WO2020252397A1 - Chimères ciblant la protéolyse à petites molécules et leurs méthodes d'utilisation - Google Patents

Chimères ciblant la protéolyse à petites molécules et leurs méthodes d'utilisation Download PDF

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WO2020252397A1
WO2020252397A1 PCT/US2020/037607 US2020037607W WO2020252397A1 WO 2020252397 A1 WO2020252397 A1 WO 2020252397A1 US 2020037607 W US2020037607 W US 2020037607W WO 2020252397 A1 WO2020252397 A1 WO 2020252397A1
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substituted
unsubstituted
compound
btk
cancer
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WO2020252397A8 (fr
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Jin Wang
Wen-hao GUO
Xiaoli Qi
Luhua Wang
Yang Liu
Krystle J. NOMIE
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Baylor College Of Medicine
Board Of Gerents, The University Of Texas System
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • Proteolysis-targeting chimeras are composed of three components, including binders for a targeted protein, an E3 ligase, and a linker bridging these two binders. Formation of the ternary complex is the prerequisite for efficient degradation of the targeted protein. Therefore, enhancing the binding affinity to the targeted protein is a strategy to improve PROTAC potency.
  • PROTACs and methods for their use as Bruton’s tyrosine kinase (BTK) degraders.
  • the small molecule PROTACs described herein are useful in treating and/or preventing BTK- related diseases, such as cancer, neurodegenerative disorders, inflammatory diseases, and metabolic disorders.
  • the methods include administering to a subject a compound as described herein.
  • a class of small molecule PROTACs as described herein includes compounds of tire following formula:
  • A is a BTK binder
  • L is a linker
  • B is an E3 ligase binder
  • L comprises a reversible covalent group
  • B comprises a CRBN ligand, a VHL ligand, a cIAPl ligand, a MDM2 ligand, a RNF2 ligand, or a DCAF15 ligand.
  • a class of small molecule PROTACs as described herein includes compounds of the following formula:
  • R 1 , R 2 , R 8 , and R 9 are each independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, substituted or unsubstituted heteroalky nyl, substituted or unsubstituted carbonyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted
  • a class of small molecule PROTACs as described herein includes compounds of the following formula:
  • R 1 , R 2 , R 3 , and R 4 are each independently selected from hydrogen, substituted or unsubstituted alkyl, and substituted or unsubstituted carbonyl; and R 5 is hydrogen, deuterium, fluoro, chloro, bromo, iodo, cyano, -OCH3, -OCDH2, -OCD2H, or - OCD3.
  • L is a substituted or unsubstituted alkyl, a substituted or unsubstituted alkenyl, a substituted or unsubstituted alkynyl, a substituted or unsubstituted alkoxy, or a substituted or unsubstituted amino.
  • L contains an amide group.
  • X and Y are each C(O).
  • R 1 , R 2 , R 3 , and R 4 are each independently selected from hydrogen and methyl.
  • the compound has the following formula:
  • the compound has the following formula;
  • the confound has the following formula:
  • a class of small molecule PROTACs as described herein includes compounds of tire following formula:
  • R 1 , R 2 , R 8 , and R 9 are each independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, substituted or unsubstituted heteroalkynyl, substituted or unsubstituted carbonyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, and substituted
  • the compound has the following formula:
  • composition including a compound as described herein and a pharmaceutically acceptable carrier.
  • kit including a compound or composition as described herein.
  • a method of treating or preventing a BTK-related disease in a subject includes administering to the subject an effective amount of a compound or composition as described above.
  • the BTK-related disease is cancer (e.g., bladder cancer, blood cancer, a bone marrow cancer, brain cancer, breast cancer, bronchus cancer, colorectal cancer, cervical cancer, chondrosarcoma, endometrial cancer, gastrointestinal cancer, gastric cancer, genitourinary cancer, head and neck cancer, hepatic cancer, hepatocellular carcinoma, leukemia, liver cancer, lung cancer, lymphoma, melanoma of the skin, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, skin cancer, testicular cancer, thyroid cancer, or uterine cancer).
  • the BTK-related disease is a neurodegenerative disorder or an inflammatory disease.
  • the methods can further include administering a second compound, biomolecule, or composition.
  • the second compound, biomolecule, or composition is a chemotherapeutic agent.
  • a method of inducing BTK degradation in a cell includes contacting a cell with an effective amount of a compound as described herein. The contacting can be performed in vitro or in vivo.
  • Fig. 1 shows a demonstration of catalytic degradation of targeted proteins by reversible covalent PROTACs.
  • Fig. 2A-D depicts BTK degradation induced by PROTACs.
  • Fig. 2A shows chemical structures of BTK degraders and their controls.
  • RC-1, RNC-1 and IRC-1 are BTK degraders with reversible covalent, reversible non-covalent, and irreversible covalent warheads, respectively.
  • RC-Ctrl, RNC-Ctrl, and IRC-Ctrl i.e., ibrutinib
  • MOLM-14 cells were incubated with RC-1, RNC-1 and IRC-1 for 24 hours. The BTK levels were quantified by Western blotting.
  • Fig. 2C shows RC-1 dose- dependent BTK degradation in MOLM-14 cells.
  • DCso compound concentration inducing 50% of protein degradation.
  • Fig. 2D MOLM-14 cells were treated with DMSO, RC-1, RC-Ctrl, Pomalidomide, RC-Ctrl + Pomalidomide, and RC-1 -Me for 24 hours. All the compound concentrations are 200 nM.
  • Fig. 3 shows the linker development for reversible covalent BTK degraders.
  • the developed linkers of the RC series of BTK degraders and their corresponding % of BTK degradation in MOLM-14 cells (200 nM, 24 h incubation) are shown.
  • Fig. 4A-D shows the target engagement for BTK degraders.
  • Fig. 4A quantitative Western blot was performed on lysate of 1 *10 6 MOLM-14 cells using BTK and CRBN recombinant proteins as the standard.
  • Fig. 4B shows the results of a CRBN in-cell target engagement assay.
  • HEK-293 cells were transiently transfected with plasmids expressing a fusion protein of CRBN and nano-luciferase (nLuc) for 24 h and then the cells w ere treated with a CRBN tracer (0.5 ⁇ ), which binds to CRBN to induce bioluminescence resonance energy transfer (BRET).
  • BRET bioluminescence resonance energy transfer
  • the target engagement ICso values for RC-1, IRC-1, and RNC-1 are 0.25, 0.86. and 1.69 ⁇ , respectively.
  • Fig. 4C shows the results of a BTK in-cell target engagement assay. This assay is the same as the CRBN in-cell target engagement assay, except BTK-nLuc fusion plasmid and BTK tracer (1.0 ⁇ ) were used.
  • the target engagement ICso values for RC-1, IRC-1, and RNC-1 are 0.043, 0.13 and 1.27 ⁇ , respectively.
  • Fig. 4D the same BTK in-cell target engagement assay as in Fig. 4C was applied to RC-1, RC-Ctrl, DD-03-171 and MT-802.
  • the target engagement ICso values for RC-1 and RC-Ctri are the same within experimental errors, demonstrating that the intracellular accumulation ofRC-1 is similar to its parent warhead molecule.
  • the target engagement IC50 values for DD-03-171 and MT-802 are 5 and 11 folds of that of RC- 1, respectively. Triplicates were performed with SEM as the error bars.
  • Fig. 5 A-B shows the results from a fluorophore phase separation-based assay for imaging ternary complex formation in living cells.
  • Fig. 5A shows a schematic diagram showing the design of the cellular assay.
  • Fig. 5B contains fluorescence images showing detection of BTK PROTACs-induced interaction between the E3 ligase cereblon and the target protein BTK. A fluorescence histogram of the line across the cells is shown below. Scale bar: 10 pm.
  • Fig. 6A-B shows that RC-1 overcomes drug resistance.
  • Fig. 6A XLA cells overexpressing wild type or C481 S mutant BTK were treated with RC-1 for 24 hours. Then Western blotting was performed to evaluate the degradation of BTK. Duplicates were performed.
  • Fig. 6B RC-1 induced degradation of BTK, pBTK, IKZF1 and 1KZF3 in Mino cells. Duplicates were performed.
  • Fig. 7A-D contains the results of aproteomic analysis showing RC-1 selectively degrades BTK MOLM-14 cells were treated with either compounds [200 nM (Fig. 7 A) RC- 1, (Fig. 7B) RNC-1, (Fig. 7C) IRC-1, or (Fig. 7D) RC-l-Me (RC-1 non-degrader control)] or DMSO for 24 hours. Lysates were treated with a TMT-10plex kit and subjected to mass spec- based proteomics analysis. Datasets represent an average of duplicates. Volcano plot shows protein abundance (log2) as a function of significance level (logio). Nonaxial vertical lines denote abundance changes from 0.7 to 1.4 (i.e.
  • Fig. 8A-C depicts molecular dynamics simulations for ternary complexes of BTK- PROTAC-CRBN.
  • a RNC-1 mediated complex shows a larger structural fluctuation compared with the other two ligands, reflected by (A) Root Mean Square Fluctuation (RMSF) per residue and (B) Root Mean Square Deviation (RMSD) calculated from the simulations of the ⁇ BTK-PROTAC-CRBN ⁇ ternary complexes.
  • Fig. 8C shows the predicted most stable conformation of ⁇ BTK-RC-l-CRBN ⁇ ternary complex.
  • Fig. 9A-B shows a comparison ofRC-1 with other reported BTK degraders, including two previously reported BTK degraders DD-03-171 and MT-802.
  • MOLM-14 cells Fig. 9A
  • Mino cells Fig. 9B
  • Alarma blue assays were performed to quantify the cellular viabilities. Data are presented as means ⁇ SEM of 5 replicates.
  • Fig. 10A-B depicts the PK/PD of RC-1.
  • Fig. 11 A-B depicts BIX degradation induced by PROTACs.
  • Fig. 11 A shows the chemical structures of BIX degraders.
  • Fig. 1 IB shows Western blots from MOLM-14 cells treated with the indicated doses of PROTACs for 24 hours. Duplicates were performed.
  • Fig. 12 is a graph shown the correlation between Kd towards TBX-1 and DC 50 .
  • Fig. 13 A-B depicts the quantification of BTK and CRBN levels in MOLM-14 cells.
  • MOLM-14 cells w r ere maintained in RPMI-1640 complete culture medium lxlO 6 cells were collected and processed for Western blot analysis of BTK and CRBN levels using their corresponding primary and secondary antibodies.
  • BTK and CRBN were quantified by normalizing the sample BTK and CRBN to their standard recombinant BTK and CRBN. The data shown were average of 3 repeat samples.
  • Fig. 14B shows the input parameters and output exact values of PROTACs based on the ternary complex formation modeling.
  • Fig. 15 shows a 9-point dose response curve for concentrations of the indicated compounds. Each concentration point was performed in duplicate.
  • Fig. 16A shows the chemical structures of IRC-l-DiMe
  • Fig. 16B is a graph showing the BTK target engagement for RC-1 , IRC-DiMe and RNC-CN-DiMe.
  • HEK-293 cells were transiently transfected with plasmids expressing a fusion protein of BTK and nano-luciferase (riLuc) for 24 hours and then the cells were treated with a BTK tracer (1.0 ⁇ ), which binds to BTK to induce energy- transfer signals. Adding PROTACs to cells would compete the BTK tracer binding to BTK, thus reducing the NanoBRET signals.
  • the IC 50 values for RC-1, IRC-DiMe, and RNC-CN- DiMe are 0.033, 0.38. and 0.93 ⁇ , respectively.
  • Fig. 17 depicts the BTK degradation by- three categories of PROTACs in XLA cells overexpressing wild type BTK (XLA-WT) or mutant C481S BTK (XLA-C481S).
  • Fig. 18A-B contains graphs showing the results of cell viability assays following treatment with BTK PROTAC degraders and their corresponding warhead controls in MOLM-14 cells (Fig. 18A) and Mino cells (Fig. 18B).
  • Fig. 19A-B shows protein degradation by BTK PROTAC RC-1 in mouse spleen. ICR mice were subjected to single intraperitoneal (IP) injection of RC-1 at t dhoese of 50 mg or 100 mg per kg body weight (N 3 - 4 per group) and spleens were harvested 24 hours after injection. The splenic p-BTK(Y233), BTK, IKZF1 and IKZF3 levels were measured with Western blot (Fig. 19A) and quantified (Fig. 19B).
  • Fig. 20 shows BTK degradation induced by RC-1 in mouse cell line. E mu-myc transgenic mouse cells were incubated with RC-1 for 24 hours. The BTK levels were quantified by Western blotting. Duplicates were performed.
  • Fig. 21 A-B show BTK degradation induced by RC-1 and MT-802.
  • MOLM-14 cells were incubated with RC-1 and MT-802 for 24 hours.
  • the BTK levels were quantified by Western blotting. Duplicates were performed with SEM as the error bars
  • Fig. 22 shows the structures of BTK PROTACs tested.
  • Fig. 23A-B show BTK degradation by the indicated PROTACs in a MOLM-14 cell line. MOLM-14 cells were treated with PROTACs for 24 hours, followed by Western blotting to measure the BTK levels. The quantification results are shown in Fig. 23 A and the
  • PROTACs and methods for their use as Bruton’s tyrosine kinase (BTK) degraders.
  • the small molecule PROTACs described herein are useful in treating and/or preventing cancer, neurodegenerative disorders, inflammatory diseases, metabolic disorders, and other BTK- related diseases.
  • PROTACs described herein are represented by the following formula:
  • A is a BTK binder (also referred to as a BTK inhibitor).
  • the BTK binders suitable for use as the A group include a Michael acceptor.
  • the L group can form a covalent bond with the A group through the Michael acceptor present in the A group.
  • Exemplary BTK binders for use as the A group are shown below:
  • L is a linker group.
  • the linker group contains a reversible covalent group.
  • L is a substituted or unsubstituted alkyl, a substituted or unsubstituted alkenyl, a substituted or unsubstituted alkynyl, a substituted or unsubstituted alkoxy, or a substituted or unsubstituted amino.
  • L contains an amide group.
  • B is an E3 ligase binder.
  • Exemplary E3 ligase binders for use as the B group include CRBN ligands, VHL ligands, cl API ligands, MDM2 ligands, RNF2 ligands, and DCAF15 ligands.
  • CRBN ligands for use as the B group are shown below:
  • VHL ligands for use as the B group are shown below:
  • cIAPl ligands for use as the B group are shown below:
  • MDM2 ligands for use as the B group are shown below:
  • RNF4 ligands for use as the B group are shown below:
  • m is 0-3.
  • n and p are each independently 0-5.
  • L is a linker.
  • L is a substituted or unsubstituted alkyl, a substituted or unsubstituted alkenyl, a substituted or unsubstituted alkynyl, a substituted or unsubstituted alkoxy, or a substituted or unsubstituted amino.
  • L contains an amide group.
  • X and Y are each independently selected from CH2, CHD, CD2, CHF, CF2, and C(O).
  • X and Y are each C(O).
  • R 1 , R 2 , R 8 , and R 9 are each independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, substituted or unsubstituted heteroalkynyl, substituted or unsubstituted carbonyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaiyl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocycloalkyl.
  • R 3 , R 4 , R 5 , each R 6 , R 7 , and each R 10 are each independently selected from hydrogen, halogen (e.g., fluoro, chloro, bromo, or iodo), cyano,
  • alkoxy, aryloxy substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, substituted or unsubstituted heteroalkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocycloalkyl.
  • L is a linker.
  • L is a substituted or unsubstituted alkyl, a substituted or unsubstituted alkenyl, a substituted or unsubstituted alkynyl, a substituted or unsubstituted alkoxy, or a substituted or unsubstituted amino.
  • L contains an amide group.
  • X and Y are each independently selected from CH 2 , CHD, CD 2 , CHF, CF 2 , and C(0).
  • X and Y are each C(0).
  • R 1 , R 2 , R 3 , and R 4 are each
  • substituted or unsubstituted alkyl e.g., methyl, ethyl, propyl, or butyl
  • substituted or unsubstituted carbonyl e.g., hydrogen, substituted or unsubstituted alkyl (e.g., methyl, ethyl, propyl, or butyl)
  • substituted or unsubstituted carbonyl e.g., methyl, ethyl, propyl, or butyl
  • R 5 is hydrogen, deuterium, fluoro, chloro, bromo, iodo, cyano, -OCH3, -OCDH2, -OCD2H, or -OCD3.
  • the compounds according to Formula I are reversible covalent (RC) compounds represented by Structure I-B:
  • Structure I-B L, X, Y, R 1 , and R 2 are as defined above for Formula I.
  • Examples of Structure I-B include the following compounds:
  • the compounds according to Formula I are irreversible covalent (IRC) compounds represented by Structure I-C:
  • Structure I-C L, X, Y, R 1 , and R 2 are as defined above for Formula I.
  • Examples of Structure I-C include the following compounds:
  • the compounds according to Formula I are reversible noncovalent (RNC) compounds represented by Structure I-D:
  • Structure I-D In Structure I-D, L, X, Y, R 1 , and R 2 are as defined above for Formula I. Examples of Structure I-D include the following compounds:
  • m is 0-3.
  • n and p are each independently 0-5.
  • L 1 and L 2 are each independently a linker.
  • L 1 and/or L 2 is a substituted or unsubstituted alkyl, a substituted or unsubstituted alkenyl, a substituted or unsubstituted alkynyl, a substituted or unsubstituted alkoxy, or a substituted or unsubstituted amino.
  • L 1 and/or L 2 contains an amide group.
  • X and Y are each independently selected from CH2, CHD, CD2, CHF, CF2, and C(O).
  • X and Y are each C(O).
  • R 1 , R 2 , R 8 , and R 9 are each independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, substituted or unsubstituted heteroalkynyl, substituted or unsubstituted carbonyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocycloalkyl.
  • R 3 , R 4 , R 5 , each R 6 , R 7 , and each R 10 are each independently selected from hydrogen, halogen, cyano, trifluoromethyl, alkoxy', aryloxy, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, substituted or unsubstituted heteroalkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocycloalkyl.
  • Structure ⁇ - ⁇ ,— is a single bond or a double bond.
  • X and Y are each independently selected from CH2, CHD, CD2, CHF, CF2, and C(O).
  • X and Y are each C(0).
  • R 1 , R 2 , R 3 , and R 4 are each
  • substituted or unsubstituted alkyl e.g., methyl, ethyl, propyl, or butyl
  • substituted or unsubstituted carbonyl e.g., hydrogen, substituted or unsubstituted alkyl (e.g., methyl, ethyl, propyl, or butyl)
  • substituted or unsubstituted carbonyl e.g., methyl, ethyl, propyl, or butyl
  • R 5 is hydrogen, deuterium, fluoro, chloro, bromo, iodo, cyano, -OCH3, -OCDH2, -OCD2H, or -OCD3.
  • alkyl, alkenyl, and alkynyl include straight- and branched- chain monovalent substituents. Examples include methyl, ethyl, isobutyl, 3-butynyl, and the like. Ranges of these groups useful with the compounds and methods described herein include C 1 -C 20 alkyl, C 2 -C 20 alkenyl, and C 2 -C 20 alkynyl.
  • Additional ranges of these groups useful with the compounds and methods described herein include C 1 -C 12 alkyl, C 2 -C 12 alkenyl, C 2 -C 12 alkynyl, C 1 -C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 1 -C 4 alkyl, C 2 -C 4 alkenyl, and C 2 -C 4 alkynyl.
  • Heteroalkyl, heteroalkenyl, and heteroalkynyl are defined similarly as alkyl, alkenyl, and alkynyl, but can contain O, S, or N heteroatoms or combinations thereof within the backbone. Ranges of these groups useful with the compounds and methods described herein include C 1 -C 20 heteroalkyl, C 2 -C 20 heteroalkenyl, and C 2 -C 20 heteroalkynyl.
  • Additional ranges of these groups useful with the compounds and methods described herein include C 1 - C 12 heteroalkyl, C 2 -C 12 heteroalkenyl, C 2 -C 12 heteroalkynyl, Ci-Ce heteroalkyl, C 2 -C 6 heteroalkenyl, C 2 -C 6 heteroalkynyl, C 1 -C 4 heteroalkyl, C 2 -C 4 heteroalkenyl, and C 2 -C 4 heteroalkynyl.
  • cycloalkyl, cycloalkenyl, and cycloalkynyl include cyclic alkyl groups having a single cyclic ring or multiple condensed rings. Examples include cyclohexyl, cyclopentylethyl, and adamantanyl. Ranges of these groups useful with the compounds and methods described herein include C 3 -C 20 cycloalkyl, C 3 -C 20 cycloalkenyl, and C 3 -C 20 cycloalkynyl.
  • Additional ranges of these groups useful with the compounds and methods described herein include C 5 -C 12 cycloalkyl, C 5 -C 12 cycloalkenyl, C 3 -C 12 cycloalkynyl, C 5 -C 6 cycloalkyl, C 5 -C 6 cycloalkenyl, and C 5 -C 6 cycloalkynyl.
  • heterocycloalkyl, heterocycloalkenyl, and heterocycloalkynyl are defined similarly as cycloalkyl, cycloalkenyl, and cycloalkynyl, but can contain O, S, or N heteroatoms or combinations thereof within the cyclic backbone. Ranges of these groups useful with the compounds and methods described herein include C 3 -C 20 heterocycloalkyl, C 3 -C 20 heterocycloalkenyl, and C 3 -C 20 heterocycloalkynyl. Additional ranges of these groups useful with the compounds and methods described herein include C 3 -C 12 heterocycloalkyl,
  • Aryl molecules include, for example, cyclic hydrocarbons that incorporate one or more planar sets of, typically, six carbon atoms that are connected by delocalized electrons numbering the same as if they consisted of alternating single and double covalent bonds.
  • An example of an aryl molecule is benzene.
  • Heteroaryl molecules include substitutions along their main cyclic drain of atoms such as O, N, or S. When heteroatoms are introduced, a set of five atoms, e.g., four carbon and a heteroatom, can create an aromatic system. Examples of heteroaryl molecules include furan, pyrrole, thiophene, imadazole, oxazole, pyridine, and pyrazine.
  • Aryl and heteroaryl molecules can also include additional fused rings, for example, benzofuran, indole, benzothiophene, naphthalene, anthracene, and quinoline.
  • the aryl and heteroaryl molecules can be attached at any position on the ring, unless otherwise noted.
  • alkoxy as used herein is an alkyl group bound through a single, terminal ether linkage.
  • aryloxy as used herein is an aryl group bound through a single, terminal ether linkage.
  • alkenyloxy, alkynyloxy, heteroalkyloxy, heteroalkeny loxy , heteroalkynyloxy, heteroaryloxy, cycloalkyloxy, and heterocycloalkyloxy as used herein are an alkenyloxy, alkynyloxy, heteroalkyloxy, heteroalkenyloxy,
  • hydroxy as used herein is represented by the formula— OH.
  • amine or amino as used herein are represented by the formula— NZ 1 Z 2 , where Z 1 and Z 2 can each be substitution group as described herein, such as hydrogen, an alkyl, halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.
  • alkoxy, aryloxy, amino, alkyl, alkenyl, alkynyl, aryl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, cycloalkyl, or heterocycloalkyl molecules used herein can be substituted or unsubstituted.
  • substituted includes the addition of an alkoxy, aryloxy', amino, alkyl, alkenyl, alkynyl, aryl, heteroalkyl, heteroalkenyl,
  • heteroalkynyl, heteroaryl, cycloalkyl, or heterocycloalkyl group to a position attached to the main chain of the alkoxy, aryloxy, amino, alkyl, alkenyl, alkynyl, aryl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, cycloalkyl, or heterocycloalkyl, e.g., the replacement of a hydrogen by one of these molecules.
  • substitution groups include, but are not limited to, hydroxy, halogen (e.g., F, Br, Cl, or I), and carboxyl groups.
  • the term unsubstituted indicates the alkoxy, aryloxy, amino, alkyl, alkenyl, alkynyl, aryl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, cycloalkyl, or heterocycloalkyl has a full complement of hydrogens, i.e., commensurate with its saturation level, with no substitutions, e.g., linear decane (-(CH 2 ) 9 -CH 3 ).
  • the compounds described herein also include isotopic substitutions (e.g., a deuterium or tritium variant) of the compounds.
  • one or more hydrogen atoms can be substituted by a hydrogen isotope (e.g., a deuterium or a tritium).
  • a methoxy group (-OCH 3 ) can be substituted with one or more isotopic groups to form, for example, - OCDH2, -OCD2H, or -OCD3.
  • the compounds described herein can be prepared in a variety of ways.
  • the compounds can be synthesized using various synthetic methods. At least some of these methods are known in the art of synthetic organic chemistry.
  • the compounds described herein can be prepared from readily available starting materials. Optimum reaction conditions can vary with the particular reactants or solvent used, but such conditions can be determined by one skilled in the art by routine optimization procedures.
  • Variations on Formula I and Formula II include the addition, subtraction, or movement of the various constituents as described for each compound. Similarly, when one or more chiral centers are present in a molecule, all possible chiral variants are included. Additionally, compound synthesis can involve the protection and deprotection of various diemical groups. The use of protection and deprotection, and the selection of appropriate protecting groups can be determined by one skilled in the art. The chemistry- of protecting groups can be found, for example, in Wuts, Greene’s Protective Groups in Organic Synthesis, 5th. Ed., Wiley & Sons, 2014, which is incorporated herein by reference in its entirety.
  • Reactions to produce tire compounds described herein can be carried out in solvents, which can be selected by one of skill in the art of organic synthesis. Solvents can be substantially nonreactive with the starting materials (reactants), the intermediates, or products under the conditions at which the reactions are carried out, i.e., temperature and pressure. Reactions can be carried out in one solvent or a mixture of more than one solvent. Product or intermediate formation can be monitored according to any suitable method known in the art.
  • product formation can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g., 1 H or 13 C) infrared spectroscopy, spectrophotometry (e.g., UV-visible), or mass spectrometry, or by chromatography such as high performance liquid chromatography (HPLC) or thin layer chromatography.
  • spectroscopic means such as nuclear magnetic resonance spectroscopy (e.g., 1 H or 13 C) infrared spectroscopy, spectrophotometry (e.g., UV-visible), or mass spectrometry
  • chromatography such as high performance liquid chromatography (HPLC) or thin layer chromatography.
  • tire pharmaceutical composition can be in the form of solid, semi-solid or liquid dosage forms, such as, for example, tablets, suppositories, pills, capsules, powders, liquids, or suspensions, preferably in unit dosage form suitable for single administration of a precise dosage.
  • the compositions will include a therapeutically effective amount of the compound described herein or derivatives thereof in combination with a pharmaceutically acceptable carrier and, in addition, may include other medicinal agents, pharmaceutical agents, carriers, or diluents.
  • pharmaceutically acceptable is meant a material that is not biologically or otherwise undesirable, which can be administered to an individual along with the selected compound without causing unacceptable biological effects or interacting in a deleterious manner with the other components of the pharmaceutical composition in which it is contained.
  • the term carrier encompasses any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations.
  • a carrier for use in a composition will depend upon the intended route of administration for the composition.
  • the preparation of pharmaceutically acceptable carriers and formulations containing these materials is described in, e.g., Remington: The Science and Practice of Pharmacy, 22d Edition, Loyd et al. eds., Pharmaceutical Press and Philadelphia College of Pharmacy at University of the Sciences (2012).
  • physiologically acceptable carriers include buffers, such as phosphate buffers, citrate buffer, and buffers with other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as
  • polyvinylpyrrolidone amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugar alcohols, such as mannitol or sorbitol; salt- forming counterions, such as sodium; and/or nonionic surfactants, such as TWEEN® (ICI, Inc.; Bridgewater, New Jersey), polyethylene glycol (PEG), and PLURONICSTM (BASF; Florham Park, NJ).
  • amino acids such as glycine, glutamine, asparagine, arginine or lysine
  • monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins chelating agents, such as EDTA
  • sugar alcohols such as mannitol or sorbitol
  • salt- forming counterions such as sodium
  • compositions containing the compound described herein or derivatives thereof suitable for parenteral injection may comprise physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions.
  • suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (propyleneglycol, polyethyleneglycol, glycerol, and the like), suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.
  • compositions may also contain adjuvants, such as preserving, wetting, emulsifying, and dispensing agents.
  • adjuvants such as preserving, wetting, emulsifying, and dispensing agents.
  • Prevention of the action of microorganisms can be promoted by various antibacterial and antifungal agents, for example, parabens,
  • chlorobutanol phenol, sorbic acid, and the like.
  • Isotonic agents for example, sugars, sodium chloride, and the like may also be included.
  • Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Solid dosage forms for oral administration of the compounds described herein or derivatives thereof include capsules, tablets, pills, powders, and granules.
  • the compounds described herein or derivatives thereof is admixed with at least one inert customary excipient (or carrier), such as sodium citrate or dicalcium phosphate, or (a) fillers or extenders, as for example, starches, lactose, sucrose, glucose, mannitol, and silicic acid, (b) binders, as for example, carboxymethylcellulose, alignates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, (c) humectants, as for example, glycerol, (d) disintegrating agents, as for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate, (e) solution retarders, as for example, paraffin, (f) absorption accelerators, as for example,
  • Solid compositions of a similar type may also be employed as fillers in soft and hard- filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight poly ethyleneglycols, and the like.
  • Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings and others known in the art They may contain opacifying agents and can also be of such composition that they release tire active compound or compounds in a certain part of the intestinal tract in a delayed manner.
  • coatings and shells such as enteric coatings and others known in the art They may contain opacifying agents and can also be of such composition that they release tire active compound or compounds in a certain part of the intestinal tract in a delayed manner.
  • embedding compositions that can be used are polymeric substances and waxes.
  • the active compounds can also be in micro-encapsulated form, if appropriate, with one or more of the above-mentioned excipients.
  • Liquid dosage forms for oral administration of the compounds described herein or derivatives thereof include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs.
  • the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents, and emulsifiers, as for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butyleneglycol,
  • oils in particular, cottonseed oil, groundnut oil, com germ oil, olive oil, castor oil, sesame oil, glycerol, tetrahydrofurfuryl alcohol, polyethyleneglycols, and fatty acid esters of sorbitan, or mixtures of these substances, and the like.
  • composition can also include additional agents, such as wetting, emulsifying, suspending, sweetening, flavoring, or perfuming agents.
  • additional agents such as wetting, emulsifying, suspending, sweetening, flavoring, or perfuming agents.
  • Suspensions in addition to the active compounds, may contain additional agents, as for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances, and the like.
  • additional agents as for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances, and the like.
  • compositions of the compounds described herein or derivatives thereof for rectal administrations are optionally suppositories, which can be prepared by mixing the compounds with suitable non-irritating excipients or carriers, such as cocoa butter, polyethyleneglycol or a suppository wax, which are solid at ordinary temperatures but liquid at body temperature and, therefore, melt in the rectum or vaginal cavity and release the active component.
  • suitable non-irritating excipients or carriers such as cocoa butter, polyethyleneglycol or a suppository wax, which are solid at ordinary temperatures but liquid at body temperature and, therefore, melt in the rectum or vaginal cavity and release the active component.
  • Dosage foms for topical administration of the compounds described herein or derivatives thereof include ointments, powders, sprays, inhalants, and skin patches.
  • the compounds described herein or derivatives thereof are admixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants as may be required.
  • Ophthalmic formulations, ointments, powders, and solutions are also contemplated as being within the scope of the compositions.
  • the compounds described herein can be contained in a drug depot.
  • a drug depot comprises a physical structure to facilitate implantation and retention in a desired site (e.g., a synovial joint, a disc space, a spinal canal, abdominal area, a tissue of the patient, etc.).
  • the drug depot can provide an optimal concentration gradient of the compound at a distance of up to about 0.1 cm to about 5 cm from the implant site.
  • a depot includes but is not limited to capsules, microspheres, microparticles, microcapsules, microfibers particles, nanospheres, nanoparticles, coating, matrices, wafers, pills, pellets, emulsions, liposomes, micelles, gels, antibody-compound conjugates, protein-compound conjugates, or other pharmaceutical delivery compositions.
  • Suitable materials for the depot include pharmaceutically acceptable biodegradable materials that are preferably FDA approved or GRAS materials. These materials can be polymeric or non-polymeric, as well as synthetic or naturally occurring, or a combination thereof.
  • the depot can optionally include a drug pump.
  • compositions can include one or more of the compounds described herein and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable salt refers to those salts of the compound described herein or derivatives thereof that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of subjects without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, and effective for their intended use, as well as the zwitterionic forms, where possible, of the compounds described herein.
  • salts refers to the relatively non-toxic, inorganic and organic acid addition salts of the compounds described herein.
  • salts can be prepared in situ during the isolation and purification of the compounds or by separately reacting the purified compound in its free base form with a suitable organic or inorganic acid and isolating the salt thus formed.
  • Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate mesylate, glucoheptonate, lactobionate, methane sulphonate, and laurylsulphonate salts, and the like.
  • alkali and alkaline earth metals such as sodium, lithium, potassium, calcium, magnesium, and the like, as well as non-toxic ammonium, quaternary ammonium, and amine cations including, but not limited to ammonium, tetramethylammonium,
  • pharmaceutically acceptable salts thereof can be carried out using therapeutically effective amounts of the compounds and compositions described herein or pharmaceutically acceptable salts thereof as described herein for periods of time effective to treat a disorder.
  • the effective amount of the compounds and compositions described herein or pharmaceutically acceptable salts thereof as described herein may be determined by one of ordinary skill in the art and includes exemplary dosage amounts for a mammal of from about 0.0001 to about 200 mg/kg of body weight of active compound per day, w hich may be administered in a single dose or in the form of individual divided doses, such as from 1 to 4 times per day.
  • the dosage amount can be from about 0.01 to about 150 mg/kg of body weight of active compound per day, about 0.1 to 100 mg/kg of body weight of active compound per day, about 0.5 to about 75 mg/kg of body w-eight of active compound per day, about 0.5 to about 50 mg/kg of body weight of active compound per day, about 0.01 to about 50 mg/kg of body weight of active compound per day, about 0.05 to about 25 mg/kg of body weight of active compound per day, about 0.1 to about 25 mg/kg of body weight of active compound per day, about 0.5 to about 25 mg/kg of body weight of active compound per day, about 1 to about 20 mg/kg of body weight of active compound per day, about 1 to about 10 mg/kg of body weight of active compound per day, about 20 mg/kg of body weight of active compound per day, about 10 mg/kg of body weight of active compound per day, about 5 mg/kg of body weight of active compound per day, about 2.5 mg/kg of body weight of active compound per day, about 1.0 mg/kg of body
  • the dosage amount is from about 0.01 mg/kg to about 5 mg/kg.
  • the dosage amount is from about 0.01 mg/kg to about 2.5 mg/kg.
  • the precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each subject's circumstances. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test sy stems. Further, depending on the route of administration, one of skill in the art would know how to determine doses that result in a plasma concentration for a desired level of response in the cells, tissues and/or organs of a subject.
  • the methods include administering to a subject an effective amount of one or more of the compounds or compositions described herein, or a pharmaceutically acceptable salt or prodrug thereof.
  • Effective amount when used to describe an amount of compound in a method, refers to the amount of a compound that achieves the desired pharmacological effect or other biological effect.
  • the effective amount can be, for example, the concentrations of compounds at which BTK is degraded in vitro, as provided herein.
  • a method that includes administering to the subject an amount of one or more compounds described herein such that an in vivo concentration at a target cell in the subject
  • the compounds and compositions described herein or pharmaceutically acceptable salts thereof are useful for treating BTK-related diseases in humans, including, without limitation, pediatric and geriatric populations, and in animals, e.g., veterinary applications.
  • the BTK-related disease is cancer.
  • the cancer is a poor prognosis cancer.
  • the term poor prognosis refers to a prospect of recovery from a disease, infection, or medical condition that is associated with a diminished likelihood of a positive outcome.
  • a poor prognosis may be associated with a reduced patient survival rate, reduced patient survival time, higher likelihood of metastatic progression of said cancer cells, and/or higher likelihood of chemoresistance of said cancer cells.
  • a poor prognosis cancer can be a cancer associated with a patient survival rate of 50% or less.
  • a poor prognosis cancer can be a cancer associated with a patient survival time of five years or less after diagnosis.
  • the cancer is an invasive cancer.
  • the cancer is a cancer that has an increased expression of BTK as compared to non-cancerous cells of the same cell type.
  • the cancer is bladder cancer, brain cancer, breast cancer (e.g, triple negative breast cancer), bronchus cancer, colorectal cancer (e.g., colon cancer, rectal cancer), cervical cancer, chondrosarcoma, endometrial cancer, gastrointestinal cancer, gastric cancer, genitourinary cancer,
  • glioblastoma head and neck cancer, hepatic cancer, hepatocellular carcinoma, leukemia, liver cancer, lung cancer, lymphoma, melanoma of the skin, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, skin cancer, testicular cancer, thyroid cancer, or uterine cancer.
  • the cancer is a cancer that affects one or more of the following sites: oral cavity and pharynx (e.g, tongue, mouth, pharynx, or other oral cavity); digestive system (e.g., esophagus, stomach, small intestine, colon, rectum, anus, anal canal, anorectum, liver and intrahepatic bile duct, gallbladder and other biliary, pancreas, or other digestive organs); respiratory system (e.g, larynx, lung and bronchus, or other respiratory organs); bones and joints; soft tissue (e.g, heart); skin (e.g., melanoma of the skin or other nonepithelial skin); breast; genital system (e.g., uterine cervix, uterine corpus, ovary, vulva, vagina and other female genital areas, prostate, testis, penis and other male genital areas); urinary system (e.g., urinary bladder,
  • lymphoma e.g., Hodgkin lymphoma and non-Hodgkin lymphoma
  • myeloma e.g., myeloma
  • leukemia e.g, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, or other leukemia
  • the cancer is a drug resistant cancer, such as an ibrutinib-resistant cancer.
  • the BTK-related disease is a metabolic disorder (e.g, obesity, diabetes, and genetic disorders).
  • the BTK-related disease is a neurodegenerative disorder.
  • the neurodegenerative disorder is Parkinson’s disease.
  • the neurodegenerative disorder is Alexander disease, Alper’s disease, Alzheimer disease, amyotrophic lateral sclerosis, ataxia telangiectasia, Batten disease (also known as
  • Corticobasal degeneration Creutzfeldt-Jakob disease, Huntington’s disease, Kennedy’s disease, Krabbe disease, Lewy body dementia, Machado- Joseph disease, Spinocerebellar ataxia type 3, multiple sclerosis, multiple system atrophy, Pelizaeus-Merzbacher disease, Pick’s disease.
  • TSE encephalopathies
  • the BTK-related disease is an inflammatory disease.
  • inflammatory disorders include, but are not limited to, respiratory or pulmonary disorders (including asthma, COPD, chronic bronchitis and cystic fibrosis); cardiovascular related disorders (including atherosclerosis, post-angioplasty, restenosis, coronary artery diseases and angina); inflammatory diseases of the joints (including rheumatoid and osteoarthritis); skin disorders (including dermatitis, eczematous dermatitis and psoriasis); post
  • autoimmune conditions including systemic lupus erythematosus, dermatomyositis, polymyositis,
  • vasculitis including Churg-Strauss syndrome, Takayasu's arteritis
  • inflammatory disorders of adipose tissue including Kaposi's sarcoma and other proliferative disorders of smooth muscle cells.
  • proliferative disorders including Kaposi's sarcoma and other proliferative disorders of smooth muscle cells.
  • the BTK-related disease is ischemia, a gastrointestinal disorder, a viral infection (e.g., human immunodeficiency virus (HIV), including HIV type 1 (HIV-1) and HIV type 2 (HIV-2)), a bacterial infection, a central nervous system disorder, a spinal cord injury, or peripheral neuropathy.
  • a viral infection e.g., human immunodeficiency virus (HIV), including HIV type 1 (HIV-1) and HIV type 2 (HIV-2)
  • HIV type 1 HIV type 1
  • HIV-2 HIV type 2
  • peripheral neuropathy e.g., peripheral neuropathy.
  • the methods of treating or preventing a BTK-related disease (e.g., cancer) in a subject can further comprise administering to the subject one or more additional agents.
  • the one or more additional agents and the compounds described herein or pharmaceutically acceptable salts or prodrugs thereof can be administered in any order, including concomitant, simultaneous, or sequential administration. Sequential administration can be administration in a temporally spaced order of up to several days apart.
  • the methods can also include more than a single administration of the one or more additional agents and/or the compounds described herein or pharmaceutically acceptable salts or prodrugs thereof.
  • the administration of the one or more additional agents and the compounds described herein or pharmaceutically acceptable salts or prodrugs thereof can be by the same or different routes and concurrently or sequentially.
  • Additional therapeutic agents include, but are not limited to, chemotherapeutic agents, anti-depressants, anxiolytics, antibodies, antivirals, steroidal and non-steroidal antiinflammatories, conventional immunotherapeutic agents, cytokines, chemokines, and/or growth factors.
  • the additional therapeutic agents can be biomolecules.
  • a chemotherapeutic agent is a compound or composition effective in inhibiting or arresting the growth of an abnormally growing cell.
  • an agent may be used therapeutically to treat cancer as well as other diseases marked by abnormal cell growth.
  • chemotherapeutic compounds include, but are not limited to, bexarotene, gefitinib, eriotinib, gemcitabine, paclitaxel, docetaxel, topotecan, irinotecan, temozolomide, carmustine, vinorelbine, capecitabine, leucovorin, oxaliplatin, bevacizumab, cetuximab, panitumumab, bortezomib, oblimersen, hexamethylmelamine, ifosfamide, CPT- 11, deflunomide, cycloheximide, dicaibazine, asparaginase, mitotant, vinblastine sulfate, carboplatin, colchicine,
  • anthracyclines such as doxorubicin, liposomal doxorubicin, and diethylstilbestrol doxorubicin, bleomycin, daunorubicin, and dactinomycin
  • antiestrogens e.g., tamoxifen
  • antimetabolites e.g., fluorouracil (FU), 5-FU, methotrexate, floxuridine, interferon alpha-2B, glutamic acid, plicamycin, mercaptopurine, and 6-thioguanine
  • cytotoxic agents e.g., carmustine, BCNU, lomustine, CCNU, cytosine arabinoside, cyclophosphamide, estramustine, hydroxyurea, procarbazine, mitomy cin, busulfan, cisplatin, vincristine and vincristine sulfate
  • hormones e.g., medroxyprogesterone, estramustine phosphate sodium, ethinyl estradiol, estradiol, megestrol acetate, methyltestosterone, diethylstilbestrol diphosphate, chlorotrianisene, and testolactone
  • nitrogen mustard derivatives e.g., mephalen, chlorambucil, mechlorethamine (nitrogen mustard) and thiotepa
  • steroids e.g., bethamethasone sodium phosphate).
  • Therapeutic agents further include, but are not limited to, levadopa, a dopamine agonist, an anticholinergic agent, a monoamine oxidase inhibitor, a COMT inhibitor, amantadine, rivastigmine, an NMDA antagonist, a cholinesterase inhibitor, riluzole, an antipsychotic agent, an antidepressant, and tetrabenazine.
  • any of the aforementioned therapeutic agents can be used in any combination with the compositions described herein.
  • Combinations are administered either concomitantly (e.g., as an admixture), separately but simultaneously (e.g., via separate intravenous lines into the same subject), or sequentially (e.g., one of the compounds or agents is given first followed by the second).
  • the term combination is used to refer to concomitant, simultaneous, or sequential administration of two or more agents.
  • a compound or therapeutic agent as described herein may be administered in combination with a radiation therapy, an immunotherapy, a gene therapy, or a surgery.
  • a therapeutically effective amount of the compounds and compositions or pharmaceutically acceptable salts thereof as described herein are administered to a subject prior to onset (e.g., before obvious signs of a BTK-related disease), during early onset (e.g., upon initial signs and symptoms of a BTK-related disease), or after the development of a BTK-related disease.
  • Prophylactic administration can occur for several days to years prior to the manifestation of symptoms of a BTK-related disease.
  • Therapeutic treatment involves administering to a subject a therapeutically effective amount of the compounds and compositions or pharmaceutically acceptable salts thereof as described herein after a BTK-related disease is diagnosed.
  • the compounds described herein are also useful in modulating BTK in a cell.
  • the compounds and compositions described herein are useful for inducing BTK degradation in a cell.
  • the methods for inducing BTK degradation in a cell includes contacting a cell with an effective amount of one or more of the compounds as described herein.
  • the contacting is performed in vivo.
  • the contacting is performed in vitro.
  • the methods herein for prophylactic and therapeutic treatment optionally comprise selecting a subject with or at risk of developing a BTK-related disease.
  • a skilled artisan can make such a determination using, for example, a variety of prognostic and diagnostic methods, including, for example, a personal or family history of the disease or condition, clinical tests (e.g., imaging, biopsy, genetic tests), and the like.
  • the methods herein can be used for preventing relapse of cancer in a subject in remission (e.g., a subject that previously had cancer).
  • kits for treating or preventing a BTK-related disease e.g., cancer, aneurodegenerative disorder, an inflammatory diseases, and/or a metabolic disorder
  • a kit can include any of the compounds or compositions described herein.
  • a kit can include one or more compounds of Formula I and/or Formula ⁇ .
  • a kit can further include one or more additional agents, such as one or more chemotherapeutic agents.
  • a kit can include an oral formulation of any of the compounds or compositions described herein.
  • a kit can include an intravenous formulation of any of the compounds or compositions described herein.
  • a kit can additionally include directions for use of the kit (e.g., instructions for treating a subject), a container, a means for administering the compounds or compositions (e.g., a syringe), and/or a carrier.
  • treatment refers to a method of reducing one or more symptoms of a disease or condition.
  • treatment can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of one or more symptoms of the disease or condition.
  • a method for treating a disease is considered to be a treatment if there is a 10% reduction in one or more symptoms or signs (e.g., size of the tumor or rate of tumor growth) of the disease in a subject as compared to a control.
  • control refers to the untreated condition (e.g., the tumor cells not treated with the compounds and compositions described herein).
  • the reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percent reduction in between 10% and 100% as compared to native or control levels. It is understood that treatment does not necessarily refer to a cure or complete ablation of the disease, condition, or symptoms of the disease or condition.
  • prevent, preventing, and prevention of a disease or disorder refer to an action, for example, administration of a composition or therapeutic agent, that occurs before or at about the same time a subject begins to show one or more symptoms of the disease or disorder, which inhibits or delays onset or severity of one or more symptoms of the disease or disorder.
  • references to decreasing, reducing, or inhibiting include a change of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater as compared to a control level.
  • subject means both mammals and non-mammals.
  • Mammals include, for example, humans; non-human primates, e.g., apes and monkeys; cattle; horses; sheep; rats; mice; pigs; and goats.
  • Non-mammals include, for example, fish and birds.
  • reaction mixture was extracted with ethyl acetate, and the organic layer was washed with water, dried over Na 2 SO 4 , and filtered. The filtrate was concentrated in vacuo and the residue was purified by flash column chromatography (0-10% MeOH in Ethyl Acetate) to give product 8a as a white solid (69 mg, 50%).
  • reaction mixture was extracted with ethyl acetate, and the organic lay a was washed with water, dried over Na2SC>4, and filtered.
  • the filtrate was concentrated in vacuo and the residue was purified by flash column chromatography (0-10% MeOH in Ethyl Acetate) to give product 12a as a colorless oil (80 mg, 70%).
  • reaction mixture was extracted with ethyl acetate, and the organic layer was washed with water, dried over Na 2 SO 4 , and filtered. The filtrate was concentrated in vacuo and the residue was purified by flash column chromatography (0-10% MeOH in Ethyl Acetate) to give product 12c as a colorless oil (56 mg, 60%).
  • a proteolysis targeting chimera is a heterobifunctional molecule that can bind both a target protein and an E3 ubiquitin ligase to facilitate the formation of a ternary complex, leading to ubiquitination and ultimate degradation of the target protein.
  • PROTACs have gained tremendous attention in recent years not only for their numerous applications in chemical biology but also as highly promising therapeutic agents. Compared with oligonucleotide and CRISPR therapeutics that face in vivo delivery challenges, PROTACs are small molecule therapeutics that provide opportunities to achieve broadly applicable bodywide protein knockdown.
  • Protein degraders have many advantages compared with traditional small molecule inhibitors.
  • small molecule inhibitors usually modulate protein functions through stoichiometrically occupying the active sites or binding pockets of targeted proteins;
  • PROTACs act as catalysts and are involved in multiple cycles of targeted protein degradation. Therefore, the degradation induced by PROTACs is sub-stoichiometric.
  • PROTACs due to the competitive nature of small molecule inhibitors, constant systemic drug exposure is necessary to maintain sufficient intracellular concentrations for therapeutic efficacy, usually leading to off-target and side effects.
  • optimized PROTACs usually achieve maximal protein degradation in a few hours and maintain the therapeutic effect (even without constant PROTAC exposure) until the targeted protein is re-synthesized in cells. Therefore, the pharmacodynamics (PD) of PROTACs is dependent on not only drug exposure (pharmacokinetic (PK) properties) but also the half-life (ti/2) of the targeted protein.
  • small molecules usually interfere with the function of one domain of a multidomain protein. However, this strategy for multidomain kinases, especially in cancer cells, may lead to compensatory feedback activation of its downstream signaling pathways via other alternative kinases. In contrast, although PROTACs only target one domain of a
  • multidomain protein they induce degradation of the full-length protein, reducing the possibility to develop drug resistance through mutations or compensatory protein
  • the specificity of small molecule inhibitors depends solely on the molecular design, which is sometimes difficult to achieve due to the presence of proteins with similar binding pockets, such as kinases.
  • the specificity of PROTACs is determined by not only the small molecule binder to targeted proteins but also the protein-protein interactions between the targeted protein and the recruited E3 ligase.
  • the targets of small molecular inhibitors are usually enzymes and receptors with defined binding pockets or active sites. However, -75% of the human proteome lacks active sites, such as transcription factors and non-enzymatic proteins, and are considered “undruggable.”
  • PROTACs can be designed to bind to any crevice on the surface of the targeted proteins to induce their degradation.
  • Fig. 1 shows a demonstration of catalytic degradation of targeted proteins by reversible covalent PROTACs.
  • the premise of this reversible covalent PROTAC design is the weak reactivity (mM Ki) between a-cyano- acrylamide group (the chemical structure shown above) and free thiols.
  • the nearby cysteine side drain can react with the a-cyano-acrylamide group to form a stable covalent bond. Once the targeted protein is degraded, the reversible covalent PROTAC can be regenerated.
  • Bruton’s tyrosine kinase (BTK) was chosen as a model target for the studies. Surprisingly, it was discovered drat cyano-acrylamide-based reversible covalent binder to BTK can significantly enhance drug accumulation and target engagement in cells.
  • RC-1 was developed as the first reversible covalent BTK PROTAC with a high target occupancy as its corresponding kinase inhibitor.
  • RC-1 is effective as a dual functional inhibitor/degrader, providing a novel mechanism-of-action for PROTACs, and forms a stable ternary complex by reducing protein conformational flexibility compared with the non-covalent PRTOAC counterparts. Importantly, it was found that this reversible covalent strategy can be generalized and applied to improve other PROTACs.
  • the MOLM-14 cell line was obtained and the Mino cells were purchased from the American Type Culture Collection (ATCC; Manassas, VA).
  • Both cell lines were cultured in RPMI 1640 medium (Thermo Fisher Scientific; Waltham, MA) supplemented with 10% fetal bovine serum (GE Healthcare; Chicago, IL) and 1% Pen/Strep (Thermo Fisher Scientific).
  • the Wild-type and C481S BTK XLA cell line was obtained and was cultured.
  • HEK 293T/17 cells ATCC were maintained in DMEM (Thermo Fisher Scientific) with 10% fetal bovine serum and 1% Pen-Strep. Cells were grown at 37 °C with 5% CO2.
  • 1.0 x 10 6 MOLM-14 cells in 2 mL of RPMI 1640 complete media were incubated with indicated doses of BTK PROTAC compounds for 24 hours, with control cells treated with 0.01% DMSO.
  • XLA cells overexpressing wild-type BTK or mutant C481S BTK were plated at the same cell number and density as the above experiments and were treated with RC-1, IRC-1, or RNC-1 at the dose of 1.6, 8.0, 40, 200, and 1000 nM for 24 hours. After completion of treatment, all the cells were collected and processed for Western blot analysis.
  • MOLM-14 cells were treated in duplicates with 200 nM of RC-1, RNC-1, IRC-1, or RC-l-Me for 24 hours, and then the cells were collected and processed for proteomics analysis (Sanford Burnham Prebys Medical Discovery Institute; La Jolla, California).
  • MOLM-14 or Mino cells were harvested in the log phase of growth and re-plated into the wells of 96- well plates at the density of 6 x 10 4 cells/ml in 100 ⁇ L of complete RPMI- 1640 culture medium After overnight recovery, cells were exposed to serially diluted BTK PROTAC compounds (from 10,000 to 0.64 nM, 5-fold dilution) for 72 hours, which was followed by adding of pre-warmed Resazurin sodium solution (Sigma; lmg/mL in PBS) in an amount equal to 10% of the volume in the well.
  • NanoBRET In-Cell Target Engagement Assay The target engagement assay for BTK or CRBN from Promega (Madison, WI) was performed as detailed below. Briefly, HEK 293/T17 cells (ATCC) were transiently transfected with BTK-NanoLuc® fusion vector or NanoLuc®-CRBN fusion vector (Promega) using a calcium phosphate transfection protocol. Forty-eight hours after transfection, the cells were re-suspended in Opti-MEM medium (Life
  • BRET signals were collected using a BioTek SYNERGY HI microplate reader equipped with a 450/80 nm BP filter for donor emission and a 610 nm LP filter for acceptor emission.
  • BRET Ratio was calculated with the equation: [(Acceptor sample / Donor sample) - (Acceptor no tracer control/Donor no tracer control)] x 1000.
  • the ICso of the compound against its BTK or CRBN tracer was calculated using GraphPad Prism software.
  • BTK kinase activity inhibition ICso was measured by PhosphoSens® Kinase Assay Kit (BioTek). This assay was performed in 384-well, white flat bottom polystyrene NBS microplates (Coming) at room temperature. Active recombinant BTK was purchased from SignalChem (Cat.# BIO-IOH-IO). All compounds (3 warhead control and 4 PROTACs) were dissolved in DMSO (10 mM).
  • Duplicate drug titrations 1000 nM, 200 nM, 40 nM, 8 nM, 1.6 nM, 0.32 nM 0.64 nM, 0.128 nM and 0.0256 nM were used to generate each ICso.
  • Typical final concentrations of each reaction component were as follows: 2.5 nM BTK, and 10 ⁇ PhosphoSens® Substrate, 54 mM HEPES, pH 7.5, ImM ATP, 1.2 mM DTT, 0.012% Brij-35, 10 mM MgCM+Hl2, 1 % glycerol and 0.2 mg/mL BSA.
  • the mixture was incubated at room temperature for 15 minutes and the fluorescence intensity (RFU) readings (Ex 360 nm/Em 492 nm) were collected for 60 minutes with 3 minute intervals in a BioTek Synergy HI fluorescence microplate reader.
  • the background fluorescence as determined with the "no kinase" control, was collected for each time point from the total signal to obtain corrected Relative Fluorescence Units (RFU) values.
  • the corrected RFU vs. time for each inhibitor concentration was collected and the initial reaction rates (slope of the linear portion) for each progress curve were determined for each inhibitor concentration. Then, the velocity (RFU corrected/minute) vs [inhibitor] was plotted and the ICso was determined using a 4-parameter logistic fit.
  • the HEK293T/17 cells were passaged in Dulbecco’s modified Eagle medium
  • DMEM fetal bovine serum
  • penicillin 100 units/mL
  • streptomycin 100 pg/mL
  • HEK293T/17 cells were transiently transfected with the plasmid using calcium phosphate transfection reagent or lipofectamine.
  • Cells were grown in 35 mm glass bottom microwell (14 mm) dishes (MatTek Coiporation). Transfection was performed when cells were cultured to ⁇ 50% confluence. For each transfection, 4.3 pg of plasmid DNA was mixed with 71 pL of IX Hanks’ Balanced Salts buffer (HBS) and 4.3 pL of 2.5 M CaCk. Cells were imaged 24 hours after transient transfection. Time-lapse imaging was performed with the aid of an environmental control unit incubation chamber (InVivo Scientific), which was maintained at 37 °C and 5% CO2.
  • Fluorescence images were acquired with an exposure time of 50 ms for EGFP. Chemical reagents, including RC-1, IRC-l and RNC-1, were carefully added to the cells in the incubation chamber when the time-lapse imaging started. Image acquisition w as controlled by the NIS-Elements Ar Microscope Imaging Software (Nikon). Images w'ere processed using NIS-Elements and ImageJ (NIH).
  • the species in a Nano-Luc based target engagement assay includes the target protein nanoLuc fusion (designated as P), the tracer (designated as T), the tracer bound target protein nanoLuc fusion (designated as PT), the drug (designated as D), and the drug bound target protein nanoLuc fusion (designated as PD).
  • P target protein nanoLuc fusion
  • T tracer
  • PT tracer bound target protein nanoLuc fusion
  • D drug bound target protein nanoLuc fusion
  • PD drug bound target protein nanoLuc fusion
  • K d,T is the dissociation equilibrium constant for the binding between the target protein nanoLuc fusion and the tracer
  • [P] is the equilibrium concentration of the target protein nanoLuc fusion
  • [T] is the equilibrium concentration of the free tracer T
  • [PT] is the equilibrium concentration of the tracer bound form of the target protein nanoLuc fusion
  • CT is the total intracellular concentration of the tracer T.
  • ⁇ d,D is the dissociation equilibrium constant for the binding between the target protein nanoLuc fusion and the drug
  • [P] is the equilibrium concentration of the target protein nanoLuc fusion
  • [D] is the equilibrium concentration of the free drug D
  • [DT] is the equilibrium concentration of the drug bound form of the target protein nanoLuc fusion
  • C D .in is the total intracellular concentration of the drug D.
  • Cp is the total concentration of the target protein nanoLuc fusion P.
  • the tracer bound nanoLuc fusion P (PT) accounts for 100% of all the nanoLuc fusion P and corresponds to the concentration CP.
  • the intracellular accumulation coefficient for drug D (AP,D) can be defined as
  • C D in is the total intracellular concentration of drug D and C D, ex is the total extracellular concentration of drug D.
  • the relative intracellular accumulation coefficient for drug D (K’ P,D ) is defined as
  • mice Female ICR mice (weighing 22-28 g) were obtained and were housed 2-4 per cage in an American Animal Association Laboratory Animal Care accredited facility' and maintained under standard conditions of temperature (22 °C ⁇ 2°C), relative humidity' (50%) and light and dark cycle (12/12 hours), and had access to food and water ad libitum. Mice were allowed to acclimate to their environment for one week before the experiments.
  • RC-1 Pharmacokinetic and Pharmacodynamic Studies of RC-1.
  • RC-1 was formulated in anon-aqueous solvent (30% PEG-400, 5% Tween 80, and 5% DMSO in deionized water) and was administered in a single intraperitoneal (IP) injection (20 mg/kg). Blood samples (25 ⁇ L) were withdrawn from the tail vein at the time points of 10 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, and up to 24 hours after dosing.
  • IP intraperitoneal
  • the blood samples were collected into 1.5 mL centrifuge tubes coated with 0.5 M EDTA and were immediately centrifuged at 12,000 x g at 4 °C for 15 minutes.
  • the resultant plasma was extracted with 9 volumes of acetonitrile and then were centrifuged at the same conditions described above.
  • the superatant was stored at -80 °C for liquid chromatography-mass spectrometry (LC-MS) analysis.
  • LC-MS liquid chromatography-mass spectrometry
  • the standard curve was established by serially diluting RC-1 in plasma collected from naive mice (from 5.0 to 0.003 ⁇ g/mL, 4-fold dilution).
  • the pharmacokinetic (PK) parameters were calculated with the Microsoft Excel PK Solver.
  • BTK/ligands/CRBN the binary' complexes of BTK/CRBN were firstly modeled.
  • the structure files of BTK/ibrutinib(PDB code: 5P9J) and that of CRBN/ lenalidomide (PDB code: 4TZ4) were obtained from RCSB.
  • Multiple residues at the entrance of the protein BTK (L408, N484 and K558) and CRBN (1371, H353 and E377) were randomly selected as the indication of active binding site, and constrained protein-protein docking simulations were performed by using ZDOCK. Three candidate binding conformations were reported and downloaded from the ZDOCK server.
  • the system was further equilibrated for 1 ns under NVT and NPT ensemble, respectively, before continuing with a production run of more than 200 ns.
  • the protein and ligand positions are constrained by a harmonic potential.
  • the temperature was set to 300K using the V-rescale thermostat separately for protein- ligand complex and solution.
  • the coupling constant of the external thermal bath was set to 0.1 ps.
  • the pressure of the system was set to 1 atm (Parrinello-Rahman coupling).
  • ibrutinib irreversibly reacts with the free cysteine residue (C481) in the active site of BTK to form a covalent bond. Ibrutinib can still bind to the C481S BTK mutant mostly through hydrogen bonding, but with >40 folds lower affinity.
  • Ibrutinib is >6 folds more potent than its Michael acceptor saturated ibrutinib analog in a kinase inhibition assay for wild type BTK (IC 50 0.72 nM vs 4.9 nM), while both compounds are equally potent towards BTK C481S mutant (ICso 4.6 nM vs 4.7 nM).
  • RC-1 reversible covalent
  • RNC reversible noncovalent
  • IRC irreversible covalent
  • RC-2, RC-3, RC-4 and RC-5 were synthesized, which are 1, 2, 5, and 8 atoms longer in the linker length, respectively, as compared with RC-1.
  • RC-1 is the most efficacious for BTK degradation compared with the PROTACs possessing longer linkers, suggesting cooperative ternary complex formation (Fig. 11).
  • RC-6, RC-7 and RC-8 were designed, which are 1, 2, and 3 atoms shorter in the linker length compared with RC-1.
  • RC-6 had an intramolecular reaction between the amide and the Michael acceptor. Comparing the BTK degradation capability in MOLM-14 cells, it was found herein that RC-7 and RC-8 are inferior to RC-1, possibly due to unfavorable steric clashes between BTK and CRBN. It was also determined in the studies described herein that the efficacy' of BTK degradation decreases significantly through only a single atom change of the thalidomide aryl amine nitrogen to an oxygen (RC-9) (Fig. 11).
  • a BTK PROTAC MT-802 has the linker placed at the C5 position on the phthalimide ring of pomalidomide instead of the C4 position as in RC-1.
  • RC-10 was synthesized by placing the linker at the C5 position of the phthalimide ring and it was found that RC-10 cannot induce any BTK degradation (Fig. 11). Therefore, RC-1 has the optimal linker length and position for BTK degradation with the BTK and CRBN binders used. Quantitative Measurements of BTK and CRBN Concentrations in Cells.
  • Quantitative Western blots were performed to measure the levels of BTK and CRBN in MOLM-14 cells using a series of concentrations of their corresponding recombinant proteins as the standards. Lysate from a million MOLM-14 cells usually produces 250 pg of protein as determined by BCA assays. Because proteins usually occupy 20-30% of cell volume (assuming 25% w/v, i.e., 250 mg/mL), the volume of 1 million MOLM-14 cells was calculated to be ⁇ 1 pL (i.e., the volume of each MOLM-14 cell is -1000 ⁇ m 3 ). Based on quantitative Western blots, the total amount of BTK and CRBN in 1 million MOLM-14 cells was determined to be 60 ng and 0.72 ng, respectively.
  • the absolute concentrations for BTK and CRBN in MOLM-14 cells are 780 nM and 13 nM, respectively (Fig. 4A and Fig. 13A-B).
  • the Kd values between PROTACs and BTK are in the range of 3.0-6.4 nM (Table I), while the ⁇ d values between the PROTACs and CRBN are in the range of 1.5-4.2 pM (Table 2).
  • the data shown in Table 1 were obtained as follows: The dissociation equilibrium constant Ki was measured using the full-length BTK protein by Eurofins DiscoverX. The reported Ki values for RC-1, IRC-1 and RC-Ctrl were measured in the absence of DTT in the buffer. D TT (6 mM) used in the standard assay condition increases the Ki values of RC-1 and RC-ctrl by 2-3 folds, while it does not affect the measurement for IRC-1. The reported Ki value for RNC-1 was measured in the standard assay buffer with the presence of DTT. Duplicates were performed. The biochemical BTK inhibition (BTK Inhibition ICso) was measured using the BTK assay kit from Assay Quant Technologies Inc. Duplicates were performed.
  • BTK Target Engagement ICso is the concentration of an unlabeled compound that results in a half-maximal inhibition binding of the BTK tracer. The target engagement of compounds was assessed following Promega’s assay protocol. Triplicates w'ere performed.
  • K' P,D is a relative intracellular accumulation coefficient for drug D and calculated as Kd/ IC 50,TE .
  • K ' P,D is an ass , ay-dependent parameter to quantify the tendency' of intracellular accumulation of a drug. Under the same assay' conditions, a greater K’ P,D value for a drug reflects its higher tendency to accumulate inside cells. Please refer to the supporting information for detailed explanation.
  • the data shown in Table 2 were obtained as follows: The dissociation equilibrium constant Ki was measured using a truncated Cereblon protein. Triplicates were performed.
  • CRBN Target Engagement ICso is the concentration of an unlabled compound that results in a half-maximal inhibition binding of the CRBN tracer. The target engagement of compounds was assessed using Promega’s assay. Triplicates were performed.
  • K' P,D is a relative intracellular accumulation coefficient for drug D and calculated as K d /IC 50
  • TE K' P,D is an assay-dependent parameter to quantify the tendency of intracellular accumulation of a drug. Under the same assay conditions, a greater K’P.D value for a drug reflects its higher tendency to accumulate inside cells. Further details are provided in the Methods and Materials section above.
  • SPPIER Synchronization of phases-based protein interaction reporter
  • BTK KD the kinase domain of BTK (amino acid residues 382 - 659, referred to as BTK KD ) was engineered into SPPIER to produce a BTK KD -EGFP-HOTag6 construct, which forms tetramers when expressing in cells (Fig. 5A).
  • BTK KD amino acid residues 382 - 659
  • PROTACs can induce ⁇ BTK-PROTAC-CRBN ⁇ ternary complex formation in cells, they' will crosslink the BTK ⁇ -EGFP-HOT ag6 tetramers and the CRBN-EGFP-HOTag3 hexamers to produce EGFP phase separation, which can be conveniently visualized with a fluorescence microscope.
  • This assay is named as BTK-SPPIER.
  • HEK293 T/17 cells were transiently transfected with both constructs. Twenty-four hours after transfection, the cells were incubated with 10 ⁇ of RC-1, IRC-1 or RNC-1. Live cell fluorescence imaging revealed that RC-1, but not IRC-1 nor RNC-1, induced appreciable green fluorescent droplets (Fig. 5B).
  • both compounds have similar ICso values (0.31 vs 0.21 ⁇ ), showing that BTK. inhibition but not degradation accounts for the toxicity in MOLM-14 cells.
  • the ICso values for RC-Ctrl, IRC-Ctrl, and RNC-Ctrl are also similar in the range of 0.3-0.5 ⁇ , suggesting that these three warheads inhibit BTK to a similar extent in cells.
  • both the ICso values for IRC-1 and RNC-1 are in the ⁇ range (2.7 and 4.1 ⁇ ).
  • a biochemical BTK kinase inhibition assay showed that IRC-1 and RNC-1 have slightly diminished inhibitory' activities ( ⁇ 3-fold) compared with their corresponding warhead controls (Table 1).
  • Nano-luciferase based bioluminescence resonance energy transfer (NanoBRET) assay developed by Promega was used.
  • HEK-293 cells were transiently transfected with plasmids expressing a fusion protein of Cereblon and nano-luciferase (nLuc) for 24 hours and then the cells were treated with a Cereblon tracer, which binds to Cereblon to induce NanoBRET signals.
  • Adding PROTACs to cells would compete the CRBN tracer binding to CRBN, thus reducing the NanoBRET signals.
  • the target engagement ICso is defined as the concentration of an unlabeled compound that results in a half-maximal inhibition binding between the fluorescent tracer and the nanoLuc fusion protein.
  • the NanoBRET assay is ratiometric and independent of the expression level of the nanoLuc fusion protein. Based on this assay, it was found that the CRBN target engagement ICso value of RC-1 is 3 and 7-fold as low as those of IRC-1 and RNC-1, respectively (Fig. 4B). It should be noted that target engagement ICso values are dependent on assay conditions, including the tracer concentration and the expression level of the nanoLuc fusion protein. Therefore, the target engagement ICso values for different compounds can only meaningfully be compared under the same assay conditions.
  • KP,D was defined as the intracellular accumulation coefficient for drug D, in which P and D denote partition and drug, respectively.
  • Kp, D is defined as the ratio between the total intracellular and extracellular concentrations of drug D and calculated as where
  • CD,in and C D,ex are the total intracellular and extracellular concentrations of drug D, respectively.
  • K' P,D K' P,D
  • K P,D is independent of assays and assay conditions but requires quantification of [P] and [PD].
  • K' P,D is an assay condition dependent parameter but provides a convenient approach to quantitatively compare the tendency of intracellular accumulation of drugs. Based on the K’ P,D values calculated by dividing the KA to CRBN with the in-cell target engagement ICso (Table 2), it was deduced that the intracellular concentration of RC-1 is 10 and 16-fold as the levels of IRC-1 and RNC-1, respectively.
  • RC-1 is a unique BTK degrader with high target occupancy.
  • PROTACs are characterized as BTK. degraders, they have warheads that can bind and inhibit BTK, essentially as dual-functional BTK inhibitors and degraders.
  • Biochemical BTK kinase inhibition assays were performed to measure the ICso values for RC-1, RNC-1 and IRC-1 and their corresponding warhead controls (Table 1 and Fig. 15).
  • IRC-Ctrl i.e. Ibrutinib
  • RC-Ctrl and RNC-Ctrl have reduced BTK inhibition activities by 7 and 45 folds, respectively.
  • the BTK PROTACs, RC-1, RNC-1, and IRC-1 have similar BTK inhibitory activities to their corresponding warheads (Table 1 and Fig. 15).
  • RNC- 1-CN-DiMe and IRC-l-DiMe were synthesized (Fig. 16A).
  • IRC-l-DiMe can be viewed as RC-1 only lacking the cyano group but maintaining the dimethyl moiety.
  • the BTK target engagement IC 50 value of RC-1 is at least an order of magnitude smaller than the values for IRC-l-DiMe and RNC-l-CN-DiMe, respectively (Table 1 and Fig. 16B).
  • the intracellular concentration of RC-1 is 5 folds of those of IRC-l-DiMe and RNC-l-CN-DiMe (Table 1), showing that the enhanced intracellular accumulation of RC-1 may not be attributed to the physical property changes caused by the additional cyano or dimethyl groups in RC-1 compared with IRC-1 and RNC-1.
  • RC-1 can achieve 50% and 90% of target engagements at 40 nM and 200 nM, respectively.
  • RC-1 can function as both a BTK inhibitor and degrader.
  • XLA cells overexpressing wild type BTK or C481S mutant BTK were treated with RC-1, RNC-1 and IRC-1 for 24 hours, followed by Western blot to compare the BTK levels (Fig. 6A and Fig. 17). Sose-dependent BTK degradation induced by RC-1 was observed regardless of its mutation status with comparable potency. This observation is consistent with the conclusion herein that altering PROTAC binding affinities to BTK within a range does not significantly change the ternary complex formation efficiency (Fig. 14A-B).
  • the potency of RC-1 is weaker in XLA cells than in MOLM-14 cells possibly because BTK is overexpressed in XLA cells.
  • IRC-1 induces much more effective degradation of the BTK C481 S mutant than its wild type in XLA cells (Fig. 17), suggesting that the irreversible covalent bond formation between IRC-1 and BTK causes the inefficient protein degradation.
  • RC-1 degrades BTK with higher specificity than IRC-1 and RNC-1.
  • MOLM-14 cells were treated with RC-1, RNC-1, IRC-1, RC-l-Me (non-degrader control), or DMSO and a quantitative multiplexed proteomic approach was employed to measure the whole cellular protein levels (Fig. 7A-D). The result showed that both in IRC-1 and RNC-1 -treated cells, seven kinases were degraded, including BTK.
  • RC-1 -treated cells only two kinases (BTK and CSK) can be degraded, showing that RC-1 has more selectivity than IRC-1 and RNC-1 for kinase degradation.
  • BTK and CSK two kinases
  • IMD immunomodulatory imide drugs
  • tire RMSD Root-mean-square deviation
  • BTK/ibrutinib and CRBN/lenalidomide From the predicted binding mode, several hydrogen bonds and the covalent bond between RC-1 and the residue C481 of BTK anchors tire ligand to the binding site of BTK Additionally, a hydrogen bond between Y355 of CRBN and anion- ⁇ interaction formed between H353, hence, further greatly stabilizing the binding of RC-1 with proteins, and helping to hold tire orientation of RC-1 to adjust and stabilize the ternary complexes (Fig. 8C).
  • RC-1 outperforms other reported BTK degraders in cell viability and target engagement assays.
  • the goal of blocking BTK signaling with either BTK inhibitors or degraders is to inhibit the growth of cancer cells.
  • BTK degraders DD-03-171 and MT-802 were compared head-to-head with RC-1, RNC-1 and IRC-1 in their abilities to inhibit cancer cell growth (Fig. 9A and 9B, and Fig. 18A and 18B).
  • RNC-1 was used as a surrogate for comparison.
  • RC-1 has the most potent inhibitory effect among all the PROTACs compared (Fig. 9A and Fig. ISA).
  • RC-1 and RC-l-Me which does not induce BTK degradation, have similar ICso values in inhibiting MOLM-14 cell growth, indicating that the growth inhibitory effect induced by PROTACs in MOLM-14 cells is due to BTK inhibition instead of its degradation.
  • the high potency of RC-1 can be due to the combinatorial effects of its high intracellular concentration and tight binding to BTK.
  • RC-1 and DD-03-171 have comparable potency for inhibiting cell growth and outperform all the other BTK PROTACs tested (Fig. 9B and Fig. 18B). Additionally, RC-1 can degrade not only BTK and phosphorylated BTK but also IKZF1 and IKZF3 (Fig. 6B), similar to DD-03-171. RC-1 is more potent than ibrutinib in Mino cells, but have similar potencies in Jeko-1 , Rec-1 and Maver-1 cells (Table 4).
  • RC-1 is more potent than MT-802 at a low
  • RC-1 is unique compared with other BTK degraders because it not only degrades BTK efficiently but also shows high target engagement to inhibit BTK in case BTK is not completely degraded. RC-1 demonstrates a novel dual mechanism of action (MO A) for BTK inhibition and degradation.
  • RC-1 has an ideal plasma half-life and degrades BTK in vivo.
  • the plasma half- life of RC-1 (20 mg/kg, i.p. injection) in ICR mice (female, 5-6 weeks, n 3) using LC-
  • RC-1 has a plasma half-life (ti/2) of 3.4 hours, Cmax of 20 ⁇ , and AUC of 72 ⁇ -h (Fig. 10A).
  • a mouse B cell lymphoma cell line derived from Eu-Myc mice was treated with RC-1 in vitro. It was found that the maximum BTK degradation is only 30-40% even dosed up to 25 ⁇ of RC-1 (Fig. 20), indicating that RC-1 is much less potent for BTK degradation in mouse cells than in human cells. Therefore, this preliminary study show ed that RC-1 has desirable PK/PD properties in vivo.
  • BTK Degradation using PROTACS Additional compounds as described herein, including RC-11, RC-12, RC-13, RC-14, IRC-l-DiMe, RNC-1 -CN-DiMe, RNC-DIS, IRC- CN, RNC-CN, and RNC-CN-Ctrl were analyzed for BTK degradation using the methods described above in this example.
  • the structures of the compounds are shown in Fig.22.
  • BTK degration of the compounds in a MOLM-14 cell line was tested as described above. Specifically, the MOLM-14 cells were treated with the PROTACs for 24 hours, followed by Western blotting to measure the BTK levels. The quantification results are shown in Fig. 23A and the Wester blots are shown in Fig. 23B.

Abstract

L'invention concerne de nouvelles chimères ciblant la protéolyse à petites molécules (PROTAC), ainsi que des méthodes pour leur utilisation en tant qu'agents de dégradation de la tyrosine kinase de Bruton (BTK). Lesdites PROTAC à petites molécules sont utiles dans le traitement et/ou la prévention de maladies associées à BTK, telles que le cancer, les troubles neurodégénératifs, les maladies inflammatoires et les troubles métaboliques. L'invention concerne également des méthodes pour induire une dégradation de BTK dans une cellule à l'aide des composés et des compositions décrits ici. Les composés préférés sont : (I).
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WO2022143856A1 (fr) * 2020-12-31 2022-07-07 Beigene, Ltd. Dégradation de la tyrosine kinase de bruton (btk) par conjugaison d'inhibiteurs de btk avec un ligand de ligase e3 et procédés d'utilisation
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CN115304606A (zh) * 2021-06-21 2022-11-08 清华大学 一种同时靶向btk和gspt1蛋白的降解剂
WO2022253250A1 (fr) * 2021-06-01 2022-12-08 正大天晴药业集团股份有限公司 Dégradation de la tyrosine kinase de bruton contenant un cycle fusionné ou un cycle spiro
WO2022268148A1 (fr) * 2021-06-24 2022-12-29 天津医科大学肿瘤医院 Composé pour dégrader la protéine btk, son procédé de préparation et son utilisation
WO2023035927A1 (fr) * 2021-09-07 2023-03-16 中国科学院成都生物研究所 Composé destiné à la dégradation ciblée de la tyrosinase, composition pharmaceutique, et procédé de synthèse de composé et utilisation associée
WO2023125907A1 (fr) * 2021-12-30 2023-07-06 Beigene, Ltd. Dégradation de la tyrosine kinase de bruton (btk) par conjugaison d'inhibiteurs de btk avec un ligand de ligase e3 et méthodes d'utilisation
WO2023227080A1 (fr) * 2022-05-25 2023-11-30 百极弘烨(南通)医药科技有限公司 Composé protac, composition pharmaceutique le contenant, son procédé de préparation et son utilisation
WO2023242597A1 (fr) * 2022-06-16 2023-12-21 Amphista Therapeutics Limited Molécules bifonctionnelles pour la dégradation ciblée de protéines
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