are each halo. Exemplary CYP51A1 inhibitors described herein include any one of the compounds in Table 1, or pharmaceutically acceptable salts thereof. Table 1. Compounds of the Invention
Figure imgf000058_0001
Figure imgf000059_0001
Figure imgf000060_0001
Figure imgf000061_0001
Figure imgf000062_0001
Figure imgf000063_0001
Figure imgf000064_0001
Figure imgf000065_0001
Figure imgf000066_0001
Figure imgf000067_0001
Figure imgf000068_0001
Figure imgf000069_0001
Figure imgf000070_0001
Figure imgf000071_0001
Figure imgf000072_0001
Figure imgf000073_0001
Figure imgf000074_0001
Figure imgf000075_0001
Figure imgf000076_0001
Figure imgf000077_0001
Figure imgf000078_0001
Figure imgf000079_0001
Figure imgf000080_0001
Figure imgf000081_0001
Figure imgf000082_0001
Figure imgf000083_0001
Figure imgf000084_0001
Figure imgf000085_0001
Figure imgf000086_0001
Figure imgf000087_0001
Figure imgf000088_0001
Figure imgf000089_0001
Figure imgf000090_0001
Figure imgf000091_0001
Figure imgf000092_0001
In some embodiments, the compound has the structure of any one of compounds 1­453 in Table 1. Other embodiments, as well as exemplary methods for the synthesis or production of these compounds, are described herein. Methods of Treatment
Suppression of CYP51A1 Activity and TDP-43 Aggregation to Treat Neurological Disorders
Using the compositions and methods described herein, a patient suffering from a neurological disorder may be administered a CYP51A1 inhibitor, such as a small molecule, antibody, antigen-binding fragment thereof, or interfering RNA molecule described herein, so as to treat the disorder and/or to suppress one or more symptoms associated with the disorder. Exemplary neurological disorders that may be treated using the compositions and methods described herein are, without limitation, amyotrophic lateral sclerosis, frontotemporal degeneration, Alzheimer’s disease, Parkinson's disease, dementia with Lewy Bodies, corticobasai degeneration, progressive supranuclear palsy, dementia parkinsonism ALS complex of Guam, Huntington’s disease, IBMPFD, sporadic inclusion body myositis, myofibrillar myopathy, dementia pugilistica, chronic traumatic encephalopathy, Alexander disease, and hereditary inclusion body myopathy, as well as neuromuscular diseases such as congenital myasthenic syndrome, congenital myopathy, cramp fasciculation syndrome, Duchenne muscular dystrophy, glycogen storage disease type II, hereditary spastic paraplegia, inclusion body myositis, Isaac's Syndrome, Kearns-Sayre syndrome, Lambert-Eaton myasthenic syndrome, mitochondrial myopathy, muscular dystrophy, myasthenia gravis, myotonic dystrophy, peripheral neuropathy, spinal and bulbar muscular atrophy, spinal muscular atrophy, Stiff person syndrome, Troyer syndrome, and Guillain-Barre syndrome.
The present disclosure is based, in part, on the discovery that CYP51A1 inhibitors, such as the agents described herein, are capable of attenuating TDP-43 aggregation in vivo. TDP-43-promoted aggregation and toxicity have been associated with various neurological diseases. The discovery that CYP51A1 inhibitors modulate TDP-43 aggregation provides an important therapeutic benefit. Using a CYP51A1 inhibitor, such as a CYP51A1 inhibitor described herein, a patient suffering from a neurological disorder or at risk of developing such a condition may be treated in a manner that remedies an underlying molecular etiology of the disease. Without being limited by mechanism, the compositions and methods described herein can be used to treat or prevent such neurological conditions, for example, by suppressing the TDP-43 aggregation that promotes pathology.
Additionally, the compositions and methods described herein provide the beneficial feature of enabling the identification and treatment of patients that are likely to respond to CYP51A1 inhibitor therapy. For example, in some embodiments, a patient (e.g., a human patient suffering from or at risk of developing a neurological disease described herein, such as amyotrophic lateral sclerosis) is administered a CYP51A1 inhibitor if the patient is identified as likely to respond to this form of treatment. Patients may be identified as such on the basis, for example, of susceptibility to TDP-43 aggregation. In some embodiments, the patient is identified is likely to respond to CYP51A1 inhibitor treatment based on the isoform of TDP-43 expressed by the patient. For example, patients expressing TDP-43 isoforms having a mutation selected from Q331 K, M337V, Q343R, N345K, R361S, and N390D, among others, are more likely to develop TDP-43-promoted aggregation and toxicity relative to patients that do not express such isoforms of TDP-43. Using the compositions and methods described herein, a patient may be identified as likely to respond to CYP51A1 inhibitor therapy on the basis of expressing such an isoform of TDP-43, and may subsequently be administered a CYP51 A1 inhibitor so as to treat or prevent one or more neurological disorders, such as one or more of the neurological disorders described herein.
Assessing Patient Response
A variety of methods known in the art and described herein can be used to determine whether a patient having a neurological disorder (e.g,, a patient at risk of developing TDP-43 aggregation, such as a patient expressing a mutant form of TDP-43 having a mutation associated with elevated TDP-43 aggregation and toxicity, for example, a mutation selected from Q331 K, M337V, Q343R, N345K, R361 S, and N390D) is responding favorably to CYP51 A1 inhibition. For example, successful treatment of a patient having a neurological disease, such as amyotrophic lateral sclerosis, with a CYP51A1 inhibitor described herein may be signaled by:
(i) an improvement in condition as assessed using the amyotrophic lateral sclerosis functional rating scale (ALSFRS) or the revised ALSFRS (ALSFRS-R), such as an improvement in the patient’s ALSFRS or ALSFRS-R score within one or more days, weeks, or months following administration of the CYP51A1 inhibitor (e.g,, an improvement in the patient’s ALSFRS or ALSFRS-R score within from about 1 day to about 48 weeks (e.g., within from about 2 days to about 36 weeks, from about 4 weeks to about 24 weeks, from about 8 weeks to about 20 weeks, or from about 12 weeks to about 16 weeks), or more, following the initial administration of the CYP51A1 inhibitor to the patient, such as within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 weeks, or more, following the initial administration of the CYP51A1 inhibitor to the patient);
(ii) an increase in slow vital capacity, such as an increase in the patient’s slow vital capacity within one or more days, weeks, or months following administration of the CYP51A1 inhibitor (e.g., an increase in the patient's slow vital capacity within from about 1 day to about 48 weeks (e.g., within from about 2 days to about 36 weeks, from about 4 weeks to about 24 weeks, from about 8 weeks to about 20 weeks, or from about 12 weeks to about 16 weeks), or more, following the initial administration of the CYP51A1 inhibitor to the patient, such as within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 weeks, or more, following the initial administration of the CYP51A1 inhibitor to the patient);
(iii) a reduction in decremental responses exhibited by the patient upon repetitive nerve stimulation, such as a reduction that is observed within one or more days, weeks, or months following administration of the CYP51 A1 inhibitor (e.g., a reduction that is observed within from about 1 day to about 48 weeks (e.g., within from about 2 days to about 36 weeks, from about 4 weeks to about 24 weeks, from about 8 weeks to about 20 weeks, or from about 12 weeks to about 16 weeks), or more, foilowing the initial administration of the CYP51A1 inhibitor to the patient, such as within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 weeks, or more, following the initial administration of the CYP51A1 inhibitor to the patient);
(iv) an improvement in muscle strength, as assessed, for example, by way of the Medical Research
Council muscle testing scale (as described, e.g., in Jagtap et al., Ann. Indian. Acad. Neurol. 17:336-339 (2014), the disclosure of which is incorporated herein by reference as it pertains to measuring patient response to neurological disease treatment), such as an improvement that is observed within one or more days, weeks, or months following administration of the CYP51A1 inhibitor (e.g., an improvement that is observed within from about 1 day to about 48 weeks (e.g., within from about 2 days to about 36 weeks, from about 4 weeks to about 24 weeks, from about 8 weeks to about 20 weeks, or from about 12 weeks to about 16 weeks), or more, following the initial administration of the CYP51A1 inhibitor to the patient, such as within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 weeks, or more, following the initial administration of the CYP51A1 inhibitor to the patient);
(v) an improvement in quality of life, as assessed, for example, using the amyotrophic lateral sclerosis-specific quality of life (ALS-specific QOL) questionnaire, such as an improvement in the patient’s quality of life that is observed within one or more days, weeks, or months following administration of the CYP51 A1 inhibitor (e.g., an improvement in the subject’s quality of life that is observed within from about 1 day to about 48 weeks (e.g., within from about 2 days to about 36 weeks, from about 4 weeks to about 24 weeks, from about 8 weeks to about 20 weeks, or from about 12 weeks to about 16 weeks), or more, following the initial administration of the CYP51A1 inhibitor to the patient, such as within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 weeks, or more, following the initial administration of the CYP51A1 inhibitor to the patient);
(vi) a decrease in the frequency and/or severity of muscle cramps, such as a decrease in cramp frequency and/or severity within one or more days, weeks, or months following administration of the CYP51 A1 inhibitor (e.g., a decrease in cramp frequency and/or severity within from about 1 day to about 48 weeks (e.g., within from about 2 days to about 36 weeks, from about 4 weeks to about 24 weeks, from about 8 weeks to about 20 weeks, or from about 12 weeks to about 16 weeks), or more, following the initial administration of the CYP51A1 inhibitor to the patient, such as within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 weeks, or more, following the initial administration of the CYP51A1 inhibitor to the patient); and/or (vii) a decrease in TDP­43 aggregation, such as a decrease in TDP­43 aggregation within one or more days, weeks, or months following administration of the CYP51A1 inhibitor (e.g., a decrease in TDP­43 aggregation within from about 1 day to about 48 weeks (e.g., within from about 2 days to about 36 weeks, from about 4 weeks to about 24 weeks, from about 8 weeks to about 20 weeks, or from about 12 weeks to about 16 weeks), or more, following the initial administration of the CYP51A1 inhibitor to the patient, such as within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 weeks, or more, following the initial administration of the CYP51A1 inhibitor to the patient. Combination Formulations and Uses Thereof The compounds of the invention can be combined with one or more therapeutic agents. In particular, the therapeutic agent can be one that treats or prophylactically treats any neurological disorder described herein. Combination Therapies A compound of the invention can be used alone or in combination with other agents that treat neurological disorders or symptoms associated therewith, or in combination with other types of treatment to treat, prevent, and/or reduce the risk of any neurological disorders. In combination treatments, the dosages of one or more of the therapeutic compounds may be reduced from standard dosages when administered alone. For example, doses may be determined empirically from drug combinations and permutations or may be deduced by isobolographic analysis (e.g., Black et al., Neurology 65:S3­S6, 2005). In this case, dosages of the compounds when combined should provide a therapeutic effect. Pharmaceutical Compositions The compounds of the invention are preferably formulated into pharmaceutical compositions for administration to human subjects in a biologically compatible form suitable for administration in vivo. Accordingly, in another aspect, the present invention provides a pharmaceutical composition comprising a compound of the invention in admixture with a suitable diluent, carrier, or excipient.
The compounds of the invention may be used in the form of the free base, in the form of salts, solvates, and as prodrugs. All forms are within the scope of the invention. In accordance with the methods of the invention, the described compounds or salts, solvates, or prodrugs thereof may be administered to a patient in a variety of forms depending on the selected route of administration, as will be understood by those skilled in the art. The compounds of the invention may be administered, for example, by oral, parenteral, buccal, sublingual, nasal, rectal, patch, pump, or transdermal administration and the pharmaceutical compositions formulated accordingly. Parenteral administration includes intravenous, intraperitoneal, subcutaneous, intramuscular, transepithelial, nasal, intrapulmonary, intrathecal, rectal, and topical modes of administration. Parenteral administration may be by continuous infusion over a selected period of time.
A. compound of the invention may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsules, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic administration, a compound of the invention may be incorporated with an excipient and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, and wafers.
A compound of the invention may also be administered parenterally. Solutions of a compound of the invention can be prepared in water suitably mixed with a surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, DMSO and mixtures thereof with or without alcohol, and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms. Conventional procedures and ingredients for the selection and preparation of suitable formulations are described, for example, in Remington's Pharmaceutical Sciences (2003, 20th ed.) and in The United States Pharmacopeia: The National Formulary (USP 24 NF19), published in 1999.
The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that may be easily administered via syringe.
Compositions for nasal administration may conveniently be formulated as aerosols, drops, gels, and powders. Aerosol formulations typically include a solution or fine suspension of the active substance in a physiologically acceptable aqueous or non-aqueous solvent and are usually presented in single or multidose quantities in sterile form in a sealed container, which can take the form of a cartridge or refill for use with an atomizing device. Alternatively, the sealed container may be a unitary dispensing device, such as a single dose nasal inhaler or an aerosol dispenser fitted with a metering valve which is intended for disposal after use. Where the dosage form comprises an aerosol dispenser, it will contain a propellant, which can be a compressed gas, such as compressed air or an organic propellant, such as fluorochlorohydrocarbon. The aerosol dosage forms can also take the form of a pump-atomizer.
Compositions suitable for buccal or sublingual administration include tablets, lozenges, and pastilles, where the active ingredient is formulated with a carrier, such as sugar, acacia, tragacanth, gelatin, and glycerine. Compositions for rectal administration are conveniently in the form of suppositories containing a conventional suppository base, such as cocoa butter. The compounds of the invention may be administered to an animal, e.g., a human, alone or in combination with pharmaceutically acceptable carriers, as noted herein, the proportion of which is determined by the solubility and chemical nature of the compound, chosen route of administration, and standard pharmaceutical practice. Dosages The dosage of the compounds of the invention, and/or compositions comprising a compound of the invention, can vary depending on many factors, such as the pharmacodynamic properties of the compound; the mode of administration; the age, health, and weight of the recipient; the nature and extent of the symptoms; the frequency of the treatment, and the type of concurrent treatment, if any; and the clearance rate of the compound in the animal to be treated. One of skill in the art can determine the appropriate dosage based on the above factors. The compounds of the invention may be administered initially in a suitable dosage that may be adjusted as required, depending on the clinical response. In general, satisfactory results may be obtained when the compounds of the invention are administered to a human at a daily dosage of, for example, between 0.05 mg and 3000 mg (measured as the solid form). Dose ranges include, for example, between 10­1000 mg. Alternatively, the dosage amount can be calculated using the body weight of the patient. For example, the dose of a compound, or pharmaceutical composition thereof, administered to a patient may range from 0.1­50 mg/kg. EXAMPLES Abbreviations: HATU (2­(1H­benzotriazol­1­yl)­1,1,3,3­tetramethyluronium hexafluorophosphate AIBN Azobisisobutyronitrile DCM Dichloromethane DIPEA N,N­Diisopropylethylamine DMAP 4­Dimethylaminopyridine DMB (group) 2,4­Dimethoxybenzyl DMF N,N­Dimethylformamide DMP Dess­Martin Periodinane DMSO Dimethylsulfoxide DPPA diphenylphosphoryl azide EDCI 1­Ethyl­3­(3­dimethylaminopropyl)carbodiimide HOBt Hydroxybenzotriazole IBX 2­Iodoxybenzoic acid MeCN/ACN Acetonitrile NMM N­Methylmorpholine PyAOP ((7­Azabenzotriazol­1­yloxy)tripyrrolidinophosphonium hexafluorophosphate) Rt Retention time Rt/RT Room temperature SGC Silica gel chromatography SPhos 2­Dicyclohexylphosphino­^ƍ^^ƍ­dimethoxybiphenyl TBAB tetrabutylammonium bromide TFA Trifluoroacetic acid XPhos [2­Dicyclohexylphosphino­^ƍ^^ƍ^^ƍ­triisopropylbiphenyl] Example 1. General Schemes General Scheme 1
Figure imgf000099_0001
An appropriately substituted nitroarene I is condensed with diethyl oxalate II under basic conditions (e.g., potassium ethoxide) to afford appropriately substituted nitroarene III. Nitroarene III is reacted with iron powder under the presence of aqueous ammonium chloride to afford appropriately substituted ester IV. Ester IV is reduced under strongly basic conditions (e.g., potassium hydroxide) to appropriately substituted carboxylic acid V. Carboxylic acid V is reacted with appropriately substituted amine VI under a variety of coupling conditions (e.g., HATU) to afford desired amide VII.
Figure imgf000100_0001
The acid­labile (e.g., tert­butyloxycarbonyl) or base­labile (e.g., fluorenylmethyloxycarbonyl) protecting group of appropriately substituted amine I is removed under acidic or basic conditions to yield deprotected amine II. Amine II is reacted with appropriately substituted carboxylic acid III under a variety of coupling conditions (e.g., HATU) to afford appropriately substituted amide IV. The acid­ or base­labile protecting group of amine IV is removed under acid or basic conditions to yield deprotected amine V. Amine V is reacted with appropriately substituted carboxylic acid VI under a variety of coupling conditions (e.g., HATU) to yield desired amide VII. General Scheme 3
Figure imgf000100_0002
Appropriately substituted alkyl halide I is reacted with tert­butyl 2­(diphenylmethyleneamino)acetate II in the presence of catalytic tetrabutylammonium bromide to afford appropriately substituted desired compound III, which is subsequently reduced under strongly acidic conditions (e.g., hydrochloric acid) to afford appropriately substituted amino acid IV. Amino acid IV is coupled appropriately substituted N­ hydroxysuccinimide ester V under basic conditions (e.g., triethylamine) to afford appropriately substituted carboxylic acid VI. Carboxylic acid VI is coupled with appropriately substituted amine VII under a variety of coupling conditions (e.g., HATU) to afford amide VIII. The enantiomers of VIII are separated via chiral­HPLC to afford desired compounds IX and X.
Figure imgf000101_0001
An appropriately substituted amino acid I is reacted with (Z)­N'­hydroxypropionimidamide II under a variety of coupling conditions (e.g., EDCI) to afford appropriately substituted oxime ester III. Oxime ester III is intramolecularly cyclized under basic conditions (e.g., potassium hydroxide) to afford appropriately substituted oxadiazole IV, which is subsequently deprotected under acidic conditions (e.g., trifluoroacetic acid) to form appropriately substituted amine V. Amine V is reacted with appropriately substituted carboxylic acid VI under a variety of coupling conditions (e.g., HATU) to form appropriately substituted amide VII. The enantiomers of VII are separated via chiral­HPLC to afford desired compounds VIII and IX.
General Scheme 5
Figure imgf000102_0001
An appropriately substituted 2­amino­aryl iodide I is coupled with 2­oxopropanoic acid II in the presence of a palladium catalyst (e.g., palladium (II) acetate) to afford appropriately substituted carboxylic acid III. Carboxylic acid III is coupled with N­hydroxysuccinimide IV under a variety of coupling conditions (e.g., hydroxybenzotriazole) to afford appropriately substituted N­hydroxysuccinimide ester V. N­ hydroxysuccinimide ester V is coupled with appropriately substituted amino acid VI to afford appropriately substituted carboxylic acid VII. Carboxylic acid VII is coupled with appropriately substituted amine VIII to afford desired amide IX. The enantiomers of IX are separated via chiral­HPLC to afford desired compounds X and XI.
Synthesis of (S)-N-(3-(2-chlorophenyl)-1-(4-hydroxypiperidin-1-yl)-1-oxopropan-2-yl)-5-methyl-1H- pyrrolo[3,2-b]pyridine-2-carboxamide (Compound 1):
Figure imgf000103_0001
Figure imgf000103_0002
Step 1: ethyl 3-(6-methyl-3-nitropyridin-2-yl)-2-oxopropanoate. A solution of diethyl oxalate (7.3 g, 50.0 mmol) in diethyl ether (50 mL) was added to a solution of potassium ethanolate (4.2 g, 50.0 mmol) at 20 ^ in diethyl ether (300 mL) and ethanol (30 mL). The mixture was stirred for 0.5 hour at 20 ^. Then a solution of 2,6­dimethyl­3­nitropyridine (7.6 g, 5.0 mmol) in diethyl ether (50 mL) was slowly added to the mixture and it was stirred at 20^ for 4 hours. The mixture was poured into crushed ice, extracted with ethyl acetate (250 mL*3). The combined organic phase was concentrated. The crude product thus obtained was purified by silica gel column chromatography (petroleum ether/ethyl acetate = 1:2) to afford the target compound (2.9 g, 11.5 mmol, yield: 23%) as a red solid. LCMS (ESI) m/z: 253.1 [M+H]+. Step 2: ethyl 5-methyl-1H-pyrrolo[3,2-b]pyridine-2-carboxylate. To a mixture of ethyl 3­(6­methyl­3­nitropyridin­2­yl)­2­oxopropanoate (2.9 g, 11.5 mmol), ethanol (50 mL), aqueous saturated ammonium chloride solution (8 mL) in tetrahydrofuran (150 mL) was added iron powder (3.8 g, 69.9 mmol). The mixture was stirred at 90 ^ for 2 hours. It was filtered and the filtrate was concentrated. The residue was purified by silica gel column chromatography (20% methanol in dichloromethane) to afford 2.0 g of a red brown solid, which was further purified by silica gel column chromatography (petroleum ether/ethyl acetate = 1:1) to afford the target compound (800 mg, 3.9 mmol) as an off­white solid. LCMS (ESI) m/z: 205.2 [M+H]+. Step 3: 5-methyl-1H-pyrrolo[3,2-b]pyridine-2-carboxylic acid. A mixture of ethyl 5­methyl­1H­pyrrolo[3,2­b]pyridine­2­carboxylate (700 mg, 3.4 mmol), potassium hydroxide (952 mg, 17.0 mmol) in water (4 mL) and ethanol (20 mL) was stirred at 80 oC for 1 hour. Ethanol was removed under reduced pressure and the residue was acidified to pH ~1 with concentrated hydrochloric acid. The precipitate thus formed was collected by filtration and dried under vacuum to afford the target compound (550 mg, 3.1 mmol, yield: 91%) as a grey solid. 1H NMR (400 MHz, DMSO­d6) į 11.89 (s, 1H), 7.71 (d, J = 8.4 Hz, 1H), 7.13 (d, J = 8.4 Hz, 1H), 7.04 (s, 1H), 2.54 (s, 3H). Step 1a: (S)-tert-butyl 3-(2-chlorophenyl)-1-(4-hydroxypiperidin-1-yl)-1-oxopropan-2-ylcarbamate. A mixture of (S)­2­(tert­butoxycarbonylamino)­3­(2­chlorophenyl)propanoic acid (1.5 g, 5.0 mmol), piperidin­4­ol (505 mg, 5.0 mmol), HATU (2.85 g, 7.5 mmol), DIPEA (1.3 g, 10.0 mmol) in DMF (10 mL) was stirred at 25 ^ for 1 hour. The mixture was poured into brine (50 mL), extracted with ethyl acetate (100 mL*2). The combined organic phase was concentrated. The residue was purified by silica gel column chromatography (10% methanol in dichloromethane) to afford the target compound (2.5g) as a red­brown oil. LCMS (ESI) m/z:383.4/385.4 [M+H]+. Step 2a: (S)-2-amino-3-(2-chlorophenyl)-1-(4-hydroxypiperidin-1-yl)propan-1-one hydrochloride. A mixture of (S)­tert­butyl 3­(2­chlorophenyl)­1­(4­hydroxypiperidin­1­yl)­1­oxopropan­2­ylcarbamate (2.5g) and HCl (1,4­dioxane, 4 mol/L, 30 mL) was stirred at 25 ^ for 3 hours. The solvent was removed under reduced pressure, triturated with ethyl ether (30 mL) and dried under vacuum to afford the target compound (2.1g) as a red­brown oil.
Figure imgf000104_0001
Step 4: (S)-N-(3-(2-chlorophenyl)-1-(4-hydroxypiperidin-1-yl)-1-oxopropan-2-yl)-5-methyl-1H- pyrrolo[3,2-b]pyridine-2-carboxamide. A mixture of 5­methyl­1H­pyrrolo[3,2­b]pyridine­2­carboxylic acid (176 mg, 1.0mmol), (S)­2­amino­3­ (2­chlorophenyl)­1­(4­hydroxypiperidin­1­yl)propan­1­one hydrochloride (318 mg, 1.0 mmol), HATU (570 mg, 1.5 mmol) and DIPEA (387 mg, 3.0 mmol) in DMF (10 mL) was stirred at 25 ^ for 1 hour. The mixture was poured into water (50 mL) and extracted with ethyl acetate(100 mL*2). The combined organic phase was concentrated and purified by silica gel column chromatography (10% methanol in ethyl acetate) to afford 300 mg of a yellow oil, which was further purified by prep­HPLC (ammonium bicarbonate as buffer) to afford the target compound (90.0 mg, 0.21 mmol, yield:21%) as a white solid.1
Figure imgf000104_0002
NMR (400 MHz, DMSO­d6) į 11.59 (d, J = 4.4 Hz, 1H), 9.03­8.98 (m, 1H), 7.62 (d, J = 8.4 Hz, 1H), 7.44­7.38 (m, 2H), 7.29 (s, 1H), 7.24­7.19 (m, 2H), 7.05 (d, J = 8.4 Hz, 1H), 5.35­5.29 (m, 1H), 4.73­4.71 (m, 1H), 4.00­3.62 (m, 3H), 3.31­2.96 (m, 4H), 2.52 (s, 3H), 1.67­1.52 (m, 2H), 1.27­1.09 (m, 2H); 441.1/443.1 [M+H]+. The following compounds were synthesized according to the protocol described for Compound 1:
Figure imgf000105_0001
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Figure imgf000136_0001
Figure imgf000137_0002
Synthesis of N-[(2S)-3-(2,4-difluorophenyl)-1-{6-hydroxy-2-azaspiro[3.3]heptan-2-yl}-1-oxopropan-2-
Figure imgf000137_0001
Step 1: Preparation of 2-azaspiro[3.3]heptan-6-ol. To a solution of tert­butyl 6­hydroxy­2­azaspiro[3.3]heptane­2­carboxylate (150 mg, 0.70 mmol) in dichloromethane (5 mL) was added trifluoroacetic acid (2 mL). The reaction mixture was stirred at room temperature for 3 hours and concentrated under reduced pressure to give the crude product 2­ azaspiro[3.3]heptan­6­ol (110 mg, 0.97 mmol, 139 %) as a light yellow oil, which used for next step without further purification. Step 2: Preparation of tert-butyl (S)-(3-(2,4-difluorophenyl)-1-(6-hydroxy-2-azaspiro[3.3]heptan-2-yl)-1- oxopropan-2-yl)carbamate. A mixture of (S)­2­((tert­butoxycarbonyl)amino)­3­(2,4­difluorophenyl)propanoic acid (200 mg, 0.66 mmol), 2­azaspiro[3.3]heptan­6­ol (75.0 mg, 0.66 mmol), HATU (376 mg, 0.99 mmol) and DIPEA (170 mg, 1.3 mmol) in DMF (5 mL) was stirred at room temperature for 1 hour. The mixture was purified directly by reversed­phase chromatography (40% acetonitrile in water, 0.1% formic acid). The product tert­butyl (S)­(3­ (2,4­difluorophenyl)­1­(6­hydroxy­2­azaspiro[3.3]heptan­2­yl)­1­oxopropan­2­yl)carbamate (150 mg, 0.38 mmol, 57%) was obtained as a colorless oil. LCMS (ESI) m/z: 397.3 [M+H]+. Step 3: Preparation of (S)-2-amino-3-(2,4-difluorophenyl)-1-(6-hydroxy-2-azaspiro[3.3]heptan-2- yl)propan-1-one. To a solution of tert­butyl (S)­(3­(2,4­difluorophenyl)­1­(6­hydroxy­2­azaspiro[3.3]heptan­2­yl)­1­ oxopropan­2­yl)carbamate (150 mg, 0.38 mmol) in dichloromethane (5 mL) was added trifluoroacetic acid (2 mL). The reaction mixture was stirred at room temperature for 3 hours and concentrated under reduced pressure. The crude product was purified directly by reversed­phase chromatography (15% acetonitrile in water, 0.1% formic acid) to give (S)­2­amino­3­(2,4­difluorophenyl)­1­(6­hydroxy­2­azaspiro[3.3]heptan­2­ yl)propan­1­one (100 mg, 0.34 mmol, 89%). LCMS (ESI) m/z: 297.3 [M+H]+. Step 4: Preparation of (S)-N-(3-(2,4-difluorophenyl)-1-(6-hydroxy-2-azaspiro[3.3]heptan-2-yl)-1- oxopropan-2-yl)-5-methyl-1H-pyrrolo[2,3-b]pyridine-2-carboxamide. A mixture of 5­methyl­1H­pyrrolo[2,3­b]pyridine­2­carboxylic acid (60.0 mg, 0.34 mmol), (S)­2­amino­ 3­(2,4­difluorophenyl)­1­(6­hydroxy­2­azaspiro[3.3]heptan­2­yl)propan­1­one (100 mg, 0.34 mmol), HATU (194 mg, 0.51 mmol) and DIPEA (88 mg, 0.68 mmol) in tetrahydrofuran (5 mL) was stirred at 70°C for 1 hour. The mixture was poured into water (20 mL), extracted with ethyl acetate (2 x 20 mL). The organics were pooled, concentrated under reduced pressure, and purified by prep­HPLC (Boston C1821*250 mm 10 µm column. The mobile phase was acetonitrile/0.01% aqueous formic acid) to afford the product (S)­N­(3­(2,4­ difluorophenyl)­1­(6­hydroxy­2­azaspiro[3.3]heptan­2­yl)­1­oxopropan­2­yl)­5­methyl­1H­pyrrolo[2,3­ b]pyridine­2­carboxamide (56.3 mg, 0.12 mmol, 37%) as a white solid.1H NMR (400 MHz, Dimethylsulfoxide­ G^^^į^^1.90 (s, 1H), 8.79 (d, J = 8.1 Hz, 1H), 8.18 (d, J = 2.0 Hz, 1H), 7.86 (dd, J = 2.1, 0.9 Hz, 1H), 7.43 ­ 7.35 (m, 1H), 7.25 ± 7.15 (m, 1H), 7.11 (s, 1H), 7.00 (td, J = 8.3, 2.6 Hz, 1H), 5.03 (dd, J = 6.2, 2.1 Hz, 1H), 4.66 (q, J = 7.5 Hz, 1H), 4.18 (dd, J = 19.4, 8.7 Hz, 1H), 3.96 ± 3.70 (m, 4H), 3.07 (dd, J = 13.7, 6.3 Hz, 1H), 2.95 (dd, J = 13.6, 8.8 Hz, 1H), 2.37 (s, 3H), 2.34 ± 2.27 (m, 1H), 1.98 ± 1.82 (m, 2H), 0.94 (d, J = 6.5 Hz, 1H); LCMS (ESI) m/z: 455.3 [M+H]+. The following compounds were synthesized similar to the protocol described for the Compound 99:
Figure imgf000139_0001
Figure imgf000140_0001
Figure imgf000141_0001
Figure imgf000142_0001
Figure imgf000143_0001
Figure imgf000144_0001
Figure imgf000145_0001
Figure imgf000146_0001
Figure imgf000147_0001
Figure imgf000148_0001
Figure imgf000149_0001
Figure imgf000150_0001
Figure imgf000151_0001
Figure imgf000152_0001
Figure imgf000153_0001
Synthesis of enantiomer 1 (Compound 146) and enantiomer 2 (Compound 145) of 5-chloro-N-(1-(3,3- difluoropyrrolidin-1-yl)-3-(2-methylthiazol-5-yl)-1-oxopropan-2-yl)-1H-pyrrolo[2,3-b]pyridine-2- carboxamide.
Figure imgf000154_0001
Step 1: Preparation of (2-methylthiazol-5-yl)methanol. To a solution of lithium aluminium hydride (42.9 mL, 42.9 mmol) in tetrahydrofuran (70 mL), was slowly added a solution of ethyl 2­methylthiazole­5­carboxylate (4.9 g, 28.6 mmol) in tetrahydrofuran (10 mL) under argon at 0oC. And the mixture was stirred at 15oC for 17 hours. The reaction mixture was cooled in an ice bath and methanol was slowly added. Aqueous potassium sodium tartarate solution was then added and the resultant mixture was extracted with ethyl acetate (20 mL*3) and then dried over anhydrous sodium sulfate and concentrated under reduced pressure to give the crude product (2­methylthiazol­5­yl)methanol (3.2 g), which was used in the next step without further purification. LCMS (ESI) m/z: 130.1 [M+H]+. Step 2: Preparation of 5-(chloromethyl)-2-methylthiazole. To a solution of (2­methylthiazol­5­yl)methanol (3.2 g, 24.8 mmol) in dichloromethane (60 mL) at 0oC was added thionyl chloride (11.8 g, 99.2 mmol) and the resultant mixture was stirred at 15°C for 17 hours. The mixture was then filtered and the filtrate was concentrated under the reduced pressure. The resultant residue was purified by silica gel column chromatography (dichloromethane: methanol =70:30) to give the desired product 5­(chloromethyl)­2­methylthiazole (2.8 g, 19.0 mmol, yield: 77.8 %) as a yellow oil. LCMS (ESI) m/z: 148.1 [M+H]+. Step 3: Preparation of tert-butyl 2-(diphenylmethyleneamino)-3-(2-methylthiazol-5-yl)propanoate. To a solution of 5­(chloromethyl)­2­methylthiazole (2.8 g, 19.0 mmol) in dichloromethane (40 mL) was added tert­butyl 2­(diphenylmethyleneamino)acetate (11.2 g, 38.0 mmol) and TBAB (612 mg, 1.9 mmol), and the mixture was stirred at 0oC for 10min, then Potassium hydroxide (50%) (5.3 g, 95.0 mmol) was added to the reaction mixture was stirred at 15°C for 17h. The mixture was then concentrated and the residue was purified by flash column chromatography (Petroleum ether: Ethyl acetate =3:1) to give the desired product tert-butyl 2-(diphenylmethyleneamino)-3-(2-methylthiazol-5-yl)propanoate (3.5 g, 8.62 mmol, yield: 45.4 %) as yellow oil. LCMS (ESI) m/z: 407.1 [M+H]+.
The remainder of the steps were performed as described for the compounds 147 and 148. The two enantiomers were separated using the chiral HPLC [instrument: SFC-80 (Thar, Waters), Column: OJ 20*250mm, 10um (Daicel) , Mobile phase: CO2/ methanol (0.2%Methanol Ammonia)= 50/50, Flow rate: 80 g/min].
Enantiomer 1 (Compound 146) was obtained as white solid. 1H NMR (400 MHz, DMSO) 6 12.41 (s, 1H), 9.17 (d, J = 8.0 Hz, 1H), 8.33 (d, J = 2.3 Hz, 1 H), 8.28 (d, J = 2.3 Hz, 1 H), 7.47 (s, 1 H), 7.27 (s, 1 H), 4.83 (ddd, J = 49.0, 13.9, 8.5 Hz, 1H), 4.21 - 3.66 (m, 3H), 3.55 (t, J = 7.3 Hz, 1 H), 3.25 (ddd, J = 18.2, 14.3, 7.4 Hz, 2H), 2.54 (s, 3H), 2.36 (d, J = 25.3 Hz, 2H); LCMS (ESI) m/z: 454.1 [M+H]+; (Rt: 1.35min).
Enantiomer 2 (Compound 145) was obtained as white solid. 1H NMR (400 MHz, DMSO) 5 12.41 (s, 1 H), 9.17 (d, J = 7.9 Hz, 1 H), 8.33 (s, 1 H), 8.28 (s, 1 H), 7.47 (s, 1 H), 7.27 (s, 1 H), 5.03 - 4.67 (m, 1 H), 3.93 (ddd, J = 75.2, 39.6, 14.4 Hz, 3H), 3.55 (d, J = 7.2 Hz, 1 H), 3.25 (d, J = 11.5 Hz, 2H), 2.54 (s, 3H), 2.47 - 2.28 (m, 2H); LCMS (ESI) m/z: 454.1 [M+HJ+; (Rt: 3.02min).
Synthesis of enantiomer 1 (Compound 148) and enantiomer 2 (Compound 147) of 5-chioro-N-(3-(4- chioro-2-cyanophenyi)~1 -(6-methoxy~2-azaspiro[3.3]heptan~2-yi)~1 -oxopropan-2~yi)-1 H-pyrroto[2,3- b]pyndine~2-carboxamide.
Figure imgf000155_0001
Step 1 : Preparation of 2-(bromomethyi)-5~chiorobenzonitriie.
To a stirred solution of 4-chloro-2-isocyano-1-methylbenzene (1 g, 6.6 mmol) in perchloromethane (10 mL) was added N-bromosuccinimide (1.4 g, 7.9 mmol), 2,2'-Azobis(2-methylpropionitrile) (213 mg, 1.3 mmol) portion wise at 0 °C. The reaction mixture was stirred at 90 'C overnight. The mixture diluted with water and extracted with ethyl acetate (10 mL * 3). The combined organic phases were dried over anhydrous sodium sulphate, filtered and concentrated under reduced pressure to get 1.5 g crude, which was used directly in next step. Step 2: Preparation of tert-butyl 3-(4-chioro-2-cyanophenyi)-2~((diphenyimethylerie)amino)propanoate.
To a solution of 2-(bromomethyl)-5-chtorobenzonitrile (1.5 g, 6.55 mmol), and tetrabutylammonium bromide (150 mg) in dichloromethane (10 mL) were added potassium hydroxide (50%) (2.2 g, 39.3 mmol), tert-butyl 2-((diphenylmethylene)amino) acetate (1.93 g, 6.55 mmol) at -10 °C. The mixture was stirred at -10 - 25 °C for 18 h. The mixture was diluted with water (100 mL), extracted with ethyl acetate (200 mL*2), The organic phase was washed with brine (100 mL), dried, concentrated and purified by flash chromatography (Petroleum ether / Ethyl acetate = 12 : 1) to afford a white solid (1 .7 g, crude), which was used directly in next step. LCMS (ESI) m/z: 445.1 [M+H]+.
Step 3: Preparation of 2-amino-3-(4-chioro~2-cyanophenyi)propanoic acid.
A mixture of tert-butyl 3-(4-chloro-2-cyanophenyl)-2-((diphenylmethylene)amino)propanoate (1.7 g, 3.8 mmol) and cone, hydrochloric acid (4 mL) in dioxane (5 mL) was stirred at room temperature overnight. The solvent was evaporated under reduce pressure and the solid was filtered, washed with petroleum ether to get the title compound (580 mg, crude), which was used directly in next stop. LCMS (ESI) m/z: 225.1 [M+H]+.
Step 4: Preparation of 2-(5-chtoro-1 H~pyrroio[2,3-b]pyridine~2-carboxamido)-3~(4-chloro-2- cyanophenyi)propanoic acid.
To a solution of 2-amino-3-(4-chloro-2-cyanophenyl)propanoic acid (480 mg, 1.85 mmol) in acetonitrile (8 mL) and water (2 mL) were added triethylamine (747 mg, 7.4 mmol) and 2,5-dioxopyrrolidin-1- yl 5-chtoro-1H-pyrrolo[2,3-b]pyridine-2-carboxylate (597 mg, 2.0 mmol) at 0 °C. The mixture was stirred at 0 ~ 25 °C for 2h. The solvent was removed under reduced pressure and the residue was neutralized with hydrochloric acid (1 M) till PH<7, the solid was filtered and dried to get the title compound (800 mg, crude), which was used directly in next step. LCMS (ESI) m/z: 403.0 [M+H]+.
Step 5: Preparation of 5-chloro-N-(3-(4-chloro-2-cyanopheny!)-1-(6-methoxy-2-azaspiro[3.3]heptan-2- yi)~1 -oxopropan-2~yi)-1 H-pyrroio[2,3-b]pyridine-2 -carboxamide.
A mixture of 2-(5-chloro-1 H-pyrrolo[2,3-b]pyridine-2-carboxamido)-3-(4-chloro-2- cyanophenyl)propanoic acid (150 mg, 0.37 mmol), 6-methoxy-2-azaspiro[3.3jheptane (67 mg, 0.41 mmol), HATU (213 mg, 0.56 mmol) and DIPEA (96.3 mg, 0.75 mmol) in DMF (5 mL) was stirred at -20°C for 1 h. The mixture was purified by PREP-HPLC (SunFire C18, 4.6*50mm, 3. Sum column Xbridge C18 3.5pm 4.6x50mm column. The elution system used was a gradient of 5%-95% over 1 .5 min at 2ml/min and the solvent was acetonitrile/0.01% aqueous NH4HCO3.) to afford the title compound (100 mg, 0.195 mmol, 52.8 % yield) as white solid. It was separated by Chiral-HPLC using the conditions described above to obtain two isomers.
Enantiomer 1 : 'H NMR (500 MHz, DMSO) 6 11.90 (s, 1H), 9.06 (d, J = 8.3 Hz, 1 H), 8.29 (dd, J =
33.8, 2.3 Hz, 2H), 8.00 (d, J = 2.2 Hz, 1 H), 7.70 (dd, J = 8.4, 2.0 Hz, 1 H), 7.56 (dd, J = 8.5, 3.6 Hz, 1 H), 7.20 (s, 1 H), 4.89 - 4.76 (m, 1 H), 4.23 (dd, J = 20.7, 9.0 Hz, 1 H), 4.02 (dd, J = 33.1 , 9.0 Hz, 1 H), 3.93 - 3.64 (m, 3H), 3.39 - 3.12 (m, 5H), 2.43 - 2.29 (m, 2H), 2.06 - 1 .85 (m, 2H); LCMS (ESi) m/z: 512.1 [M+H]+;(Rt: 1.936mm, ee: 100%).
Enantiomer- 2: 1H NMR (500 MHz, DMSO) 5 12.13 (s, 1H), 9.06 (d, J = 8.3 Hz, 1 H), 8.29 (dd, J = 33.4, 2.3 Hz, 2H), 8.02 (t, J = 17.9 Hz, 1 H), 7.70 (dd, J = 8.4, 1.9 Hz, 1 H), 7.56 (dd, J = 8.5, 3.6 Hz, 1 H), 7.20 (s, 1 H), 4.88 - 4.72 (m, 1 H), 4.22 (dd, J = 20.7, 9.0 Hz, 1 H), 4.02 (dd, J = 33.0, 8.9 Hz, 1H), 3.93 - 3.61 (m, 3H), 3.40 - 3.29 (m, 2H), 3.08 (d, J = 1 .4 Hz, 3H), 2.46 - 2.23 (m, 2H), 2.11 - 1 .79 (m, 2H); LCMS (ESI) m/z: 512.1 [M+HJ+; (Rt: 2.864min, ee: 100%).
The following compounds were synthesized according to the protocol described for the Compounds 147 and 148:
Figure imgf000157_0001
Figure imgf000158_0001
Figure imgf000159_0001
Figure imgf000160_0001
Figure imgf000161_0001
Figure imgf000162_0001
Figure imgf000163_0001
Figure imgf000164_0001
Figure imgf000165_0001
Figure imgf000166_0001
Figure imgf000167_0001
Figure imgf000168_0001
Figure imgf000169_0001
Figure imgf000170_0001
Figure imgf000171_0001
Example: Synthesis of enantiomer 1 (Compound 196) and enantiomer 2 (compound 197) of 5-chtoro~ N-[3-(4-cyanophenyl)~1 -(3,3~difluoroazetidin-1 -yi)-1 -oxopropan-2-yl]~1 H-pyrrolo[2,3~b]pyndine~2- carboxamide.
Figure imgf000172_0001
Step 1 : Preparation of 5-chloro-1H-pyrro!o[2,3-b]pyridme-2-carboxy!ic acid.
A mixture of 5-chtoro-3-iodopyridin-2-amme (12.0 g, 47 mmoi), 2-oxopropanoic acid (12.5 g, 140 mmoi), palladium (II) acetate (0.538 g, 2.4 mmol), bicyclo[2.2,2]octane (15.9 g, 140 mmol) in DMF (100 mL) was stirred at 110°C under nitrogen atmosphere for 16 hours. The mixture was poured into water (400 mL) and a precipitate was formed. The precipitate was collected by filtration and dried under vacuum to afford 5- chloro-1 H-pyrrolo[2,3-b]pyridine-2-carboxylic acid as a grey solid, which was carried onto the next step without further purification. LCMS (ESI) m/z: 197.1 [M+Hp.
Step 2: Preparation of 2,5-daoxopyrrobdm-l-yl S-chSoro-IH-pyrrolo^^-bJpyndme^-carboxylate™
To a mixture of S-chloro-I H-pyrrolop^-bjpyrldine^-carboxylic acid (12.0 g, 61 mmol), EDCI (23.5 g, 120 mmol), HOBt (8.3 g, 61 mmol) and N-hydroxysuccinimide (14.0 g, 120 mmol) in DMF (180 mL) was added DMAP (373 mg, 3.06 mmol) and the reaction was stirred at room temperature for 3 hours. The resultant mixture was poured into water (300 mL) and the resuitant precipitate was collected by filtration which was dried to obtain crude 2,5-dioxopyrrolidin-l-yl S-chloro-I H-pyrrolop^-bjpyridine^-carboxylate (25.0 g, 85 mmol) as a brown solid, which was used in the next step without further purification. LCMS (ESI)
20 m/z: 294.1 [M+Hp. Step 3: Preparation of 2-(5-chtoro-1H-pyrroio[2,3-b]pyndine-2~carboxamido)~3-(4- cyanopheny!)propanoic acid.
To a solution of 2,5-dioxopyrroiidin-1 -yl 5-chloro-1 H-pyrrolo[2,3-b]pyndine-2-carboxylate (1.00 g, 3.4 mmol) and 2-amino-3-(4-cyanophenyi)propanoic add (0.300 g, 1.5 mmol) in acetonitrile (50 mL) and water (25 mL) was added triethylamine (0.758 g, 7.5 mmol) at 0°C. The reaction mixture was allowed to warm up to room temperature over 2 hours, then was adjusted to between pH 1-2 with concentrated hydrochloric acid. The resultant precipitate was collected by filtration which was purified by prep-HPLC (25% methanol in dichloromethane) to obtain 2-(5-chloro-1 H-pyrrolo[2,3-b]pyridine-2-carboxamido)-3-(4-cyanophenyl)propanoic acid (450 mg, 1 .2 mmol, 81 %) as a grey solid. LCMS (ESI) m/z: 369.0 [M+H]+.
Step 4: Synthesis of enantiomer 1 (Compound 196) and enantiomer 2 (compound 197) of 5-ch toro-N- [3-(4-cyanophenyi)-1 -(3,3~difiuoroazetidin-1 -yi)-1 -oxopropan~2-yi]~1 H-pyrroto[2,3~b]pyndine-2- carboxamide.
A mixture of 2-(5-chloro-1 H-pyrrolo[2,3-bjpyridine-2-carboxamido)-3-(4-cyanophenyl)propanoic acid (400 mg, 1.1 mmol), 3,3-difluoroazetidine hydrochloride (142 mg, 1.1 mmol), HATH (501 mg, 1.3 mmol), DIPEA (426 mg, 3.3 mmol) and DMF (10 mL) was stirred at room temperature for 1 hour. The mixture was then poured into water (100 mL) and the resultant precipitate was collected by filtration. The crude product thus obtained was purified by prep-HPLC to afford 5-chloro-N-(3-(4-cyanophenyl)-1-(3,3-difluoroazetidin-1- yl)-1-oxopropan-2-yl)-1 H-pyrrolo[2,3-b]pyridine-2-carboxamide (100 mg, 0.22 mmol, 20 %) as a white solid. This racemate was separated by chiral-HPLC to give two enantiomers:
Enantiomer 1 (Compound 196): ’H NMR (400 MHz, Dimethylsulfoxide-d6) 6 12.37 (s, 1 H), 9.11 (d, J = 8.0 Hz, 1 H), 8.32 (d, J = 2.0 Hz, 1 H), 8.26 (d, J = 2.4 Hz, 1H), 7.76 (d, J = 8.4 Hz, 2H), 7.56 (d, J = 8.4 Hz, 2H), 7.21 (d, J = 1.6 Hz, 1H), 4.75-4.59 (m, 3H), 4.38-4.27 (m, 2H), 3.21-3.10 (m, 2H); LCMS (ESI) m/z: 443.9 [M+H]*; (Rt: 1.16min).
Enantiomer 2 (Compound 197): 1H NMR (400 MHz, Dimethylsulfoxide-d6) 5 12.37 (s, 1 H), 9.11 (d, J = 7.6 Hz, 1 H), 8.32 (d, J = 2.0 Hz, 1 H), 8.26 (d, J = 2 4 Hz, 1 H), 7.76 (d, J = 8.0 Hz, 2H), 7.56 (d, J = 8.4 Hz, 2H), 7.21 (d, J = 2.0 Hz, 1 H), 4.75-4.59 (m, 3H), 4.41-4.23 (m, 2H), 3.21-3.10 (m, 2H); LCMS (ESI) m/z: 443.9 [M+HF; (Rt: 1.67mm).
The following compounds were synthesized according to the protocol described for the Compounds 196 and 197:
Figure imgf000174_0001
Figure imgf000175_0001
Figure imgf000176_0001
Figure imgf000177_0001
Figure imgf000178_0001
Figure imgf000179_0001
Figure imgf000180_0001
Figure imgf000181_0001
Figure imgf000182_0001
Figure imgf000183_0001
Figure imgf000184_0001
Figure imgf000185_0001
Figure imgf000186_0001
Figure imgf000187_0001
Figure imgf000188_0001
Figure imgf000189_0001
Figure imgf000190_0001
Figure imgf000191_0001
Figure imgf000192_0001
Enantiomer 1 (Compound 259) and enantiomer s (Compound 260) of 5-chtoro-N-(3-(2-cyanophenyl)~1-
(3,3-difluoroazetidin-1 -yl)-1 -oxopropan-2-yl)-1 H-pyrroto[2,3-b]pyndine-2 -carboxamide.
Figure imgf000193_0001
, ,
Enantiomer 2 Compound 280
Step 1 : 2-(bro:nomethyl)benzomtriie.
A. mixture of 2-methylbenzon Strife (4.5 g, 38.4 mmol), N-bromosuccinimide (8.2 g, 46.0 mmol), AIBN
(623 mg, 3.8 mmol) in acetonitrile (100 ml) was stirred at 85°C for 4 hours. The mixture was poured into water (200 mL) and extracted with ethyl acetate (200 mL*2). The combined organic phase was concentrated and the crude product was purified by silica gel column chromatography (petroleum ether/ethyl acetate = 9:1) to afford the target compound (8.5 g, crude) as a yellow oil, which was used in the next step without further purification. 1H NMR (500 MHz, DMSO-ds) 6 7.69-7.55 (m, 3H), 7.45-7.41 (m, 1H), 4.64 (s, 2H).
Step 2: tert-buty! 3-(2-cyanophenyi)~2-(diphenyimethyleneamino)propanoate.
To a solution of 2-(bromomethyl)benzonitrile (8.5 g, 47.7 mmol), tert-butyl 2-
(diphenylmethyleneamino)acetate (14.1 g, 47.7 mmol) and tetrabutylammonium bromide (966 mg) in dichloromethane (250 mL) was added potassium hydroxide (50% in water, 32.0 g, 286.2 mmol) at 0 °C and the resultant mixture was slowly brought to room temperature over a period of 4h with stirring. Then the mixture was diluted with water (300 mL) and extracted with dichloromethane (250 mL*2). The combined organic phase was concentrated and the crude product was purified by silica gel column chromatography
(petroleum ether/ethyl acetate=9:1 ) to afford the target compound (4.5 g, crude) as a yellow solid. LCMS
(ESI) m/z: 41 1.1 [M+Hp. Step 3: 2-amino-3-(2-cyanophenyl)propanoic acid hydrochloride. A solution of tert­butyl 3­(2­cyanophenyl)­2­(diphenylmethyleneamino)propanoate (4.5g) in 36% hydrochloric acid (20 mL) was stirred at 15 oC for 2 hours. The solution was concentrated and the resulting solid was washed with ethyl acetate (100 mL) to afford the target compound ( 2.8 g, benzophenone contained) as a grey solid, which was used in the next step without further purification. LCMS (ESI) m/z: 191.1 Step 4: 2-(tert-butoxycarbonylamino)-3-(2-cyanophenyl)propanoic acid. To a solution of 2­amino­3­(2­cyanophenyl)propanoic acid hydrochloride (2.5 g, crude), sodium hydroxide (1.2 g, 30.0 mmol), tetrahydrofuran (40 mL) and water (10 mL) at 0 oC was added slowly di­tert­ butyl dicarbonate (2.1 g, 30.0 mmol). After the addition, the mixture was stirred for another 2 hours and the resultant mixture was acidified to pH~1 with 36% hydrochloric acid and extracted with ethyl acetate (150 mL*3). The combined organic phase was concentrated and the crude product was purified by silica gel column chromatography (25% methanol in dichloromethane) to afford the target compound (950 mg, 3.2 mmol) as a grey oil. LCMS (ESI) m/z: 313.0 [M+Na]+. Step 5: Synthesis of enantiomer 1 and enantiomer 2 of tert-butyl 3-(2-cyanophenyl)-1-(3,3- difluoroazetidin-1-yl)-1-oxopropan-2-ylcarbamate. A mixture of 2­(tert­butoxycarbonylamino)­3­(2­cyanophenyl)propanoic acid (500 mg, 1.7 mmol), 3,3­ difluoroazetidine hydrochloride (220 mg, 1.7 mmol), HATU (950 mg, 2.5 mmol), DIPEA (658 mg, 5.1 mmol) and DMF (8 mL) was stirred at 15^ for 1 hour. The mixture was poured into water and extracted with ethyl acetate (80 mL*2). The combined organic phase was concentrated and the crude product was purified by silica gel column chromatography (30% ethyl acetate in petroleum ether) to afford 450 mg of a grey solid, which was further washed with methanol (15 mL) to afford the racemic compound (200 mg, 0.54 mmol) as a white solid. This racemic derivative was subjected to chiral prep­HPLC to afford enantiomer 1 (85 mg, 0.23 mmol, Rt: xxxmin) and enantiomer 2 (85 mg, 0.23 mmol, Rt: xxxmin) of tert­butyl 3­(2­cyanophenyl)­1­(3,3­ difluoroazetidin­1­yl)­1­oxopropan­2­ylcarbamate as white solids. LCMS (ESI) m/z: 310.2 [M+H­56]+. Step 6: 2-(2-amino-3-(3,3-difluoroazetidin-1-yl)-3-oxopropyl)benzonitrile (from enantiomer 2 of step-5). To a solution of enantiomer 2 (70 mg, 0.2 mmol) from step­5 in dichloromethane (4 mL) was added trifluoroacetic acid (1.5 mL) and the mixture was stirred at 15^ for 2 hours. The mixture was then concentrated to afford the target compound (95 mg, crude) as a colorless oil, which was used in the next step without further purification. LCMS (ESI) m/z: 266.3 [M+H]+. Step 7: 5-chloro-N-(3-(2-cyanophenyl)-1-(3,3-difluoroazetidin-1-yl)-1-oxopropan-2-yl)-1H-pyrrolo[2,3- b]pyridine-2-carboxamide (Compound 259, from enantiomer 2 of step-5). A mixture of 5­chloro­1H­pyrrolo[2,3­b]pyridine­2­carboxylic acid (80 mg, 0.4 mmol), step­6 product (80 mg, crude), HATU (182 mg, 0.48 mmol), DIPEA (155 mg, 1.2 mmol) and DMF (4 mL) was stirred at 15^ for 1 hour. The mixture was poured into water (50 mL) and the resultant precipitate was collected by filtration. The resulting solid was triturated with methanol (25 mL) to afford the desired compound (16.3 mg, 0.036 mmol) as a grey solid.1H NMR (400 MHz, DMSO­d6) į 12.39 (s, 1H), 9.18 (d, J = 7.6 Hz, 1H), 8.32 (d, J = 2.0 Hz, 1H), 8.27 (d, J = 2.0 Hz, 1H), 7.81 (d, J = 8.0 Hz, 1H), 7.62­7.59 (m, 2H), 7.44­7.41 (m, 1H), 7.21 (s, 1H), 4.90­4.84 (m, 1H), 4.75 (dd, J = 22.8, 12.0 Hz, 1H), 4.56 (dd, J = 23.6, 11.6 Hz, 1H), 4.40­4.29 (m, 2H), 3.39­3.22 (m, 2H); LCMS (ESI) m/z: 444.1/446.1 [M+H]+; (Rt: 1.65min). Compound 260 was synthesized similarly form enantiomer 1 from step-5 product. 1H NMR (500 MHz, DMSO­d6) į 12.39 (s, 1H), 9.19 (d, J = 7.5 Hz, 1H), 8.32 (d, J = 2.0 Hz, 1H), 8.26 (d, J = 2.0 Hz, 1H), 7.81 (d, J = 7.5 Hz, 1H), 7.63­7.59 (m, 2H), 7.42 (d, J = 7.5 Hz, 1H), 7.21 (s, 1H), 4.89­4.84 (m, 1H), 4.75 (dd, J = 23.0, 12.0 Hz, 1H), 4.56 (dd, J = 24.0, 12.0 Hz, 1H), 4.41­4.28 (m, 2H), 3.38­ 3.23 (m, 2H); LCMS (ESI) m/z: 443.9.445.9 [M+H]+; (Rt: 1.381min). Synthesis of enantiomer 1 (Compound 261) and enantiomer 2 (Compound 262) of 5-chloro-N-(3-(4- cyano-2-fluorophenyl)-1-oxo-1-(2-oxa-6-azaspiro[3.3]heptan-6-yl)propan-2-yl)-1H-pyrrolo[2,3- b]pyridine-2-carboxamide.
Figure imgf000195_0001
Step 1: Preparation of 2-(5-chloro-1H-pyrrolo[2,3-b]pyridine-2-carboxamido)-3-(4-cyano-2- fluorophenyl)propanoic acid. To a cold solution of (S)­2­amino­3­(4­cyano­2­fluorophenyl)propanoic acid hydrochloride (200 mg, 0.82 mmol) in acetonitrile (8 mL) and water (2 mL) was added triethylamine (331 mg, 3.28 mmol) and 2,5­ dioxopyrrolidin­1­yl 5­chloro­1H­pyrrolo[2,3­b]pyridine­2­carboxylate (240 mg, 0.82 mmol) and the resultant mixture was stirred at 0 ~ 25 oC for 2h. The mixture was concentrated and quenched with hydrochloric acid (1M) till PH<7, filtered the solid and dried under reduce pressure to get 2­(5­chloro­1H­pyrrolo[2,3­b]pyridine­ 2­carboxamido)­3­(4­cyano­2­fluorophenyl)propanoic acid (180 mg, 0.466 mmol, 57% yield) as yellow solid (crude product), which was used directly in next step. LCMS (ESI) m/z: 387.1 [M+H]+. Step 2: Preparation of enantiomer 1 and enantiomer 2 of 2-(5-chloro-1H-pyrrolo[2,3-b]pyridine-2- carboxamido)-3-(4-cyano-2-fluorophenyl)propanoic acid. An amount of 180mg of 2­(5­chloro­1H­pyrrolo[2,3­b]pyridine­2­carboxamido)­3­(4­cyano­2­ fluorophenyl)propanoic acid was separated by Chiral­HPLC to give two stereoisomers: enantiomer 1 (80 mg, 0.207 mmol, 44.4 % yield) and enantiomer 2 (70 mg, 0.181 mmol, 38.9 % yield). Step 3: Synthesis of Compound 261: (from enantiomer 1 of step-2) A mixture of enantiomer 1 (70 mg, 0.18 mmol) of 2­(5­chloro­1H­pyrrolo[2,3­b]pyridine­2­ carboxamido)­3­(4­cyano­2­fluorophenyl)propanoic acid from step­2, 2­oxa­6­azaspiro[3.3]heptane (17.9 mg, 0.18 mmol), PyAOP (141.7 mg, 0.27 mmol) and DIPEA (70.2 mg, 0.54 mmol) in DMF (2 mL) was stirred at 0oC for 1 h. The resultant mixture was subjected to prep­HPLC (BOSTON pHlex ODS 10um 21.2×250mm120A. The mobile phase was acetonitrile/0.1% Ammonium bicarbonate) to obtain the desired product as white solid (34 mg, 0.073 mmol, 40% yield). 1H NMR (400 MHz, DMSO­d6^^į^^2.33 (s, 1H), 8.99 (d, J=8.4 Hz, 1H), 8.32 (d, J=2.4 Hz, 1H), 8.25 (d, J=2.8 Hz, 1H), 7.82±7.79 (m, 1H), 7.62­7.54 (m, 2H), 7.18 (d, J=1.2 Hz, 1H), 4.79±4.77 (m, 1H), 4.65± 4.61 (m, 4H), 4.37 (d, J=9.2 Hz, 1H), 4.25 (d, J=9.2 Hz, 1H), 4.10­4.06 (m, 2H), 3.23±3.20 (m, 1H), 3.06­3.03 (m, 1H). LCMS (ESI) m/z: 468.1 [M+H]+. (Rt=1.93 min, ee%=96.62%). Step 4: Synthesis of Compound 262 (from enantiomer 2 of step-2). A mixture of enantiomer 2 of 2­(5­chloro­1H­pyrrolo[2,3­b]pyridine­2­carboxamido)­3­(4­cyano­2­ fluorophenyl)propanoic acid (60 mg, 0.16 mmol) from step­2, 2­oxa­6­azaspiro[3.3]heptane (15.4 mg, 0.16 mmol), PyAOP (121.5 mg, 0.23 mmol) and DIPEA (60.2 mg, 0.47 mmol) in DMF (2 mL) was stirred at 0oC for 1 h. The mixture was subjected to prep­HPLC (BOSTON pHlex ODS 10um 21.2×250mm120A. The mobile phase was acetonitrile/0.1% Ammonium bicarbonate) to get the desired product as white solid (53.1 mg, 0.114 mmol, 73.1% yield). 1H NMR (400 MHz, DMSO­d6^^į^^2.33 (s, 1H), 9.00 (d, J=8.4 Hz, 1H), 8.32 (d, J=2.4 Hz, 1H), 8.25 (d, J=2.0 Hz, 1H), 7.82±7.79 (m, 1H), 7.62­7.54 (m, 2H), 7.18 (s, 1H), 4.80±4.78 (m, 1H), 4.65±4.63 (m, 4H), 4.38 (d, J=9.6 Hz, 1H), 4.25 (d, J=9.2 Hz, 1H), 4.10­4.03 (m, 2H), 3.24±3.20 (m, 1H), 3.06­3.03 (m, 1H). LCMS (ESI) m/z: 468.1 [M+H]+; (Rt=1.28 min, ee%=94.66%). The following compounds were synthesized according to the protocol described above. The chiral separation was done after the first amide coupling and the 2nd amide coupling was done separately. The retention times assigned for the following compounds were based on the same HPLC conditions.
Figure imgf000197_0001
Figure imgf000198_0001
Synthesis of enantiomer 1 (Compound 265) and enantiomer 2 (Compound 266) of 5-chtoro-N-(3-(2- cyano-4-fiuorophenyl)-1 -(3,3-difiuoroazetidin~1 -y!)-1 -oxopropan-2~yi)-1 H-pyrroio[2,3-b]pyridine~2- carboxamide.
Figure imgf000199_0001
Enantiomer 1 Enantiomer 2
The compound 265 and 266 were synthesized according to the protocol described above. However, the enantiomers were separated at the last step.
Enantiomer 1 (Compound 265): 'H NMR (400 MHz, DMSO) 6 12.39 (s, 1 H), 9.18 (d, J = 8.0 Hz, 1 H), 8.33 (d, J = 2.2 Hz, 1H), 8.27 (d, J = 2.2 Hz, 1H), 7.90 - 7.74 (m, 1 H), 7.71 - 7.59 (m, 1 H), 7.53 (t, J = 8.6 Hz, 1 H), 7.21 (s, 1 H), 4.86 (d, J = 5.3 Hz, 1 H), 4.74 (d, J = 12.0 Hz, 1 H), 4.60 (d, J = 12.3 Hz, 1 H), 4.36 (dd, J = 22.5, 12.1 Hz, 2H), 3.29 - 3.09 (m, 2H); LCMS (ESI) m/z: 462.1 [M+H]+; (Rt: 0.91 min).
Enantiomer 2 (Compound 266): 1H NMR (400 MHz, DMSO) 5 12.40 (s, 1 H), 9.18 (d, J = 8.0 Hz, 1 H), 8.33 (d, J = 2.4 Hz, 1 H), 8.26 (d, J = 2.4 Hz, 1 H), 7.82 (dd, 8.6, 2.7 Hz, 1 H), 7.64 (dd, J = 8.7, 5.5 Hz, 1 H), 7.53 (td, J = 8.7, 2.8 Hz, 1 H), 7.21 (s, 1 H), 4.93 - 4.81 (m, 1 H), 4.73 (t, J = 11.8 Hz, 1 H), 4.60 (dd, J = 23.4, 11.8 Hz, 1 H), 4.36 (dd, J = 22.5, 12.0 Hz, 2H), 3.34 - 3.18 (m, 2H); LCMS (ESI) m/z: 462.1 [M+H]+; (Rt: 1.17min).
Synthesis of enantiomer 1 (Compound 267) and enantiomer 2 (Compound 268) of 5-chioro-N-(3-(2- chioro-4-cyanophenyl)-1 -oxo-1 -(2-oxa-6^azaspiro[3.3]hepten-6-y!)propan-2-yi)-1 H-pyrroio[2,3- b]pyndine~2-carboxamide.
Figure imgf000199_0002
Enantiomer 1 Enantiomer 2
The compounds 267 and 268 were synthesized starting from 4-(bromomethyi)-3-chlorobenzonitrile according to the protocol described for compounds 265 and 266. Compound 267 (enantiomer 1): 1H NMR (400 MHz, DMSO­d6) į 12.31 (s, 1H), 9.01 (d, J = 8.4 Hz, 1H), 8.32 (d, J = 2.4 Hz, 1H), 8.25 (d, J = 2.4 Hz, 1H), 8.04 (d, J = 1.2 Hz, 1H), 7.72 (dd, J = 8.0, 2.0 Hz, 1H), 7.59 (d, J = 8.0 Hz, 1H), 7.18 (d, J = 1.6 Hz, 1H), 4.89­4.83 (m, 1H), 4.66­4.62 (m, 4H), 4.40 (d, J = 9.2 Hz, 1H), 4.28 (d, J = 9.6 Hz, 1H), 4.08 (dd, J = 16.0, 14.0 Hz, 2H), 3.28 (dd, J = 14.0, 5.2 Hz, 1H), 3.12 (dd, J = 13.6, 10.4 Hz, 1H); LCMS (ESI) m/z: 484.1/486.0 [M+H]+; (Rt: 2.848min). Compound 268 (enantiomer 2): 1H NMR (400 MHz, DMSO­d6) į 12.32 (s, 1H), 9.01 (d, J = 8.4 Hz, 1H), 8.32 (d, J = 2.0 Hz, 1H), 8.25 (d, J = 2.4 Hz, 1H), 8.04 (d, J = 1.2 Hz, 1H), 7.72 (dd, J = 8.0, 1.6 Hz, 1H), 7.59 (d, J = 8.0 Hz, 1H), 7.18 (d, J = 1.6 Hz, 1H), 4.88­4.84 (m, 1H), 4.66­4.62 (m, 4H), 4.41 (d, J = 8.8 Hz, 1H), 4.28 (d, J = 9.2 Hz, 1H), 4.08 (dd, J = 16.0, 10.8 Hz, 2H), 3.28 (dd, J = 13.6, 4.8 Hz, 1H), 3.12 (dd, J = 13.2, 10.4 Hz, 1H); LCMS (ESI) m/z: 484.0/486.1 [M+H]+; (Rt: 4.116min). The following compounds were synthesized using 2­amino­3­(2­chloro­4­cyano­phenyl)propanoic acid described in the synthesis of compounds 265 and 266.
Figure imgf000200_0001
Figure imgf000201_0001
Figure imgf000202_0002
Synthesis of enantiomer 1 (Compound 275) and enantiomer 2 (Compound 276) of 5-chloro-N-(3-(5- chloropyridin-2-yl)-1-(3,3-difluoroazetidin-1-yl)-1-oxopropan-2-yl)-1H-pyrrolo[2,3-b]pyridine-2- carboxamide:
Figure imgf000202_0001
Step 1: Preparation of (5-chloropyridin-2-yl)methanol. To a solution of methyl 5­chloropicolinate (2 g, 11.7 mmol) in methanol (30 mL) was added Sodium borohydride (442 mg, 11.7 mmol), the mixture was stirred at 15oC for 17 h. The solvent was removed under the reduced pressure and to the residue was added water (10 mL). It was extracted with ethyl acetate (10 mL*2), the combined organic layer was washed with brine (10 mL), dried over sodium sulfate, filtered and concentrated. The residue was purified by silica gel column chromatography (dichloromethane: methol =20:1) to give the desired product (5­chloropyridin­2­yl)methanol (1.3 g, 9.09 mmol, yield: 77.8 %) as a yellow solid. LCMS (ESI) m/z: 144.1
Figure imgf000203_0001
Step 2: Preparation of 5-chloro-2-(chloromethyl)pyridine. To a solution of (5­chloropyridin­2­yl)methanol (1.2 g, 8.39 mmol) in dichloromethane (20 mL) at 0 oC was added thionyl chloride (4 g, 33.6 mmol). The resultant mixture was stirred at 15oC for 17 hour and concentrated and the resultant residue was purified by silica gel column chromatography (Petroleum ether: Ethyl acetate =6:1) to give the desired product 5­chloro­2­(chloromethyl)pyridine (1 g, 6.17 mmol, yield: 73.5 %) as a yellow oil. Step 3: Preparation of tert-butyl 3-(5-chloropyridin-2-yl)-2-(diphenylmethyleneamino)propanoate. To a solution of 5­chloro­2­(chloromethyl)pyridine (1 g, 6.17 mmol) in dichloromethane (30 mL) were added tert­butyl 2­(diphenylmethyleneamino)acetate (3.6 g, 12.3 mmol) and TBAB (200 mg, 0.62 mmol) and the mixture was stirred at 0oC for 10min. Then Potassium hydroxide (50%) (1.7 g, 30.8 mmol) was added to the reaction mixture and stirred further at 15oC for 17h. The mixture was filtered and the solvent was removed under the reduced pressure. The obtained residue was purified by flash column chromatography (Petroleum ether: Ethyl acetate =4:1) to give the desired product tert­butyl 3­(5­chloropyridin­2­yl)­2­ (diphenylmethyleneamino)propanoate (0.92 g, 2.19 mmol, yield: 35.4 %) as a yellow oil. LCMS (ESI) m/z: 421.1 [M+H]+. Step 4: Preparation of 2-amino-3-(5-chloropyridin-2-yl)propanoic acid hydrochloride. A mixture of tert­butyl 3­(5­chloropyridin­2­yl)­2­(diphenylmethyleneamino)propanoate (500 mg, 1.19 mmol) in water (8 mL) and conc. hydrochloric acid (8 mL) was stirred at 15 oC for 4 h. The solution was diluted with water (15 mL) and toluene (15 mL) and the organic layer was separated. The aqueous phase was concentrated to afford a white solid 2­amino­3­(5­chloropyridin­2­yl)propanoic acid hydrochloride (220 mg). The crude product was used in the next step without further purification. LCMS (ESI) m/z: 201.1 [M+H]+. Step 5: Preparation of 2-(5-chloro-1H-pyrrolo[2,3-b]pyridine-2-carboxamido)-3-(5-chloropyridin-2- yl)propanoic acid. To a solution of 2­amino­3­(5­chloropyridin­2­yl)propanoic acid hydrochloride (220 mg, 1.1 mmol) in acetonitrile (10 mL) and water (2 mL) were added 2,5­dioxopyrrolidin­1­yl 5­chloro­1H­pyrrolo[2,3­b]pyridine­ 2­carboxylate (645 mg, 2.2 mmol) and triethylamine (222 mg, 2.2 mmol) and the resultant mixture was stirred 15oC for 2 h. The mixture was then concentrated, water (10 mL) was added and the pH was adjusted to 2 with 1 N. hydrochloric acid. The resultant mixture was filtered and the solvent was removed under the reduced pressure. The crude product thus obtained was purified by prep-TLC (dichloromethane: methol =15:1) to give the desired product 2-(5-chloro-1H-pyrroto[2,3-b]pyridine-2-carboxamido)-3-(5-chloropyridin-2- yl)propanoic acid (140 mg, 0.37 mmol, yield: 33.6 %) as a yellow solid. LCMS (ESI) m/z: 379.1 [M+H]+.
Step 6: Synthesis of enantiomer 1 (Compound 275) and enantiomer 2 (Compound 276) of 5-chioro-N- (3-(5-chloropyridin-2-yi)-1 -(3,3-difiuoroazetidin-1 -y $)-1 -oxopropan-2-yi)-1 H-pyrroio[2,3-b]pyridine-2- carboxamide
To a solution of 2-(5-chloro-1 H-pyrrolo[2,3-b]pyridine-2-carboxamido)-3-(5-chloropyridin-2- yl)propanoic acid (100 mg, 0.26 mmol) in DMF (15 mL) were added 3,3-difluoroazetidine (50 mg, 0.39 mmol), HATU (148 mg, 0.39 mmol) and DIPEA (101 mg, 0.78 mmol) and the mixture was stirred at 15°C for 1 h. The mixture was filtered and the solvent was removed under the reduced pressure and the residue was purified by prep-HPLC (SunFire C18, 4.6*50mm, 3. Sum column Xbridge C18 3.5pm 4.6x50mm column. The elution system used was a gradient of 5%~95% over 1.5 min at 2ml/min and the solvent was acetonitrile/0.01% aqueous NH4HCO3) to give the desired racemic product (40 mg, yield: 33.9 %) as a white solid. 40 mg of this product was subjected to chiral separation using HPLC ( instrument: SFC-80 (Thar, Waters), Column: AS 20*250mm, 10um (Daicel) , Mobile phase: CO2/ methanol (0.2%Methanol Ammonia)^ 70/30, Flow rate: 80 g/min, Sample solution: 40 mg dissolved in 10 ml Methanol) to afford two isomers:
Enantiomer 1 (Compound 275) was obtained (5.8 mg, 0.05 mmol) as white solid. 1H NMR (500 MHz, DMSO) 6 12.40 (s, 1H), 9.10 (d, J = 7.5 Hz, 1 H), 8.57 (t, J = 10.9 Hz, 1 H), 8.32 (d, J = 2.2 Hz, 1H), 8.24 (t, J = 13.1 Hz, 1 H), 7.86 (dd, J = 8.3, 2.5 Hz, 1 H), 7.41 (d, J = 8.4 Hz, 1H), 7.20 (s, 1 H), 4.96 (dd, J = 14.9, 7.6 Hz, 1H), 4.78 (d, J = 11.9 Hz, 1 H), 4.63 (d, 11.3 Hz, 1H), 4.45 - 4.14 (m, 2H), 3.32 - 3.26 (m, 1 H), 3.22
(dd, J = 14.3, 8.4 Hz, 1 H); LCMS (ESI) m/z: 454.1 [M+H]+.
Enantiomer 2 (Compound 276) was obtained (5 mg, 0.04 mmol) as white solid. 1H NMR (500 MHz, DMSO) 5 12.40 (s, 1 H), 9.08 (s, 1 H). 8.55 (d, J = 2.3 Hz, 1 H), 8.30 (s, 1 H), 8.23 (s, 1 H), 7.86 (dd, J = 8.3, 2.5 Hz, 1H), 7.41 (d, J = 8.4 Hz, 1 H), 7.17 (s, 1 H), 5.07 - 4.86 (m, 1H), 4.78 (d, 11.2 Hz, 1H), 4.63 (d, J =
11.4 Hz. 1 H). 4.43 - 4.12 (m, 2H), 3.32 - 3.26 (m, 1 H), 3.22 (dd, J = 14.3, 8.4 Hz, 1 H); LCMS (ESI) m/z: 454.0 [M+H]+.
Synthesis of enantiomer 1 (Compound 277) and enantiomer 2 (Compound 278) of 5-chloro-N-(3-(5- chloropyridin-2-yl)-1-(4-hydroxy-4-methylpiperidin-1-yl)-1-oxopropan-2-yl)-1H-indole-2-carboxamide:
Figure imgf000205_0001
Step 1: (5-chloropyridin-2-yl)methanol. To the solution of methyl 5­chloropicolinate (3.43 g, 20 mmol) in methanol (50 ml) was added sodium borohydride (1.52 g, 40 mmol) at room temperature and the reaction mixture was stirred for 1h and concentrated. The resultant residue was redissolved in dichloromethane (200 mL), washed with brine (100 mL), dried over anhydrous sodium sulfate and concentrated in vacuum to get a colorless liquid (2.85 g, 19.9 mmol, 90%), which was directly used in the next step. LCMS (ESI) m/z: 144.1 [M+H]+. Step 2: 5-chloro-2-(chloromethyl)pyridine hydrochloride. To a 0 °C solution of (5­chloropyridin­2­yl)methanol (2350 mg, 16.37 mmol) in dichloromethane (50 mL), was added sulfurous dichloride (2922 mg, 24.55 mmol) followed by N,N­dimethylformamide (100 uL), after which the reaction was warmed to room temperature and stirred at that temperature for 16 hours. The reaction mixture was then concentrated to give a yellow solid (3.25 g, crude), which was directly used in the next step. LCMS (ESI) m/z: 162.2 [M+H]+. Step 3: tert-butyl 3-(5-chloropyridin-2-yl)-2-(diphenylmethyleneamino)propanoate. To a solution of 5­chloro­2­(chloromethyl)pyridine hydrochloride (2.25 g, 11.25 mmol), and TBAB (200 mg) in dichloromethane (100 mL) were added potassium hydroxide (50%) (3.78 g, 67.5 mmol) and tert­ butyl 2­(diphenylmethyleneamino) acetate (4 g, 13.5 mmol) at ­10 oC and the resultant mixture was stirred for 2h between ­10 ~ 25 oC. Water (100 mL) was added to the reaction mixure and extracted with ethyl acetate (200 mL). The organic phase was washed with brine (100 mL), dried, concentrated and purified by SGC (Petroleum ether / Ethyl acetate = 10 : 1) to afford the desired product as a light yellow solid ( 3 g, 7.13 mmol, 36%). LCMS (ESI) m/z: 421.2 [M+H]+. Step 4: 2-ammo-3-(5-chioropyridin-2~yi)propanoic acid hydrochioride.
A solution of tert-butyl 3-(5-chtoropyridin-2-yl)-2-(diphenylmethyieneamino)propanoate (2.58 g, 6.13 mmol) in cone hydrogen chloride (20 mL) was stirred at 25 c,C for 4h. The mixture was concentrated to afford the desired product as a white solid (1 ,5g, crude). LCMS (ESI) m/z: 201.1 [M+Hj*.
The remaining steps were carried out to similar to the amide coupling methods described before. The final racemic derivative was subjected to chiral-HPLC (column: OJ, 20*250 mm, 10pm. co-solvent: methonal (0.2% methanol ammonia)) io obtain enantiomer 1 and enantiomer 2 of 5-chloro-N-(3-(5-chloropyridin-2-yl)-1-(4- hydroxy-4-methylpiperidin-1-yi)-1-oxopropan-2-yl)-1H-indole-2-carboxamide.
Compound 277 (enantiomer 1):!H NMR (500 MHz, DMSO) 611.74 (s, 1H), 8.94 (dd, J = 8.5, 3.6 Hz, 1H), 8.54 (t, J = 2.0 Hz, 1 H), 7.85 - 7.77 (m, 1 H), 7.70 (s, 1 H), 7.43 - 7.33 (m, 2H), 7.22 (d, J = 6.6 Hz, 1 H), 7.17 (d, J = 8.7 Hz, 1H), 5.40 (dd, J = 15.1, 7.3 Hz, 1H), 4.40 (d, J = 2.2 Hz, 1H), 4.05-3.83 (m, 1H), 3.80-3.68 (m, 1H), 3.45-3.34 (m, 1H), 3.26-3.16 (m, 1H), 3.15-2.94 (m, 2H), 1.50- 1.27 (m, 3H), 1.25-1.15 (m, 1H), 1.09 (d,J = 9.1 Hz, 3H); LCMS(ESi)m/z=475.1 (M+H)+; (Rt:1.41min).
Compound 278 (enantiomer 2):1H NMR (400 MHz, DMSO) b 11.74 (s, 1H), 8.94 (dd, J = 8.5, 2.6 Hz, 1H), 8.54 (t, J = 2.0 Hz, 1 H), 7.85 - 7.78 (m, 1 H), 7.70 (d, J = 1.6 Hz, 1 H), 7.43 - 7.33 (m, 2H), 7.22 (d, J = 4.8 Hz, 1H), 7.17 (dd, J = 8.7, 1.1 Hz, 1H), 5.40 (dd, J = 15.0, 8.1 Hz, 1H), 4.40 (d, J= 1.7 Hz, 1H), 4.05-3.83 (m, 1H), 3.80-3.68 (m, 1H), 3.45-3.34 (m, 1H), 3.26-3.16 (m, 1H), 3.15-2.93 (m, 2H), 1.49-1.27 (m, 3H), 1.26-1.14 (m, 1H), 1.09 (d, J = 7.3 Hz, 3H); LCMS(ESi) m/z=475.1 (M+H)*: (Rt: 2.29min).
The following compounds were synthesized according to the protocol described above.
Figure imgf000206_0001
Figure imgf000207_0001
Figure imgf000208_0001
Figure imgf000209_0001
Synthesis of enantiomer 1 (Compound 289) and enantiomer 2 (Compound 290) of 5-chloro-N-(3-(4- chlorothiazol-2-yl)-1-(6-hydroxy-2-azaspiro[3.3]heptan-2-yl)-1-oxopropan-2-yl)-1H-indole-2- carboxamide.
Figure imgf000210_0001
Step 1: Preparation of methyl 4-chlorothiazole-2-carboxylate. To a solution of 2,4­dichlorothiazole (6 g, 39.2 mmol) in methanol (100 mL) under carbon monoxide atmosphere were added [1,1'­bis(diphenylphosphino)ferrocene]dichloropalladium(II) (850 mg, 1.17 mmol) and triethylamine (7.9 g, 78.4 mmol), the mixture was stirred at 80oC for 17 h. The mixture was filtered and the solvent was removed under the reduced pressure and the residue was purified by silica gel column chromatography (Petroleum ether: Ethyl acetate =8:1) to give the desired product methyl 4­chlorothiazole­2­ carboxylate (1.1 g, 6.21 mmol, yield: 15.9 %) as a yellow solid. LCMS (ESI) m/z: 178.1 [M+H]+ Step 2: Preparation of (4-chlorothiazol-2-yl)methanol. To a solution of methyl 4­chlorothiazole­2­carboxylate (2.1 g, 11.9 mmol) in methanol (30 mL) was added sodium borohydride (450 mg, 11.9 mmol) and the mixture was stirred at 25oC for 17 h. The solvent was removed under the reduced pressure and to the residue was added water (20 mL) and the mixture was extracted with ethyl acetate (10 mL*2). The organic layer was washed with brine (10 mL), dried over sodium sulfate, filtered and concentrated. The crude product (4­chlorothiazol­2­yl)methanol (1.1 g) was used in the next step without further purification. LCMS (ESI) m/z: 150.1 [M+H]+. Step 3: Preparation of 4~chioro-2-(chioromethyi)thiazole.
To a solution of (4-c-hlorothiazol-2-yl)methanol (1.6 g, 10.7 mmol) in dichloromethane (30 mL) at 0°C was added thionyl chloride (2.5 g, 21.4 mmol) and the resultant mixture was stirred at 25°C for 17 hour. It was then filtered and the solvent was removed under reduced pressure. The resultant residue was purified by silica gel column chromatography (Petroleum ether: Ethyl acetate =8:1) to give the desired product 4-chloro- 2-(chloromethyl)thiazo!e (1.1 g, 6.55 mmol, yield: 61.8 %) as a yellow oil.
Step 4: Preparation of tert-butyl 3-(4-ch!oroth!azol-2-yl)-2-(dipheny!methyieneamino)propanoate.
To a solution of 4-chloro-2-(chloromethyl)thiazole (1 g, 5.95 mmol) in dichloromethane (30 mL) was added tert-butyl 2-(diphenylmethyleneamino)acetate (3.5 g, 11.9 mmol) and TBAB (190 mg, 0.59 mmol) and the resultant mixture was stirred at 0°C for 10min. The potassium hydroxide (50%) (1 .7 g, 29.7 mmol) was added to the reaction mixture and it was stirred at 25°C for 2h. The mixture was concentrated and purified by flash column chromatography (Petroleum ether: Ethyl acetate =8:1) to give the desired product tert-butyl 3-(4- chlorothiazol-2-yl)-2-(diphenylmethyleneamino)propanoate (1.6 g, 3.75 mmol, yield: 64 %) as yellow oil. LCMS (ESI) m/z: 427.1 [M+H]+.
Step 5: Preparation of 2-amino-3-(4-chiorothsazoi~2-y!)propanoic acid.
A solution of tert-butyl 3-(4-chlorothiazol-2-yl)-2-(diphenylmethyleneamino)propanoate (1 .4 g, 3.29 mmol) in water (10 ml) and cone, hydrochloric acid (10 mL) was stirred at 25 °C for 4 h. To the reaction mixture was added water (10 mL) and Petroleum ether (10 mL) filtered and the solvent was removed under the reduced pressure. The crude product 2-amino-3-(4-chlorothiazol-2-yl)propanoic acid (500 mg) was used in the next step without further purification. LCMS (ESI) m/z: 207.0 [M+H]+.
The reminder of the steps were carried out similar to protocols described earlier. Chiral separation led to the isolation of Compound 289 and Compound 290 as white solids.
Compound 289 (enantiomer 1): 1H NMR (400 MHz, DMSO) 6 11.82 (s, 1 H), 9.04 (d, J = 8.3 Hz, 1 H), 7.73 (d, J = 1.9 Hz, 1 H), 7.57 (s, 1 H), 7.41 (d, J = 8.7 Hz, 1 H), 7.32 - 7.10 (m, 2H), 5.03 (dd, J = 6.2, 2.9 Hz, 1 H), 4.96 - 4.77 (m, 1 H), 4.17 (ddd, J = 29.3, 18.3, 8.7 Hz, 2H), 3.94 (dd, J = 13.9, 6.9 Hz, 1 H), 3.90 - 3.69 (m, 2H), 3.45 (dd, J = 14.9, 6.2 Hz, 1 H), 3.31 (s, 1 H), 2.43 - 2.31 (m, 2H), 2.03 - 1.83 (m, 2H); LCMS (ESI) m/z: 479.0[M+H]+; (Rt: 1.58min)
Compound 290 (enantiomer 2): !H NMR (400 MHz, DMSO) 6 11.82 (s, 1 H), 9.04 (d, J = 8.4 Hz, 1 H), 7.73 (d, J = 1.9 Hz, 1 H), 7.57 (s, 1 H), 7.41 (d, J = 8.8 Hz, 1 H), 7.28 - 7.11 (m, 2H), 5.03 (dd, J = 6.2, 2.9 Hz, 1 H), 4.93 - 4.78 (m, 1 H), 4.17 (ddd, J = 29.6, 18.4, 8.8 Hz, 2H), 3.94 (dd, J = 13.7, 6.9 Hz, 1H), 3.80 (dt, J = 18.2, 9.0 Hz, 2H), 3.45 (dd, J = 14.8, 6.2 Hz, 1 H), 3.31 (s, 1 H), 2.43 - 2.30 (m, 2H), 2.02 - 1.81 (m, 2H); LCMS (ESI) m/z: 480.9[M+H]+; (Rt: 2.05min) Synthesis of enantiomer 1 (Compound 291) and enantiomer 2 (Compound 292) of 5-chloro-N-(3-(5- chlorothiazol-2-yl)-1-(6-hydroxy-2-azaspiro[3.3]heptan-2-yl)-1-oxopropan-2-yl)-1H-indole-2- carboxamide:
Figure imgf000212_0001
Step 1: Preparation of methyl 5-chlorothiazole-2-carboxylate. To a solution of 2­bromo­5­chlorothiazole (5 g, 25.5 mmol) in methanol (60 mL) under carbon monoxide atmosphere were added [1,1'­bis(diphenylphosphino)ferrocene]dichloropalladium(II) (1.9 g, 2.55 mmol) and triethylamine (5.1 g, 51 mmol) and the mixture was stirred at 40oC for 17 h. The resultant mixture was filtered and the solvent was removed under the reduced pressure and the residue was purified by silica gel column chromatography (Petroleum ether: Ethyl acetate =6:1) to give the desired product methyl 5­ chlorothiazole­2­carboxylate (1.1 g, 6.21 mmol, yield: 24.4 %) as a yellow solid. LCMS (ESI) m/z: 178.0 Step 2: Preparation of (5-chlorothiazol-2-yl)methanol. To a solution of methyl 5­chlorothiazole­2­carboxylate (3.3 g, 18.6 mmol) in methanol (50 mL) was added sodium borohydride (703 mg, 18.6 mmol) and the mixture was stirred at 25oC for 17 h. The solvent was removed under the reduced pressure and to the residue was added water (30 mL), extracted with ethyl acetate (20 mL*2). The organic layer was washed with brine (20 mL), dried over sodium sulfate, filtered and concentrated. The residue was purified by silica gel column chromatography (Petroleum ether: Ethyl acetate =2:1) to give the desired product (5­chlorothiazol­2­yl)methanol (1.8 g, yield: 64.9 %) as a yellow oil. LCMS (ESI) m/z: 150.1 [M+H]+. Step 3: Preparation of 5-chloro-2-(chioromettiyi)thiazole.
To a solution of (5-chlorothiazol-2-yl)methanol (1.8 g, 12.1 mmol) in dichloromethane (30 mL) at 0°C was added thionyl chloride (2.9 g, 24.2 mmol) and the residue was stirred at 25°C for 17hours. The resultant mixture was filtered and the solvent was removed under the reduced pressure and the residue was purified by silica gel column chromatography (Petroleum ether: Ethyl acetate =8:1) to give the desired product 5- chloro-2-(chloromethyl)thiazole (1.1 g, 6.55 mmol, yield: 55 %) as a yellow oil.
Step 4: Preparation of tert-butyl 3-(5-ch!oroth!azoi-2-yl)-2-(dipheny!methyleneamino)propanoate.
To a solution of 5-chloro-2-(chloromethyl)thiazole (1 g, 5.95 mmol) in dichloromethane (20 mL) were added tert-butyl 2-(diphenylmethyleneamino)acetate (3.5 g, 11.9 mmol) and TBAB (190 mg, 0.59 mmol) and the resultant mixture was stirred at 0°C for Wmin. Potassium hydroxide (50%) (1.7 g, 29.7 mmol) was then added to the reaction mixture and the mixture was stirred at 20°C for 2h. It was then concentrated and purified by flash column chromatography (Petroleum ether: Ethyl acetate =9:1) to give the desired product tert-butyl 3-(5-chlorothiazol-2-yl)-2-(diphenylmethyleneamino)propanoate (1.8 g, 4.22 mmol, yield: 71.1 %) as yellow oil. LCMS (ESI) m/z: 427.1 [M+HJ+.
Step 5: Preparation of 2-ammo-3-(5-chlorothiazol~2-y!)propanoic acid.
A solution of tert-butyl 3-(5-chlorothiazol-2-yl)-2-(diphenylmethyleneamino)propanoate (1.7 g, 3.99 mmol) in water (15 mL) and cone, hydrochloric acid (15 mL) was stirred at 20 °C for 4 h. To the reaction mixture was added water (10 mL) and Petroleum ether (10 mL), the solids were filtered off and the filtrate was concentrated under the reduced pressure to obtain 2-amino-3-(5-chlorothiazol-2-yl)propanoic acid (820 mg). This product was used in the next step without further purification. LCMS (ESI) m/z: 207.0 [M+H]+. The reminder of the steps were performed as described earlier to obtain a racemic compound, which was chirally separated io obtain Compound 291 and Compound 292:
Compound 291 (enantiomer 1): 1H NMR (400 MHz, DMSO) 6 11.83 (s, 1 H), 9.04 (d, J = 8.3 Hz, 1 H), 7.80 - 7.63 (m, 2H), 7.42 (d, J = 8.7 Hz, 1 H), 7.30 - 7.13 (m, 2H), 5.03 (dd, J = 6.2, 1.9 Hz, 1 H), 4.93 - 4.79 (m, 1 H), 4.14 (ddd, J = 32.4, 19.5, 8.8 Hz, 2H), 3.94 (dd, J = 12.3, 6.4 Hz, 1 H), 3.80 (dd, J = 17.7, 14.3 Hz, 2H), 3.51 - 3.40 (m, 1 H), 3.28 (s, 1 H), 2.43 - 2.31 (m, 2H), 2.06 - 1.86 (m, 2H); LCMS (ESI) m/z: 479.1 [M+H]+; (Rt: 1.47min).
Compound 292 (enantiomer 2): !H NMR (400 MHz, DMSO) 6 11.83 (s, 1 H), 9.04 (d, J = 8.5 Hz, 1 H), 7.73 (d, J = 5.6 Hz, 2H), 7.42 (d, J = 9.0 Hz, 1 H), 7.20 (d, J = 12.6 Hz, 2H), 5.03 (d, J = 6.2 Hz, 1 H), 4.87 (s, 1 H), 4.36 - 4.01 (m, 2H), 3.93 (s, 1 H), 3.82 (d, J = 17.3 Hz, 2H), 3.42 (d, J = 8.8 Hz, 1 H), 3.30 - 3.14 (m, 1 H), 2.38 (s, 2H), 1.94 (s, 2H); LCMS (ESI) m/z: 479.0[M+H]+; (Rt: 1.85min).
The following compounds were synthesized according to the protocol described above:
Figure imgf000214_0001
Figure imgf000215_0001
Synthesis of enantiomer 1 (Compound 299) and enantiomer 2 (Compound 300) of 5-chloro-N-(1-(4- hydroxy-4-methylpiperidin-1-yl)-3-(5-methylthiophen-2-yl)-1-oxopropan-2-yl)-1H-indole-2- carboxamide:
Figure imgf000216_0001
Step 1: (5-methylthiophen-2-yl)methanol. To the solution of 5­methylthiophene­2­carbaldehyde (1.26 g, 1 mmol) in methanol (30 ml) was added sodium borohydride (757 mg, 20 mmol) at 25oC and the reaction mixture was stirred for 1 h. It was concentrated and the residue was dissolved in dichloromethane (200 mL), washed with brine (100 mL), dried over anhydrous sodium sulfate and concentrated in vacuum to get 1.2 g of the crude product as yellow liquid. Step 2: 2-(chloromethyl)-5-methylthiophene. To a 0 °C solution of (5­methylthiophen­2­yl)methanol (1.2 g, 9.4 mmol) in dichloromethane (20 mL), was added sulfurous dichloride (1.67 g, 14 mmol) followed by DMF (100 uL), after which the reaction was warmed to 25oC and stirred at that temperature for 16 hours. The reaction mixture was then concentrated to give an yellow solid (1.4 g, crude), which was directly used for the next step. Step 3: tert-butyl 2-(diphenylmethyleneamino)-3-(5-methylthiophen-2-yl)propanoate. To a stirred solution of 2­(chloromethyl)­5­methylthiophene (1.54 g, 10.5 mmol), and TBAB (100 mg) in dichloromethane (30 mL) were added potassium hydroxide (50%) (3.53 g, 63 mmol) and tert­butyl 2­ (diphenylmethyleneamino) acetate (3.1 g, 10.5 mmol) at ­10 oC and the mixture was slowly warmed up to 25 oC over a period of 2h. The resultant mixture was diluted with water (100 mL), extracted with ethyl acetate (200 mL), washed with brine (100 mL), dried over sodium sulfate and concentrated. The crude product was purified by SGC (Petroleum ether / Ethyl acetate = 10 : 1) to afford tert­butyl 2­(diphenylmethyleneamino)­3­ (5­methylthiophen­2­yl)propanoate as a yellow oil (2 g, 4.93 mmol, 47%) . LCMS (ESI) m/z: 406.1 [M+H]+. Step 4: 2-amino-3-(5-methyithiophen~2-yi)propanoic acid hydrochloride.
A solution of tert-butyl 2-(diphenylmethyleneamino)-3-(5-methyithiophen-2-yi)propanoate (1.6 g, 3.94 mmol) in cone. HCI (10 mL) was stirred at 25 °C for 4 h. The resultant mixture was concentrated io afford a white solid (800 mg, crude), which was directly used for the next step. LCMS (ESI) m/z: 186.1 [M+H]L
The remaining steps were performed as described earlier. The final racemic product was chirally separated to obtain two enantiomers;
Compound 299: (enantiomer 1): 1H NMR (500 MHz, DMSO) 1H NMR (500 MHz, DMSO) 6 11.79 (s, 1 H), 8.93 (s, 1H), 7.71 (s, 1 H), 7.41 (d, J = 8.6 Hz, 1 H), 7.25 (s, 1 H), 7.18 (d, J = 7.3 Hz, 1H), 6.70 (d, J = 3.2 Hz, 1H), 6.58 (d, J = 9.7 Hz, 1 H), 5.09 (s, 1H), 4.38 (s, 1 H), 3.98 (dd, J = 50.9, 12.8 Hz, 1 H), 3.69 (t, J = 12.1 Hz, 1 H), 3.40-3.27 (m, 1 H), 3.22 (td, J = 14.6, 6.4 Hz, 1 H), 3.14-2.95 (m, 2H), 2.33 (d, J = 3.3 Hz, 3H), 1.49 - 1.22 (m, 4H), 1.08 (d, J = 2.8 Hz, 3H); LCMS (ESI) m/z: 460.2 [M+H]+; (Rt: 1.34min).
Compound 300: (enantiomer 2): 1H NMR (500 MHz, DMSO) 6 11.79 (s, 1 H), 8.93 (dd, J = 13.8, 8.4 Hz, 1 H), 7.72 (s, 1 H), 7.41 (d, J = 6.7 Hz, 1 H), 7.26 (s, 1 H), 7.18 (dd, J = 8.7, 1.9 Hz, 1 H), 6.70 (d, J = 3.2 Hz, 1 H), 6.58 (d, J = 9.8 Hz, 1 H), 5.19 - 4.94 (m, 1 H), 4.38 (d, J = 9.0 Hz, 1 H), 3.98 (dd, J = 51.7, 12.2 Hz, 1 H), 3.69 (t, J = 12.0 Hz, 1 H), 3.39 - 3.27 (m, 1 H), 3.22 (td, J = 14.6, 6.1 Hz, 1 H), 3.14-2.96 (m, 2H), 2.33 (d, J = 3.1 Hz, 3H), 1.50 - 1.25 (m, 4H), 1.08 (d, J = 3.0 Hz, 3H); LCMS(ESI) m/z:460.2(M+H)*; (Rt:2.02rnin).
Synthesis of enantiomer 1 (Compound 301) and enantiomer 2 (Compound 302) of 5-chloro-N-(1-(3,3- difiuoropyrrolidin-1 -yi)-3~(5-methyM H-imidazol-4-yi)-1 -oxopropan-2-yl)-1 H-indoie-2 -carboxamide,
Figure imgf000217_0001
Enantiomer 1 Enantiomer 2
Step 1 : Preparation of 4-(chioromethyl)-5-metiiyi-1 H-imidazoie.
To a solution of (5-methyi-1H-imidazol-4-yl)methanol (5,0 g, 34 mmol) in chloroform (30 mL) was added thionyl chloride (12 mL) at 0°C under argon. The mixture was stirred at 25°C for 24 h. The resultant white precipitate was collected by filtration, washed with chloroform (30 mL) to give the desired product 4­ (chloromethyl)­5­methyl­1H­imidazole as a white solid (5.6 g ,33.5 mmol, yield: 99%). Step 2: Preparation of diethyl 2-acetamido-2-((5-methyl-1H-imidazol-4-yl)methyl)malonate. To a solution of diethyl 2­acetamidomalonate (3.52 g, 16.2 mmol) in tetrahydrofuran (50 mL) was added sodium hydride (1.35 g, 33.8 mmol) at 0oC under nitrogen. The mixture was stirred at 25oC for 1h. Then 4­ (chloromethyl)­5­methyl­1H­imidazole (2.26 g, 13.5 mmol) was added and the mixture was stirred at 25oC for 16 h. The mixture was then quenched with water and extracted with ethyl acetate (80mL*2). The organic layer was dried over sodium sulfate, filtered and concentrated. The crude product (3.1 g, yield:74%) was obtained as yellow oil which was used in next step directly. LCMS (ESI) m/z: 312.3 [M+H]+. Step 3: Preparation of 2-amino-3-(5-methyl-1H-imidazol-4-yl)propanoic acid hydrochloride. A mixture of diethyl 2­acetamido­2­((5­methyl­1H­imidazol­4­yl)methyl)malonate (3.1g, 9.96 mmol) in concentrated hydrochloric acid(10 mL) was stirred at 100oC for 5 h. The mixture was concentrated and the crude product (brown oil) was used in next step directly. (1.5 g crude, 7.3 mmol, yield: 73 %). LCMS (ESI) m/z: 170.1 [M+H]+. Step 4: Preparation of 5-chloro-1H-pyrrolo[2,3-b]pyridine-2-carbonyl chloride. To a solution of 2­amino­3­(5­methyl­1H­imidazol­4­yl)propanoic acid hydrochloride(650 mg, 3.2 mmol) in methanol(20 mL) was added thionyl chloride (761 mg, 6.4 mmol) slowly at room temperature under nitrogen. The mixture was stirred at 70oC for 4 h and concentrated. The crude product was used in next step directly. LCMS (ESI) m/z: 184.2 [
Figure imgf000218_0001
Step 5: Preparation of 2-(5-chloro-1H-indole-2-carboxamido)-3-(5-methyl-1H-imidazol-4-yl)propanoic acid. To a solution of 5­chloro­1H­indole­2­carboxylic acid (320 mg, 1.64 mmol), methyl 2­amino­3­(5­ methyl­1H­imidazol­4­yl)propanoate (300 mg crude, 1.64 mmol) and DIPEA (636 mg, 4.92 mmol) in DMF (5 mL) was added HATU (937 mg, 2.46 mmol) at room temperature under argon. The mixture was stirred at room temperature for 1h, diluted with ethyl acetate (50 mL) and washed brine(30 mL*3).The organic layer was dried over sodium sulfate, filtered and concentrated. The resultant crude product was purified by Prep­HPLC (Boston C1821*250mm 10µm column. The mobile phase was acetonitrile/0.01% aqueous trifluoroacetic acid.) to give the desired 2­(5­chloro­1H­indole­2­carboxamido)­3­(5­methyl­1H­imidazol­4­yl)propanoic acid (170 mg, 0.49 mmol, yield: 30 %) as a white solid. LCMS (ESI) m/z: 347.1[
Figure imgf000218_0002
. Step 6: Preparation of 5-chloro-N-(1-(3,3-difluoropyrrolidin-1-yl)-3-(5-methyl-1H-imidazol-4-yl)-1- oxopropan-2-yl)-1H-indole-2-carboxamide. To a solution of 2­(5­chloro­1H­indole­2­carboxamido)­3­(5­methyl­1H­imidazol­4­yl)propanoic acid (140 mg, 0.4 mmol), 3,3­difluoropyrrolidine hydrochloride (86 mg, 0.6 mmol) and DIPEA (155 mg, 1.2 mmol) in DMF (5 mL) was added HATU (160 mg, 0.42 mmol) at ­20oC under argon. The mixture was stirred at ­20oC for 24h, diluted with ethyl acetate (50 mL) and washed brine(30 mL*3).The organic layer was dried over sodium sulfate, filtered and concentrated. The crude product was purified by Prep-HPLC (Boston C1821*250mm 10pm column. The mobile phase was acetonitrile/0.01% aqueous trifluoroacetic acid.) to give the desired product 5- chtoro-N-(1-(3,3-difluoropyrrolidin-1-yl)-3-(5-methyl-1 H-imidazol-4-yl)-1-oxopropan-2-yl)-1H-indote-2- carboxamide (135 mg, 0.31 mmol, yield: 77 %) as a white solid. LCMS (ESI) m/z: 346.1 [M+H]+.
Step 7: Separation of enantiomer 1 (Compound 301) and enantiomer 2 (Compound 302) of S-ch!oro-N- (1 -(3,3-difiuoropyrroiidin-l -yi)~3-(5-methyi-1 H-imidazoi-4-yl)~1 -oxopropan-2-yi)-1 H-indoie-2- carboxamide.
100 mg o off racemic 5-chloro-N-(1-(3,3-difiuoropyrrolidin-1-yl)-3-(5-methyl-1H-imidazoi-4-yl)-1- oxopropan-2-yl)-1 H-indole-2-carboxamide was subjected to chiral separation (instrument: SFC-80 (Thar, Waters), Column: OJ 20*250mm, 10um (Daicel) Column temperature: 35 °C Mobile phase: CO2/ MeOH(0.2%Methanol Ammonia) = 75/25 Flow rate: 80 g/min Back pressure: 100 bar, Detection wavelength: 214 nm, Cycle time: 4.3 min, Sample solutiomWO mg dissolved in 35 ml Methanol .Injection volume: 1 .9 ml) to afford two enantiomers.
Enantiomer 1 was obtained (30.8 mg, 0.07 mmol) as white solid. 1H NMR (400 MHz, DMSO) 6 1 1.81 (d, J = 9.6 Hz, 1H), 11.64 (s, 1 H), 8.97 (dd, Ji = 7.2 Hz, J2 = 17.6 Hz, 1 H), 7.73 (s, 1 H), 7.43-7.41 (m, 2H), 7.25 (s, 1 H), 7.19 (dd, Ji = 2.0 Hz, J2 = 8.8 Hz, 1 H), 4.97-4.79(m,1H), 4.12-3.85 (m,1 H), 3.71-3.36 (m,3H), 3.01- 2.79 (m,2H), 2.50-2.28 (m,2H), 2.08 (s,3H); LCMS (ESI) m/z: 435.9[M+H]+; (Rt: 1.34min).
Enantiomer 2 was obtained (41.7 mg, 0.10 mmol) as white solid. 1H NMR (400 MHz, DMSO) 6 11.81 (d, J = 9.2 Hz, 1H), 11.70 (s, 1 H), 8.97 (dd, Ji = 7.2 Hz, J2 = 17.2 Hz, 1 H), 7.73 (s, 1 H), 7.45-7.41 (m, 2H), 7.25 (s, 1 H), 7.19 (dd, Ji = 1.6 Hz, J2 = 8.8 Hz, 1 H), 4.97-4.79(m,1H), 4.13-3.85 (m,1 H), 3.71-3.38 (m,3H), 3.03- 2.79 (m,1 H), 2.87-2.81 (m,1 H), 2.50-2.25 (m,2H), 2.07 (s,3H); LCMS (ESI) m/z: 435.9[M+H]+; (Rt: 2.02min).
Synthesis of enantiomer 1 (Compound 303) and enantiomer 2 (Compound 304) of N-(1-(6-oxa-3- azabicyclo[3.1.1]heptan-3-y!)-3-(3-chioropyndin-4-yl)-1 -oxopropan-2-yl)-5-chioro-1 H-pyrroto[2,3- bJpyridine-2 -carboxamide
Figure imgf000219_0001
■N P d
Enantiomer 2
The racemic N-(1-(6-oxa-3-azabicyclo[3.1 .1]heptan-3-yl)-3-(3-chloropyndin-4-yl)-1-oxopropan-2-yl)-5- chloro-1 H-pyrrolo[2,3-b]pyridine-2-carboxamide was synthesized according to the protocol described earlier. It was then subjected to chiral separation ( instrument: SFC-80 (Thar, Waters), Column: AS 20*250mm, 10um (Daicel) , Mobile phase: CO2/ methanol (0.2%Methanol Ammonia)= 60/40, Flow rate: 80 g/min, Sample solution: 50 mg dissolved in 15 ml Methanol) to afford two isomers:
Enantiomer 1: ’H NMR (400 MHz, DMSO) 512.36 (s, 1H), 9.19 (d, J = 8.0 Hz, 1H), 8.58 (d, J = 3.6
Hz, 1H), 8.40 (dd, J = 4.7, 3.0 Hz, 1H), 8.32 (t, J = 2.2 Hz, 1H), 8.26 (d, J = 2.2 Hz, 1H), 7.50 (t, J = 4.5 Hz, 1H), 7.24 (5, 1H), 5.27 (s, 1H), 4.58 (d, J = 6.1 Hz, 2H), 4.00 (dd, J = 62.4, 11.7 Hz, 1H), 3.74 (d, J = 14.3 Hz, 1H), 3.68-3.57 (m, 1H), 3.44 (dd, J = 21.6, 12.9 Hz, 1H), 3.30-3.14 (m, 2H), 3.11 -2.95 (m, 1H), 1.62 (dd, J = 58.1, 8.9 Hz, 1H); LCMS (ESI) m/z: 460.1 [M+H]+; (Rt: 1.43min).
Enantiomer 2: 1H NMR (400 MHz, DMSO) 612.36 (s, 1H), 9.19 (d, J = 8.1 Hz, 1H), 8.58 (d, J= 3.6 Hz, 1H), 8.40 (dd, J= 4.9, 3.0 Hz, 1H), 8.32 (t, J = 2.3 Hz, 1H), 8.26 (t, J = 2.3 Hz, 1H), 7.50 (t, J = 4.5 Hz, 1H), 7.24 (s, 1H), 5.26 (d, J = 6.1 Hz, 1H), 4.58 (d, J = 5.8 Hz, 2H), 4.00 (dd, J = 63.1, 11.6 Hz, 1H), 3.74 (d, J = 14.0 Hz, 1H), 3.70-3.59 (m, 1H), 3.44 (dd, J = 20.9, 13.9 Hz, 1H), 3.30-3.14 (m, 2H), 3.04 (dd, J = 14.1 , 6.9 Hz, 1H), 1.77- 1.48 (m, 1H); LCMS (ESI) m/z: 460.0 [M+H]+; (Rt: 2.04min).
The following compound were synthesized according to the protocol described above:
Figure imgf000220_0001
Figure imgf000221_0001
Figure imgf000222_0001
Figure imgf000223_0001
Figure imgf000224_0001
Figure imgf000225_0001
Synthesis of enantiomer 1 (Compound 321) and enantiomer 2 (Compound 322) of 5-chloro-N-(1-(1- (2,4-diduorophenyl)cyctopropyl)~2-(4-hydroxypiper!din-1 ~yi)-2~oxoethyl)-1 H-pyrro!o[2,3-b]pyndine-2~ carboxamide.
F O
Figure imgf000226_0001
Enantiomer 1 Enantiomer 2
Slept : 1-(2,4-difluorophenyi)cyctopropanecarbonitri!e,
To a suspension of sodium hydride (1.72 g, 43.0 mmol) in N,N-dimethylformamide (50 mL) at 0°C was added a solution of 2-(2,4-difluorophenyl)acetonitrile (3.3 g, 21.5 mmol) in N,N-dimethylformamide (10 mL). After the addition, the mixture was stirred for another 0.5 hour, followed by the addition of 1 ,2- dibromoethane (6.0 g, 32.3 mmoi). The resulting mixture was stirred at 0°C for another 4 hours. The mixture was quenched with crushed ice, extracted with ethyl acetate (250 mL*2). The combined organic phase was concentrated. The obtained crude product was purified by silica gel column chromatography (30% ethyl acetate in petroleum ether) to afford the target compound (2.5 g, 13.9 mmoi, 64.6%) as a light yellow oil. !H NMR (400 MHz, CDCb) 6 7.36-7.30 (m, 1 H), 6.90-6.84 (m, 2H), 1 .70 (dd, J = 7.2, 5.2 Hz, 2H), 1 .37 (dd, J = 7.2, 5.2 Hz, 2H); LCMS (ESI) m/z: 311.3 [M+Hp.
Step 2: 1-(2,4-dif!uorophenyi)cyctopropanecarba!dehyde.
To a solution of 1-(2,4-difiuorophenyi)cyclopropanecarbonitrile (2.5 g, 13.9 mmol) in toluene (50 mL) at 0°C was added a solution of diisobutylaluminium hydride (2.5 M in hexane, 17.0 mL, 41.7 mmol). After the addition, the mixture was stirred for another 3 hours. The resultant mixture was quenched with ice, acidified io pH 1-2 with 36% hydrochloric acid and then stirred at 15°C for another 2 hours. The mixture was extracted with ethyl acetate (200 mL*2) and the combined organic phase was dried over sodium sulfate and concentrated to afford the target compound (2.5g) as a light yellow oil. TH NMR (400 MHz, CDCb) 6 9.03 (s, 1 H), 7.22-7.18 (m, 1 H), 6.90-6.84 (m, 2H), 1.65 (dd, J = 7.2, 4.4 Hz, 2H), 1.41 (dd, J = 7.2, 4.4 Hz, 2H); LCMS (ESI) m/z: 183.1 [M+H]T Step 3: 5-(1-(2,4-difluorophenyl)cyclopropyl)imidazolidine-2,4-dione. A solution of 1­(2,4­difluorophenyl)cyclopropanecarbaldehyde (1.5 g, 8.2 mmol), NaCN (2.0 g, 41.0 mmol) and ammonium carbonate (7.9 g, 82.0 mmol) in water (20 mL) was stirred at 100^ for 16 hours. The mixture was poured into brine (200 mL), extracted with ethyl acetate (350 mL*2). The combined organic phase was concentrated. The crude product was purified by silica gel column chromatography (30% methanol in dichloromethane) to afford the target compound (900 mg, 3.5 mmol, yield :42.6 %) as a grey solid. LCMS (ESI) m/z: 253.2 [M+H]+. Step 4: 2-amino-2-(1-(2,4-difluorophenyl)cyclopropyl)acetic acid. A mixture of 5­(1­(2,4­difluorophenyl)cyclopropyl)imidazolidine­2,4­dione (900 mg, 3.6 mmol) in aqueous sodium hydroxide (6.0N, 6 mL) and ethane­1,2­diol (20 mL) was stirred at 130^ for 6 hours. The mixture was acidified to pH 1~2 with 36% hydrochloric acid. The resultant precipitate was filtered off. The filtrate was concentrated under vacuum to afford the target compound (5.5 g, crude) as a gum, which was used in the next step without further purification. LCMS (ESI) m/z: 228.1 [M+H]+. Step 5: 2-(tert-butoxycarbonylamino)-2-(1-(2,4-difluorophenyl)cyclopropyl)acetic acid. To a mixture of 2­amino­2­(1­(2,4­difluorophenyl)cyclopropyl)acetic acid (900 mg, 3.6 mmol), sodium hydroxide (2.0 g, 50.0 mmol), tetrahydrofuran (40 mL) and water (10 mL) at 0^ was added dropwise di­tert­ butyl dicarbonate (1.1 g, 5.0 mmol). After the addition, the mixture was stirred for another 1 hour. The mixture was then poured into crushed ice and acidified to pH 1~2 with 36% hydrochloric acid, extracted with ethyl acetate (150 mL*3). The combined organic phase was concentrated and the crude product was purified by silica gel column chromatography (5% methanol in dichloromethane) to afford the target compound (1.7g) as a brown oil. LCMS (ESI) m/z: 350.0 [M+Na]+. Step 6: tert-butyl 1-(1-(2,4-difluorophenyl)cyclopropyl)-2-(4-hydroxypiperidin-1-yl)-2- oxoethylcarbamate. A mixture of 2­(tert­butoxycarbonylamino)­2­(1­(2,4­difluorophenyl)cyclopropyl)acetic acid (700 mg, 2.1 mmol), piperidin­4­ol (253 mg, 2.5 mmol), HATU (950 mg, 2.5 mmol), DIPEA (813 mg, 6.3 mmol) and DMF (8 mL) was stirred at 15 ^ for 1 hour. The mixture was poured into water, extracted with ethyl acetate (100 mL*2). The combined organic phase was concentrated and the obtained crude product was purified by silica gel column chromatography (petroleum ether/ethyl acetate = 2:1) to afford the target compound (800 mg) as a light yellow oil. LCMS (ESI) m/z: 411.2 [M+H]+. Step 7: 2-amino-2-(1-(2,4-difluorophenyl)cyclopropyl)-1-(4-hydroxypiperidin-1-yl)ethenone. A mixture of tert­butyl 1­(1­(2,4­difluorophenyl)cyclopropyl)­2­(4­hydroxypiperidin­1­yl)­2­ oxoethylcarbamate (700mg), trifluoroacetic acid (0.5 mL) and dichloromethane (5 mL) was stirred at 15 ^ for 2 hours. The mixture was concentrated and the residue was purified by prep­HPLC to afford the target compound (120 mg, 0.38 mmol) as a white solid. LCMS (ESI) m/z: 311.3 [M+H]+. Step 8: Synthesis of compounds 321 and 322: A mixture of 5­chloro­1H­pyrrolo[2,3­b]pyridine­2­carboxylic acid (63 mg, 0.32 mmol), 2­amino­2­(1­ (2,4­difluorophenyl)cyclopropyl)­1­(4­hydroxypiperidin­1­yl)ethanone (100 mg, 0.32 mmol), PyAOP (203 mg, 0.39 mmol), DIPEA (124 mg, 0.96 mmol) and DMF (4 mL) was stirred at 15 ^ for 1 hour. The crude product from the mixture was purified by prep­HPLC to afford 5­chloro­N­(1­(1­(2,4­difluorophenyl)cyclopropyl)­2­(4­ hydroxypiperidin­1­yl)­2­oxoethyl)­1H­pyrrolo[2,3­b]pyridine­2­carboxamide (40 mg, 0.082 mmol, yield: 31.5%) as a white solid. An amount of 105mg (combined with an additional batch for step­8) of 5­chloro­N­(1­(1­(2,4­ difluorophenyl)cyclopropyl)­2­(4­hydroxypiperidin­1­yl)­2­oxoethyl)­1H­pyrrolo[2,3­b]pyridine­2­carboxamide was resolved by chiral prep­HPLC to afford 47.8mg of enantiomer 1 (Compound 321) and 42.2mg of enantiomer 2 (Compound 322) as white solids. (47.8 mg; 42.2 mg) as a white solid respectively. Enantiomer 1: 1H NMR (400 MHz, DMSO­d6) į 12.47 (s, 1H), 8.61 (dd, J = 20.4 , 8.4 Hz, 1H), 8.34 (d, J = 2.8 Hz, 1H), 8.24 (t, J = 2.8 Hz, 1H), 7.57­7.49 (m, 1H), 7.26 (d, J = 6.0 Hz, 1H), 7.21­7.15 (m, 1H), 7.02­6.96 (m, 1H), 5.43 (dd, J = 8.8, 5.2 Hz, 1H), 4.80 (dd, J = 8.0, 4.4 Hz, 1H), 3.98­3.63 (m, 3H), 3.39­3.31 (m, 0.5H), 3.24­3.15 (m, 1H), 2.99­2.92 (m, 0.5H), 1.81­1.68 (m, 2H), 1.42­1.02 (m, 4H), 0.82­0.73 (m, 2H); LCMS (ESI) m/z: 489.1/491.1 [M+H]+. Enantiomer 2: 1H NMR (400 MHz, DMSO­d6) į 12.47 (s, 1H), 8.61 (dd, J = 20.4 , 8.4 Hz, 1H), 8.34 (d, J = 2.4 Hz, 1H), 8.24 (t, J = 2.8 Hz, 1H), 7.57­7.49 (m, 1H), 7.26 (d, J = 5.2 Hz, 1H), 7.21­7.15 (m, 1H), 7.01­6.96 (m, 1H), 5.43 (dd, J = 8.4, 5.2 Hz, 1H), 4.80 (dd, J = 8.0, 4.0 Hz, 1H), 3.98­3.67 (m, 3H), 3.39­3.31 (m, 0.5H), 3.24­3.15 (m, 1H), 2.99­2.93 (m, 0.5H), 1.77­1.66 (m, 2H), 1.39­1.03 (m, 4H), 0.82­0.74 (m, 2H); LCMS (ESI) m/z: 489.1/491.1 [M+H]+. Synthesis of enantiomer 1 (Compound 323) and enantiomer 2 (Compound 324) of 5-chloro-N-(1-(2- chlorophenyl)-4-(4-hydroxypiperidin-1-yl)-4-oxobutan-2-yl)-1H-indole-2-carboxamide:
Figure imgf000228_0001
Step 1: Preparation of 5-(2-(2-chlorophenyl)-1-hydroxyethylidene)-2,2-dimethyl-1,3-dioxane-4,6-dione. To a solution of 2­(2­chlorophenyl)acetic acid (1.7 g, 10 mmol), 2,2­dimethyl­1,3­dioxane­4,6­dione (1.44 g, 10 mmol) and 4­dimethylaminopyridine (1.34 g, 11 mmol) in dry dichloromethane (100 mL) at 0°C, was added slowly a solution of dicyclohexylcarbodiimide (2.27 g, 11 mol) in dry dichloromethane (20 mL). After the addition, the reaction was stirred at 0°C for 8 h. The solids were filtered off and the filtrate was washed subsequently with 1M aq. potassium bisulfate (40 mL) and brine (40 mL), dried over sodium sulfate, filtered and concentrated to afford the crude 5­(2­(2­chlorophenyl)­1­hydroxyethylidene)­2,2­dimethyl­1,3­ dioxane­4,6­dione (3.1g, quant. yield) as pale yellow oil, which was used directly in next step without further purification. LCMS (ESI) m/z: 239.1 [M­58+H]+. Step 2: Preparation of ethyl 4-(2-chlorophenyl)-3-oxobutanoate. A solution of 5­(2­(2­chlorophenyl)­1­hydroxyethylidene)­2,2­dimethyl­1,3­dioxane­4,6­dione (3 g, 10 mmol) in ethanol (40 mL) was stirred at 80°C for 4h. The reaction was concentrated and the residue was purified by Combi­Flash (Biotage, 40 g silica gel, eluted with ethyl acetate in petro ether from 5% to 10%) to afford ethyl 4­(2­chlorophenyl)­3­oxobutanoate (2.15 g, 8.96 mmol, 89.5% yield) as yellow oil. LCMS (ESI) m/z: 241.1 [M+H]+. Step 3: Preparation of methyl 3-amino-4-(2-chlorophenyl)butanoate. To a solution of ethyl 4­(2­chlorophenyl)­3­oxobutanoate (2 g, 8.33 mmol) in methanol (40 mL) were added ammonium acetate (6.4 g, 83.3 mmol), magnesium sulfate (3 g, 25 mmol) and sodium cyanoborohydride (1.05 g, 16.67 mmol). Then the mixture was stirred at 70°C (reflux) for 16h. The mixture was cooled down and poured into water (120 mL) and the solution was extracted with ethyl acetate (3 X 50 mL). The combined organic layers were concentrated and the residue was diluted with ethyl acetate (60 mL) and extracted with 1N hydrochloric acid (60 mL). The aqueous phase was separated and 50% aqueous sodium hydroxide was added to adjust the pH to 8~9. The basic solution was extracted with ethyl acetate (3 X 50 mL) and the combined organic layers were dried over sodium sulfate, filtered and concentrated to afford methyl 3­amino­4­(2­chlorophenyl)butanoate (1.17 g, 5.15 mmol, 61.9% yield) as yellow oil. LCMS (ESI) m/z: 228.1 [M+H]+. Step 4: Preparation of methyl 3-(5-chloro-1H-indole-2-carboxamido)-4-(2-chlorophenyl)butanoate. To a solution of methyl 3­amino­4­(2­chlorophenyl)butanoate (1 g, 4.4 mmol), 5­chloro­1H­indole­2­ carboxylic acid (0.86 g, 4.4 mmol) and HATU (2.5g, 6.6 mmol) in DMF (25 mL) was added DIPEA (1.7 g, 13.2 mmol) drop­wise under nitrogen. After the addition, the reaction was stirred at 20°C for 2 hour. The reaction mixture was diluted with ethyl acetate/water (30 mL/30 mL), extracted with ethyl acetate (2 X 30 mL). The combined organic layers were washed with brine (40 mL), and concentrated. The residue was purified by Combi­Flash (Biotage, 40 g silica gel, eluted with 7N ammonia in methanol:dichloromethane(1:10) in dichloromethane from 25% to 40%) to afford methyl 3­(5­chloro­1H­indole­2­carboxamido)­4­(2­ chlorophenyl)butanoate (1.6 g, 3.96 mmol, 89.9% yield) as yellow solid. LCMS (ESI) m/z: 405.0 [M+H]+. Step 5: Preparation of 3-(5-chloro-1H-indole-2-carboxamido)-4-(2-chlorophenyl)butanoic acid. To a solution of methyl 3­(5­chloro­1H­indole­2­carboxamido)­4­(2­chlorophenyl)butanoate (0.7 g, 1.73 mmol) in tetrahydrofuran/methanol/water (24 mL/24 mL/ 6mL) was added lithium hydroxide monohydrate (87 mg, 2.08 mmol) in one portion, then the reaction was stirred at 20°C for 16 hours. The reaction mixture was concentrated, diluted with water (30 mL) and acidified with 4N hydrochloric acid. The precipitate was filtered, washed with water and dried to afford 3­(5­chloro­1H­indole­2­carboxamido)­4­(2­ chlorophenyl)butanoic acid (0.45 g, 1.15 mmol, 66.6% yield) as white solid. LCMS (ESI) m/z: 391.0 [M+H]+. Step 6: Synthesis of enantiomer 1 and enantiomer 2 of 5-chloro-N-(1-(2-chlorophenyl)-4-(4- hydroxypiperidin-1-yl)-4-oxobutan-2-yl)-1H-indole-2-carboxamide: To a solution of 3­(5­chloro­1H­indole­2­carboxamido)­4­(2­chlorophenyl)butanoic acid (0.2 g, 0.51 mmol), piperidin­4­ol (52 mg, 0.51 mmol) and HATU (0.29 g, 0.77 mmol) in DMF (20 mL) was added DIPEA (0.2 g, 1.54 mmol) drop­wise under nitrogen. After the addition, the reaction was stirred at 20°C for 2 hour. The reaction mixture was then diluted with ethyl acetate/water (30 mL/30 mL) and extracted with ethyl acetate (2 X 30 mL). The combined organic layers were washed with brine (40 mL), and concentrated. The residue was purified by Combi­Flash (Biotage, 40 g silica gel, eluted with 7N ammonia in methanol :dichloromethane (1:10) in dichlormethane from 25% to 40%) to afford the racemic compound 5­chloro­N­(1­ (2­chlorophenyl)­4­(4­hydroxypiperidin­1­yl)­4­oxobutan­2­yl)­1H­indole­2­carboxamide (0.23 g, 0.48 mmol, 95% yield) as white solid.100 mg of this product was separated by chiral prep­HPLC ( instrument: SFC­80 (Thar, Waters), Column: OJ 20*250mm, 10um (Daicel) , Mobile phase: CO2/ methanol (0.2%Methanol Ammonia)= 40/60, Flow rate: 80 g/min, Sample solution: 100mg dissolved in 25 ml Methanol) to afford two isomers: Enantiomer 1 was obtained as white solid.1H NMR (400 MHz, DMSO­d6) į 11.70 (s, 1H), 8.46 (t. J=8.4 Hz, 1H), 7.71 (d, J=1.6 Hz, 1H), 7.42­7.34 (m,3H), 7.24­7.13 (m, 3H), 7.05 (s, 1H), 4.73 (d, J=4 Hz, 1H), 4.62 (brs, 1H), 3.95­3.62 (m, 3H), 3.28­2.90 (m, 4H), 2.83­2.73 (m, 1H), 2.63­2.53 (m, 1H), 1.84­1.59 (m, 2H), 1.47­1.15 (m, 2H). LCMS (ESI) m/z: 474.1 [M+H]+.; (Rt:3.22min). Enantiomer 2 was obtained as white solid.1H NMR (400 MHz, DMSO­d6) į 11.70 (s, 1H), 8.46 (t. J=8.4 Hz, 1H), 7.71 (d, J=1.6 Hz, 1H), 7.44­7.33 (m,3H), 7.25­7.13 (m, 3H), 7.05 (s, 1H), 4.73 (d, J=3.6 Hz, 1H), 4.63 (brs, 1H), 3.96­3.62 (m, 3H), 3.28­2.91 (m, 4H), 2.83­2.72 (m, 1H), 2.63­2.53 (m, 1H), 1.85­1.58 (m, 2H), 1.47­1.15 (m, 2H). LCMS (ESI) m/z: 474.0 [M+H]+.: (Rt: 4.41min).
Synthesis of stereoisomer 1 (Compound 325), stereoisomer 2 (Compound 326), stereoisomer 3 (Compound 327) and stereoisomer 4 (Compound 328) of (S)-N-(5-chioro-1 H~indoi-2-yi)~3-(2- ch!orophenyi)~2-((T*,4*)-4-hydroxycyciohexyiamino)propenamide.
Figure imgf000231_0001
Step 1 : tert-butyi 5-chtoro-1 H~indoi-2-yicarbamate.
Dry tert-butyl alcohol (40 mL), triethylamine (10 g, 100 mmol), and diphenylphosphoryl azide (7.5 g, 27.5 mmol) were added to a solution of 5-chloro-1H-indole-2-carboxylic acid (5 g, 25 mmol) in dioxane (140 mL) under Argon. The mixture was heated at 100 °C for 5 h. Upon cooling, the mixture was evaporated under vacuum, diluted in ethyl acetate, washed with a 5% aqueous citric acid, a 5% aqueous sodium bicarbonate, water, and brine, dried over magnesium sulfate, and concentrated under vacuum, The crude product was purified by SGC (Petroleum ether / Ethyl acetate = 10 : 1) to provide desired compound as yellow solid (1.75 g, 6.56 mmol, 26.2%). 1 H NMR (400 MHz, DMSO) 6 10.88 (s, 1H), 10.14 (s, 1 H), 7.35 (d, J = 8.5 Hz, 1 H), 7.32 (d, J = 2.0 Hz, 1 H), 6.89 (dd, J = 8.5, 2.1 Hz, 1 H), 5.87 (d, J = 1.6 Hz, 1 H), 1.51 (s, 9H); LCMS (ESI) m/z: 267.1 [M+H]+.
Step 2: 5-chloro-1 H-indo!-2-amine.2,2,2-tnfluoroacetate.
A. solution of tert-butyl 5-chloro-1H-indol-2-ylcarbamate (1 .437 g, 5.39 mmol) in trifluoroacetic acid (5 mL) and dichloromethane (10 mL) was stirred at 25 °C for 16h and the mixture was concentrated to afford a white solid (1 ,5g, crude), which was directly used in the next step. LCMS (ESI) m/z: 167.1 [M+H]+.
Step 3: (S)-fert-bufyl 1 -(5-chioro-1 H-indoi-2-ylammo)-3-(2~chtorophenyi)-1 -oxopropan-2~y!carbamate.
To a solution of (S)-2-(tert-butoxycarbonylamino)-3-(2-chlorophenyl)propanoic acid (852 mg, 2.85 mmol), DIPEA (732 mg, 5.68 mmol) and HATU (1 .3 g, 3.41 mmol) in DMF (8 mL) was added a solution of 5- chloro-1 H-indol-2-amine.TFA (400 mg, 1.42 mmol) in N,N-Dimethylformamide (4 mL) and the resultant mixture was stirred at room temperature for 5 min. 200rnL of water was added to the reaction mixture and the resultant grey solid was filtered and dried. The crude product was purified by Prep-HPLC (BOSTON pHlex ODS 10um 21 ,2x250mm120A. The mobile phase was acetonitrile/0.1 % trifluoroacetate) to get (S)-tert- butyl 1-(5-chloro-1H-indol-2-ylamino)-3-(2-chloropheny!)-1-oxopropan-2-ylcarbamate as white solid (200 mg, 0.356 mmol, 25%). 1H NMR (400 MHz, DMSO) 6 11.12 (s, 1 H), 10.69 (d, 1 H), 7.48-7.38 (m, 3H), 7.38 - 7.33 (m, 1 H), 7.32 - 7.16 (m, 3H), 6.95 (dd, J = 8.6, 2.0 Hz, 1 H), 6.11 (s, 1 H), 4.46 (dd, J = 14.0, 7.6 Hz, 1 H), 3.51 (s, 1 H), 3.22 (dd, J = 14.4, 5.3 Hz, 1 H), 3.02 (dd, J = 13.5, 9.5 Hz, 1H), 1.26 (d, 9H); LCMS (ESI) m/z: 448.2 [M+Hp.
Step 4: (S)-2-amino-N-(5-chioro-1 H-indoi-2~yi)-3~(2-chtorophenyl)propenamide,
A solution of (S)-tert-butyi 1-(5-chloro-1 H-indol-2-ylammo)-3-(2-chlorophenyl)-1-oxopropan-2- ylcarbamate (330 mg, 0.737 mmol) in TFA (1 .5 mL) and DCM (4 mL) was stirred at rt for 3h. The reaction mixture was diluted with dichloromethane (150 mL) and washed with saturated sodium bicarbonate solution (80 mL) and dried over sodium sulfate. The solution was concentrated to obtain (330 mg, crude) of the crude product as yellow oil. This was directly used for the next step. LCMS (ESI) m/z: 348.1 [M+H]+.
Step 5: synthesis of diastereomer 1 and diastereomer 2 of (S)-N-(5-chloro-1 H~indoi-2-yl)~3-(2- ch!orophenyi)-2-((T*,4*)-4-hydroxycyclohexylamino)propenamide.
A solution of (S)-2-amino-N-(5-chtoro-1 H-indol-2-yl)-3-(2-chlorophenyl)propanamide (330 mg, 0.737 mmol), 4-hydroxycyclohexanone (168 mg, 1.474 mmol), sodium borohydride (280 mg, 7.37 mmol) and acetic acid (1 drop) in methanol (15 mL) was stirred at room temperature for 18 h. The crude mixture was purified by Prep-HPLC (BOSTON pHlex ODS 10um 21 ,2x250mm120A. The mobile phase was acetonitrile/0.1 % Ammonium bicarbonate) to get two products: diastereomer 1 (70 mg, 0. 157 mmol, 21.3%). LCMS (ESI) m/z: 446.1 [M+HJ+ and diastereomer 2 (100 mg, 0.224 mmol, 30.4%). LCMS (ESI) m/z: 446.1 [M+H]+.
The above diastereomers were chirally separated to afford 4 enantimerically pure compounds. Stereoisomers 1 and 2 were obtained from diastereomer 1 and stereoisomers 3 and 4 were obtained from diastereomer 2:
Coinpound 325 (stereoisomer 1): 'H NMR (500 MHz, DMSO) 6 11.30 (s, 1 H), 10.81 (s, 1 H), 7.48 -
7.31 (m, 4H), 7.28 - 7.19 (m, 2H), 6.94 (dd, J = 8.5, 2.1 Hz, 1 H), 6.13 (s, 1 H), 4.42 (s, 1H), 3.63 (t, J = 7.0 Hz, 1 H), 3.06 (dd, 13.7, 6.9 Hz, 1 H), 2.98 (dd, J = 13.6, 7.5 Hz, 1 H), 2.24 (s, 1 H), 1.75 (dd, J = 37.3, 10.5 Hz, 4H), 1.14 - 0.88 (m, 4H): LCMS (ESI) m/z: 446.1 [M+H]+: (Rt: 3.246min).
Compound 326 (Stereoisomer 2): 'H NMR (500 MHz, DMSO) 6 11.25 (s, 1 H), 10.72 (s, 1 H), 7.47 - 7.33 (m, 4H), 7.28-7.19 (m, 2H), 6.95 (dd, J = 8.5, 1 .9 Hz, 1 H), 6.13 (s, 1 H), 4.43 (s, 1 H), 3.63 (t, J = 7.0 Hz, 1 H), 3.06 (dd, 13.6, 6.8 Hz, 1 H), 2.98 (dd, J = 13.7, 7.5 Hz, 1 H), 2.24 (s, 1 H), 1.75 (dd, J = 37.9, 12.1 Hz, 4H), 1.14 - 0.88 (rn, 4H); LCMS (ESI) m/z: 446.1 [M+H]+; (Rt: 4.515min).
Compound 327 (stereoisomer 3): ’H NMR (500 MHz, DMSO) 6 11.22 (s, 1 H), 10.67 (s, 1H), 7.48 -
7.32 (m, 4H), 7.30 - 7.19 (m, 2H), 6.95 (dd, J = 8.5, 2.1 Hz, 1 H), 6.12 (d, 1.5 Hz, 1 H), 4.27 (d, J = 3.3 Hz,
1 H), 3.63 (t, J = 7.1 Hz, 1 H), 3.56 (d, J = 2.6 Hz, 1 H), 3.08 (dd, J = 13.6, 6.7 Hz, 1 H), 3.00 (dd, J = 13.6, 7.7 Hz, 1 H), 2.36 (s, 1 H), 1 .57 - 1 .23 (m, 8H). LCMS (ESI) m/z: 446.1 [M+H]+ : (Rt:0.828min). Compound 328 (stereoisomer 4): NMR (500 MHz, DMSO) 6 11.29 (s, 1 H), 10.75 (s, 1 H), 7.48 - 7.32 (m, 4H), 7.30 - 7.18 (m, 2H), 6.94 (dd, J = 8.5, 2.1 Hz, 1 H), 6.13 (s, 1 H), 4.26 (d, J = 3.2 Hz, 1 H), 3.63 (t, J = 7.1 Hz, 1H), 3.56 (d, J = 2.6 Hz, 1 H), 3.07 (dd, J = 13.7, 6.7 Hz, 1 H), 3.00 (dd, J = 13.7, 7.7 Hz, 1H), 2.36 (s, 1 H), 1 .56 - 1 .20 (m, 8H). LCMS (ESI) m/z: 446.2 [M+H]* ; (Rt:0.938min).
Synthesis of enantiomer 1 (Compound 329) and enantiomer 2 (Compound 330) of 5-ch!oro-N-(2-(2,4- difluoropheriyi)-! -(3-ethyi-1,2,4-oxadiazoi-5-yi)ethyi)-1H-pyrrolo[2,3-b]pyridine-2-carboxamide:
Figure imgf000233_0001
Enantiomer 1 Enantiomer 2
Note: The reaction conditions used here ted to the racemization of (S)-amino acid used and the final product was chirally separated using HPLC.
Step 1 : Preparation of (S.Z)-tert-buty! 1-(1-aminopropySideneaminooxy)-3-(2,4-difiuorophenyi)-1- oxopropan-2-yicarbamate.
To a mixture of (Z)-N'-hydroxypropionimidamide (132 mg, 1 .5 mmoi) in acetone (15 mL) was added 1-(3-Dimethyiaminopropyi)-3-ethyicarbodiimide hydrochloride (575 mg, 3.0 mmol). The mixture was stirred at 10°C for O.Sh and then (S)-2-(tert-butoxycarbonylamino)-3-(2,4-difIuorophenyl)propanoic add (300 mg, 1.0 mmol) was added. The mixture was stirred further at 10 °C for 17 h. It was then filtered and the solvent was removed under the reduced pressure and the residue was purified by TLC (dichloromethane: metho! =15:1) to give the desired product (S,Z)-tert-butyl 1-(1-aminopropylideneaminooxy)-3-(2,4-difluorophenyl)-1- oxopropan-2-ylcarbamate (220 mg, 0.59 mmoi, yield: 59.3%) as a yellow oil. LCMS (ESI) m/z: 372.1
Step 2: Preparation of (S)-tert-butyl 2-(2,4-difiuoropheny!)-1-(3~ethy!-1,2,4-oxadiazol-5- yi)ethyicarbamate.
To a mixture of (S.Z)-tert-butyl 1-(1-aminopropylideneaminooxy)-3-(2,4-difluorophenyl)-1-oxopropan- 2-yicarbamate (200 mg, 0.54 mmol) in DMSO (8 ml) was added potassium hydroxide (31 mg, 0.54 mmol). The mixture was stirred at 10°C for 1 h, filtered and the solvent was removed under the reduced pressure. The crude product thus obtained was purified by prep-HPLC (SunFire C18, 4.6*50mm, 3. Sum column Xbridge C18 3.5pm 4.6x50mm column.The elution system used was a gradient of 5%-95% over 1 .5 min at 2ml/min and the solvent was aceton itrile/0.01% aqueous NH4HCO3) to give the desired product (S)-tert-butyl 2-(2,4-difluorophenyl)-1-(3-ethyl-1 ,2,4-oxadiazol-5-yl)ethylcarbamate (110 mg, 0.31 mmol, yield:
57.7 %) as a white solid. LCMS (ESI) m/z: 354.0 [M+H]+.
Step 3: Preparation of (S)-2-(2,4-difluorophenyl)-1-(3-ethyl-1 ,2,4-oxadiazoL5-yi)ethanamine.
A solution of (S)-tert-butyl 2-(2,4-difluorophenyl)-1 -(3-ethyi-1 ,2,4-oxadiazol-5-yl)ethylcarbamate (90 mg, 0.25 mmol) in trifluoroacetic acid (2 mL) and dichloromethane (8 ml) was stirred at 10°C for 2h. The solvent was removed under the reduced pressure to obtained the desired product (60 mg) which was used in the next, step without further purification. LCMS (ESI) m/z: 254.1 [M+H]+.
Step 4: Preparation of enantiomer 1 and enantiomer 2 of 5~ctsioro~N-(2-(2,4~difkiorophenyi}-1~(3-ettsyl- 1 ,2,4-oxadiazoi-5-yl)ethyi)-1 H-pyrroio[2,3-b]pyridine-2-carboxamide,
To a solution of (S)-2-(2,4-difluorophenyl)-1-(3-ethyl-1 ,2,4-oxadiazol-5-yl)ethanamine (60 mg, 0.24 mmol) in DMF (10 mL) were added 5-chioro-1H-pyrrolo[2,3-b]pyridine-2-carboxylic acid (71 mg, 0.36 mmol) and HATU (137 mg, 0.36 mmol) and DIPEA. (93 mg, 0.72 mmol) and the resultant mixture was stirred at 70 °C for 1 h. It was filtered and the solvent was removed under the reduced pressure and the residue was purified by prep-HPLC (SunFire C18, 4.6*50mm, 3. Sum column Xbridge C18 3.5pm 4.6x50mm column.The elution system used was a gradient of 5%-95% over 1 .5 min at 2ml/min and the solvent was acetonitrile/0.01% aqueous NH4HCO3) to give the desired product (70 mg, 0.16 mmol, yield: 67.9 %) as a white solid. 70 mg of this product was subjected to chiral separation ( instrument: SFC-80 (Thar, Waters), Column: OJ 20*250mm, 10um (Daicel) , Mobile phase: CO2/ methanol (0.2%Methanol Ammonia)^ 75/25, Flow rate: 80 g/min, Sample solution: 200 mg dissolved in 40 ml Methanol) to afford two isomers:
Compound 329 (enantiomer 1): 1H NMR (400 MHz, DMSO) 6 12.42 (s, 1H), 9.43 (d, J = 8.2 Hz, 1H), 8.31 (dd, J = 19.8, 2.3 Hz, 2H), 7.45 (dd, J = 15.4, 8.5 Hz, 1 H), 7.19 (dd, J = 11.5, 7.1 Hz, 2H), 7.02 (t, J = 8.5 Hz, 1H), 5.53 (dd, J = 14.5, 9.2 Hz, 1H), 3.52 - 3.38 (m, 2H), 2.79 - 2.65 (m, 2H), 1.22 (t, J = 7.5 Hz, 3H); LCMS (ESI) m/z: 432.1 [M+H]+; (Rt: 1.40min).
Compound 330 (enantiomer 2): 1H NMR (400 MHz, DMSO) 0 12.42 (s, 1 H), 9.43 (d, J = 8.0 Hz, 1 H), 8.31 (d, J = 18.1 Hz, 2H), 7.45 (d, J = 7.0 Hz, 1 H), 7.20 (d, J = 15.5 Hz, 2H), 7.03 (d, J = 6.6 Hz, 1 H), 5.53 (d, J = 5.6 Hz, 1 H), 3.42 (dd, J = 21.9, 16.1 Hz, 2H), 2.72 (q, J = 7.5 Hz, 2H), 1.22 (t, J = 7.5 Hz, 3H); LCMS (ESI) m/z: 432.1 [M+H]+; (Rt: 1.69min).
Synthesis of (S)-N-(1-(4-acetylpiperazin-1-yl)-3-(3-chlorophenyl)-1-oxopropan-2-yl)-5-chloro-1H-indole- 2-carboxamide (Compound 331):
Figure imgf000235_0001
Step 1: (S)-tert-butyl 1-(4-acetylpiperazin-1-yl)-3-(3-chlorophenyl)-1-oxopropan-2-ylcarbamate. A mixture of (S)­2­(tert­butoxycarbonylamino)­3­(3­chlorophenyl)propanoic acid (600 mg, 2.0 mmol), 1­(piperazin­1­yl)ethanone (202 mg, 2.0 mmol), HATU (1.14 g, 3.0 mmol) and DIPEA (774 mg, 6.0 mmol) in DMF (8 mL) was stirred at 25 ^ for 1 h. The mixture was then poured into brine (50 mL) and extracted with ethyl acetate (100 mL*2). The combined organic phase was concentrated and the resultant residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate=1:4) to afford the target compound (1.0g) as a light yellow oil. LCMS (ESI) m/z:410.2/412.0 [M+H]+. Step 2: (S)-1-(4-acetylpiperazin-1-yl)-2-amino-3-(3-chlorophenyl)propan-1-one hydrochloride. A mixture of (S)­tert­butyl 1­(4­acetylpiperazin­1­yl)­3­(3­chlorophenyl)­1­oxopropan­2­ylcarbamate (1.0g) and HCl (1,4­dioxane, 4 mol/L, 10 mL) was stirred at 25 ^ for 3h and then concentrated. The resultant residue was triturated with the mixed solvent of petroleum ether/ethyl acetate (100 mL/30mL) to afford the target compound (600 mg) as a light yellow oil. LCMS (ESI) m/z:310.2/312.1 [M+H]+. Step 3: (S)-N-(1-(4-acetylpiperazin-1-yl)-3-(3-chlorophenyl)-1-oxopropan-2-yl)-5-chloro-1H-indole-2- carboxamide. A mixture of (S)­1­(4­acetylpiperazin­1­yl)­2­amino­3­(3­chlorophenyl)propan­1­one hydrochloride (450 mg, 1.3 mmol), 5­chloro­1H­indole­2­carboxylic acid (254 mg, 1.3 mmol), HATU (741 mg, 1.95 mmol), DIPEA (839 mg, 6.5 mmol) and DMF (8 mL) was stirred at 25 ^ for 1 h. The mixture was then poured into water and extracted with ethyl acetate (70 mL*2). The combined organic phase was concentrated and the crude product thus obtained was purified by silica gel column chromatography (100% ethyl acetate) to afford 300 mg of a yellow oil, which was triturated with the mixed solvent of petroleum ether/ethyl acetate (50 mL/10 mL) to afford the target compound (180 mg, 0.37 mmol, yield:28.5%) as a white solid. 1H NMR (400 MHz, DMSO­d6) į 11.74 (s, 1H), 9.00 (t, J = 9.6, Hz, 1H), 7.72 (s, 1H), 7.43­7.38 (m, 2H), 7.29­7.22 (m, 4H), 7.18 (dd, J = 8.8, 1.6 Hz, 1H), 5.17­5.13 (m, 1H), 3.61­3.44 (m, 8H), 3.05­2.99 (m, 4H), 1.99 (s, 3H); LCMS (ESI) m/z: 487.0/489.1 [M+H]+. The following compounds were synthesized according to the protocol described for the Compound 331:
Figure imgf000236_0001
Figure imgf000237_0001
Figure imgf000238_0001
Figure imgf000239_0001
Figure imgf000240_0001
Figure imgf000241_0001
Figure imgf000242_0001
Figure imgf000243_0001
Figure imgf000244_0001
Figure imgf000245_0001
Figure imgf000246_0001
Figure imgf000247_0001
Figure imgf000248_0001
Figure imgf000249_0001
Figure imgf000250_0001
Figure imgf000251_0001
Figure imgf000252_0001
Figure imgf000253_0001
Figure imgf000254_0002
Synthesis of (S)-N-(1-(6-acetyl-2,6-diazaspiro[3.3]heptan-2-yl)-3-(2-chlorophenyl)-1-oxopropan-2-yl)-5-
Figure imgf000254_0001
Step 1 : Preparation of tert-butyl 6-acetyl-2,6-diazaspiro[3.3]heptane~2-carboxylate.
To a mixture of tert-butyl 2,6-diazaspiro[3.3]heptane-2-carboxylate (250 mg, 0.5 mmol, as oxalate) and DIPEA (2.1 mmol, 0.3 mL) in DCM (8 mL) was added acetyl chloride (1.1 mmol, 0.1 mL) at 0 °C. The mixture was stirred at room temperature for 1 h. Water (5 mL) was added and the product was extracted with DCM (2 x 30 mL). The organic phases was dried over NazSO^, filtered and concentrated to give the desired product as brown oil (97%). LCMS (ESI) m/z: 241 [M+H]+.
Step 2: Preparation of 1-(2,6-diazaspiro[3.3]heptan-2-yl)ethan-1-one.
To a solution of tert-butyl 6-acetyl-2,6-diazaspiro[3.3]heptane-2-carboxylate (240 mg, 1.0 mmol) in DCM (6 mL) was added TFA (2 mL) at 0 °C. The mixture was stirred at room temperature for 1h and concentrated. Water (5 mL) and saturated NaHCOs were added and the mixture was extracted with DCM (2 x 30 mL). The organic phases was dried over NazSCX filtered and concentrated to give the desired product as brown oil (57%). LCMS (ESI) m/z: 140 [M+H]+.
Step 3: Preparation of tert-butyl (S)-(1-(6-acetyi-2,6-diazaspiro[3.3]beptan-2-yi)-3-(2-cblorophenyi)-1- oxopropan-2-yl)carbamate.
To a mixture of 1-(2,6-diazaspiro[3.3]heptan-2-yl)ethan-1-one (80 mg, 0.57 mmol), (S)-2-((tert- butoxycarbonyl)amino)-3"(2-chlorophenyl)propanoic acid (205 mg, 0.68 mmol) and DIPEA (2.85 mmol, 0.5 mL) in DMF (8 mL) was added HATU (0.86 mmol, 325 mg) at 0 °C. The mixture was stirred at room temperature for 1h, water (5 mL) was added, and extracted with DCM (2 x 30 mL). The organic phases was dried over NazSCX concentrated and purified by column chromatography (5% MeOH in DCM) to give the desired product as brown solid (42%). LCMS (ESI) m/z: 422 [M +Hf .
Step 4: Preparation of (S)-1-(6~acestyl-2,6-diazaspiro[3.3]hepfan-2-yl)-2-amino-3-(2- chlorophenyi)propan-1 -one.
To a solution of tert-butyl (S)-(1-(6-acetyl-2,6-diazaspiro[3.3]heptan-2-yl)-3-(2-chlorophenyl)-1- oxopropan-2-yl)carbamate (100 mg, 0.24 mmol) in DCM (5 mL) was added TFA. (2 mL) at 0 °C. The mixture was stirred at room temperature for 1h and concentrated. Water (5 mL) and saturated NaHCOs (5 mL) were added and the resultant mixture was extracted with DCM (20 mL). The organic phase was dried over NazSCM, filtered and concentrated to give the desired product as brown solid (92%). LCMS (ESI) m/z: 322 [M +H]+.
Step S: Preparation of (S)-M-(1-(6-acetyl-2,8-diazaspiro[3.3]beptan~2-yl)~3-(2-chioropheriyl)~1- oxopropan-2-yl)-5-chioro-1H-indoie-2-carboxamide.
A. mixture of (S)-1-(6-acetyi-2,6-diazaspiro[3.3jheptan-2-yl)-2-amino-3-(2-chlorophenyl)propan-1-one (70 mg, 0.24 mmol), 5-chloro-1H-indole-2-carboxylic acid (50 mg, 0.28 mmol), DIPEA (0.1 mL) and HATU (78 mg, 0.28 mmol) in DMF (5 mL) was stirred at room temperature for 1 h. The mixture was purified by Prep- HPLC (SunFire C18, 4,6*50mm, 3. Sum column Xbridge C18 3.5pm 4.6x50mm column.The elution system used was a gradient of 5%-95% over 1.5 min at 2ml/min and the solvent was acetonitrile/0.01% aqueous ammonium bicarbonate.) to give the desired product as white solid (24.1 mg, yield 32%).
1H NMR (400 MHz, DMSO-cfe) 6 11.69 (s, 1H), 8.91 (d, J = 8.0 Hz, 1 H), 7.72(s, 1 H), 7.71-7.38 (m, 3H), 7.24-7.17 (m, 4H), 4.81-4.80 (m, 1 H), 4.41 - 4.37 (m, 1 H), 4.20-3.84 (m, 7H), 3.21 - 3.18 (m, 1H), 3.09 - 3.05 (m, 1 H), 1.70 (s, 3H); LCMS (ESI) m/z: 499 [M+H]+.
The following compounds were synthesized according to the protocol described for the Compound
387:
Figure imgf000256_0001
Preparation of (S)-5~ch toro-N-(3~(2~ch toropheny!)-1 -oxo-1 ~(2~oxo~1 ,2-dihydropyridin-4~yl)propan-2-yi)~
1 H-mdole-2-carboxamide (Compound 399):
Figure imgf000257_0001
Step 1 : Preparation of (S)-methy! 2-(tert-butoxycarbonylamino)-3-(2~chlorophenyi)propanoate.
To a solution of (S)-2-(tert-butoxycarbonylamino)-3-(2-chlorophenyl)propanoic acid (12 g, 40.1 mmol) and K2CO3 (11 g, 80.2 mmol) in MeCN (300 mL) was added iodomethane (6.8 g, 48.2 mmol) under ice-bath and the mixture was stirred for 16h at 25 °C. It was then filtered and concentrated to give the crude product, which was purified by flash column (EtOAc/PE 1 :2 )to give (S)-methyl 2-(tert-butoxycarbonylamino)-3-(2- chlorophenyl)propanoate as a white soild (10 g, 31.9 mmol, yield: 79.6 %).
Step 2: Preparation of (S)-tert-butyl 1 -(2-cb!orophei"syi)-3-hydroxypropai"s-2-yicarbamate.
To a solution of (S)-methyl 2-(tert-butoxycarbonyiamino)-3-(2-chlorophenyl)propanoate (7.2 g, 3.8 mmol) in EtOH (100.0 mL) was added NaBH* (954 mg, 25.2 mmol) under ice-bath condition and the mixture was warmed up to 25 °C and stirred further for 3h. Water (30 mL) was added and the mixture was extracted with EtOAc (300mL*3). The organic layer was dried over NazSO*, filtered and concentrated. The crude product thus obtained was purified by flash column (EtOAc/PE 1 :2 )to give (S)-tert-butyl 1-(2-chlorophenyl)-3- hydroxypropan-2-ylcarbamate as a white soild ( 5.3 g, 18.6 mmol, yield: 81.6 %).
Step 3: Preparation of (S)-tert-butyl 1 -(2-ch!orophenyl)-3-oxopropan~2-yicarbamate.
To a solution of (S)-tert-butyl 1-(2-chlorophenyl)-3-hydroxypropan-2-ylcarbamate (1.8 g, 6.3 mmol) in DMSO (10.0 mL) was added IBX (1.77 g, 6.3 mmol) under ice-bath condition and the mixture was warmed up io 25 °C and stirred for 16h. Water (10 mL) was added and the mixture was extracted with EtOAc (30mL*2). The organic layer was dried over NazSOt, filtered and concentrated, The crude residue was purified by flash column (EtOAc/PE 1 :2 )to give (S)-tert-butyl 1-(2-chlorophenyl)-3-oxopropan-2-ylcarbamate as a white soild ( 830 mg, 2.9 mmol, yield: 46 %). Step 4: Preparation of tert-butyl (2S)~3-(2-chtorophenyl)~1 -hydroxy-1 -(2-methoxypyridin~4-yi)propan-2- yicarbamate,
A solution of 4-bromo-2-methoxypyridine (570 mg, 3.0 mmol) in THF (100.0 mL) was cooled to -70c,C under argon atmosphere and BuLi (3.5 ml, 8.7 mmol) was added carefully. Then a solution of (S)-tert-butyl 1- (2-chlorophenyl)-3-oxopropan-2-ylcarbamate (800 mg, 2.8 mmol) in THF (10 mL ) was added and the mixture was stirred at a temperature of -65°C to 20cC for 2h. The reaction was then quenched with 4 mL concentrated HCI in 5 mL H2O and 20 mL of EteO. The organic layer was washed with brine and dried over anhydrous MgSOa followed by filtration and removal of solvent with rotary evaporation. The crude product was purified by Prep-HPLC to give tert-butyl (2S)-3-(2-chlorophenyl)-1-hydroxy-1-(2-methoxypyridin-4- yl)propan-2-yicarbamate as a white soiid (360 mg, 0.92 mmol, yield: 33 %) 1H NMR (400 MHz, DMSO) 6 8.17 - 7.96 (m, 1 H), 7.33 (d, 11.2 Hz, 2H), 7.28 - 7.07 (m, 2H), 6.93 (dd, 30.4, 5.3 Hz, 1 H), 6.75 (t, J
= 17.7 Hz, 1 H), 6.44 (d, J = 9.4 Hz, 1 H), 5.74 (dd, J = 30.7, 5.1 Hz, 1 H), 4.66 - 4.52 (m, 1 H), 4.00 (s, 1 H), 3.82 (d, J = 2.8 Hz, 3H), 3.07 - 2.93 (m, 1 H), 2.69 (dd, J = 25.2, 11.3 Hz, 1 H), 1.16 (d, J = 4.5 Hz, 9H).
Step 5: Preparation of (2S)-2-amino-3-(2-chtorophenyi)-1-(2-methoxypyridin-4-yl)propan-1-oi hydrochloride.
To a solution of tert-butyl (2S)-3-(2-chloropheny!)-1-hydroxy-1-(2-methoxypyridin-4-yl)propan-2- ylcarbamate (220 mg, 0.56 mmol) in DCM (10.0 mL) was added HCI in dioxane solution (0.36 mL,4.0 M) and the resultant mixture was stirred at 25°C for 2 h under N2. The white solid thus formed was filtered and washed with petroleum ether (30 mL*2) to give the crude product as light yellow solid (180 mg, 0.53 mmol, ye lid 94%).
Step 6: Preparation of 5-chioro-N-((2S)-3~(2-chlorophenyl)-1 -hydroxy-1 -(2-methoxypyndsn-4-yi)propan- 2-yl)-1 H-indoie-2 -carboxamide.
To a solution of 2,5-dioxopyrrolidin-1-yl 5-chloro-1 H-indole-2-carboxylate (300 mg, 1.0 mmol) and (2S)-2-amino-3-(2-chlorophenyj)-1-(2-methoxypyridin-4-yl)propan-1-ol hydrochloride (360 mg, 360 mmol) in ACN (30 mL) and H2O (10 mL) was added EtsN (303 mg, 3.0 mmol) under ice-bath condition and the reaction mixture was warmed up to 25 °C and stirred for 17h . Then the pH of the mixture was adjusted to 2 ~ 3 with 1M HCi and extracted with EtOAc (100mL*3). The organic layer was dried over Na2SO4, filtered and concentrated. The crude product was purified by Prep-HPLC to give 5-chloro-N-((2S)-3-(2-chlorophenyl)-1- hydroxy-1-(2-methoxypyridin-4-yl)propan-2-yl)-1 H-indole-2-carboxamide as a while solid ( 260 mg, 0.55 mmol, 55 %) JH NMR (400 MHz, DMSO) 6 11.59 (d, J = 10.4 Hz, 1H), 8.50 (d, J = 8.9 Hz, 1 H), 8.18 (d, J = 9.5 Hz, 1 H), 8.05 (dd, J = 13.3, 5.2 Hz, 1 H), 7.70 (dd, J = 8.1 , 2.0 Hz, 1 H), 7.42 (dd, J = 9.2, 5.3 Hz, 1 H), 7.33 (ddd, J = 14.1 , 11.3, 6.2 Hz, 2H), 7.13 (ddd, J = 35.0, 17.2, 13.8 Hz, 4H), 7.04 (d, J = 5.7 Hz, 1 H), 6.97 (d, J = 5.0 Hz, 1 H), 6.80 (d, J = 33.0 Hz, 1 H), 4.78 (dd, J = 17.8, 4.4 Hz, 1 H), 4.62 (s, 1 H), 4.40 (s, 1 H), 3.79 (d, J = 11.4 Hz, 3H), 3.15 (d, J = 14.0 Hz, 1 H), 2.94 (dd, J = 26.6. 15.6 Hz, 1 H). Step 7: Preparation of 5-chioro-N-((2S)-3~(2-chtorophenyl)-1-hydroxy~1-(2-oxo-1,2-dihydropyridsn-4~ yi)propafi-2-yi)-1 H-sndoie-2 -carboxamide.
A mixture of 5-chloro-N-((2S)-3-(2-chlorophenyl)-1-hydroxy-1-(2-methoxypyridin-4-yl)propan-2-yl)-1H- indole-2-carboxamide (100 mg, 0.2 mmol) and pyridine hydrochloride (500 mg, 4.3 mmol) was heated to 150 °C for 1 h. Then the resultant mixture was diluted with ethyl acetate (100 mL) and washed with brine (10 mL*3). The organic layer was dried over NasSO-s, filtered and concentrated to give the crude product, which was purified by Prep-HPLC to give 5-chloro-N-((2S)-3-(2-chlorophenyl)-1-hydroxy-1-(2-oxo-1 ,2- dihydropyridin-4-yl)propan-2-yl)-1 H-indole-2-carboxamide as a white solid (50 mg, 0.11 mmol, yield: 55 %).
Step 8: Preparation of (S)-5~chioro~N-(3-(2-chioropheriyl)~1-oxo-1-(2-oxo-1 ,2-dibydropyridm-4- y!)propan-2-yl)-1 H-ii"sdole-2-carboxainide.
To a solution of 5-chloro-N-((2S)-3-(2-chlorophenyl)-1-hydroxy-1-(2-oxo-1 ,2-dihydropyridin-4- yl)propan-2-yl)-1 H-indoie-2-carboxamide (50 mg, 0.11 mmol) in DCM (10.0 ml ) was added a solution of DMP (46 mg, 0.11 mmol) in DMSO (1.0 mL) dropwise over Smin and the resultant mixture solution was stirred at 25 °C for 2h. Then the mixture was diluted with ethyl acetate (100 mL) and washed with brine (10 mL*3). The organic layer was dried over NazSCX filtered and concentrated to give the crude product, which was purified by Prep-HPLC to give (S)-5-chloro-N-(3-(2-chloropheny!)-1-oxo-1-(2-oxo-1 ,2-dihydropyridin-4- yl)propan-2-yl)-1 H-indole-2-carboxamide as a white solid (11 mg, 0.024 mmol, yield: 22 %). 1H NMR (400 MHz, DMSO) 6 11.89 (s, 1 H), 11.76 (s, 1 H), 9.19 (d, J = 8.1 Hz, 1 H), 7.71 (d, J = 1.9 Hz, 1 H), 7.53 - 7.30 (m, 4H), 7.27 - 7.12 (m, 3H), 7.08 (d, J = 1.5 Hz, 1 H), 6.86 (d, J = 1.1 Hz, 1 H), 6.44 (d, J = 6.7 Hz, 1 H), 5.46 (td, J = 9.9, 4.8 Hz, 1 H), 3.40 - 3.34 (m, 1 H), 3.17 (dd, J = 13.9, 10.1 Hz, 1 H). LCMS(ESI) m/z: 454.1 [M+H]+.
Synthesis of (S)-5~chioro~N-(3-(2-chioropheriyl)~1 -(6-methoxypyiidm~3-yi)-1 -oxopropan-2~yi)-1 H- mdoie-2 -carboxamide (Compound 391 ), (R)-5-cbtoro-N-(3-(2-ch torophenyi)-! -(6-methoxypyridin-3-yl)~ 1 -oxopropan-2-yi)-1 H-indoie-2-carboxamide (Compound 392), (S)-5-ch!oro-N-(3-(2-ch!orophenyi)-1 - oxo-1-(6-oxo-1,6-dihydropyridm-3-yi)propan-2~yl)-1H~mdoie-2~carboxamide (Compound 393) and (R)~ 5-ch!oro-N-(3-(2-ch!orophenyi)-1 -oxo-1 -(6-oxo-1,6-dihydropyridin-3-y!)propan-2-yl)-1 H-indo!e-2- carboxamide (Compound 394),
Figure imgf000260_0001
Mote: Even though the reaction procedure used a enantiomerically pure starting material, alter step-4, minor amounts of the other enantiomer were found, which necessitated the chiral separation.
Step 1 : (S)-tert-butyi 3~(2-chlorophenyl)-1-(methoxy(methyi)amino)-1-oxopropan-2-ylcarbamate.
A solution of (S)-2-(tert-butoxycarbonylamino)-3-(2-chtorophenyl)propanoic acid (3 g, 10 mmol), N,O- dimethylhydroxylamine hydrochloride (975 mg, 10 mmol), DIPEA (5.16 mg, 40 mmol) and HATU (4.56 g, 12 mmol) in dichloromethane (100 ml) was stirred at RT for 2h. The mixture was concentrated under vacuum and purified by flash (Petroleum ether / Ethyl acetate = 3: 1) to get a colorless oil (3.1 g, 9 mmol, 90%), LCMS (ESI) m/z: 287.1 [M-55]\
Step 2: (S)-tert-butyl 3-(2-cb iorophenyl)-1 -(6-methoxypyndm~3-yi)~1 -oxopropan-2~yicarbamate,
5-bromo-2-methoxypyridine (733 mg, 3.9 mmol) was slowly added to a suspension of sodium hydride (156 mg, 3.9 mmol) in tetrahydrofuran (16 mL) at 0°C. After the mixture was stirred for 10 minutes, n- butyllithium (1.56 mL 2.5 M in tetrahydrofuran) was added drop wise over a period of 15 min at -78°C. The mixture was stirred for 20 minutes at -78°C and (S)-tert-butyl 3-(2-chlorophenyl)-1-(methoxy(methyl)amino)-1- oxopropan-2-ylcarbamate (1.34 g, 3.9 mmol) in tetrahydrofuran (16 mL) was added dropwise. After 2h the reaction was quenched with 10 mL concentrated ammonium chloride and 150 mL of diethyl ether. The organic layer was washed with brine and dried with anhydrous sodium sulfate followed by filtration and removal of solvent with rotary evaporation. The crude product was purified by Prep-HPLC (BOSTON pHlex ODS 10um 21.2x250mm120A. The mobile phase was acetonitrile/0.1 % Ammonium bicarbonate) to give (S)- tert-butyl 3-(2-chlorophenyl)-1-(6-methoxypyridin-3-yl)-1-oxopropan-2-ylcarbamate as a white solid (920 mg, 2.35mmol, yield: 40 %). LCMS (ESI) m/z: 391.2
Step 3: (S)-2-x3mino-3-(2-chioropheny!)-1-(6-methaxypyndin-3-yl)propan-1-one hydrochioride and (S)- 5-(2-amino-3-(2-chiorophenyi)propanoyi)pyridin-2(1 H)~one hydrochioride.
A solution of (S)-tert-butyl 3-(2-chlorophenyl)-1-(6-methoxypyridin-3-yl)-1-oxopropan-2-yicarbamate (400 mg, 1 mmol) in hydrochloric acid /dioxane (4M, 5 mL) and dichloromethane (20 mL) was stirred at room temperature for 3h. The mixture was then concentrated to get a mixture of products (S)-2-amino-3-(2- chtorophenyl)-1-(6-methoxypyridin-3-yl)propan-1-one hydrochloride and (S)-5-(2-amino-3-(2- chlorophenyl)propanoyl)pyridin-2(1 H)-one hydrochloride.. LCMS (ESI) m/z: 291.1 [M+H]+, 277.2[M+H]+.
Step 4: 5-ch toro-N-(3~(2-ch torophenyl)-1 -(6~methoxypyridm-3~yi)-1 -oxopropan~2-yi)~1 H-indoie-2- carboxamide (S isomer enriched) and 5-cbtoro-N-(3-(2-chtorophenyi)-1-oxo-1-(6-oxo-1 ,6- dshydropyridin-3-y!)propan-2~yi)-1 H-indo!e-2-carboxamide (S isomer enriched).
A solution of 5-chloro-1H-indole-2-carboxylic acid (196 mg, 1 mmol), DIPEA (516 mg, 4 mmol), the mixture of (S)-2-amino-3-(2-chlorophenyl)-1-(6-methoxypyridin-3-yl)propan-1-one hydrochloride and (S)-5-(2- amino-3-(2-chlorophenyl) propanoyi)pyridin-2(1H)-one hydrochloride (340 mg, 1 mmol), and HATU (456 mg, 1 .2 mmol) in DMF (5 mL) was stirred at RT for 2h. The crude product thus obtained was purified by Prep- HPLC (BOSTON pHlex ODS 10um 21 2x250mm120A. The mobile phase was acetonitrile/0.1 % Ammonium bicarbonate) to get two white solids, 5-chloro-N-(3-(2-chlorophenyl)-1-(6-methoxypyridin-3-yl)-1-oxopropan-2- yl)-1 H-indole-2-carboxamide (S isomer enriched) (180 mg, 0.384 mmol, 36.6%), LCMS (ESI) m/z:
468.1 [M+HP; and 5-chloro-N-(3-(2-chlorophenyl)-1-oxo-1-(6-oxo-1 ,6-dihydropyridin-3-yl)propan-2-yl)-1H- indoie-2-carboxamide (S isomer encirhced) (100 mg, 0.22 mmol, 21 %), LCMS (ESI) m/z: 454.0[M+H]+.
Step 5: (S)-5-chioro-N-(3-(2-chioropheny!)-1 -(6-methoxypyndin-3-yi)-1 -oxapropan-2-yl)-1 H-indofe-2- carboxamide (Compound 391 ) and (R)~5-chloro-N~(3-(2-chiorophenyi)-1~(6-methoxypyridin-3-yl)-t- oxopropan-2-yl)-1 H-mdo!e-2-cart3oxamide (Compound 392).
A. solution of 5-chloro-N-(3-(2-chlorophenyi)-1-(6-methoxypyridin-3-yi)-1-oxopropan-2-yl)-1H-indole-2- carboxamide (100 mg, 0.214 mmol) in methanol was separated by Chiral-HPLC (column: OD, 20*250 mm, 10pm. co-solvent: methonal (0.2% methanol ammonia)) to give (S)-5-chloro-N-(3-(2-chlorophenyl)-1-(6- methoxypyridin-3-yl)-1-oxopropan-2-yl)-1H-indole-2-carboxamide (66.5 mg, 66.5%) as white solid and (R)-5- chtoro-N-(3-(2-chlorophenyl)-1-(6-methoxypyridin-3-yl)-1-oxopropan-2-yl)-1H-indole-2-carboxamide (3.8 mg, 3.8%)
Compound 391: 1H NMR (400 MHz, DMSO) 611.75 (s, 1H), 9.21 (d, J= 7.3 Hz, 1H), 8.87 (d, J = 2.2 Hz, 1 H), 8.22 (dd, J = 8.8, 2.5 Hz, 1 H), 7.70 (d, J = 2.0 Hz, 1 H), 7.49 - 7.43 (m, 1 H), 7.43 - 7.33 (m, 2H), 7.27-7.15 (m, 3H), 7.12 (s, 1H), 6.92 (d, J = 8.8 Hz, 1H), 5.68 (s, 1H), 3.91 (s, 3H), 3.38 (dd, J = 14.1, 4.9 Hz, 1H), 3.25 - 3.16 (m, 1H). LCMS (ESI) m/z: 468.1 (M+H)*; (Rt:4.74min).
Compound 392: 1H NMR (400 MHz, DMSO) 611.82 (s, 1H), 9.27 (s, 1H), 8.86 (d, J = 2.3 Hz, 1H), 8.22 (dd, J= 8.8, 2.2 Hz, 1H), 7.69 (d, J = 1.7 Hz, 1H), 7.50 -7.43 (m, 1H), 7.43-7.33 (m, 2H), 7.27-7.14 (m, 4H), 7.11 (s, 1H), 6.92 (d, J = 8.8 Hz, 1H), 5.67 (s, 1H), 3.90 (s, 3H), 3.40 (dd, J =14.4, 4.7 Hz, 1H), 3.21 (dd, J = 13.8, 10.1 Hz, 1H). LCMS (ESI) m/z: 468.1 [M+H]+; (Rt: 1.80min).
Step 6: (R)-5~ctiioro~N-(3-(2~ctiiorophenyl)~1 -oxo-1 -(6~oxo-1 ,6-dihydropyridin-3~yi)propan-2-yl)-1 H~ indole-2 -carboxamide (Compound 394) and (S)-5-chloro-N-(3-(2-chlorophenyi)-1-oxo-1-(8-oxo-1,6- dihydropyridin~3-yi)propan-2~yi)-1 H-indoie-2-carboxamide (Compound 393).
A solution of 5-chloro-N-(3-(2-chlorophenyl)-1-oxo-1-(6-oxo-1,6-dihydropyridin-3-yl)propan-2-yl)-1H- indole-2-carboxamide (100 mg, 0.22 mmol) in methanol was separated by Chiral-HPLC (column: ID, 20*250 mm, 10pm. co-solvent: methonal (0.2% methanol ammonia)) to give (R)-5-chloro-N-(3-(2-chlorophenyl)-1- oxo-1-(6-oxo-1,6-dihydropyridin-3-yl)propan-2-yl)-1H-indole-2-carboxamide (Compound 394) (4.8 mg, 4.8%) and (S)-5-chloro-N-(3-(2-chlorophenyl)-1-oxo-1-(6-oxo-1,6-dihydropyridin-3-yl)propan-2-yl)-1H-indole-2- carboxamide (Compound 393) (17.5 mg, 17.5 %) as white solids.
Compound 394: 1H NMR (400 MHz, DMSO) 511.72 (s, 1H), 9.11 (d, J= 8.3 Hz, 1H), 8.34 (d, J = 2.5 Hz, 1H), 7.86 (dd, J = 9.7, 2.6 Hz, 1H), 7.70 (d, J = 1.8 Hz, 1H), 7.49 -7.43 (m, 1H), 7.42-7.35 (m, 2H), 7.27-7.13 (m, 4H), 6.31 (d, J= 9.6 Hz, 1H), 5.58 (d, J=5.G Hz, 1H), 3.27 (d, J = 5.0 Hz, 1H), 3.16 (dd, J = 14.6, 9.4 Hz, 1H). LCMS (ESI) m/z: 454.0 [M+H]*.; (Ri: 1.09min).
Compound 393: 1H NMR (400 MHz, DMSO) 611.72 (s, 1H), 9.13 (d, J= 8.6 Hz, 1H), 8.32 (s, 1H), 7.87 (dd, J= 9.7, 2.4 Hz, 1H), 7.71 (s, 1H), 7.50 -7.42 (m, 1H), 7.38 (d, J = 8.4 Hz, 2H), 7.25-7.12 (m, 4H), 6.35 (d, J = 9.7 Hz, 1H), 5.57 (id, J= 8.9, 4.9 Hz, 1H), 3.28 (d, J = 4.6 Hz, 1H), 3.16 (dd, J = 13.7, 10.0 Hz, 1H). LCMS (ESI) m/z: 454.0 [M+H]*;(Rt:2.07min).
Synthesis of 5-chloro-N~(3-(2,4-difiuorophenyl)~1 -(2~methoxypyndin-4-yl)-1 -oxopropan-2-yl)-1 H-indoie- 2-carboxamide (Compound 395) and 5-chloro-N-(3~(2,4-difluorophenyl)~1-oxo-1-(2~oxo-1,2- dihydropyridin-4-yi)propan-2-y!)-1 H-indote-2-carboxamide (Compound 396):
Figure imgf000263_0001
Step 1 : (S)-2-ximino-3-(2,4-difluorophenyl)-1 -(2-methoxypyndin-4-yl)propan-1 -one hydrochloride.
A. solution of (S)-tert-butyl 3-(2,4-difluorophenyl)-1-(2-methoxypyridin-4-yl)-1-oxopropan-2- ylcarbamate (146 mg, 0.372 mmol) in hydrogen chloride/ dioxane (3 mL, 4M) was stirred at 20 °C for 16 h and the mixture was concentrated to afford (S)-2-amino-3-(2,4-difluorophenyl)-1-(2-methoxypyridin-4- yl)propan-1-one hydrochloride as a white solid (120 mg, crude), which was directly used for the next step. LCMS (ESI) m/z: 293.1 [M+H]
Step 2: 5-chioro-N-(3-(2,4-difluorophenyi)-1 -(2~methoxypyridin-4-y!)-1 -oxopropan-2-yi)~1 H-indo!e-2- carboxamide (Compound 395).
To solution of (S)-2-amino-3-(2,4-difluorophenyl)-1-(2-methoxypyridin-4-yl)propan-1-one hydrochloride (40 mg, 0.12 mmol), DIPEA (76 mg, 0.6 mmol) and 5-chloro-1H-indole-2-carboxylic acid (24 mg, 0.12 mmol) in DMF (1 mL) was slowly added HATU (60 mg, 0.156 mmol) at 20°C and the resultant mixture was stirred at 20°C for 1 h. The resultant crude product was purified by Prep-HPLC (BOSTON pHlex ODS 10um 21.2x250mm120A. The mobile phase was acetonitrile/0.1 % Formic acid) for two times to get (10 mg, 0.021 mmol, 14.2 %) as white solid. NMR (400 MHz, DMSO) 6 11.75 (s, 1 H), 9.19 (d, J = 8.2 Hz, 1 H), 8.31 (d, J = 5.3 Hz, 1 H), 7.69 (d, J = 1.9 Hz, 1 H), 7.44 (dd, J = 15.4, 8.6 Hz, 1 H), 7.39-7.33 (m, 2H), 7.24 (s, 1 H), 7.19 - 7.09 (m, 2H), 7.05 (d, J = 1.4 Hz, 1 H), 6.98 (td, J = 8.5, 2.3 Hz, 1 H), 5.51 (td, J = 9.8, 4.7 Hz, 1 H), 3.85 (s, 3H), 3.28 (dd, J = 14.1 , 4.5 Hz, 1 H), 3.11 (dd, J = 13.9, 10.1 Hz, 1H). LCMS (ESI) m/z: 470.0[M+H]+. Step 3: 5-chloro-N-(3-(2,4-difluorophenyl)-1 -oxo-1 ~(2-oxo~1 ,2-dihydropyridin-4-yl)propan~2-yi)~1 H- indoie-2~carboxamide (Compound 396).
To a solution of iodotrimethylsilane (30 mg, 0.149 mmol) in acetonitrile (25 mL) at 0 °C was added a solution of 5-chloro-N-(3-(2,4-difluorophenyl)-1-(2-methoxypyridin-4-yl)-1-oxopropan-2-yl)-1H-indole-2- carboxamide (35 mg, 0.07411 mmol) in acetonitrile (5 mL). The resulting mixture was stirred at 80 °C for 1 h. Then the mixture was cooled to room temperature. LCMS showed 82% product. The mixture with CP- 0022561-166 was purified by Prep-HPLC (BOSTON pHlex ODS 10um 21.2x250mm120A. The mobile phase was acetonitrile/0.1% Formic acid) to get 5-chtoro-N-(3-(2,4-difluorophenyl)-1-oxo-1-(2-oxo-1 ,2- dihydropyridin-4-yl)propan-2-yl)-1 H-indole-2-carboxamide (25 mg, 0.055 mmol, 64.5%) as white solid. 1H NMR (400 MHz, DMSO) 6 1 1.87 (s, 1 H), 1 1.79 (s, 1 H), 9.18 (d, J = 8.2 Hz, 1 H), 7.71 (d, J = 1.8 Hz, 1 H), 7.49 - 7.40 (m, 2H), 7.37 (d, J = 8.7 Hz, 1 H), 7.16 (ddd, J = 12.0, 9.4, 2.3 Hz, 2H), 7.07 (d, J = 1.3 Hz, 1 H), 6.98 (td, J = 8.5, 2.5 Hz, 1 H), 6.85 (s, 1 H), 6.44 (d, J = 5.6 Hz, 1 H), 5.39 (td, J = 9.7, 4.9 Hz, 1 H), 3.24 (dd, J = 13.9, 4.3 Hz, 1 H), 3.07 (dd, 13.9, 10.2 Hz, 1 H); LCMS (ESI) m/z: 456.0 [M+H]+.
Synthesis of (S)-N-(3-(2-chtorophenyl)-1 -(4-hydroxypiperidin-1 -yl)-1 -oxopro pan-2 -yl)-4-cyc!opiopyi- 1 H-pyrrole-2 -carboxamide (Compound 397):
Figure imgf000264_0001
Step 1 : l-tert-butyi 2 -methyl 4-bromo-1 H-pyiToie-1 ,2-dicarboxylate.
A. mixture of methyl 4-bromo-1H-pyrrole-2-carboxylate (1 .015 g, 5 mmol}, 4-Dimethylaminopyridine (31 mg, 0.25 mmol), triethylamine (1515 mg, 15 mmol) and Di-tert-butyi dicarbonate (1.31 g, 6 mmol) in dichloromethane (25 mL) was stirred at 25"C for 16 hrs. The mixture was concentrated and purified by flash chromatography eluting with 0-30% ethyl acetate in petro ether to afford 1 -tert-butyl 2-methyl 4-bromo-1 H- pyrrole-1 ,2-dicarboxylate (1.5 g, 4.95 mmol, 99% yield) as yellow oil. LCMS (ESI) m/z: 247.9, 249.9[M- 56+HJ*. Step 2: 1-tert-butyl 2 -methyl 4-cyciopropyM H-pyrroie-1 ,2-dicarboxylate.
A mixture of 1-tert-butyl 2-methyl 4-bromo-1 H-pyrrole-1 ,2-dicarboxylate (0.7 g, 2.3 mmol), cyclopropylboron ic acid (0.59 g, 6.9 mmol), tetrakis(triphenylphosphine)palladium (0.4 g, 0.34 mmol) and potassium carbonate (0.73 g, 6.9 mmol) in toluene (60 mL) was stirred at 110°C under inert, atmosphere (nitrogen) for 16 h. The reaction was cooled down, diluted with ethyl acetate (40 mL) and filtered through a pad of Celite. The filtrate was concentrated and the residue was purified by Combi-Flash (Biotage, 40 g silica gel, eluted with ethyl acetate in petro ether from 5% to 10%) io afford 1-tert-butyl 2-methyl 4-cyclopropyl-1 H- pyrrole-1 ,2-dicarboxylate (0.33 g, 1.25 mmol, 54.1 % yield) as yellow oil. LCMS (ESI) m/z: 210.1[M-56+H]*.
Step 3: 4-cydopropyM H~pyrroie~2-carboxyiic acid.
To a solution of 1 -tert-butyl 2-methyl 4-cyclopropyl-1 H-pyrrole-1 ,2-dicarboxylate (0.3 g, 1 .13 mmol) in ethanol/water (5 mL/ 5 mL) was added sodium hydroxide (90 mg, 2.26 mmol). The reaction mixture was stirred at 60°C for 3 h and concentrated. The residue was diluted with ethyl acetate/water (10 mL/ 10 mL), neutralized with 1 N Hydrochloric acid aqueous solution and extracted with ethyl acetate (10 mL) twice. The combined organic phase was washed with brine (15 mL), dried over sodium sulfate, filtered and concentrated to afford 4-cyclopropyl-1H-pyrrole-2-carboxylic acid (0.14 g, 0.93 mmol, 82.3% yield) as yellow oil. LCMS (ESI) m/z: 152.1 [M+H]+.
Step 4: (S)-N-(3-(2~chlorophenyi)~1 -(4-hydroxypiperidin-1 -yi)-1 -oxopropan~2-y!)~4-cyclopropyl-1 H- pyrroie-2 -carboxamide.
To a solution of (S)-2-amino-3-(2-chlorophenyl)-1-(4-hydroxypiperidin-1-yl)propan-1-one (0.12 g, 0.4 mmol), 4-cyclopropyl-1H-pyrrole-2-carboxylic acid (60 mg, 0.4 mmol), HATU (0.31 g, 0.6 mmol) in DMF (5 mL) was added DIPEA (0.1 g, 0.8 mmol) and the mixture was stirred at 20°C for 16 h. The reaction mixture was diluted with ethyl acetate/water (10 mL/ 10 mL), extracted with ethyl acetate (10 mL) twice. The combined organic phase was washed with brine (10 mL), dried over sodium sulate, filtered and concentrated. The residue was purified by Combi-Flash (Biotage, 20 g silica gel, eluted with 7N ammonia methanol:dichloromethane=1 :10 in dichloromethane from 20% to 40%) to afford 30 mg crude product which was further purified by PREP-HPLC to afford (S)-N-(3-(2-chlorophenyl)-1-(4-hydroxypiperidin-1-yl)-1- oxopropan-2-yl)-4-cyclopropyl-1 H-pyrrole-2-carboxamide (15.7 mg, 0.038 mmol, 9.5% yield) as white solid. !H NMR (400 MHz, d-DMSO) 6 11.06 (brs, 1H), 8.23 (d, J = 8.8 Hz, 1 H), 7.44-7.33 (m, 2H), 7.27-7.16 (m, 2H), 6.67-6.61 (m, 2H), 5.27-5.17 (m, 1 H), 4.75-4.68 (m, 1 H), 3.96-3.55 (m, 3H), 3.28 - 2.91 (m, 4H), 1.74- 1.42 (m, 3H), 1.32-0.95 (m, 2H), 0.80-0.70 (m, 2H), 0.45-0.35 (m, 2H). LCMS (ESI) m/z: 416.0[M+H}+; (RL1.073 min, ee=100%). Synthesis of (S)-N-(3~(1 H-imidazol-4-yl)-1 -morphoimo-1 -oxopropan-2-yl)-6-chioro~1 H-indoie-2- carboxamide (Compound 398):
Figure imgf000266_0001
Step 1 : Preparation of (tert-butoxycarbonyi)-L-histidine.
To a solution of L-histidine (500 mg, 3.2 mmol) in tetrahydrofuran (5 mL) were added di-tert-butyl dicarbonate (774 mg, 3.5 mmol) and NaOH (256 g, 6.4 mmol) in water (5 mL). The mixture was stirred at 25°C for 2h and concentrated to remove organics. The pH of the resultant solution was adjusted to 4 with 1 N. HCI followed by extraction with dichioromethane (20 mL*3). The organics were washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated. The crude product (260 mg) was used in the next step without further purification. LCMS (ESI) m/z: 300.1 [M+100-55-f-H]+.
Step 2: Preparation of tert-butyl (S)-(3-(1 H-imidazoM-yl)-1-morphoiino-1 -oxopropan-2-yl)carbamate.
A. mixture of (tert-butoxycarbonyl)-L-histidine (150 mg, 0.59 mmol), morpholine (56 mg, 0.65 mmol), HATU (338 mg, 0.89 mmol) and DIPEA (152 mg, 1 .2 mmol) in DMF (5 mL) was stirred at rt for 1 h. The mixture poured into water (25 mL) and the resultant precipitate was filtered to get the crude solid, which was used in next step directly. LCMS (ESI) m/z: 325.2 [M+H]+.
Step 3: Preparation of (S)-(3-(1 H-imidazoi-4~yl)-1 ~morphoiino-1 ~oxopropan-2-yi)carbamate,
A mixture of tert-butyl (S)-(3-(1H-imidazo!-4-yl)-1-morpho!ino-1-oxopropan-2-yl)carbamate (crude) in trifluoroacetic acid (2 ml) and dichioromethane (5 mL) was stirred at rt for 2h. The mixture was then concentrated under reduced pressure, and io the residue was added ammonia methanol solution till pH>7. It was extracted with ethyl acetate (10 mL * 3), organics were dried over sodium sulphate, filtered and concentrated under reduced pressure to obtain the product 200 mg. LCMS (ESI) m/z: 225.1 Step 4: Preparation of (S)-N-(3-(1 H-imidazoM-yi)-1-morphotino-1 ~oxopropan-2-yl)-6-chioro-1 H-mdoie-
2-carboxamide,
A mixture of 6-chloro-1H-indole-2-carboxylic acid (50 mg, 0.26 mmol), (S)-(3-(1 H-imidazol-4-yl)-1- morpholino-1-oxopropan-2-yl)carbamate (63 mg, 0.28 mmol), HATU (148 mg, 0.39 mmol) and DIPEA (67 mg, 0.52 inmol) in DMF (5 mL) was stirred at rt for 1 h. The crude product from the mixture was purified by PREP-HPLC (SunFire Cl 8, 4,6*50mm, 3. Sum column Xbridge C18 3.5pm 4.6x50mm column. The elution system used was a gradient of 5%-95% over 1.5 Enin at 2snl/min and the solvent was acetOEiitrile/0.01% aqueous NH4HCO3.) to afford (S)-N-(3-(1H-imidazol-4-yl)-1-morpholino-1-oxopropan-2-yl)-6-chtoro-1 H- indole-2-carboxamide (2.6 mg, 0.0065 Enmol, 2.5% yield) as white solid.
1 H NMR (400 MHz, DMSO) 6 12.44 (s, 1 H), 11.72 (s, 1 H), 8.88 (d, J = 8.0 Hz, 1 H), 7.75 (s, 1 H), 7.66 (d, J = 8.6 Hz, 1 H), 7.42 (s, 1 H), 7.27 (s, 1 H), 7.16 - 6.99 (m, 1 H), 6.92 (d, J = 17.9 Hz, 1 H), 5.18 (q, J = 7.6 Hz, 1 H), 3.49 (dd, J = 33.7, 5.1 Hz, 8H), 3.06 - 2.88 (m. 2H); LCMS (ESI) m/z: 402.1 [M+H]+; (Rt: 1.91 min, ee%=95.72).
Synthesis of (S)-5-chioro-N-(2-(2,4-dif!uorophei"syi)-1 -(5-ethyM ,3,4-oxadiazo!-2-yi)ethy!)-1 H- pyrroio[2,3-b]pyridine"2~carboxamide (Compound 399),
Figure imgf000267_0001
Step 1 : (S)-terf-butyl 3-(2,4-difluorophenyl)-1 -oxo-1 -(2-propionyihydraziny i)propan~2-yicarbamate.
To solution of (S)-2-(tert-butoxycarbonylamino)-3-(2,4-difluorophenyl)propanoic acid (450 mg, 1.5 mmol), DIPEA (580 Eng, 4.5 mmol), propionohydrazide (158 mg, 1.8 Enmol) in DMF (10 mL) was slowly added HATU (741 mg, 1 .95 mmol) at -2Q°C and then stirred at -20°C for 1 h. The mixture was poured in to water (200 ml) and the resultant precipitate was filtered and dried to get crude product (S)-tert-butyl 3-(2,4- difluorophenyl)-1-oxo-1-(2-propionylhydrazinyl)propan-2-ylcarbamate (500 mg, 1.35 mmol, 84.1%) as a white solid. LCMS (ESI) m/z: 316.1 [M-SSj*.
Step 2: (S)-tert-butyl 2-(2,4-difiuorophenyl)-1-(5-ethy -1 ,3,4-oxadiazol-2-yl)ethyicarbamate.
The Burgess reagent ((Methoxycarbonylsulfamoyl)triethylammonium hydroxide) (428 mg, 1.8 mmol) was added to a solution of (S)-tert-butyl 3-(2,4-difluorophenyj)-1-oxo-1-(2-propionylhydrazinyi)propan-2- ylcarbamate (450 mg, 1 .2 mmol) in tetra hydrofuran (20 mL) and the resultant mixture was stirred at 70°C for 2 h. The reaction was then quenched with water and concentrated to remove the organics and ethyl acetate (100 mL) was added. The organic layer was collected, washed with water and brine, dried over sodium sulfate and concentrated to get the crude product (450 mg) as a white solid, which was directly used for the next step. LCMS (ESI) m/z: 354.2(M+H)*.
Step 3: (S)-2-(2,4-dif!tiorophenyi)-1 -(5-ethyM ,3,4-oxadiazo!-2-yi)ef hanamine.
A solution of (S)-tert-butyl 2-(2,4-difluorophenyl)-1-(5-ethyl-1 ,3,4-oxadiazol-2-yl)ethylcarbamate (140 mg, 0.4 mmol) in TFA (1 mL) and dichloromethane (4 mL) was stirred at 0-20 °C for 2h and concentrated. The resultant crude product was purified by Prep-HPLC(BOSTON pHlex ODS 10um 21 .2x250mm120A. The mobile phase was acetonitrile/0.1 % Ammonium bicarbonate) to afford (S)-2-(2,4-difluorophenyl)-1-(5-ethyl- 1 ,3,4-oxadiazol-2-yl)ethanamine as a white solid (90 mg, 0.354 mmol, 70.9%). LCMS (ESi) m/z: 254.1 [M+Hp.
Step 4: (S)-5~chloro-N-(2~(2,4-difluorophenyl)-1 -(5-ethyM ,3,4-oxad!azoi-2-yl)ethyi)-1 H-pyrroio[2,3- bJpyndine-2 -carboxamide.
To a solution of (S)-2-(2,4-difluorophenyl)-1-(5-ethyl-1 ,3,4-oxadiazol-2-yl)ethanamine (80 mg, 0.32 mmol), DIPEA (163 mg, 1.26 mmol), 5-chloro-1 H-pyrrolo[2,3-b]pyridine-2-carboxylic acid (63 mg, 0.32 mmol) in DMF (3 mL) was slowly added HATU (129 mg, 0.34 mmol) at 0°C and then stirred further for 1 h. The mixture was concentrated and the crude product was purified by Prep-HPLC (BOSTON pHlex ODS l Oum 21 ,2x250mm120A. The mobile phase was acetonitrile/0.1% Ammonium bicarbonate) to get (S)-5-chloro-N- (2-(2,4-difluorophenyl)-1-(5-ethyl-1 ,3,4-oxadiazol-2-yl)ethyl)-1 H-pyrrolo[2,3-b]pyridine-2-carboxamide (16.5 mg, 0.038 mmol, 10.7%) as a white solid. 1H NMR (500 MHz, DMSO) 6 12.39 (s, 1 H), 9.34 (d, J = 8.4 Hz, 1 H), 8.33 (d, J = 2,4 Hz, 1H), 8.27 (d, J = 2.3 Hz, 1 H), 7.45 (dd, J = 15.3, 8.5 Hz, 1H), 7.22 - 7.15 (m, 2H), 7.00 (td, J = 8.5, 2.4 Hz, 1H), 5.56 (td, J = 9.0, 6.2 Hz, 1 H), 3.43 (dd, J = 14.0, 6.0 Hz, 1H), 3.30 (d, J = 9.4 Hz, 1 H), 2.84 (q, J = 7.5 Hz, 2H), 1.23 (t, J = 7.5 Hz, 3H). LCMS (ESI) m/z: 432.1 [M+H]+: (Rt: 0.897 min, 66=97.68%).
Synthesis of (S)-5-cti!oro-N-(3-(2,4-difitioropheny!)-1 -oxo-1 -(2-oxa-6-azaspiro[3.3]heptan-6-y!)propan- 2-yi)~N-methyi-1 H-pyrrolo[2,3-b]pyndine-2~carboxamide (Compound 408):
Figure imgf000268_0001
Step 1 : Preparation of (S)~2-((tert-butoxycarbonyi)(methyl)amirio)~3-(2,4-difluorophenyi)propanoic acid.
To a solution of (S)-2-((tert-butoxycarbonyl)amino)-3-(2,4-difluorophenyl)propanoic acid (1.2 g, 4.0 mmol) in tetra hydrofuran (40 mL) was added sodium hydride (320 mg, 8.0 mmol) at 0°C under nitrogen. The mixture was stirred at 25°C for 2h and iodomethane (1 .14 g, 8.0 mmol) was added. The resultant mixture was stirred for 48h, diluted with ethyl acetate (100mL) and washed with water(30mL*3). The organic layer was dried over sodium sulfate, filtered and concentrated. The crude product thus obtained was used in next step directly (1 .3 g brown oil, Yield:100%). LCMS (ESI) m/z: 216.1 [M+Na]+.
Step 2: Preparation of (S)-3~(2,4-difluoropbenyl)-2-(methyiamino)propanoic acid bydrochioride.
A mixture of (S)-2-((tert-butoxycarbonyl)(methyl)amino)-3-(2,4-difiuorophenyi)propanoic acid (1 .3 g crude, 4.0 mmol) in hydrochloric acid in dioxane(4M) (20 mL) was stirred at 25°C for 2 h and concentrated. The crude product was triturated with petroleum ether to give the desired product (S)-3-(2,4-difluorophenyl)- 2-(methylamino)propanoic acid hydrochloride as a brown solid (1.6g, yield: 75%). LCMS (ESI) m/z: 216.1 [M+H]+.
Step 3: Preparation of methyi (S)-3~(2,4-difiuorophenyl)~2-(methylamino)propanoate.
To a solution of (S)-3-(2,4-difluorophenyl)-2-(methylamino)propanoic acid hydrochloride (1.6g, 4.0 mmol) in methanol(20 mL) was added thionyl chloride (952 mg, 8.0 mmol) slowly at room temperature under nitrogen. The mixture was stirred at 70°C for 3h and concentrated. To the resultant residue was added, a solution of ammonia in methanol (7N) and concentrated again. The resultant crude product was purified by silica gel column (petroleum ether: acetic ester from 1 :1) to give the desired product methyl (S)-3-(2,4- difluorophenyl)-2-(methylamino)propanoate as a yellow oil (423 mg , yield: 46%). LCMS (ESI) m/z: 230.1 [M+HJ+.
Step 5: Preparation of (S)-2-(5-chtoro-N-methyMH-pyrroio[2,3-b]pyiidine-2-carboxamido)-3-(2,4- difiuorophenyl)propanoic acid hydrochloride.
To a solution of 5-chloro-1 H-pyrroio[2,3-b]pyridine-2-carbonyl chloride (1.83 mmol) In tetrahydrofuran(20 mL) was added methyl (S)-3-(2,4-difluorophenyi)-2-(methylamino)propanoate(350 mg, 1.53 mmol) at 0°C under nitrogen. The mixture was stirred at 0°C for 30min, then 1 QmL of water was added and stirred further for 1h. The mixture was concentrated to remove the organics and the pH of the aqueous phase was adjusted to 3 with 1 N hydrochloric acid solution. The resultant solid was collected by filtration and dried in vacuo to give the desired product (S)-2-(5-chloro-N-methyl-1 H-pyrrolo[2,3-b]pyridine-2- carboxamido)-3-(2,4-difluorophenyl)propanoic acid hydrochloride as a brown solid (500 mg , yield: 83%). LCMS (ESI) m/z: 394.1 [M+H]+. Step 6: Preparation of (S)~5-cbioro-M~(3~(2,4-difiuorophenyi)~1-oxo-1 -(2~oxa-6~azaspiro[3.3]heptan~6- yi)propan-2-yi)-N-methyi~1 H-pyrroio[2,3-b]pyr!dine-2 -carboxamide.
To a solution of (S)-2-(5-ch!oro-N-methyl-1H-pyrrolo[2,3-b]pyridine-2-carboxamido)-3-(2,4- difiuorophenyi)propanoic acid hydrochloride(100 mg, 0.23 mmol), 2-oxa-6-azaspiro[3.3)heptane (35 mg, 0.35 mmol) and DIPEA (89 mg, 0.69 mmol) in tetra hydrofuran (5 mL) was added HATU (133 mg, 0.25 mmol) at - 60°C under argon. The mixture was stirred at -60°C for 0.5 h and then warmed to room temperature and stirred for 1 h. The mixture was then concentrated and resultant crude product was purified by Prep-TLC (dichloromethane: methanol= 20:1) to give the desired (S)-5-chloro-N-(3-(2,4-difluorophenyl)-1-oxo-1-(2-oxa- 6-azaspiro[3.3]heptan-6-yl)propan-2-yl)-N-methyl-1 H-pyrrolo[2,3-b]pyridine-2-carboxamide (29.3 mg, 0.06 mmol, yield: 27 %) as a white solid.
1H NMR (400 MHz, DMSO-ds) 6 12.35(s, 1 H), 8.32 (d, J = 2.0 Hz, 1 H), 8.16 (s, 1 H), 7.40 (dd, J,- = 8.4 Hz, J2 =: 16.0 Hz, 1 H), 7.19-7.14 (m, 1 H), 7.01-6.96 (m, 1 H), 6.75 (s, 1 H), 5.39-5.36(m, 1 H), 4.62(s, 4H), 4.25-4.23(m, 1 H), 4.17-4.15(m, 1 H), 4.08(s, 2H), 3.17-3.12(m, 3H), 3.04-2.98(m, 2H); LCMS (ESI) m/z: 475.2 [IVHH]+
Preparation of (S)-5~ch toro-N-(3~(2~ch torophenyi)-1 -(4~hydroxypiperidm-1 -yi)-1 -oxopropan~2-yi)-N- methyM H-mdoie-2-carboxamide (Compound 401).
Figure imgf000270_0001
Step 1 : (S)-2-(tert-biitoxycarbonyi(methyi)amirio)~3-(2-chlorophenyi)propanoic acid.
Sodium hydride (264 mg, 6.6 mmol) was added slowly to a solution of (S)-2-(tert- butoxycarbonyiamino)-3-(2-chlorophenyl)propanoic acid (1.0 g, 3.3 mmol) in tetrahydrofuran (60 mL) at 0 °C. After the addition, the mixture was stirred for another 24 hours. Another portion of sodium hydride (264 mg, 6.6 mmol) was added slowly to the reaction mixture at 0°C. After the addition, the mixture was stirred for another 24 hours. The mixture was poured into crushed ice, acidified to pH~1 with 2M hydrochloric acid and extracted with ethyl acetate (150 mL*3). The combined organic phase was dried and concentrated to afford the target compound (1.2 g, crude) as a brown oil. LCMS (ESI) m/z: 214.1/216.1 [M+H-WOp; 336.0 [M+Na]+ Step 2: (S)-tert-butyl 3-(2-chtorophenyl)-1-(4~hydroxypipendin-1-yl)~1-oxopropan-2- yl(methyl)carbamate.
A mixture of (S)-2-(tert-butoxycarbonyl(methyl)amino)-3-(2-chlorophenyl)propanoic acid (1.0 g, 3.2 mmol), piperidin-4-ol (324 mg, 3.2 mmol), HATU (1.8 g, 4.8 mmol), DIPEA (826 mg, 6.4 mmol) in DMF (10 mb) was stirred at 25 °C for 1 hour. The mixture was then poured into brine (100 mb) and extracted with ethyl acetate (150 mb*2). The combined organic phase was concentrated and the resultant residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate =1 :4) to afford the target compound (2.0g) as a light yellow oil. bCMS (ESI) m/z:397.2/399.1 [M+H]+.
Step 3: (S)-3-(2-ch!orophei"syi)-1 -(4-hydroxypiperidm-l -y!)-2-(methy!amino)propan-1 -one hydrochloride.
A mixture of (S)-tert-butyl 3-(2-chlorophenyl)-1-(4-hydroxypiperidin-1-yl)-1-oxopropan-2- yl(methyl)carbamate (2.0g) and MCI (1 ,4-dioxane, 4 mol/L, 20 mb) was stirred at 25 °C for 3 hours. The solvent was removed under reduced pressure and dried under vacuum to afford the target compound (3.0 g, N,N-dimethylformamide residual) as a grey gum, which was used in the next, step without further purification. bCMS (ESI) m/z: 297.1/299.2 [M+H]+.
Step 4: (S)-5-chloro-N-(3-(2-chtorophenyl)-1 -(4-hydroxypiperidin-l -yl)-1 -oxopropan-2-yl)-N-methyM H- indole-2 -carboxamide.
A mixture of 5-chloro-1H-indole-2-carboxylic acid (588 mg, S.Ommol), (S)-3-(2-chtorophenyi)-1-(4- hydroxypiperidin-1-yl)-2-(methylamino)propan-1-one hydrochloride (1.0 g, crude), HATU (1.71 g, 4.5 mmol) and DIPEA (1.17 g, 9.0 mmol) in DMF (10 mb) was stirred at 25 °C for 1 hour. The mixture was poured into water (50 mb) and extracted with ethyl acetate (100 mL*2). The combined organic phase was concentrated and purified by silica gel column chromatography (10% methanol in ethyl acetate) to afford 800 mg of a yellow oil, which was further purified by prep-HPLC(ammonium bicarbonate as buffer) to afford the target compound (40.3 mg, 0.085 mmol, yield: 2.8%) as a white solid. 1H NMR (400 MHz, DMSO-ds) 5 11 .68 (s, 1 H), 7.67-7.66 (m, 1 H), 7.39-7.37 (m, 3H), 7.20-7.18 (m, 3H), 6.92-6.91 (m, 1 H), 5.90-5.89 (m, 1 H), 4.73 (s, 1H), 4.13-3.60 (m, 3H), 3.31-2.87 (m, 7H), 1.75-1.53 (m, 2H), 1.20-1.01 (m, 2H); 474.1/476.1 [M+H]+.
Synthesis of 5-chloro-N-(3-(2-chloro-4-fiuoiophei"syl)-1 -(4-hydroxypiperidm-l -y 6)-1 -oxopropan-2-yl)-1 H- indole-2-carboxamide (Compound 492).
Figure imgf000271_0001
F
This compound was synthesized according to the protocols described above.
1H NMR (400 MHz, DMSO-ds) 6 11.71 (d, J = 3.2 Hz, 1 H), 8.97 (dd, J = 12.8, 0.8 Hz, 1 H), 7.71 (s,
1H), 7.48-7.44 (m, 1 H), 7.40-7.36 (m, 2H), 7.25 (s, 1 H), 7.18 (dd, 8.8, 1.6 Hz, 1 H), 7.12-7.06 (m, 1 H), 5.27 (dd, J = 14.4, 8.8 Hz, 1 H), 4.73 (dd, J = 9.2, 4.0 Hz, 1 H), 4.03-3.63 (m, 3H), 3.31-2.95 (m, 4H), 1.69-1.57 (m, 2H), 1.28-1.14 (m, 2H); LCMS (ESI) m/z: 478.1/480.0 [M+Hp.
Synthesis of (S)-5~chioro~N-(1 -morphoiino-1 -oxo-3-(pyridm~4-y!)propan-2-yi)-1 H-indo!e-2-carboxamide (Compound 403):
Figure imgf000272_0001
Step 1: Preparation of (S)~2-(5-chioro-1 H~indoie-2~carboxamido)~3-(pyndin-4-yl)propanoic acid.
To a solution of (S)-2-amino-3-(pyridin-4-yl)propanoic acid (200 mg, 1.2 mmol) in acetonitrile (15 mL) and water (3 mL) was added 2,5-dioxopyrrolidin-1-yl 5-chloro-1 H-indole-2-carboxylate (701 mg, 2.4 mmol) and triethylamine (242 mg, 2.4 mmol) and the resultant mixture was stirred 15 °C for 17 h. It was then concentrated followed by the addition of water (10 mL) and adjusted to pH = 2 with 1 N. hydrochloric, acid. The mixture was filtered and the solvent was removed under the reduced pressure. The crude product (S)-2-(5-chloro-1 H-indole- 2-carboxamido)-3-(pyridin-4-yl)propanoic acid (300 mg) thus obtained was used in the next step without further purification; LCMS (ESI) m/z: 344.1 [M+H]+.
Step 2: Preparation of (S)-5-chiaro-N-(1-moipholino-1-oxo-3-(pyndin-4-yl)propan-2-y!)-1 H-indate-2- carboxamide.
To a solution of (S)-2-(5-chloro-1H-indole-2-carboxamido)-3-(pyridin-4-yl)propanoic acid (120 mg, 0.35 mmol) in DMF (15 mL) were added morpholine (45 mg, 0.52 mmol), PyAOP (271 mg, 0.52 mmol) and DiPEA (135 mg, 1.05 mmol) and the mixture was stirred at 15°C for 17 h. The mixture was filtered and the solvent was removed under the reduced pressure and the residue was purified by prep-HPLC (SunFire Cl 8, 4.6*50mm, 3. Sum column Xbridge C18 3.5pm 4.6 x50mm column.The elution system used was a gradient of 5%-95% over 1.5 min at 2ml/min and the solvent was acetonitrile/0.01% aqueous NHaHCOa) to give the desired product (S)-5-chloro-N-(1-morphoiino-1-oxo-3-(pyridin-4-yl)propan-2-yi)-1 H-indole-2-carboxamide (18.1 mrngg,, 0.04 mmol, yield: 12.5 %) as a white solid.
1H NMR (400 MHz, DMSO) 6 11.74 (s, 1 H), 9.03 (d, J = 8.4 Hz, 1 H), 8.42 (d, J = 5.8 Hz, 2H), 7.72 (d, J = 1.8 Hz, 1 H), 7.37 (dd, J = 16.9, 7.3 Hz, 3H), 7.24 (s, 1 H), 7.18 (dd, J = 8.7, 2.0 Hz, 1 H), 5.19 (dd, J = 14.8, 8.5 Hz, 1 H), 3.69 - 3.37 (m, 8H), 3.17 - 2.89 (m, 2H); LCMS (ESI) m/z: 413.1 [M+H]+. The following compounds were synthesized according to the protocol described above.
Figure imgf000273_0001
Figure imgf000274_0001
Figure imgf000275_0001
Figure imgf000276_0001
Figure imgf000277_0001
Figure imgf000278_0001
Synthesis of 2-(2-tert-butylpyrimidm~5-yi)-N-[(1S)-1 ~[(3~chioro~4-pyridyl)methyi]-2~(2-oxa-6~ azaspiro[3.3]heptan-6-yl)-2-oxo-ethyl]acetamide (Compound 427): p
Br
Figure imgf000279_0001
2. Pd(OAc)2, X-Phos, DMF 25 °C, 12 h
Figure imgf000279_0002
Step 1 : methyl (2S)-2-(tert-butoxycarbonylamino)-3-(3-chioro-4-pyridyi)propanoate.
Into a 500-mL 3-necked round-bottom flask purged and maintained with an inert atmosphere of nitrogen, was placed Zn (8.66 g, 132.44 mmol), DMF (200 mL) and iz (5.28 g, 20.79 mmol). To this was added O4-tert-buty! O1 -methyl (2R)-2-(iodomethyl)butanedioate (20.46 g, 62.36 mmol) in several batches at 0 °C. The mixture was warmed upand stirred at 20 °C for 0.5 h. To the mixture were added 4-bromo-3-chloro- pyridine (8 g, 41.57 mmol), Pd(OAc)z (467 mg, 2.08 mmol) and XPhos (1.98 g, 4.16 mmol) under ice bath conditions. The resulting solution was stirred for 20 h at 12 °C and then at 20 °C for 24 h. The solids were filtered out and the filtrate was diluted with 300 mL of HzO. The resulting solution was extracted with ethyl acetate (4 * 80 mL), the organic layers were combined, dried over anhydrous sodium sulfate and concentrated. The crude product was purified by flash column (ISCO 100 g silica, 0-15 % ethyl acetate in petroleum ether, gradient over 20 min) to obtain methyl (2S)-2-(tert-butoxycarbonylamino)-3-(3-chioro-4- pyridyl)propanoate (8 g, 20.33 mmol, 49%) as a yellow oil. SFC (Rt=1 .420) method: IG„MeOH„IPAm„10„50„34„35„4min. 'H NMR (400 MHz, CHLOROFORM-d) 6 8.55 (s, 1 H), 8.40-8.39 (d, J = 4.8 Hz, 1 H), 7.16 (d, J = 4.8 Hz, 1 H), 5.19 - 5.08 (m, 1 H), 4.74 - 4.62 (m, 1 H), 3.74 (s, 3H), 3.32 (dd, 5.6 Hz, 13.6 Hz, 1 H), 3.15 - 3.02 (m, 1 H), 1.38 (s, 9H).
Step 2: (2S)-2~(tert-butoxycarbonylamino)-3~(3-chtoro-4~pyr!dyl)propanoic acid.
To a solution of methyl (2S)-2-(tert-butoxycarbonylamino)-3-(3-chloro-4-pyridyl)propanoate (2 g, 6.35 mmol) in MeOH (4 mL) and THF (12 mL) was added LiOH.HzO (533 mg, 12.71 mmol) in H2O (4 mL) and the mixture was stirred at 20 °C for 2 h. The reaction mixture was then concentrated to obtain (2S)-2-(tert- butoxycarbonylamino)-3-(3-chloro-4-pyridyl)propanoic acid (1.8 g, crude) as a pale yellow solid. LCMS (ESI) m/z: 301 .2 [M+Hp. Step 3: tert-butyl N-[(1S)-1 ~[(3-chioro~4-pyi'idyl)methyi]-2~(2~oxa-6~azaspiro[3.3]heptan-6-yi)-2-oxo- ethyijcarbamate.
To a solution of (2S)-2-(tert-butoxycarbonylamino)-3-(3-chloro-4-pyridyl)propanoic acid (400 mg, 1.33 mmol) in DMF (6 mL) were added NMM (404 mg, 3.99 mmol), EDCI (306 mg, 1.60 mmol), HOBt (216 mg, 1.60 mmol) and 2-oxa-6-azaspiro[3.3]heptane;oxalic acid (377 mg, 2.00 mmol) and the resultant mixture was stirred at 20 °C for 12 h. The reaction mixture was diluted with H2O (5 mL) and extracted with ethyl acetate (20 mL *3). The combined organic layers were dried over NazSO-i, filtered and concentrated under reduced pressure to give the crude product which was purified by flash column chromatography (ISCO 40 g silica, 0- 30 % Methanol in ethyl acetate, gradient over 20 min). The ccompound tert-butyl N-[(1S)-1-[(3-chloro-4- pyridyl)methyl]-2-(2-oxa-6-azaspiro[3.3]heptan-6-yl)-2-oxo-ethyl]carbamate (450 mg, 1.12 mmol, 42%) was obtained as a colourless gum. ^H NMR (400 MHz, CHLOROFORM-d) 6 8.57 (s, 1 H), 8.39 (d, J = 4.8 Hz, 1 H), 7.17 (d, J = 4.8 Hz, 1 H), 5.28 - 5.26 (m, 1 H), 4.76 - 4.70 (m, 3H), 4.65 - 4.62 (m, 1 H), 4.59 - 4.48 (m, 1 H), 4.42 (br d, J = 9.4 Hz, 1 H), 4.18 (br d, J = 10.8 Hz, 1 H), 4.05 - 3.96 (m, 2H), 3.08 - 2.93 (m, 2H), 1.35 (s, 9H); LCMS (ESI) m/z: 382.2 [M+Hp.
Step 4: (2S)~2-ammo~3-(3-chioro~4-pyiidyi)-1 ~(2~oxa-6~azaspiro[3.3]heptan-6-yi)propan-1 -one.
To a solution of tert-butyl N-[(1S)-1-[(3-chloro-4-pyridyl)methyl]-2-(2-oxa-6-azaspiro[3,3]heptan-6-yl)- 2-oxo-ethyl]carbamate (60 mg, 157 umol) in DCM (1 mL) was added TFA (277 mg, 2.43 mmol) and the mixture was stirred at 20 °C for 2 h. The mixture was then concentrated to obtain the crude product (2S)-2- amino-3-(3-chloro-4-pyridyl)-1-(2-oxa-6-azaspiro[3.3]heptan-6-yl)propan-1-one.TFA (60 mg, 152 umol, 97%) as a yellow gum. LCMS (ESI) m/z: 282.2 [M+H]*. This was used in the next step without further purification.
Step 5: 2-(2-tert-butylpynmsdin~5-yi)~N-[(1S)-1 -[(3-chtoro-4-pyridyl)methyl]-2-(2~oxa-6- azaspiro[3.3]heptan-6-yl)-2-oxo-ethyi]acetamide.
To a solution of 2-(2-tert-butylpyrimidm-5-yl)acetic acid (30 mg, 152 umol) in DMF (1 mL) were added DEA (59 mg, 455 umol) and T3P (116 mg, 182 umol) followed by (2S)-2-amino-3-(3-chloro-4-pyridyl)-1-(2- oxa-6-azasplro[3.3jheptan-6-yl)propan-1-one.TFA (60 mg, 152 umol) and the resultant mixture was stirred at 20 °C for 2h. The reaction mixture was concentrated and the residue was purified by prep-HPLC (Waters Xbridge Prep OBD C18 150*40mm*10um column; 15-35% acetonitrile in an a 10mM ammonium bicarbonate solution in water and in an a 0.05% ammonia solution in water, 8 min gradient) to obtain 2-(2-tert- butylpyrimidin-5-yl)-N-[(1S)-1-[(3-chloro-4-pyridyi)methyip2-(2-oxa-6-azaspiro[3.3]heptan-6-yi)-2-oxo- ethyljacetamide (38 mg, 83 umol, 55%) as a white solid. SFC (Rt =2.366) method:AD EtOH IPAm 5 50 34 35 3min.
'H NMR (400 MHz, CHLOROFORM-d) 6 8.55 - 8.54 (m, 3H), 8.35 (d, J = 4.8 Hz, 1 H), 7.09 (d, J = 4.8 Hz, 1 H), 7.05 - 7.03 (m, 1 H), 4.84 - 4.80 (m, 1 H), 4.71 (m, 3H), 4.62 - 4.59 (m, 1 H), 4.43 - 4.40 (m, 1 H), 4.21 - 4.16 (m, 1 H), 4.07 - 4.04 (m, 1 H), 3.90 - 3.86 (m, 1 H), 3.42 (s, 2H), 3.07 - 3.06 (m, 2H), 1.39 (s, 9H). LCMS (ESI for C23H28CIN5O3) [M+H]+: 458.2.
The following compounds were synthesized according to the protocol described above.
Figure imgf000281_0001
Figure imgf000282_0001
Figure imgf000283_0002
Synthesis of 5-chioro-N~[(1 R)-2-(3~methoxy-3~methyi~azet!din-1 -yi)-1 ~[(4~methoxy-3~pyridyl)methyl]~2- oxo-ethyl]-1 H-pyrroio[2,3-b]pyndme-2-carboxamide (Compound 437):
Figure imgf000283_0001
Step 1 : methyl (2R)-2-(tert~butoxycarbonyiamino)~3-(4-methoxy-3-pyndyi)propanoate.
Into a 100-mL 3-necked round-bottom flask purged and maintained with an inert atmosphere of nitrogen was placed Zn (522 mg, 7.98 mmol), DMF (3 ml) and h (202 mg, 798 nmol). To this was added methyl (2S)-2-(tert-butoxycarbonylamino)-3-iodo-propanoate (963 mg, 2.93 mmol) in several batches at 0°C for 30 min. To the resultant mixture was added 3-bromo-4-methoxy-pyridine (500 mg, 2.66 mmol), Pd(OAc>2 (30 mg, 133 nmol) and XPhos (127 mg, 266 umol) in ice bath. The resulting solution was stirred for 15 h at 25CC and the solids were filtered out. The resulting solution was diluted with 15 ml of H2O and extracted with ethyi acetate (3*10 mL). The organic layers were pooled, washed with 15 mL of brine, dried over anhydrous sodium sulfate and concentrated. The crude product was purified by flash column (ISCO 10 g silica, 0-90 % ethyl acetate in petroleum ether, gradient over 20 min) to obtain methyl (2R)-2-(tert-butoxycarbonylamino)-3- (4-methoxy-3-pyridyl)propanoate (450 mg, 1 ,16 mmol, 44%) as yellow gum. LCMS (ESI) m/z: 311.1 [M+H]+
Step 2: (2R)-2-(tert-butoxycarbonylam!no)-3-(4-methoxy~3-pyridyl)propanoic acid.
To a solution of methyl (2R)-2-(tert-butoxycarbonylamino)-3-(4-methoxy-3-pyridyl)propanoate (900 mg, 2,90 mmol) in THF (6 mL) and MeOH (2 mL), was added LiOH.HzO (243 mg, 5.80 mmol) in HzO (2 mL) at 0°C.The mixture was stirred at 25 °C for 2h and poured into water (10 mL). The resulting mixture was extracted with ethyl acetate (5mL*3) and the aqueous phase was freeze-dried to give the crude product (2R)-2-(tert-butoxycarbonylamino)-3-(4-methoxy-3-pyridyl)propanoic acid (530 mg, 1.79 mmol, 62%) as pale yellow solid.
1H NMR (400 MHz, DEUTERIUM OXIDE) 6 8.23 (br d, J = 5.7 Hz, 1 H), 8.11 - 8.04 (m, 1 H), 7.02 - 6.93 (rn, 1 H), 4.21 - 4.12 (m, 1 H), 3.93 - 3.83 (m, 3H), 3.28 (s, 1 H), 2.69 - 2.61 (m, 2H), 1.84 (s, 2H), 1.21 (s, 3H). LCMS (ESI) m/z: 151.3 [M+HJT
Step 3: tert-butyl N~[(1 R)-2-(3~methoxy-3-methyl-azet!din-1 -y S)-1 ~[(4~methoxy-3~pyr!dyl)methyl]~2-oxo~ ethyijcarbamate.
To a solution of (2R)-2-(tert-butoxycarbonylamino)-3-(4-methoxy-3-pyridyl)propanoic acid (250 mg, 843.69 umol) in DMF (2.5 mL) was added NMM (256 mg, 2.53 mmol), EDCI (194 mg, 1.01 mmol), HOBt (136 mg, 1.01 mmol) and 3-methoxy-3-methyl-azetidine;hydrochloride (128 mg, 928 umol). The mixture was stirred at 25°C for 15h and poured into ice water (5 mL). The resulting mixture was extracted with dichloromethane (3mL*5), the combined organic layers were washed with brine (5 mL), dried over NazSCU and concentrated. The product tert-butyl N-[(1R)-2-(3-methoxy-3-methyl-azetidin-1-yl)-1-[(4-methoxy-3- pyridyl)methyl]-2-oxo-ethyl]carbamate (190 mg, 501 umol, 59%) was obtained as yellow oil.
!H NMR (400 MHz, CHLOROFORM-d) 6 8.44 - 8.39 (m, 1 H), 8.21 (s, 1 H), 6.83 - 6.77 (m, 1 H), 5.31 - 5.24 (m, 1 H), 4.51 - 4.44 (m, 1 H), 4.11 - 3.84 (m, 3H), 3.86 (m, 1 H), 3.79 - 3.77 (m, 1 H), 3.27 - 3.25 (m, 2H), 3.20 - 2.19 (m, 3H), 2.96 - 2.88 (m, 1 H), 2.87 - 2.86 (m, 1H), 1.50 - 1.42 (m, 3H), 1.37 - 1.29 (m, 9H). LCMS (ESI) m/z: 297.1 [M+H]+.
Step 4: (2R)-2-amino-1 ~(3~methoxy-3~methyl~azetidin-1 -yl)-3-(4-methoxy~3-pyi'idyl)propan-1~one.
To a solution of tert-butyl N-[(1R)-2-(3-methoxy-3-methyl-azetidin-1-yl)-1-[(4-methoxy-3- pyridyl)methyl]-2-oxo-ethyl]carbamate (183 mg, 483 umol) in DCM (1.2 mL) was added TFA (616 mg, 5.40 mmol). The mixture was stirred at 25 °C for 15h and concentrated. The crude product was dissolved in MeOH (5mL) and then was added AMbersep 900(OH),ion exchange resin(SOOmg) to adjust the pH to 8. The mixture was filtered and the filtrate was concentrated to obtain (2R)-2-amino-1-(3-methoxy-3-methyl-azetidin-1-yl)-3- (4-methoxy-3-pyridyl)propan-1-one (100 mg, 358 umol, 74%) as yellow oil. LCMS (ESI) m/z: 280.2 [M+H]+ Step S: S-chloro-N-[(1 R)-2-(3-methoxy-3-methyl-azetidin-1 -yl)-1 -[(4-rnethoxy-3-pyridyi)rnethyi]-2-oxo- ethy!]-1 H-pyrrolo[2,3-b]pyridme-2 -carboxamide.
To a solution of 5-chloro-1 H-pyrrolo[2,3-b]pyridine-2-carboxylic acid (67 mg, 338 umol) in DMF (1 ml) were added NMM (163 mg, 1.61 mmol), EDCI (74 mg, 387 umol), HOBt (52 mg, 387 umol) and (2R)-2- amino-1-(3-methoxy-3-methyl-azetidin-1-yl)-3-(4-methoxy-3-pyridyl)propan-1-one (90 mg, 322 umol). The resultant mixture was stirred at 25 °C for 2h and filtered. The filtrate was purified by prep-HPLC (Phenomenex Gemini-NX C18 75*30mm*3um column; 20-50 % acetonitrile in an a 0.05% ammonium hydroxide and 10mM ammonium bicarbonate solution in water, 8 min gradient) to obtain 5-chloro-N-[(1R)-2- (3-methoxy-3-methyl-azetidin-1-yl)-1-[(4-methoxy-3-pyridyl)methyl]-2-oxo-ethyl]-1 H-pyrrolo[2,3-b]pyridine-2- carboxamide (40 mg, 88 umol, 27%) as pale yellow solid.
1H NMR (400 MHz, DMSO-d6) 6 12.33 (s, 1 H), 8.95 (dd, 7.9, 13.4 Hz, 1 H), 8.33 - 8.31 (m, 2H), 8.27 - 8.25 (m, 2H),7.21 (s, 1 H), 7.02 (dd, 3.2, 5.5 Hz, 1 H), 4.77 - 4.67 (m, 1 H), 4.17 (d, J = 8.9 Hz, 1 H),
3.90 (s, 3H), 3.81 - 3.65 (m, 2H), 3.55 (d, 10.1 Hz, 1 H), 3.13 (d, J = 2.8 Hz, 3H), 3.10 - 3.00 (m, 1 H), 2.91
(ddd, J = 5.1 , 9.0, 13.7 Hz, 1 H), 1.39 -1.29 (d, 3H). LCMS (ESI for C22H24CIN5O4) 458.1 ; (Rt:
0.81 Omin).
Synthesis of 5-chioro-N-[(1 R)-2-(3-methoxy~3-methy!-azetidin~1 -yi)~1 -[(3-methoxy~4-pyridyi)methyi]-2~ oxo-ethyl]-1 H-pyrro!o[2,3-b]pyndine-2-carboxamide (Compound 438):
Figure imgf000285_0001
Step 1 : methyl (2R)-2~(tert-butoxycarbonyiamino)-3~(3-methoxy-4-pyndy!)propanoate.
In a 100-mL 3-necked round-bottom flask purged and maintained with an inert atmosphere of nitrogen, was placed Zn (522mg, 7.98 mmol) in DMF (6 ml) followed by Iz (243mg, 957 umol). To this was added, methyl (2S)-2-(tert-butoxycarbonylamino)-3-lodo-propanoate (1 .26 g, 3.83 mmol) in several batches at 0°C for 30 min. To the mixture was added 4-bromo-3-methoxy-pyridine (600 mg, 3.19 mmol), Pd2(dba)s (146 mg, 159 umol) and SPhos (131 mg, 319 umol) in ice bath. The resulting solution was stirred for 15 h at 20°C and filtered. The filtrate was diluted with 10 mL of water and the resulting mixture was extracted with ethyl acetate (10 mL*3). The combined organic layers were washed with brine (5 ml), dried over NazSO4 and concentrated. The crude product was purified by flash column (ISCO 12 g silica, 0-100 % ethyl acetate in petroleum ether, gradient over 20 min) to obtain methyl (2R)-2-(tert-butoxycarbonylamino)-3-(3-methoxy-4- pyridyl)propanoate (250 mg, 483 umol, 15%) as yellow oil.
Step 2: (2R)-2-(tert-butoxycarbonyiamino)-3-(3~methoxy-4~pyndyi)propanoic acid.
To a solution of methyl (2R)-2-(tert-butoxycarbonylamino)-3-(3-methoxy-4-pyridyl)propanoate (230 mg, 444 umol) in THF-water-MeOH mixture (5mL, 3:1 :1) was added L1OH.H2O (37 mg, 889 umol) at O’C. The mixture was warmed up and stirred at 20°C for 2h. The mixture was concentrated and 5mL of water was added and the aqueous phase was extracted with ethyl acetate (15 mL*3). The aqueous phase was lyophilized io give crude product (2R)-2-(tert-butoxycarbonylamino)-3-(3-methoxy-4-pyridyl)propanoic acid (250 mg, 421 umol) as a yellow solid.
Step 3: tert-butyl N-[(1 R)-2-(3-methoxy-3-methyi-azetidin~1 -yl)~1 -[(3-methoxy~4-pyridyi)methyi]-2~oxo- ethyljcarbamate.
To a solution of (2R)-2-(tert-butoxycarbonylamino)-3-(3-methoxy-4-pyridyl)propanoic acid (220 mg, 371 umol, 50% purity, 1 eq) in DMF (3 ml) was added NMM (112 mg, 1.11 mmol, 122 uL, 3 eq), EDCI (85 mg, 445 umol, 1.2 eq), HOBt (60 mg, 445 umol, 1.2 eq) and 3-methoxy-3-methyl-azetidine (51 mg, 371 umol, 1 eq, HCI) at 0°C. The mixture was stirred at 20°C for 2h. LC-MS showed the starting material was consumed completely and one main peak with desired mass was detected. The mixture was poured into ice-water(SmL), The aqueous phase was extracted with Ethyl acetate (5 mL*3). The combined organic phase was washed with brine (5 ml), dried with anhydrous NasSO^, filtered and concentrated in vacuum to afford crude product. The 10 mg residue was purified by prep-HPLC (column: Phenomenex Gemini-NX C18 75*30mm*3um;mobile phase: [water(0.05%NH3H20+10mM NH4HCO3)-ACNj;B%: 10%-40%,8min) to run SFC. Compound tertbutyl N-[(1R)-2-(3-methoxy-3-methyl-azetidin-1-yl)-1-[(3-methoxy-4-pyridyl)methyl]-2-oxo-ethyi]carbamate (120 mg, 316 umol, 85%) was obtained as a yellow oil which was used directly.
Step 4: (2R)-2-arnmo-1 -(3-methoxy-3-rnethyl-azetidm-1 -yl)-3-(3-rnethoxy-4-pyiidyl)propan-1-one.
To a solution of tert-butyl N-[(1R)-2-(3-methoxy-3-methyl-azetidin-1-yl)-1-[(3-methoxy-4- pyridyl)methyi]-2-oxo-ethyl)carbamate (120 mg, 316 umol, 1 eq) in DCM (1.2 mb) was added TFA (434mg, 3.81 mmol, 282 uL, 12.06 eq). The mixture was stirred at 20°C for 15 h. LCMS showed the reaction was complete. The reaction mixture was concentrated to dryness to give the crude. The crude was washed with toluene (2mL * 3). The solution was concentrated to dryness and was dissolved with MeOH(3mL),then was added AMbersep 900(OH),ion exchange resin(2g) to adjust pH to 8. The mixture was filtered and the filtrate was concentrated to dryness to give the product. (2R)-2-amino-1-(3-methoxy-3-methyl-azetidin-1-yl)-3-(3- methoxy-4-pyridyl)propan-1-one (98 mg, 315 umol, 99%) was obtained as yellow oil.
Step 5: 5-cbioro~N-[(1 R)-2~(3-methoxy-3-methyl-azetsdifi-1 ~yl)-1 -[(3~methoxy-4-pyridy!)metby!]-2-oxo- ethyi]-1 H-pyrrolo[2, 3-b]pyridme-2 -carboxamide.
To a solution of 5-chioro-1 H-pyrroto[2,3-b]pyridine-2-carboxylic acid (70 mg, 357 umol, 1.05 eq) in
DMF (1.2 mL) was added NMM (172 mg, 1.70 mmol, 186 uL, 5 eq), EDCI (78 mg, 408 umol, 1.2 eq), HOBt (55 mg, 408umol, 1.2 eq) and (2R)-2-amino-1-(3-methoxy-3-methyl-azetidin-1-yl)-3-(3-methoxy-4- pyridyl)propan-1-one (95 mg, 340 umol, 1 eq).The mixture was stirred at 20°C for 4h. LCMS showed the starting material was consumed completely and 30% of desired ms was detected. The reaction mixture was filtered and the filtrate was purified by prep-HPLC (Phenomenex Gemini-NX C18 75*30mm*3um column; 15%-45% acetonitrile in an a 0.05% ammonium hydroxide and 10mM ammonium bicarbonate solution in water, 8 min gradient. 5-chloro-N-[(1R)-2-(3-methoxy-3-methyl-azetidin-1-yl)-1-[(3-methoxy-4-pyridyl)methyl]- 2-oxo-ethyl]-1H-pyrrolo[2,3-b]pyridine-2-carboxamide (51 mg, 108 umoi, 32%) was obtained as pale yellow solid.
1H NMR (400 MHz, CHLOROFORM-d) 6 8.27 (s, 1H), 8.26(d, 1 H),8.24 (br d, J = 4.6 Hz, 1H), 7.96 (br s, 1 H), 7.39 (br s, 1 H), 6.86 (s, 1 H), 4.88 (br d, J = 8.1 Hz, 1 H), 4.62(br s, 0.4H), 4.33 - 4.30(m, 1 H), 4.11- 4.09 (m, 1 H), 3.97-3.95(d, 3.6H), 3.93(d, 0.5H), 3.88-3.85(d,0.5H), 3.67-3.65(s,1.5H),3.45-3.32(s,3.5H), 1.67 (s, 1.5H) 1.45 (s, 1.5H). LCMS (ESI for C22H24CIN5O4) [M+Hf: 458.2. SFC method OD„MeOH_IPAm„5„50„34„35„3min, RT=1 .152.
Synthesis of 5-chtoiO-N-[1 -[(3-ch toro-4-pyridyi)inethyi]-2-(4-hydroxy-4-methyl-1 -pipendy!)-2-oxo- ethyi]-hi~methyM H-pyi”rolo[2,3~b]pyndine-2-carboxamlde (Compound 439):
Figure imgf000287_0001
Note: During step 3, the conditions used ted the complete racemization of the R-enantiomer that was used as a starting material. Step 1 : methyl (2R)-2-(tert~butoxycarbonyiammo)~3-(3-chioro-4-pyridlyi)propa8TOate.
Into a 2 liter 3-necked round-bottom flask purged and maintained with an inert atmosphere of nitrogen, was placed Zn (30.72 g, 469.80 mmol), DMF (800 mL) and I2 (19.78 g, 77.95 mmol) at 0 °C. To this was added methyl (2S)-2-(tert-butoxycarbonylamino)-3-iodo-propanoate (76.96 g, 233.84 mmol) in several batches at 0°C. The mixture was warmed and stirred at 20°C for 1 h. The resultant mixture was cooled in an ice bath and were added 4-bromo-3-chloro-pyridine (30 g, 155.89 mmol), Pd(OAc)z (1 .75 g, 7.79 mmol) and XPhos (7.43 g, 15.59 mmol) and was stirred for 15 h at 20°C. The solids were filtered out and the resulting solution was diluted with 500 mL of water and extracted with ethyl acetate (4 * 150mL). The combined organics were dried over anhydrous sodium sulfate and concentrated. The crude product thus obtained was purified by flash column (ISCO 120 g silica, 0-20 % ethyl acetate in petroleum ether, gradient over 60 min) to obtain methyl (2R)-2-(tert-butoxycarbonylamino)-3-(3-chloro-4-pyridyl)propanoate (20 g, 50.83 mmol, 33 %) as yellow oil. LCMS (ESI) for 315.1
Step 2: methyl (2R)-2~amino-3-(3-chioro-4~pyridyl)propanoate.
A solution of methyl (2R)-2-(tert-butoxycarbonylamino)-3-(3-chloro-4-pyridyl)propanoate (2.2 g, 6.99 mmol) in HCI/EtOAc (20 mL) was stirred at 20°C for 2h. The solid was collected by filtration, rinsed with EtOAc (2 mL * 3) and dried under reduced pressure to give methyl (2R)-2-amino-3-(3-chloro-4- pyridyl)propanoate.HCI (1.6 g, 6.37 mmol, 91%) as white solid. LCMS (ESI) for [M+H]+: 215.1
Step 3: methyl 3-(3-chtoro-4-pyridyl)-2-[(2,4-dimefhoxyphenyi)methyl-methyl-amino]propanoate. To a solution of methyl (2R)-2-amino-3-(3-chloro-4-pyridyl)propanoate.HCI (2.2 g, 8.76 mmol) in
MeOH (25 mL) was added TEA (887 mg, 8.76 mmol) at 0“C. Then to the mixture were added 2,4- dimethoxybenzaldehyde (1.82 g, 10.95 mmol) and AcOH (658 mg, 10.95 mmol) where the pH of the reaction mixture was 4. The mixture was stirred at 20°C for 3h, cooled to 0c,C and then NaBHsCN (1 .10 g, 17.52 mmol) and formaldehyde (1 .42 g, 17.52 mmol) were added. The resulting mixture was warmed up and stirred at 20°C for 5h. The mixture was diluted with water (10 mL) and extracted with ethyl acetate (40 mL * 3). The combined organics were dried over NazSCX filtered and the filtrate was concentrated to dryness to give the crude product. It was purified by flash column (ISCO 10 g silica, 0-25 % ethyl acetate in petroleum ether, gradient over 15 min) to obtain methyl 3-(3-chloro-4-pyridyl)-2-[(2,4-dimethoxyphenyl)methyl-methyl- amino]propanoate (1.6 g, 4.22 mmol, 48) as colorless oil. LCMS (ESI) for [M+H]+: 379.1
Step 4: 3-(3-chioro-4-pyridyi)-2-[(2,4-dimethoxypheny!)methy!-methy!-amino]propanoic acid.
To a solution of methyl 3-(3-chloro-4-pyridyl)-2-[(2,4-dimethoxyphenyi)methyl-methyl- amino)propanoate (1.8 g, 4.75 mmol) in THF (10 mL) and MeOH (5 mL) was added LIOH.HzO (399 mg, 9.50 mmol) in HsO (5 mL) and then the mixture was stirred at 20 °C for 2 h. The reaction mixture then was concentrated to obtain the crude product 3-(3-chloro-4-pyridyl)-2-[(2,4-dimethoxyphenyl)methyl-methyl- aminojpropanoic acid (2.2 g, crude, LiOH) as a white solid. LCMS (ESI) m/z: 365.3 [M+Hp. It was taken io the next, step without further purification. Step 5: 3-(3-cbioro-4~pyridyi)~2-[(2,4-d!methoxyphenyi)methyi~methyi~amino]-1-(4~hydroxy-4-methyi-1- piperidyl)propan-1 -one.
To a solution of 3-(3-chloro-4-pyridyl)-2-[(2,4-dimethoxyphenyl)methyl-methyl-aminojpropanoic acid (0.9 g, 2.47 mmol) and 4-methylpiperidin-4-ol (369 mg, 3.21 mmol) in THF (20 mL) was added HATH (1.88 g, 4.93 mmol) and DIPEA (1.28 g, 9.87 mmol) and the resultant mixture was stirred at 20 °C for 2 h. 10mL of water was added to the reaction mixture and extracted with ethyl acetate (30 mL *3). The combined organic layers were dried over NazSCh, filtered and concentrated to obtain the crude product. It was purified by flash column (ISCO 40 g silica, 90-100 % ethyl acetate in petroleum ether, gradient over 20 min) to obtain 3-(3- chtoro-4-pyridyl)-2-[(2,4-dimethoxyphenyl)methyl-methyl-amino]-1-(4-hydroxy-4-methyl-1-piperidyl)propan-1- one (1.1 g, 1.19 mmol, 48%) as a pale yellow solid. LCMS (ESI) m/z: 462.2 [M+H]’.
Step 6: 3-(3-cbioro-4~pyridyi)~1-(4-tiydroxy~4-methyl-1 -pipendyi)-2~(mettiylamino)propan-1~one.
To a solution of 3-(3-chloro-4-pyridyl)-2-[(2>4-dimethoxyphenyl)methyl-methyl-amino]-1-(4-hydroxy-4- methyl-1-piperidyl)propan-1-one (1 g, 2.16 mmol) in DCM (10 mL) was added TFA (7.70 g, 67.53 mmol) and the mixture was stirred at 40 °C for 12 h. The reaction mixture was concentrated, redissolved in HzO (30 mL) and the mixture was washed with EtOAc (20 mL*3). The aqueous phase was then concentrated in vacuum to obtain the crude product 3-(3-chloro-4-pyridyl)-1-(4-hydroxy-4-methyl-1-piperidyl)-2-(methylamino)propan- 1-one.TFA (0.9 g, crude) as a pale yellow gum. LCMS (ESI) m/z: 312.2 [M+H]+. This product was used in the next step without further purification.
Step 7: 5-chtoro~N-[1-[(3-chioro-4~pyndyl)methyl]~2-(4-hydroxy~4-methyM ~piperidyi)-2~oxo-ethyl]-N- methyl-1 H-pyrrolo[2,3-b]pyridirie-2-carboxamide.
To a solution of 5-chloro-1 H-pyrroto[2,3-b]pyridine-2-carboxylic acid (139 mg, 705 umol) in DMF (5 mL) were added EDCI (162 mg, 845 umol), HOBt (114 mg, 845 umol), NMM (218 mg, 2.11 mmol) followed by 3-(3-chloro-4-pyridyl)-1-(4-hydroxy-4-methyl-1-piperidyl)-2-(methyiamino)propan-1-one.TFA (300 mg, 705 umol) and the mixture was stirred at 20 °C for 1 h. The reaction mixture was concentrated and purified first by prep-HPLC (Waters Xbridge Prep OBD C18 150*40mm*10um column; 20-50% acetonitrile in an a 10mM ammonium bicarbonate solution in water and in an a 0.05% ammonia solution in water, 8 min gradient) and then by prep-HPLC (Waters Xbridge Prep OBD C18 150*40mm*10um column; 20-50% acetonitrile in an a 10mM ammonium bicarbonate solution in water and in an a 0.05% ammonia solution in water, 8 min gradient) to obtain 5-chloro-N-[1-[(3-chloro-4-pyridyl)methyl]-2-(4-hydroxy-4-methyl-1-piperidyl)-2-oxo-ethyl]- N-methy!-1 H-pyrrolo[2,3-b]pyridine-2-carboxamide (62 mg, 127 umol, 18%) as a white solid.
1H NMR (400 MHz, CHLOROFORM-d) 6 10.98 - 10.80 (m, 1 H), 8.56 (d, J = 3.3 Hz, 1 H), 8.41 (t, J = 2.6 Hz, 1 H), 8.38 - 8.32 (m, 1 H), 7.98 (d, J = 2.0 Hz, 1 H), 7.30 (br d, J = 5.1 Hz, 1 H), 6.82 - 6.74 (m, 1 H), 6.21 - 6.04 (m, 1 H), 4.34 - 4.14 (m, 1 H), 3.81 - 3.63 (m, 1 H), 3.55 - 3.36 (m, 5H), 3.36 - 3.12 (m, 2H), 1.66 - 1 .58 (m, 1 H), 1 .56 - 1 .46 (m, 2H), 1 .38 - 1 .30 (m, 1 H), 1 .23 (d, J = 11 .3 Hz, 3H). LCMS (ESI for C23H25CI2N5O3) [M+H]*: 490.1. The foliowing compounds were synthesized according to the protocol described above:
Figure imgf000290_0001
Figure imgf000291_0001
Synthesis of 5-ch!oro-hi~(1 -(3,3-difiuoropyrroiidin-1 ~yi)-3~(2-methyi-1 H-imidazoi-4~yi)-1 -oxopropan-2- yi)-1 H-indo!e-2-carboxamide (Compound 447):
Figure imgf000292_0001
Step 1 : Preparation of methyl 2-methyM H-imidazoie-5-carboxylate,
To a solution of 2-methyl-1 H-imidazole-5-carboxylic acid (3.6 g, 28.5 mmol) in methanol (30 mL) was added concentrated hydrochloric acid (10 mL) at 25°C under argon. The mixture was stirred at 80°C for 24 h and cooled to room temperature. It was diluted with ethyl acetate (30 mL), and adjusted to pH=10 with 1 N sodium hydroxide solution. Phases separated and the aqueous phase was extracted with ethyl acetate (100 mL'‘5), the combined organics was dried over sodium sulfate, filtered and concentrated to give the desired product methyl 2-methyi-1 H-imidazole-5-carboxylate as a off-white solid (3.0 g ,21.4 mmol, yield: 75%).
Step 2: Preparation of (2-methyi-1 H-im!dazoi-5-yi)methanoi.
To a solution of methyl 2-methyi-1H-imidazole-5-carboxylate (2.2 g, 15.7 mmol) In tetrahydrofuran (150 mL) was added lithium aluminium hydride solution (47mL, 47 mmol) slowly at 0°C under argon. The mixture was stirred at 25°C for 16h and the mixture was cooled to 0°C , diluted with diethyl ether (20 mL) and then the mixture was quenched with water(2 mL). To the resultant mixture, 15% sodium hydroxide solution(2 mL) and water (3mL) was added and the mixture was stirred further for 1 h. The mixture was filtered and washed with ethyl acetate (300 mL) and the filtrate was concentrated to give the desired product (2-methyi- 1 H-imidazol-5-yl)methanol as a yellow solid (1.67 g ,14.9 mmol, yield: 95%).
Step 3: Preparation of 5-(chtoromethyl)-2-methyM H~imidazoie hydrochloride.
To a solution of (2-methyl-1 H-imidazol-5-yl)methanol (1.67 g, 14.9 mmol) in chloroform (15 mL) was added thionyl chloride (6 mL) at 0°C under argon. The mixture was stirred at 25°C for 24h and concentrated io give the desired product 5-(chloromethyl)-2-methyl-1 H-imidazole hydrochloride as a brown solid (2.6 g crude ,14.9 mmol, yield: 100%). Step 4: Preparation of diethyi 2-acetamido-2~((2-methyi~1 H~imidazol~5-yi)methyi)maionate.
To a solution of diethyl 2-acetamidomalonate (4.4 g, 20.1 mmol) in N,N-dimethylformamide (40 mL) was added sodium hydride (1 .68 g, 42.0 mmol) at 0°C under nitrogen. The mixture was stirred at 25°C for 1h. Then 5-(chloromethyl)-2-methyMH-imidazole hydrochloride (2.8 g, 16.8 mmol) was added and the mixture was stirred at 25°C for 16 h. It was then diluted with ethyl acetate (200 mL) and washed with brine(50 mL*3). The organic layer was dried over sodium sulfate, filtered and concentrated. The crude product (3.3 g, 10.6 mmoi, yield:63%) was obtained as brown oil which was used in next step directly. LCMS (ESI) m/z: 312.1
Step 5: Preparation of 2-amino-3~(2-methyi-1 H-imidazol-4-yl)propanoic acid hydrochloride.
A mixture diethyi 2-acetamido-2-((2-methyl-1 H-imidazol-5-yl)methyl)malonate(3.3 g crude, 10.6 mmol) in concentrated hydrochloric acid(10 mL) was stirred at 100°C for 3 h. The resultant mixture was concentracted and the crude product (1.8 g crude, 10.6 mmol, yield: 100 %, as brown oil) was used in next step directly. LCMS (ESI) m/z: 170.2 [M+H]+.
Step 6: Preparation of methyl 2~amino-3-(2-methyM H-lmidazoi-4~yl)propanoate hydrochloride.
To a solution of 2-amino-3-(2-methyl-1 H-imidazol-4-yl)propanoic- acid hydrochloride (1.8 g crude, 10.6 mmol) in methanol (20 mL) was added thionyl chloride (2.52 g, 21.2 mmol) slowly at room temperature under nitrogen. The mixture was stirred at 70°C for 4h and concentrated in vacuo. The crude product methyl 2-amino-3-(2-methyl-1 H-imidazol-4-yl)propanoate hydrochloride (3.0g, 10.6 mmol, yield: 100%) was used in next, step directly. LCMS (ESI) m/z: 184.2 [M+H]+.
Step 7: Preparation of 2~(5-chloro-1 H-indole~2-carboxamido)-3~(2-methyl-1 H-imidazoi-4-yl)propanoic acid.
To a solution of 5-chloro-1 H-indole-2-carboxylic acid (880 mg, 4.5 mmol), methyl 2-amino-3~(2- methyl-1H-imidazol-4-yl)propanoate hydrochloride (549 mg crude, 3.0 mmol) and DIPEA (1.16 g, 9.0 mmol) in DMF (10 mL) was added HATU (1 .71 g, 4.5 mmol) at room temperature under argon. The mixture was stirred at room temperature for 1 h and the product in the mixture was purified by Prep-HPLC (Boston C18 21*250mm 10pm column. The mobile phase was acetonitrile/0.01 % aqueous trifluoroacetic acid.) to give the desired 2-(5-chloro-1 H-indole-2-carboxamido)-3-(2-methyl-1 H-imidazoi-4-yl)propanoic acid (275 mg, 0.8 mmol, yield: 26 %) as a off-white solid. LCMS (ESI) m/z: 347.1 [M+H]+.
Step 8: Preparation of 5-chioro-N~(1 -(3,3-diflnoropyrroiidin-l -yl)-3~(2-methyi-1 H-imidazoi-4~yl)-1 - oxopropan-2-yl)-1 H-indole-2-carboxamide.
To a solution of 2-(5-chloro-1H-indole-2-carboxamido)-3-(2-methyl-1H-imidazol-4-yl)propanolc acid (140 mg, 0.4 mmol), 3,3-difiuoropyrrolidine hydrochloride (86 mg, 0.6 mmol) and DIPEA (155 mg, 1.2 mmol) in DMF (5 mL) was added PyAOP (250 mg, 0.48 mmol) at 25°C under argon. The mixture was stirred at 25°C for 1 h and the desired product in the mixture was purified by Prep-HPLC (Boston C18 21*250mm 10pm column. The mobile phase was acetonitrile/0.01 % aqueous trifluoroacetic acid.). The product 5-chloro-N-(1- (3,3-difluoropyrrolidin-1-yl)-3-(2-methyl-1 H-imidazol-4-yl)-1-oxopropan-2-yl)-1 H-indole-2-carboxamide (58.7 mg, 0.13 mmol, yield: 34 %) was obtained as a white solid. 1H NMR (400 MHz, DMSO-ds) 6 11 .81 (d, J = 10.8 Hz, 1H), 11.48 (d, J = 14.4 Hz, 1 H),8.95 (dd, J, = 7.2 Hz, J2 = 16.8Hz, 1 H), 7.73 (s,1 H), 7.42(d, J = 8.8 Hz, 1 H), 7.25(5,1 H), 7.19(dd, J, = 2.0 Hz, J2 = 8.8Hz, 1 H), 6.68(s,1 H), 4.95-4.77(m,1H), 4.20-4.14 (m,0.5H), 3.99-3.90(m,1 H), 3.74-3.41 (m,2.5H), 2.98-2.84(m,2H), 2.47-2.25(m,2H),2. 21(s,3H); LCMS (ESI) m/z: 436.1 [M+H]+.
Synthesis of enantiomer 1 (Compound 448) and enantiomer 2 (Compound 449) of 5-chioro-N-(3-(3- chioropyridin-4-y!)-1 -(4-8iydroxy-4-methylpiperidin-1 -yl)-1 -oxopropan-2-yi)-N-methyM H-indoie-2- carboxamide.
Figure imgf000294_0001
Enantiomer 1 Enantiomer 2
The racemic compound 5-chloro-N-[1-[(3-chloro-4-pyridyl)methyl]-2-(4-hydroxy-4-methyl-1-piperidyl)- 2-oxo-ethyl]-N-methyl-1 H-indole-2 carboxamide (Compound 440, 118 mg, 242umol) was synthesized as described above and was subjected to chiral HPLC using SFC conditions (REGIS(S,S)WHELK-O1 250*25mm, 10pm column, eluting with 50% methanol containing Neu-ETOH in a flow of 70 g/min CO2 at 100 bar, 10 min gradient) to obtain enantiomer 1 (Compound 448, 41mg, 34%) and enantiomer 2 (Compound 449, 41 mg, 34%) as white solids.
Compound 448: 1H NMR (400MHz, CHLOROFORM-d) 6 9.28 (bs, 1 H), 8.55 (bs, 1 H), 8.34 (d, J = 4.5Hz, 1 H), 7.64 (s, 1 H), 7.36 - 7.29 (m, 1 H), 7.25 (s, 2H), 6.83 (d, J = 6.6Hz, 1H), 6.09 - 6.02 (m, 1 H), 4.33 - 4.12 (m, 1 H), 3.74 - 3.56 (m, 1H), 3.49 - 3.12 (m, 7H), 1.65 - 1.44 (m, 4H), 1.26 - 1.21 (m, 3H). LCMS (ESI for C24H26CI2N4O3) [M+H]+: 489.2. (Rt: 1.542min).
Compound 449: 1H NMR (400MHz, CHLOROFORM-d) 6 9.31 (s, 1H), 8.55 (s, 1H), 8.34 (d, J = 3.3Hz, 1 H), 7.63 (s, 1 H), 7.35 - 7.29 (m, 1 H), 7.24 (s, 1H), 6.83 (d, J = 6.3Hz, 1 H), 6.12 - 6.01 (m, 1 H), 4.33 - 4.14 (m, 1 H), 3.73 - 3.57 (m, 1H), 3.49 - 3.11 (m, 7H), 1.69 - 1.41 (m, 4H), 1.22 (d, 14.8Hz, 3H). LCMS
(ESI for C24H26CI2N4O3) [M+Hp: 489.2. (Rt: 1.642min).
Synthesis of enantiomer 1 (Compound 450) and enantiomer 2 (Compound 451 ) of 5-chloro-N-(3-(3- chioropyridin-4-yi)~1 -(4-methoxypiperidin-1 -yl)-1 -oxopropan-2-yl)-N-metbyl-1 H-pyrrolo[2,3-b]pyridine- 2-carboxamide.
Figure imgf000295_0002
The racemic compound 5-chloro-N-[1-[(3-chloro-4-pyridy5)methyl]-2-(4-methoxy-1-piperidyl)-2-oxo- ethyl]-N-methyl-1 H-pyrrolo[2,3-b]pyridine-2-carboxamide (Compound 446, 60 mg, 122umol) was synthesized as described above and subjected to chirai HPLC using SFC conditions (Phenomenex- Cellulose-2 250mm*30mm,10um column, 40°C, eluting with 60% ethanol containing 0.1% ammonium hydroxide in a flow of 80 g/min COz at 100 bar) to obtain enantiomer 1 (Compound 459, 32mg, 53%) and enantiomer 2 (Compound 451 , 22mg, 37%) as white solids.
Compound 450: 1H NMR (400 MHz, CHLOROFORM-d) 6 10.64 - 10.35 (s, 1 H), 8.55 (s, 1 H), 8.47 - 8.40 (m, 2H), 7.98 (s, 1 H), 7.38 - 7.30 (m, 1 H), 6.85 - 6.68 (m, 1 H), 6.22 - 5.95 (m, 1 H), 4.01 - 3.93 (m, 0.5H), 3.73 - 3.26 (m, 12.5H), 1.86 - 1.73 (m, 2H), 1.63 - 1.30 (m, 3H). LCMS (ESI for C23H25CI2N5O3) [M+Hf: 490.2. (Rt: 1.823min).
Compound 451 : 1H NMR (400 MHz, CHLOROFORM-d) 6 10.88 (s, 1 H), 8.56 (s, 1 H), 8.40 - 8.34 (m, 2H), 7.98 - 7.96 (m, 1 H), 7.28 (d, J = 1.5Hz, 1 H), 6.78 - 6.76 (m, 1H), 6.10 (s, 1 H), 4.18 - 3.95 (m, 1 H), 3.74 - 3.73 (m, 0.5H), 3.44 - 3.23 (m, 12.5H), 1.75 - 1.64 (m, 2H), 1.53 - 1.49 (m, 2H). LCMS (ESI for C23H25CI2N5O3) 490.2. (Rt: 2.299min).
Synthesis of enantiomer 1 (Compound 452) and enantiomer 2 (Compound 453) of 5-chloro-N-[1-[(3- chloro-4-pyndyl)mefhyl]-2-(4-hydroxy-4-mesthyi-1 -piperidyi)-2-oxo-efhyl]-M-methyl-1 H-pyrroto[2,3- bJpyridine-2 -carboxamide.
Figure imgf000295_0001
Enantiomer 1 Enantiomer 2
The racemic compound 5-chloro-N-[1-[(3-chloro-4-pyridyl)methyl]-2-(4-hydroxy-4-methyl-1-piperidyl)-
2-oxo-ethylj-N-methyl-1 H-pyrrolo[2,3-b]pyridine-2-carboxamide (Compound 439, 60 mg, 122.35umol) was synthesized as described above and was subjected to chiral separation using SFC conditions (REGIS(S,S)WHELK-01 (250mm*25mm,10um), 40°C, eluting with 40% isopropanol in a flow of 70 g/min CO2 at 100 bar) to obtain enantiomer 1 (Compound 452, 23mg, 37%) and enantiomer 2 (Compound 453, 24mg, 40%) as white solids.
Compound 452: 1H NMR (400 MHz, CHLOROFORM-d) 6 11.20 - 11.12 (m, 1 H), 8.56 (d, J = 4.2Hz, 1H), 8.41 (t, J = 2.7Hz, 1 H), 8.35 (d, J = 5.0Hz, 1 H), 7.98 (d, J = 2.0Hz, 1H), 7.28-7.26 (m, 1H), 6.77 (d, J = 9.0Hz, 1 H), 6.20 - 6.02 (m, 1 H), 4.33 - 4.13 (m, 1 H), 3.80 - 3.61 (m, 1 H), 3.47 - 3.19 (m, 7H), 1 .65 - 1 .47 (m, 3H), 1.41 - 1.30 (m, 1H), 1.23 (d, 12.9Hz, 3H) LCMS SI for C23H25CI2N5O3) m/z: 490.1 [M+HJ+. (Rt:
1.59min).
Compound 453: 1H NMR (400 MHz, CHLOROFORM-d) 6 11.31 - 11.23 (m, 1 H), 8.56 (d, J = 4.4Hz, 1 H), 8.41 (dd, J = 3.1 , 2.4Hz, 1 H), 8.35 (d, J = 5.0Hz, 1 H), 7.99 (d, J = 2.0Hz, 1 H), 7.28-7.26 (m, 1 H), 6.77 (d, J = 9.2Hz, 1 H), 6.18 - 6.07 (m, 1 H), 4.36 - 4.15 (m, 1 H), 3.79 - 3.62 (m, 1 H), 3.53 - 3.09 (m, 7H), 1.71 - 1.41 (m, 3H), 1.40 - 1.28 (m, 1 H), 1.26 - 1.19 (m, 3H) LCMS SI for C23H25CI2N5O3) m/z: 490.1 [IVH-H]+. (Rt: 1.698min).
Example s. Inhibition of CYP51A1 by Coinpounds of the Invention
Method: Recombinant human CYP51A1 (lanosterol-14a-demethylase) enzyme was co-expressed with CYP reductase in bacterial membranes and the fluorescent substrate BOMCC (a non-natural substrate that causes increases in fluorescence upon CYP51A1 -dependent demethylation) was used to obtain 8-point dose concentration-response curves for each compound.
Results: As shown in Table 9, the compounds of the invention inhibit CYP51 A1 .
Table 9.
Figure imgf000296_0001
Figure imgf000297_0001
Figure imgf000298_0001
Example 7. inhibition of CYP51A1 moduiates TDP-43 aggregation
Introduction
Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease, is an aggressive, debilitating disease in which affected patients succumb within two to five years after diagnosis. ALS presents with heterogeneous clinical features but has a common underlying pathology of motor neuron loss that limits the central nervous system’s ability to effectively regulate voluntary and involuntary muscle activity. Additionally, without neuronal trophic support muscles being to atrophy, further exacerbating motor deterioration. Cellular and tissue degeneration results in motor impairment such as fasciculations and weakening in the arms, legs and neck, difficulty swallowing, slurred speech and ultimately failure of the diaphragm muscles that control breathing.
At the cellular level, 97% of all ALS cases have the common pathological feature of misfolded and aggregated TAR-DNA binding protein (TDP)-43 in spinal motor neuron inclusions. TDP-43 is a DNA/RNA binding protein involved in RNA splicing and is typically localized to the nucleus but can be translocated to the cytoplasm under conditions of cell stress. Nuclear clearing and cytoplasmic accumulation of misfolded and aggregated TDP-43 are hallmarks of degenerating motor neurons in ALS, but it remains unclear if mechanism of toxicity is due to aggregation-dependent loss of TDP-43 function or if the aggregates acquire toxic gain of function. Aggregates of TDP-43 accumulate in discrete cellular domains known as stress granules, which are also enriched with translationally inactive mRNAs. Stress granules are observed in multiple cellular types and are thought to be directly related to TDP-43-dependent toxicity in ALS and FTD. Dysfunction in DNA/RNA binding protein activity plays a crucial role in susceptible motor neurons in ALS, as familial cases have also been traced to mutations in the protein Fused in Sarcoma (FUS), a DNA/RNA binding protein that recently has been shown to be involved in gene silencing. Preclinical studies suggest that FUS mutations promote a toxic gain of function that may be causative in motor neuron degeneration.
Mutations in the TDP-43 gene (TARDBP) have also been causally linked to familial forms of ALS. A common TDP-43 mutation is known as Q331 K, in which glutamine (Q) 331 has been mutated to a lysine (K). This mutation results in a TDP-43 protein that is more aggregation prone and exhibits enhanced toxicity. A recent study has also demonstrated that the Q331 K mutation can confer a toxic gain of function in a TDP-43 knock-in mouse, which exhibits cognitive deficits and histological abnormalities similar to that which occurs in frontotemporal dementia (FTD). FTD refers to a group of degenerative disorders that are characterized by atrophy in the frontal and temporal cortices due to progressive neuron loss. Due to the functional nature of the brain regions impacted in FTD, the most common symptoms involve noticeable alterations in personality, behavior and linguistic ability and can also present with loss of speech. The pathological basis of FTD appears to be multifactorial involving mutations in genes such as C9orf72, progranulin (GRN) and MART, but intracellular inclusions of aggregated TDP-43, FUS and tau have been observed. Although ALS and FTD may have different genetic and molecular triggers and occur in different cell types, similar protein misfolding and degenerative mechanisms may operate in multiple diseases.
The toxic gain of function features of TDP-43 can be faithfully recapitulated in the simple model organism, budding yeast, where the protein also localizes to stress granules. Human disease mutations in TDP-43 enhance toxicity and yeast genetic screens have revealed key connections that are conserved io humans. The yeast model thus provides a robust cell­based screening platform for small molecules capable of ameliorating toxicity. To validate compounds from such phenotypic screens, it is imperative to test compounds in a mammalian neuronal context. In an effort to develop TDP­43­related mammalian models of neuron loss that occurs in ALS and FTD, primary cultures of rat cortical neurons were transfected with human wild type or Q331K mutant TDP­43. These cells were compared to cells which received an empty expression vector control. Validation studies have demonstrated that cells expressing either wild type or Q331K TDP­43 have are more susceptible to dying over time in culture. In the experiments described in this example, this model system is used to interrogate new therapeutic approaches to ameliorate TDP­43 toxicity. Results From the TDP­43 yeast model, a compound with known mode of action was identified that restored viability to TDP­43­expressing yeast (FIG.1A). Fluconazole is an antifungal known to inhibit Erg11, the yeast lanosterol 14­alpha demethylase (FIG.1B). Inhibition of Erg11 reduces ergosterol synthesis (yeast equivalent of cholesterol), while increasing lanosterol levels, the substrate of Erg11 (FIG.1C). The human homolog of Erg11 is Cyp51A1, a member of the cytochrome P450 superfamily of enzymes but does not appear to have a role in detoxification of xenobiotics. CYP51A1 has also been known as lanosterol 14­alpha demethylase, which describes its function in removing the 14­alpha­methyl group from lanosterol to generate 4,4­dimethylcholesta­8(9),14,24­trien­3β­ol, which is a critical step in the cholesterol biosynthetic pathway. To evaluate the potential role of CYP51A1 in TDP­43 pathology, the aforementioned primary rat cortical neuron TDP­43 models were utilized to test the efficacy of published inhibitors (FIG.2). Rat cortical neurons transfected with wild type human TDP­43 exhibited a significant reduction in survival compared to neurons transfected with empty vector control, and this reduction in survival was partially alleviated by treatment with compound A (FIGS.3A and 3B). Compound A has the structure:
Figure imgf000300_0001
A similar survival benefit was conferred by compound A when applied to cells transfected with Q331K mutant TDP­43 (FIGS.4A and 4B). A similar effect in rescuing a survival deficit was observed for a structurally differentiated compound, compound B, when applied to cells transfected with wild­type TDP­43 (FIGS.5A and 5B). Compound B has the structure:
Figure imgf000300_0002
These studies demonstrate that inhibition of Erg11 in yeast and inhibition of Cyp51A1 has a beneficial effect of rescuing cells from wild type and mutant TDP­43 toxicity and promotes cell survival. This is the first demonstration that inhibition of CYP51A1 is beneficial in treating and preventing TDP­43 pathological processes and represents a novel therapeutic approach for the treatment of ALS. Other Embodiments While the present invention has been described with reference to what are presently considered to be the preferred examples, it is to be understood that the invention is not limited to the disclosed examples. To the contrary, the invention is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims. All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety. Where a term in the present application is found to be defined differently in a document incorporated herein by reference, the definition provided herein is to serve as the definition for the term. Other embodiments are in the claims.

Claims

CLAIMS 1. A compound, or a pharmaceutically acceptable salt thereof, having the structure:
Figure imgf000302_0001
Formula I wherein m is 0, 1, 2, 3, or 4; X1 is CH, S, or N; X2 and X3 are, independently, N, CH, or CR1; X4 is NH or S; each R1 is, independently, halo, optionally substituted C1­C6 alkyl, or optionally substituted C1­C6 alkoxy; Ar is optionally substituted C6­C10 aryl or optionally substituted C2­C9 heteroaryl; L1 is ­CONR­ or ­NRCO­; R is hydrogen or optionally substituted C1­C6 alkyl; L2 is optionally substituted C1­C6 alkylene or optionally substituted C3­C8 cycloalkylene; RA is ­CH2CONHR2, ­CONHR2, or ­COR2; R2 is optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cyclohexyl, or has the structure:
Figure imgf000302_0002
Formula II Formula III wherein R3 is optionally substituted C1­C3 alkyl; R4 is optionally substituted C2­C6 alkyl, optionally substituted C2­C6 heteroalkyl, optionally substituted C3­C8 cycloalkyl, optionally substituted C2­C9 heterocyclyl, or optionally substituted C6­C10 aryl C1­C6 alkyl; n is 0 or 1; o is 0, 1, or 2; p is 0, 1, 2, 3, 4, 5, 6, 7, or 8; X5 is NR5, CR5R6, O, or SR5R6; R5 is absent, halo, oxo, hydrogen, hydroxy, optionally substituted amino, cyano, optionally substituted C1­C6 alkyl, optionally substituted C1­C6 alkoxy, ­SO2­optionally substituted C1­C6 alkyl, optionally substituted C2­C9 heterocyclyl, or optionally substituted C2­C9 heteroaryl; and R6 is absent, halo, oxo, hydroxy, optionally substituted amino, cyano, optionally substituted C1­C6 alkyl, optionally substituted C1­C6 alkoxy, ­SO2­optionally substituted C1­C6 alkyl, optionally substituted C2­C9 heterocyclyl, or optionally substituted C2­C9 heteroaryl; or R5 and R6 combine with the atoms to which they are attached to form an optionally substituted C2­C9 heterocyclyl, or optionally substituted C2­C9 heteroaryl; and each R7 is, independently, halo, optionally substituted C1­C6 heteroalkyl, or optionally substituted C1­C6 alkyl, and/or two R7, together with the atoms to which they are attached, combine to form an optionally substituted C2­C9 heterocyclyl; or R5 is absent, halo, oxo, hydrogen, hydroxy, optionally substituted amino, cyano, optionally substituted C1­C6 alkyl, optionally substituted C1­C6 alkoxy, ­SO2­optionally substituted C1­C6 alkyl, optionally substituted C2­C9 heterocyclyl, or optionally substituted C2­C9 heteroaryl; R6 and one R7, together with the atoms to which they are attached, combine to form an optionally substituted C2­C9 heterocyclyl or optionally substituted C2­C9 heteroaryl; and the remaining R7 groups, if present, are independently halo, optionally substituted C1­C6 heteroalkyl, or optionally substituted C1­C6 alkyl; or R6 is absent, halo, oxo, hydroxy, optionally substituted amino, cyano, optionally substituted C1­C6 alkyl, optionally substituted C1­C6 alkoxy, ­SO2­optionally substituted C1­C6 alkyl, optionally substituted C2­C9 heterocyclyl, or optionally substituted C2­C9 heteroaryl; R5 and one R7, together with the atoms to which they are attached, combine to form an optionally substituted C2­C9 heterocyclyl or optionally substituted C2­C9 heteroaryl; and the remaining R7 groups, if present, are independently halo, optionally substituted C1­C6 heteroalkyl, or optionally substituted C1­C6 alkyl; wherein if X5 is S, then each of R5 and R6 is, independently, absent or oxo; wherein if Ar is optionally substituted C6­C10 aryl, one of n and o is 1 and the other of n and o is 0 or 1, then X5 is NR5, wherein R5 is hydrogen, hydroxy, optionally substituted amino, cyano, optionally substituted C1­C6 alkoxy, ­SO2­optionally substituted C1­C6 alkyl, optionally substituted C2­C9 heterocyclyl, or optionally substituted C2­C9 heteroaryl; each R7 is, independently, halo, optionally substituted C1­C6 heteroalkyl, or optionally substituted C1­C6 alkyl, and/or two R7, together with the atoms to which they are attached, combine to form an optionally substituted C2­C9 heterocyclyl; or R5 and one R7, together with the atoms to which they are attached, combine to form an optionally substituted C2­C9 heterocyclyl or optionally substituted C2­C9 heteroaryl; X5 is CR5R6, wherein R5 is halo, hydrogen, hydroxy, optionally substituted amino, cyano, optionally substituted C1­C6 alkyl, optionally substituted C1­C6 alkoxy, ­SO2­optionally substituted C1­ C6 alkyl, optionally substituted C2­C9 heterocyclyl, or optionally substituted C2­C9 heteroaryl, and R6 is halo, optionally substituted amino, cyano, optionally substituted C1­C6 alkyl, optionally substituted C1­ C6 alkoxy, ­SO2­optionally substituted C1­C6 alkyl, optionally substituted C2­C9 heterocyclyl, or optionally substituted C2­C9 heteroaryl, and each R7 is, independently, halo, optionally substituted C1­ C6 heteroalkyl, or optionally substituted C1­C6 alkyl, and/or two R7, together with the atoms to which they are attached, combine to form an optionally substituted C2­C9 heterocyclyl; or R5 is halo, hydrogen, hydroxy, optionally substituted amino, cyano, optionally substituted C1­C6 alkyl, optionally substituted C1­C6 alkoxy, ­SO2­optionally substituted C1­C6 alkyl, optionally substituted C2­C9 heterocyclyl, or optionally substituted C2­C9 heteroaryl; R6 and one R7, together with the atoms to which they are attached, combine to form an optionally substituted C2­C9 heterocyclyl or optionally substituted C2­C9 heteroaryl; and the remaining R7 groups, if present, are independently halo, optionally substituted C1­C6 heteroalkyl, or optionally substituted C1­C6 alkyl; or R6 is halo, hydroxy, optionally substituted amino, cyano, optionally substituted C1­C6 alkyl, optionally substituted C1­C6 alkoxy, ­SO2­optionally substituted C1­C6 alkyl, optionally substituted C2­C9 heterocyclyl, or optionally substituted C2­C9 heteroaryl; R5 and one R7, together with the atoms to which they are attached, combine to form an optionally substituted C2­C9 heterocyclyl or optionally substituted C2­C9 heteroaryl; and the remaining R7 groups, if present, are independently halo, optionally substituted C1­ C6 alkyl, or optionally substituted C1­C6 alkyl; X is O, wherein p is 1, 2, 3, 4, 5, 6, 7, or 8; or X is SR5R6, wherein each of R5 and R6 is oxo. 2. The compound of claim 1, wherein X1 is CH. 3. The compound of claim 1, wherein X1 is N. 4. The compound of claim 1, wherein X1 is S. 5. The compound of any one of claims 1 to 4, wherein X2 is CR1 and X3 is N. 6. The compound of claim 5, wherein the compound has the structure:
Figure imgf000304_0001
Formula 1a or a pharmaceutically acceptable salt thereof. 7. The compound of any one of claims 1 to 4, wherein X2 is N and X3 is CR1. 8. The compound of claim 7, wherein the compound has the structure:
Figure imgf000304_0002
. Formula 1b or a pharmaceutically acceptable salt thereof.
9. The compound of any one of claims 1 to 3, wherein the compound has the structure:
Figure imgf000305_0001
or a pharmaceutically acceptable salt thereof, wherein each R1A is independently H or R1. 10. The compound of any one of claims 1 to 3, wherein X2 and X3 are CR1. 11. The compound of any one of claims 1 to 10, wherein at least one R1 is halo. 12. The compound of claim 11, wherein halo is chloro. 13. The compound of any one of claims 1 to 10, wherein at least one R1 is optionally substituted C1­ C6 alkyl. 14. The compound of claim 13, wherein optionally substituted C1­C6 alkyl is methyl. 15. The compound of any one of claims 1 to 10, wherein at least one R1 is optionally substituted C1­ C6 alkoxy. 16. The compound of claim 15, wherein optionally substituted C1­C6 alkoxy is methoxy or ethoxy. 17. The compound of any one of claims 1 to 16, wherein Ar is optionally substituted C6­C10 aryl. 18. The compound of claim 17, wherein optionally substituted C6­C10 aryl is phenyl, 2­chloro­phenyl, 3­chloro­phenyl, 4­chloro­phenyl, 2­fluoro­phenyl, 3­fluoro­phenyl, 4­fluoro­phenyl, 2­methyl­phenyl, 4­ benzoxy­phenyl, 2­methoxy­phenyl, 3­methoxy­phenyl, 4­methoxy­phenyl, 2­cyano­phenyl, 3­cyano­phenyl, 4­cyano­phenyl, 2­chloro­4­fluoro­phenyl, 2,4­fluoro­phenyl, 2­chloro­3­fluoro­phenyl, 2­chloro­4­cyano­ phenyl, 2­cyano­4­fluoro­phenyl, 2­cyano­4­chloro­phenyl, 2,3­fluoro­phenyl, 2­fluoro­4­cyano­phenyl, 2­ chloro­6­fluoro­phenyl, 2­fluoro­4­chloro­phenyl, 2,6­fluoro­phenyl, 2,5­fluoro­phenyl, or 2­fluoro­4­methoxy­ phenyl. 19. The compound of any one of claims 1 to 16, wherein Ar is optionally substituted C2­C9 heteroaryl.
Figure imgf000306_0001
21. The compound of claim 20, wherein the compound has the structure:
Figure imgf000306_0002
Formula 1d or a pharmaceutically acceptable salt thereof. 22. The compound of any one of claims 1 to 20, wherein R2 has the structure of Formula II. 23. The compound of any one of claims 1 to 20, wherein R2 has the structure of Formula III. 24. The compound of claim 23, wherein R2 has the structure:
Figure imgf000306_0003
Figure imgf000307_0001
R10 is optionally substituted C1­C6 alkyl; and each R8 is, independently, halo, hydroxy, optionally substituted C1­C6 alkyl, or optionally substituted C1­C6 alkoxy.
25. The compound of any one of claims 1 to 24, wherein R is hydrogen. 26. The compound of claim 6, wherein the compound has the structure:
Figure imgf000308_0001
or a pharmaceutically acceptable salt thereof, wherein R11 is Cl or F; and R12 is optionally substituted 2­oxa­6­azaspiro[3.3]heptan­6­yl. 27. The compound of claim 6, wherein the compound has the structure:
Figure imgf000308_0002
Formula 1e or a pharmaceutically acceptable salt thereof, wherein R13 is optionally substituted pyridin­4­yl or optionally substituted phenyl; and R14 is optionally substituted piperidin­4­yl, optionally substituted 2­oxa­6­azaspiro[3.3]heptan­6­yl, 2­ azaspiro[3.3]heptan­2­yl substituted with hydroxy. 28. The compound of claim 27, wherein R13 is optionally substituted phenyl. 29. The compound of claim 28, wherein optionally substituted phenyl is 2,4­difluorphenyl. 30. The compound of claim 27, wherein R13 is optionally substituted pyridin­4­yl. 31. The compound of claim 30, wherein the optionally substituted pyridin­4­yl is
Figure imgf000308_0003
. 32. The compound of claim 6, wherein the compound has the structure:
Figure imgf000308_0004
Formula 1f or a pharmaceutically acceptable salt thereof, wherein R15 is optionally substituted 4­azaspiro[2.4]heptan­4­yl. 33. The compound of claim 6, wherein the compound has the structure:
Figure imgf000309_0001
Formula 1g or a pharmaceutically acceptable salt thereof, wherein R16 is optionally substituted 2­oxa­5­azabicyclo[2.2.1]heptan­5­yl, or optionally substituted 3­ azabicyclo[3.1.0]hexan­3­yl. 34. The compound of claim 6, wherein the compound has the structure:
Figure imgf000309_0002
or a pharmaceutically acceptable salt thereof, wherein R17 and R18 are each, independently, H or F; and R19 is 2­oxa­5­azabicyclo[2.2.1]heptan­5­yl, or 3­azabicyclo[3.1.0]hexan­3­yl. 35. The compound of claim 6, wherein the compound has the structure:
Figure imgf000309_0003
or a pharmaceutically acceptable salt thereof, wherein R20 is optionally substituted 2­oxa­6­azaspiro[3.3]heptan­6­yl. 36. The compound of claim 6, wherein the compound has the structure:
Figure imgf000310_0001
Formula 1j or a pharmaceutically acceptable salt thereof, wherein R21 is optionally substituted pyridinyl; and R22 is piperidin­1­yl optionally substituted with methoxy; azetidin­1­yl optionally substituted with methyl, methoxy, or fluoro; 2­oxa­5­azabicyclo[2.2.1]heptan­5­yl; or optionally substituted morpholin­4­yl. 37. The compound of claim 36, wherein optionally substituted pyridinyl
Figure imgf000310_0002
,
Figure imgf000310_0003
, 38. A compound having the structure:
Figure imgf000310_0004
Formula 2 or a pharmaceutically acceptable salt thereof, wherein m is 0, 1, 2, 3, or 4; X6 is CH, or N; X7 is NH or S; Ar1 is optionally substituted C6­C10 aryl; R23 is hydrogen, halo, or optionally substituted C1­C6 alkoxy; L3 is ­NR24CO­ or ­CONR24­, R24 is H or optionally substituted C1­C6 alkyl; RB is ­CH2CONHR25, or ­COR25 or NR25; R25 is optionally substituted heteroaryl, optionally substituted cyclohexyl, or has the structure
Figure imgf000310_0005
Formula II Formula III wherein R26 is optionally substituted C1­C3 alkyl; R27 is optionally substituted C1­C6 alkyl, or optionally substituted C1­C6 heteroalkyl; n is 0 or 1; o is 0 or 1 or 2; X8 is NR28, CR28R29, or O; R28 is absent, hydrogen, hydroxy, optionally substituted amino, optionally substituted C1­C6 alkyl, OR optionally substituted C1­C6 alkoxy; R29 is absent, hydrogen, hydroxy, optionally substituted amino, optionally substituted C1­C6 alkyl, OR optionally substituted C1­C6 alkoxy; each R25 is, independently, halo or optionally substituted C1­C6 alkyl. 39. The compound of claim 38, wherein X6 is CH. 40. The compound of claim 38, wherein X6 is N. 41. The compound of claim 38, wherein X7 is N. 42. The compound of claim 38, wherein X7 is S. 43. The compound of claim 38, wherein the compound has the structure:
Figure imgf000311_0001
or a pharmaceutically acceptable salt thereof. 44. The compound of claim 38, wherein the compound has the structure:
Figure imgf000311_0002
Formula 2b or a pharmaceutically acceptable salt thereof. 45. The compound of claim 38, wherein the compound has the structure:
Figure imgf000312_0001
or a pharmaceutically acceptable salt thereof. 46. The compound of claim 38, wherein the compound has the structure:
Figure imgf000312_0002
Formula 2d or a pharmaceutically acceptable salt thereof. 47. The compound of claim 38, wherein the compound has the structure:
Figure imgf000312_0003
Formula 2e or a pharmaceutically acceptable salt thereof. 48. The compound of any one of claims 38 to 47, claim 15, wherein Ar1 is phenyl, 2­chloro­phenyl, 4­ chloro­phenyl, 2­fluoro­phenyl, 3­fluoro­phenyl, 4­fluoro­phenyl, 2­methyl­phenyl, 2­methoxy­phenyl, 3­ methoxy­phenyl, 4­methoxy­phenyl, 2­chloro­4­fluoro­phenyl, 2,4­fluoro­phenyl, 2­chloro­3­fluoro­phenyl, 2­ cyano­4­fluoro­phenyl, 2­cyano­4­chloro­phenyl, 2,3­difluoro­phenyl, 2­chloro­6­fluoro­phenyl, or 2­fluoro­4­ chloro­phenyl. 49. The compound of any one of claims 38 to 48, wherein R25 has the structure
Figure imgf000312_0004
,
Figure imgf000312_0005
50. The compound of any one of claims 38 to 48, wherein R25 has the structure:
Figure imgf000313_0001
51. A compound having the structure:
Figure imgf000313_0002
Formula 3 or pharmaceutically acceptably salt thereof, Wherein n is 0, 1, 2, 3, or 4; L4 is optionally substituted C1­C6 alkylene or optionally substituted C3­C8 cycloalkylene; X9 is N and X10 is CH, or X9 is CH and X10 is N; X11 is N or CH; each R30 is, independently, halo, optionally substituted C1­C6 alkyl, or optionally substituted C1­C6 alkoxy; ArC is optionally disubstituted C6­C10 aryl, or C6­C10 aryl optionally monosubstituted with chloro, optionally substituted C1­C6 heteroalkyl, cyano, meta­fluoro, or ortho­fluoro; RC is COR31, or ­R31; R31 is optionally substituted C2­C5 heteroaryl, or has the structure:
Figure imgf000313_0003
Formula II Formula III wherein R32 is optionally substituted C1­C3 alkyl; R33 is optionally substituted C1­C6 alkyl C6 aryl, optionally substituted C1­C6 alkyl C2­C5 heteroaryl, optionally substituted C5 cycloalkyl, optionally substituted C3­C6 alkyl, or optionally substituted C3 heteroalkyl. n is 0 or 1; o is 0 or 1 or 2; p is 0, 1, 2, 3, 4, 5, 6, 7, or 8; X12 is NR34, CR34R35, O, or SR34R35; R34 is absent, halo, oxo, hydrogen, hydroxy, optionally substituted amino, cyano, optionally substituted C1­C6 alkyl, optionally substituted C1­C6 heteroalkyl, ­SO2­optionally substituted C1­C6 alkyl, optionally substituted C2­C9 heterocyclyl, optionally substituted C6­C10 aryl, or optionally substituted C2­C9 heteroaryl, or R33 and R34 combine with the atoms to which they are attached to form an optionally substituted C2­C9 heteroaryl; R35 is absent, halo, oxo, hydroxy, optionally substituted amino, cyano, optionally substituted C1­C6 alkyl, optionally substituted C1­C6 heteroalkyl, ­SO2­optionally substituted C1­C6 alkyl, optionally substituted C2­C9 heterocyclyl, optionally substituted C6­C10 aryl or optionally substituted C2­C9 heteroaryl, or R33 and R35 combine with the atoms to which they are attached to form an optionally substituted C2­C9 heteroaryl; and each R33 is, independently, halo, oxo, or optionally substituted C1­C6 alkyl, or two R33 combine with the atoms to which they are attached to form an optionally substituted C4 cycloalkyl. 52. The compound of claim 51, wherein X9 is N and X10 is CH. 53. The compound of claim 51, wherein X9 is CH and X10 is N. 54. The compound of claim 51, wherein X11 is N. 55. The compound of claim 51, wherein X11 is CH. 56. The compound of claim 51, wherein the compound has the structure:
Figure imgf000314_0001
Formula 3a or a pharmaceutically acceptable salt thereof. 57. The compound of claim 51, wherein the compound has the structure:
Figure imgf000314_0002
Formula 3b or a pharmaceutically acceptable salt thereof. 58. The compound of any one of claims 51 to 57, wherein at least one R30 is halo. 59. The compound of claim 58, wherein halo is chloro. 60. The compound of any one of claims 51 to 57, wherein at least one R30 is optionally substituted C1­C6 alkyl. 61. The compound of claim 58, wherein optionally substituted C1­C6 alkyl is methyl.
62. The compound of any one of claims 51 to 57, wherein at least one R30 is optionally substituted ­ alkoxy. 63. The compound of claim 62, wherein optionally substituted C1­C6 alkoxy is methoxy or ethyoxy. 64. The compound of any one of claims 51 to 63, wherein ArC is 2­chloro­phenyl, 3­chloro­phenyl, 4­ chloro­phenyl, 2­fluoro­phenyl, 3­fluoro­phenyl, 4­benzoxy­phenyl, 2­cyano­phenyl4­cyano­phenyl, 2­chloro­ 4­fluoro­phenyl, 2,4­difluoro­phenyl, 2­chloro­3­fluoro­phenyl, 2­chloro­4­cyano­phenyl, 2­cyano­4­fluoro­ phenyl, 2­cyano­4­chloro­phenyl, 2,3­difluoro­phenyl, 2­fluoro­4­cyano­phenyl, 2­chloro­6­fluoro­phenyl, 2­ fluoro­4­chloro­phenyl, 2,6­difluoro­phenyl, 2,5­difluoro­phenyl, 2­chloro­3­fluoro­phenyl, 3,4­difluoro­phenyl, 2,3­difluoro­phenyl, or 2­fluoro­4­methoxy­phenyl. 65. The compound of any one of claims 51 to 64, wherein R31 has the structure
Figure imgf000315_0001
or
Figure imgf000315_0002
. 66. The compound of any one of claims 51 to 64, wherein R31 has the structure:
Figure imgf000315_0003
wherein p is 0, 1, 2, 3, 4, or 5; q is 0, 1, 2, 3, or 4; and each R36 is, independently, halo, hydroxy, optionally substituted C1­C6 alkyl, or optionally substituted C1­C6 alkoxy. 67. The compound of claim 56, wherein the compound has the structure:
Figure imgf000316_0001
Formula 3c or a pharmaceutically acceptable salt thereof, wherein R37 is cyano and R38 is fluoro, or R37 is fluoro and R38 is cyano; and R39 is or 3,3­difluoro­azetidin­1­yl. 68. The compound of claim 51, wherein the compound has the structure:
Figure imgf000316_0002
Formula 3d or a pharmaceutically acceptable salt thereof, wherein l is 0 or 1; L4 is is optionally substituted C1­C6 alkylene or optionally substituted C3­C8 cycloalkylene; and R40 is 4­hydroxy­piperidin­1­yl, 3­methoxy­piperidin­1­yl, optionally substituted diazapen­1­yl, triazolopiperazinyl substituted with methyl, 4,4­difluoro­piperidin­1­yl, 1,1­dioxothiomorpholin­4­yl, 2­ (methoxymethyl)­pyrroloin­1­yl, tetrahydro­1,3­oxazin­3­yl, 4­isopropyl­piperazin­1­yl, 4­(2­oxazolidin­3­yl)­ piperidin­1­yl, optionally substituted 1,2,4 oxadizol­5­yl, or optionally substituted 1,3,4 oxadizol­2­yl. 69. The compound of claim 68, wherein l is 1. 70. The compound of claim 56, wherein the compound has the structure: .
Figure imgf000316_0003
or a pharmaceutically acceptable salt thereof, wherein R41 is piperidin­1­yl substituted with optionally substituted dialkylamino. 71. The compound of claim 56, wherein the compound has the structure:
Figure imgf000317_0001
Formula 3f or a pharmaceutically acceptable salt thereof, wherein R43 is F or CN; and R42 is optionally substituted azetidin­1­yl. 72. The compound of claim 71, wherein optionally substituted azetidine­1­yl is 3,3­difluoro­azetidin­1­ yl. 73. The compound of claim 56, wherein the compound has the structure:
Figure imgf000317_0002
Formula 3g or a pharmaceutically acceptable salt thereof, wherein R44 is optionally substituted azetidin­1­yl. 74. the compound of claim 73, wherein optionally substituted azetidine­1­yl is 3,3­difluoro­azetidin­1­ yl. 75. A compound, or pharmaceutically acceptable salt thereof, having the structure:
Figure imgf000317_0003
Formula 4 or a pharmaceutically acceptable salt thereof, wherein n is 0, 1, 2, 3, or 4; R45 is halo; and R46 is optionally substituted azetidinyl. 76. The compound of claim 75, wherein the compound has the structure:
Figure imgf000318_0001
Formula 4a or a pharmaceutically acceptable salt thereof. 77. The compound of claim 75, wherein the compound is:
Figure imgf000318_0002
, or a pharmaceutically acceptable salt thereof. 78. A compound, or pharmaceutically acceptable salt thereof, having the structure:
Figure imgf000318_0003
Formula 5 or a pharmaceutically acceptable salt thereof, wherein p is 0 or 1; X13 is a single bond or O; R48 is optionally substituted C6­C10 aryl, optionally substituted C2­C5 heteroaryl, or trifluoromethyl; R49 is H or optionally substituted C1­C6 alkyl; R50 is optionally substituted C6­C10 aryl or optionally substituted C2­C5 hetetoaryl; and R51 is optionally substituted C2­C5 heterocyclyl. 79. The compound of claim 78, wherein p is 0. 80. The compound of claim 78, wherein p is 1. 81. The compound of claim 78, wherein X13 is O.
82. The compound of claim 78, wherein R48 is optionally substituted C6­C10 aryl. 83. The compound of claim 78, wherein optionally substituted C6­C10 aryl is
Figure imgf000319_0001
, ,
Figure imgf000319_0002
84. The compound of claim 78, wherein R48 is optionally substituted C2­C5 heteroaryl. 85. The compound of claim 79, wherein optionally substituted C2­C5 heteroaryl is
Figure imgf000319_0003
,
Figure imgf000319_0004
86. The compound of claim 78, wherein R49 is H. 87. The compound of claim 78, wherein R49 is C1­C6 alkyl. 88. The compound of claim 87, wherein C1­C6 alkyl is methyl. 89. The compound of claim 78, wherein R50 is optionally substituted C6­C10 aryl. 90. The compound of claim 89, wherein optionally substituted C6­C10 aryl is
Figure imgf000319_0005
Figure imgf000319_0006
. 91. The compound of claim 78, wherein R50 is optionally substituted C2­C5 heteroaryl.
92. The compound of claim 86, wherein optionally substituted C2­C5 heteroaryl is
Figure imgf000320_0001
. 93. The compound of claim 87, wherein R51 is morpholin­4­yl, 2­oxa­6­azaspiro[3.3]heptan­6­yl, or 4­ hydroxy­piperidin­4­yl. 94. The compound of claim 1, wherein the compound has the structure:
Figure imgf000320_0002
Formula 1k or a pharmaceutically acceptable salt thereof, wherein
Figure imgf000320_0003
95. The compound of claim 1, wherein the compound has the structure:
Figure imgf000320_0004
Formula 1l or a pharmaceutically acceptable salt thereof, wherein
Figure imgf000320_0005
Figure imgf000321_0001
96. The compound of claim 1, wherein the compound has the structure:
Figure imgf000321_0002
Formula 1m or a pharmaceutically acceptable salt thereof,
Figure imgf000321_0003
97. The compound of claim 96, wherein X14 is N. 98. The compound of claim 96, wherein X14 is CH. 99. The compound of claim 38, wherein the compound has the structure:
Figure imgf000321_0004
Formula 2f or a pharmaceutically acceptable salt thereof, wherein R57 is halo. 100. The compound of claim 99, wherein halo is chloro.
101. The compound of claim 51, wherein the compound has the structure:
Figure imgf000322_0001
Formula 3h or a pharmaceutically acceptable salt thereof, wherein R57 and R58 are each halo. 102. The compound of claim 101, wherein R57 and R58 are each fluoro. 103. The compound of claim 1, wherein the compound has the structure:
Figure imgf000322_0002
,
Figure imgf000323_0001
104. The compound of claim 1, wherein the compound has the structure:
Figure imgf000323_0002
,
Figure imgf000323_0003
pharmaceutically acceptable salt thereof. 105. The compound of claim 1, wherein the compound has the structure:
Figure imgf000323_0004
,
Figure imgf000323_0005
, or a pharmaceutically acceptable salt thereof.
106. The compound of claim 38, wherein the compound has the structure:
Figure imgf000324_0001
. 108. A compound having the structure of any one of compounds 1­198, 356, 373, 386, 419, 435, or 436 in Table 1, or a pharmaceutically acceptable salt thereof. 109. A compound having the structure of any one of compounds 199­355, 357­372, 374­385, 387­ 418, 420­434, or 437­453 in Table 1, or a pharmaceutically acceptable salt thereof. 110. A pharmaceutical composition comprising a compound of any one of claims 1 to 109, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient. 111. A method of treating a neurological disorder in a subject in need thereof, the method comprising administering an effective amount of a compound, or pharmaceutically acceptable salt thereof, of any one of claims 1 to 109 or a pharmaceutical composition of claim 110. 112. A method of inhibiting toxicity in a cell related to a protein, the method comprising administering an effective amount of a compound of any one of claims 1 to 109 or a pharmaceutical composition of claim 110. 113. The method of claim 112, wherein the toxicity is TDP­43­related toxicity. 114. The method of claim 112 or 113, wherein the cell is a mammalian neural cell.
115. A method of treating a CYP51A1­associated disorder in a subject in need thereof, the method comprising administering an effective amount of a compound, or pharmaceutically acceptable salt thereof, of any one of claims 1 to 109 or a pharmaceutical composition of claim 110. 116. The method of claim 115, wherein the CYP51A1­associated disorder is ALS. 117. A method of inhibiting CYP51A1, the method comprising contacting a cell with an effective amount of a compound of any one of claims 1 to 109 or a pharmaceutical composition of claim 110.
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