WO2024002289A1 - Protein degradation compounds and methods of use - Google Patents

Protein degradation compounds and methods of use Download PDF

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Publication number
WO2024002289A1
WO2024002289A1 PCT/CN2023/104153 CN2023104153W WO2024002289A1 WO 2024002289 A1 WO2024002289 A1 WO 2024002289A1 CN 2023104153 W CN2023104153 W CN 2023104153W WO 2024002289 A1 WO2024002289 A1 WO 2024002289A1
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group
optionally substituted
substituted
unsubstituted
alkyl
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English (en)
French (fr)
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Chu-Chiang Lin
Hung-Chuan Chen
Pei-Chin Cheng
Chih-Chang Chou
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Anhorn Medicines Co Ltd
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Anhorn Medicines Co Ltd
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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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/4545—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring hetero atom, e.g. pipamperone, anabasine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/4965—Non-condensed pyrazines
    • A61K31/497—Non-condensed pyrazines containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/50—Pyridazines; Hydrogenated pyridazines
    • A61K31/501—Pyridazines; Hydrogenated pyridazines not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A61K31/551—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having two nitrogen atoms, e.g. dilazep
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/10—Anti-acne agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/14—Drugs for dermatological disorders for baldness or alopecia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • 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
    • 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
    • C07D495/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms
    • C07D495/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
    • C07D495/04—Ortho-condensed 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

  • bivalent compounds e.g., bi-functional small molecule compounds
  • compositions comprising one or more of the bivalent compounds
  • methods of use the bivalent compounds for the treatment of certain disease in a subject in need thereof e.g., bi-functional small molecule compounds
  • TBM target binding moiety
  • CLM cereblon E3 ubiquitin ligase binding moiety
  • W1 and W2 are each independently selected from C, CRC2 and N;
  • G is selected from the group consisting of H, OH, CH2OH, RC3OCOORC4, RC3OCONRC4RC5, and 2- (trimethylsilyl) ethoxymethyl group;
  • Q1 to Q7 are each independently C, O, S, N, CRC2 or NRC2; at least one of W1, W2, Q1, Q2, Q3, Q4, Q5, Q6 and Q7 comprises a heteroatom;
  • K is selected from the group consisting of H, an unsubstituted alkyl group, an alkyl group substituted by RC2, an unsubstituted cycloalkyl group, and a cycloalkyl group substituted by RC2; K is bound to the 6-membered ring with a stereospecific bond or a non-stereospecific bond;
  • RC1 is selected from the group consisting of an unsubstituted alkyl group, an alkyl group substituted by RC2, an unsubstituted aryl group, an aryl group substituted by RC2, an unsubstituted alkyl-aryl group, an alkyl-aryl group substituted by RC2, an unsubstituted alkoxy group, and an alkoxy group substituted by RC2;
  • RC2 is selected from the group consisting of H, halo, CH2OH, CRC4, NRC4RC5, 2- (trimethylsilyl) ethoxymethyl, an alkoxyl group, an unsubstituted alkyl group, an alkyl group substituted by one or more halo groups, an unsubstituted cycloalkyl group, a cycloalkyl group substituted by one or more halo groups, an unsubstituted aryl group, an aryl group substituted by one or more halo groups, an unsubstituted heteroaryl group, a heteroaryl group substituted by one or more halo groups, an unsubstituted heterocyclyl group, and a heterocyclyl group substituted by one or more halo groups;
  • RC3 is selected from the group consisting of an unsubstituted alkylene group, and an alkylene group substituted by RC2;
  • RC4 and RC5 are independently selected from the group consisting of an unsubstituted alkyl group, an alkyl group substituted by RC2, an unsubstituted cycloalkyl group, a cycloalkyl group substituted by RC2, an unsubstituted heterocyclyl group, a heterocyclyl group substituted by RC2, an unsubstituted aryl group, an aryl group substituted by RC2, an unsubstituted heteroaryl group, and a heteroaryl group substituted by RC2; and n is 0, 1, 2, 3 or 4, or a pharmaceutically acceptable salt or analog thereof, wherein the TBM is not selected from the group consisting of:
  • the TBM is not selected from the group consisting of:
  • A1 is selected from Cl, F, Br or CF3;
  • A2 is selected from O, NH, N-methyl or N-ethyl; and
  • A3, A4, A5 and A6 are each independently CH or N.
  • the TBM is not selected from the group consisting of:
  • Z1 is selected from the group consisting of an aryl group, a heteroaryl group, a bicyclic group, and a bi-heterocyclic group, each independently substituted by one or more substituents selected from the group consisting of a halo group, a hydroxyl group, a nitro group, CN, C ⁇ CH, an unsubstituted C1-6 alkyl group, a C1-6 alkyl group substituted by a C1-6 alkoxyl group or one or more halo groups, an unsubstituted C1-6 alkoxyl group, a C1-6 alkoxyl group substituted by one or more halo groups, an unsubstituted C2-6 alkenyl, a C2-6 alkenyl substituted by one or more halo groups, an unsubstituted C2-6 alkynyl, and a C3-6 alkynyl substituted by one or more halo groups;
  • Y1, Y2, Y6 are each independently NRY1, O or S;
  • M is a 3-to 6-membered ring with 0 to 4 heteroatoms, which is unsubstituted or substituted by 1 to 6 RM groups;
  • each RM group is independently selected from the group consisting of H, an unsubstituted C1-6 alkyl group, a C1-6 alkyl group substituted by a C1-6 alkoxyl group or one or more halo groups, halogen, and a C1-6 alkoxy group; or two RM groups are taken together with the atom they are attached to and form a 3-to 8-membered ring system containing 0 to 2 heteroatoms;
  • Ra, Rb, Rc, Rd, RY1, RY2 are each independently selected from the group consisting of H, an unsubstituted C1-6 alkyl group, a C1-6 alkyl group substituted by a C1-6 alkoxyl group or one or more halo groups, halogen, a C1-6 alkoxy group, a cyclic group, and a heterocyclic group; or Ra, Rb are taken together with the atom they are attached to and form a 3-to 8-membered ring system containing 0 to 2 heteroatoms;
  • Z2 is selected from the group consisting of a bond, a C1-6 alkyl group, a C1-6 heteroalkyl group, O, an aryl group, a heteroaryl group, an alicyclic group, a heterocyclic group, a biheterocyclic group, a biaryl group, and a biheteroaryl group, each of which is unsubstituted or substituted by 1 to 10 RZ2 groups;
  • each R Z2 group is independently selected from the group consisting of H, halo, an unsubstituted C1-6 alkyl group, a C1-6 alkyl group substituted by one or more F, -ORZ2A, a C3-6 cycloalkyl group, a C4-6 cycloheteroalkyl group, an unsubstituted C1-6 alkyl group, a C1-6 alkyl group substituted by a C1-3 alkyl group or a C1-6 alkoxyl group or one or more halo groups, an unsubstituted heterocyclic group, a heterocyclic group substituted by a C1-3 alkyl group or a C1-6 alkoxyl group or one or more halo groups, an unsubstituted aryl group, an aryl group substituted by a C1-3 alkyl group or a C1-6 alkoxyl group or one or more halo groups, an unsubstituted heteroaryl group, aryl group substituted by a C1-3
  • R Z2A is selected from the group consisting of H, a C1-6 alkyl group, and a C1-6 heteroalkyl group, each of which is unsubstituted or substituted by a cycloalkyl group, a cycloheteroalkyl group, an aryl group, a heterocyclic group, a heteroaryl group, halo, or a OC1-3 alkyl group.
  • the TBM is capable of binding to a protein degradable by a cereblon E3 ubiquitin ligase.
  • Q1 is NRC2.
  • RC2 is an unsubstituted C1-6 alkyl group, a C1-6 alkyl group substituted by one or more halo groups, an unsubstituted C1-6 cycloalkyl group, a C1-6 cycloalkyl group substituted by one or more halo groups.
  • RC2 is an unsubstituted C1-6 alkyl group.
  • RC2 is an unsubstituted C1-4 alkyl group.
  • RC2 is methyl or ethyl.
  • W1 and W2 are each C and connected together through a double bond.
  • Q2, Q3, Q4, Q5, Q6 and Q7 together form a benzene or heterobenzen ring, optionally substituted with one or more RC2. In some embodiment, Q2, Q3, Q4, Q5, Q6 and Q7 together form an unsubstituted benzene or heterobenzen ring. In some embodiment, Q2, Q3, Q4, Q5, Q6 and Q7 together form an unsubstituted benzene ring.
  • K is H.
  • n is 0, 1, or 2. In some embodiment, n is 0 or 1. In some embodiment, n is 0.
  • G is selected from the group consisting of H, CH2OH, RC3OCOORC4, and RC3OCONRC4RC5. In some embodiment, G is selected from the group consisting of H, CH2OH, and RC3OCOORC4. In some embodiment, G is H. In some embodiment, G is CH2OH. In some embodiment, G is RC3OCOORC4. In some embodiment, RC3 is selected from the group consisting of an unsubstituted alkylene group. In some embodiment, RC3 is an unsubstituted C1-6 alkylene group. In some embodiment, RC3 is an unsubstituted C1-4 alkylene group. In some embodiment, RC3 is methylene or ethylene. In some embodiment, RC3 is methylene.
  • RC4 is an unsubstituted alkyl group or an alkyl group substituted by RC2. In some embodiment, RC4 is an unsubstituted alkyl group. In some embodiment, RC4 is an unsubstituted C1-6 alkyl group. In some embodiment, RC4 is an unsubstituted C1-4 alkyl group. In some embodiment, RC4 is methyl or ethyl. In some embodiment, RC4 is methyl.
  • the CLM is represented with Formula (II) -2:
  • Q1 is O, S or NRC2;
  • Q3 to Q5 are each independently C or CRC2;
  • G is selected from the group consisting of H, OH, CH2OH, RC3OCOORC4 and 2- (trimethylsilyl) ethoxymethyl group;
  • RC2 is selected from the group consisting of H, CH2OH, CRC4, 2- (trimethylsilyl) ethoxymethyl, an unsubstituted C1-6 alkyl group, a C1-6 alkyl group substituted by one or more halo groups, a unsubstituted C1-6 cycloalkyl group, a C1-6 cycloalkyl group substituted by one or more halo groups;
  • RC3 is selected from the group consisting of a methylene group and a methylene group substituted by RC2;
  • RC4 is selected from the group consisting of an unsubstituted alkyl group, an alkyl group substituted by RC2, an unsubstituted cycloalkyl group, a cycloalkyl group substituted by RC2, an unsubstituted heterocyclyl group, a heterocyclyl group substituted by RC2.
  • Q1 is NRC2.
  • RC2 is an unsubstituted C1-6 alkyl group, a C1-6 alkyl group substituted by one or more halo groups, an unsubstituted C1-6 cycloalkyl group, a C1-6 cycloalkyl group substituted by one or more halo groups.
  • RC2 is an unsubstituted C1-6 alkyl group.
  • RC2 is an unsubstituted C1-4 alkyl group.
  • RC2 is methyl or ethyl.
  • Q3-Q5 are each C.
  • G is selected from the group consisting of H, CH2OH, RC3OCOORC4, and RC3OCONRC4RC5. In some embodiment, G is selected from the group consisting of H, CH2OH, and RC3OCOORC4. In some embodiment, G is H. In some embodiment, G is CH2OH. In some embodiment, G is RC3OCOORC4. In some embodiment, RC3 is selected from the group consisting of an unsubstituted alkylene group. In some embodiment, RC3 is an unsubstituted C1-6 alkylene group. In some embodiment, RC3 is an unsubstituted C1-4 alkylene group. In some embodiment, RC3 is methylene or ethylene. In some embodiment, RC3 is methylene.
  • RC4 is an unsubstituted alkyl group or an alkyl group substituted by RC2. In some embodiment, RC4 is an unsubstituted alkyl group. In some embodiment, RC4 is an unsubstituted C1-6 alkyl group. In some embodiment, RC4 is an unsubstituted C1-4 alkyl group. In some embodiment, RC4 is methyl or ethyl. In some embodiment, RC4 is methyl.
  • the TBM is connected to the CLM through a bond or a linker moiety (L) . In some embodiment, the TBM is connected to the CLM through a linker moiety (L) . In some embodiment, the TBM is connected to the CLM through Q5. In some embodiment, the TBM is connected to the CLM through Q4. In some embodiment, the TBM is connected to the CLM through Q3.
  • the linker moiety is of Formula (III) :
  • A, W, and B, at each occurrence, are independently selected from null, CO, CO 2 , C (O) NR 1 , C (S) NR 1 , O, S, SO, SO 2 , SO 2 NR 1 , NR 1 , NR 1 CO, NR 1 CONR 2 , NR 1 C (S) , optionally substituted C 1 -C 8 alkyl, optionally substituted C 1 -C 8 alkoxy, optionally substituted C 1 -C 8 alkoxyC 1 -C 8 alkyl, optionally substituted C 1 -C 8 haloalkyl, optionally substituted C 1 -C 8 hydroxyalkyl, optionally substituted C 2 -C 8 alkenyl, optionally substituted C 2 -C 8 alkynyl, optionally substituted C 3 -C 8 cycloalkyl, optionally substituted C 3 -C 8 cycloalkoxy, optionally substituted 3-8 membered heterocyclyl, optionally substituted
  • R 1 and R 2 are independently selected from hydrogen, optionally substituted C 1 -C 6 alkyl, optionally substituted C 3 -C 6 cycloalkyl, optionally substituted C 3 -C 6 cycloalkoxy, optionally substituted 3-6 membered heterocyclyl, optionally substituted C 1 -C 6 alkoxy, optionally substituted C 1 -C 6 alkoxyalkyl, optionally substituted C 1 -C 6 haloalkyl, optionally substituted C 1 -C 6 hydroxyalkyl, optionally substituted C 1 -C 6 alkylamino, and optionally substituted C 1 -C 6 alkylaminoC 1 -C 6 alkyl; and
  • m 0 to 15.
  • the linker moiety is of Formula (III) -1:
  • R 1 , R 2 , R 3 , and R 4 are independently selected from hydrogen, halogen, CN, OH, NH 2 , optionally substituted C 1 -C 6 alkyl, optionally substituted C 3 -C 6 cycloalkyl, optionally substituted C 3 -C 6 cycloalkoxy, optionally substituted 3-6 membered heterocyclyl, optionally substituted C 1 -C 6 alkoxy, optionally substituted C 1 -C 6 alkoxyalkyl, optionally substituted C 1 -C 6 haloalkyl, optionally substituted C 1 -C 6 hydroxyalkyl, optionally substituted C 1 -C 6 alkylamino, and optionally substituted C 1 -C 6 alkylaminoC 1 -C 6 alkyl;
  • A, W, and B, at each occurrence, are independently selected from null, CO, CO 2 , C (O) NR 5 , C (S) NR 5 , O, S, SO, SO 2 , SO 2 NR 5 , NR 5 , NR 5 CO, NR 5 CONR 6 , NR 5 C (S) , optionally substituted C 1 -C 8 alkyl, optionally substituted C 1 -C 8 alkoxy, optionally substituted C 1 -C 8 alkoxyC 1 -C 8 alkyl, optionally substituted C 1 -C 8 haloalkyl, optionally substituted C 1 -C 8 hydroxyalkyl, optionally substituted C 2 -C 8 alkenyl, optionally substituted C 2 -C 8 alkynyl, optionally substituted C 3 -C 8 cycloalkyl, optionally substituted C 3 -C 8 cycloalkoxy, optionally substituted 3-8 membered heterocyclyl, optionally substituted
  • R 5 and R 6 are independently selected from hydrogen, optionally substituted C 1 -C 6 alkyl, optionally substituted C 3 -C 6 cycloalkyl, optionally substituted C 3 -C 6 cycloalkoxy, optionally substituted 3-6 membered heterocyclyl, optionally substituted C 1 -C 6 alkoxy, optionally substituted C 1 -C 6 alkoxyalkyl, optionally substituted C 1 -C 6 haloalkyl, optionally substituted C 1 -C 6 hydroxyalkyl, optionally substituted C 1 -C 6 alkylamino, and optionally substituted C 1 -C 6 alkylaminoC 1 -C 6 alkyl;
  • n 0 to 15;
  • each n is 0 to 15;
  • o 0 to 15.
  • the linker moiety is of Formula (III) -2:
  • each R 1 , and each R 2 are independently selected from hydrogen, halogen, CN, OH, NH 2 , and optionally substituted C 1 -C 6 alkyl, optionally substituted C 3 -C 6 cycloalkyl, optionally substituted C 3 -C 6 cycloalkoxy, optionally substituted 3-6 membered heterocyclyl, optionally substituted C 1 -C 6 alkoxy, optionally substituted C 1 -C 6 alkoxyalkyl, optionally substituted C 1 -C 6 haloalkyl, optionally substituted C 1 -C 6 hydroxyalkyl, optionally substituted C 1 -C 6 alkylamino, or C 1 -C 6 alkylaminoC 1 -C 6 alkyl;
  • each A and each B are independently selected from null, CO, CO 2 , C (O) NR 3 , C (S) NR 3 , O, S, SO, SO 2 , SO 2 NR 3 , NR 3 , NR 3 CO, NR 3 CONR 4 , NR 3 C (S) , and optionally substituted C 1 -C 8 alkyl, optionally substituted C 1 -C 8 alkoxy, optionally substituted C 1 -C 8 alkoxyC 1 -C 8 alkyl, optionally substituted C 1 -C 8 haloalkyl, optionally substituted C 1 -C 8 hydroxyalkyl, optionally substituted C 2 -C 8 alkenyl, optionally substituted C 2 -C 8 alkynyl, optionally substituted C 3 -C 8 cycloalkyl, optionally substituted C 3 -C 8 cycloalkoxy, optionally substituted 3-8 membered heterocyclyl, optionally substituted aryl, optionally
  • R 3 and R 4 are independently selected from hydrogen, and optionally substituted C 1 -C 6 alkyl, optionally substituted C 3 -C 6 cycloalkyl, optionally substituted C 3 -C 6 cycloalkoxy, optionally substituted 3-6 membered heterocyclyl, optionally substituted C 1 -C 6 alkoxy, optionally substituted C 1 -C 6 alkoxyalkyl, optionally substituted C 1 -C 6 haloalkyl, optionally substituted C 1 -C 6 hydroxyalkyl, optionally substituted C 1 -C 6 alkylamino, or C 1 -C 6 alkylaminoC 1 -C 6 alkyl;
  • each m is 0 to 15;
  • n 0 to 15.
  • the linker moiety is of FORMULA (III) -3:
  • X is selected from O, NH, and NR 7 ;
  • R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 are independently selected from hydrogen, halogen, CN, OH, NH 2 , optionally substituted C 1 -C 6 alkyl, optionally substituted C 3 -C 6 cycloalkyl, optionally substituted C 3 -C 6 cycloalkoxy, optionally substituted 3-6 membered heterocyclyl, optionally substituted C 1 -C 6 alkoxy, optionally substituted C 1 -C 6 alkoxyalkyl, optionally substituted C 1 -C 6 haloalkyl, optionally substituted C 1 -C 6 hydroxyalkyl, optionally substituted C 1 -C 6 alkylamino, and optionally substituted C 1 -C 6 alkylaminoC 1 -C 6 alkyl;
  • a and B are independently selected from null, CO, CO 2 , C (O) NR 7 , C (S) NR 7 , O, S, SO, SO 2 , SO 2 NR 7 , NR 7 , NR 7 CO, NR 7 CONR 8 , NR 7 C (S) , optionally substituted C 1 -C 8 alkyl, optionally substituted C 1 -C 8 alkoxy, optionally substituted C 1 -C 8 alkoxyC 1 -C 8 alkyl, optionally substituted C 1 -C 8 haloalkyl, optionally substituted C 1 -C 8 hydroxyalkyl, optionally substituted C 2 -C 8 alkenyl, optionally substituted C 2 -C 8 alkynyl, optionally substituted C 3 -C 8 cycloalkyl, optionally substituted C 3 -C 8 cycloalkoxy, optionally substituted 3-8 membered heterocyclyl, optionally substituted aryl,
  • R 8 and R 8 are independently selected from hydrogen, optionally substituted C 1 -C 6 alkyl, optionally substituted C 3 -C 6 cycloalkyl, optionally substituted C 3 -C 6 cycloalkoxy, optionally substituted 3-6 membered heterocyclyl, optionally substituted C 1 -C 6 alkoxy, optionally substituted C 1 -C 6 alkoxyalkyl, optionally substituted C 1 -C 6 haloalkyl, optionally substituted C 1 -C 6 hydroxyalkyl, optionally substituted C 1 -C 6 alkylamino, and optionally substituted C 1 -C 6 alkylaminoC 1 -C 6 alkyl;
  • each m is 0 to 15;
  • each n is 0 to 15;
  • o 0 to 15;
  • p 0 to 15.
  • the linker moiety comprises a 3 to 13 membered ring, a 4 to 13 membered fused ring, a 5 to 13 membered bridged ring, and a 5 to13 membered spiro ring. In some embodiment, the linker moiety comprises a ring selected from the group consisting of Formula C1, C2, C3, C4 and C5:
  • the linker moiety is of Formula (IV) :
  • Z is selected from the group consisting of a 3-to 8-membered ring, a 5-to 12-membered bicyclic ring, an 8-to 15-membered tricyclic ring and a 6-to 12-membered spiro bicyclic ring, each independently having 0-4 heteroatoms;
  • RL1 is selected from the group consisting of H, an unsubstituted C1-6 alkyl group, a C1-6 alkyl group substituted by a C1-6 alkoxyl group or one or more halo groups, halogen, a C1-6 alkoxy group, a keto group, or an oxide group; wherein, two RL1 groups are optionally taken together to form a 3-8 membered ring system containing 0-2 heteroatoms;
  • RL2 is a bond or an ethynylene group
  • X1 is selected from the group consisting of a methylene group and an ethylene group
  • X3 is selected from the group consisting of an unsubstituted C1-8 alkylene group, a C1-8 alkylene group substituted by 1 to 6 RL1, an unsubstituted C1-8 heteroalkylene group, a C1-8 heteroalkylene group substituted by 1 to 6 RL1, an unsubstituted 3-to 8-membered arylene group, a 3-to 8-membered arylene group substituted by 1 to 6 RL1, an unsubstituted 3-to 8-membered heteroarylene group with 1 to 3 hetero atoms, and a 3-to 8-membered heteroarylene group with 1 to 3 hetero atoms and substituted by 1 to 6 RL1, an unsubstituted 3-to 7-membered cyclic alkylene group, a 3-to 7-membered cyclic alkylene group substituted by 1 to 6 RL1, an unsubstituted 3-to 7-membered heterocyclic alkylene group with 1 to 2 hetero atom
  • RL3 is hydrogen
  • RL4 and RL5 are independently selected from the group consisting of H, an unsubstituted C1-6 alkyl group, and a C1-6 alkyl group substituted by a C1-6 alkoxyl group or one or more halo groups;
  • n1 0, 1, 2, 3, 4, 5 or 6;
  • n2 is 0 or 1
  • n3 is 0 or 1;
  • n4 1;
  • m5 is 0 or 1.
  • heteroatoms in Formula (IV) are each independently selected N, O and S.
  • the Z ring comprises one or two heteroatoms selected from N, O and S.
  • Z is selected from the group consisting of a 3-to 8-membered ring, a 6-to 10-membered bicyclic ring and a 8-to 10-membered spiro bicyclic ring, each independently having 1-2 heteroatoms.
  • RL1 is selected from the group consisting of H, an unsubstituted linear C1-3 alkyl group, a keto group, or an oxide group;
  • RL2 is a bond or an ethynylene group.
  • X1 is selected from the group consisting of a methylene group and an ethylene.
  • X3 is selected from the group consisting of an unsubstituted C1-6 alkylene group, an unsubstituted 3-to 7-membered cyclic alkylene group, and an unsubstituted 3-to 7-heterocyclic alkylene group with 0 to 2 hetero atoms; or, X3 is a 10-to 12-membered spiro bicyclic ring having 0-2 heteroatoms.
  • X4 is selected from the group consisting of a methylene group, CONRL3 and O.
  • m1 is 0, 1, 2 or 3.
  • RL1 is selected from the group consisting of H, an unsubstituted C1-3 alkyl group, a C1-3 alkyl group substituted by a C1-3 alkoxyl group or one or more halo groups, halogen, a C1-3 alkoxy group, a keto group, or an oxide group; wherein, two RL1 groups are optionally taken together to form a 3-8 membered ring system containing 0-2 heteroatoms.
  • RL1 is an oxide group which is attached to one heteroatom N on the Z ring to form an N-oxide group (N + –O - ) .
  • RL1 is an alkyl selected from the group consisting of a linear C1-6 alkyl and a branched C1-6 alkyl, each of which is unsubstituted or substituted by a C1-6 alkoxyl group or one or more halo groups. In some embodiment, RL1 is an alkyl selected from the group consisting of a linear C1-3 alkyl and a branched C1-3 alkyl, each of which is unsubstituted or substituted by a C1-6 alkoxyl group or one or more halo groups.
  • X3 is selected from the group consisting of a bond, an unsubstituted C1-6 alkylene group, a C1-6 alkylene group substituted by 1 to 6 RL1, an unsubstituted C1-6 heteroalkylene group, a C1-6 heteroalkylene group substituted by 1 to 6 RL1. In some embodiment, X3 is selected from the group consisting of an unsubstituted C1-5 alkylene group.
  • RL4 and RL5 are independently an alkyl selected from the group consisting of a linear C1-6 alkyl and a branched C1-6 alkyl, each of which is unsubstituted or substituted by a C1-6 alkoxyl group or one or more halo groups. In some embodiment, RL4 and RL5 are independently an alkyl selected from the group consisting of a linear C1-3 alkyl and a branched C1-3 alkyl, each of which is unsubstituted or substituted by a C1-6 alkoxyl group or one or more halo groups.
  • linker moiety is selected from the group consisting of:
  • f is an integer of 0, 1, 2, 3 or 4; and g is an integer of 0, 1, 2 or 3.
  • the bivalent compound selected from the group consisting of:
  • compositions comprising a bivalent compound disclosed herein, and a pharmaceutically acceptable carrier.
  • Another aspect of the present disclosure relates to a method of degrading a protein associated with a disease or condition, by contacting the protein with the bivalent compound disclosed herein.
  • the present disclosure is based, at least in part, on the discovery that protein-protein interactions are difficult to be targeted using small molecules because proteins have large contact surfaces and the shallow grooves or flat interfaces thereon may get involved in the interactions.
  • tagging the pathogenic protein with ubiquitin and the eventual degradation by the 26S proteasome system has demonstrated that this modality can provide an extended and thorough removal of the cause of disease (Sun et al., Signal Transduct. Target Ther. 2019, 4: 64) .
  • E3 ubiquitin ligases also known as E3 ligases
  • their substrate recognition proteins confer the substrate specificity for ubiquitination.
  • E3 ligases such as cereblon (CRBN) E3 ligase, von Hippel-Lindau disease tumor suppressor (VHL) E3 ligase, double minute 2 protein (MDM2) E3 ligase, and cell inhibitor of apoptosis protein (cIAP) E3 ligase have been utilized successfully for small molecule protein degrader design.
  • CBN cereblon
  • VHL von Hippel-Lindau disease tumor suppressor
  • MDM2 double minute 2 protein
  • cIAP cell inhibitor of apoptosis protein
  • E3 ligase One E3 ligase with therapeutic potential is cereblon E3 ligase, a protein in humans that is encoded by the CRBN gene. CRBN orthologs are highly conserved from plants to humans. Cereblon forms an E3 ubiquitin ligase complex with damaged DNA binding protein 1 (DDB1) , Cullin-4A (CUL4A) , and Regulator of Cullins 1 (ROC 1) . This complex ubiquitinates a number of other proteins (Vriend et al., Front Mol Biosci. 2018, 5: 19) .
  • DDB1 DNA binding protein 1
  • CUL4A Cullin-4A
  • ROC 1 Regulator of Cullins 1
  • an element means one element or more than one element.
  • a reference to "A and/or B" when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than ⁇ ) ; in another embodiment, to ⁇ only (optionally including elements other than A) ; in yet another embodiment, to both A and ⁇ (optionally including other elements) ; etc.
  • the phrase "at least one, " in reference to a list of one or more elements, should be understood to mean at least one element selected from anyone or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements.
  • This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified.
  • “at least one of A and B" can refer, in one embodiment, to at least one, optionally including more than one, A, with no ⁇ present (and optionally including elements other than ⁇ ) ; in another embodiment, to at least 5 one, optionally including more than one, ⁇ , with no A present (and optionally including elements other than A) ; in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, ⁇ (and optionally including other elements) ; etc.
  • Alkyl refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, containing no unsaturation.
  • An alkyl may comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or sixteen carbon atoms.
  • an alkyl comprises one to fifteen carbon atoms (e.g., C 1 -C 15 alkyl) .
  • an alkyl comprises one to thirteen carbon atoms (e.g., C 1 -C 13 alkyl) .
  • an alkyl comprises one to eight carbon atoms (e.g., C 1 -C 8 alkyl) .
  • an alkyl comprises five to fifteen carbon atoms (e.g., C 5 -C 15 alkyl) . In other embodiments, an alkyl comprises five to eight carbon atoms (e.g., C 5 -C 8 alkyl) .
  • the alkyl is attached to the rest of the molecule by a single bond, for example, methyl (Me) , ethyl (Et) , n-propyl, 1-methylethyl (iso-propyl) , n-butyl, n-pentyl, 1, 1-dimethylethyl (t-butyl) , pentyl, 3-methylhexyl, 2-methylhexyl, and the like.
  • Alkenyl refers to a straight or branched hydrocarbon chain radical group consisting solely of carbon and hydrogen atoms, containing at least one double bond.
  • An alkenyl may comprise two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or sixteen carbon atoms.
  • an alkenyl comprises two to twelve carbon atoms (e.g., C 2 -C 12 alkenyl) .
  • an alkenyl comprises two to eight carbon atoms (e.g., C 2 -C 8 alkenyl) .
  • an alkenyl comprises two to six carbon atoms (e.g., C 2 -C 6 alkenyl) . In other embodiments, an alkenyl comprises two to four carbon atoms (e.g., C 2 -C 4 alkenyl) .
  • the alkenyl is attached to the rest of the molecule by a single bond, for example, ethenyl (i.e., vinyl) , prop-1-enyl (i.e., allyl) , but-1-enyl, pent-1-enyl, penta-1, 4-dienyl, and the like.
  • alkynyl refers to a straight or branched hydrocarbon chain radical group consisting solely of carbon and hydrogen atoms, containing at least one triple bond.
  • An alkynyl may comprise two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or sixteen carbon atoms.
  • an alkynyl comprises two to twelve carbon atoms (e.g., C 2 -C 12 alkynyl) .
  • an alkynyl comprises two to eight carbon atoms (e.g., C 2 -C 8 alkynyl) .
  • an alkynyl has two to six carbon atoms (e.g., C 2 -C 6 alkynyl) . In other embodiments, an alkynyl has two to four carbon atoms (e.g., C 2 -C 4 alkynyl) .
  • the alkynyl is attached to the rest of the molecule by a single bond. Examples of such groups include, but are not limited to, ethynyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, and the like.
  • alkoxy means an alkyl group as defined herein which is attached to the rest of the molecule via an oxygen atom.
  • alkoxy means an alkyl group as defined herein which is attached to the rest of the molecule via an oxygen atom. Examples of such groups include, but are not limited to, methoxy, ethoxy, n-propyloxy, iso-propyloxy, n-butoxy, iso-butoxy, tert-butoxy, pentyloxy, hexyloxy, and the like.
  • aryl refers to a radical derived from an aromatic monocyclic or multicyclic hydrocarbon ring system by removing a hydrogen atom from a ring carbon atom.
  • the aromatic monocyclic or multicyclic hydrocarbon ring system contains only hydrogen and carbon atoms.
  • An aryl may comprise from six to eighteen carbon atoms, where at least one of the rings in the ring system is fully unsaturated, i.e., it contains a cyclic, delocalized (4n+2) ⁇ –electron system in accordance with the Hückel theory.
  • an aryl comprises six to fourteen carbon atoms (C 6 -C 14 aryl) .
  • an aryl comprises six to ten carbon atoms (C 6 -C 10 aryl) .
  • groups include, but are not limited to, phenyl, fluorenyl and naphthyl.
  • heteroaryl refers to a radical derived from a 3-to 18-membered aromatic ring radical that comprises two to seventeen carbon atoms and from one to six heteroatoms selected from nitrogen, oxygen and sulfur.
  • the heteroaryl radical may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, wherein at least one of the rings in the ring system is fully unsaturated, i.e., it contains a cyclic, delocalized (4n+2) ⁇ –electron system in accordance with the Hückel theory.
  • Heteroaryl includes fused or bridged ring systems.
  • the heteroatom (s) in the heteroaryl radical is optionally oxidized.
  • One or more nitrogen atoms, if present, are optionally quaternized.
  • the heteroaryl is attached to the rest of the molecule through any atom of the ring (s) .
  • groups include, but not limited to, pyridinyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, quinolinyl, isoquinolinyl, indolyl, benzimidazolyl, benzofuranyl, cinnolinyl, indazolyl, indolizinyl, phthalazinyl, pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl, thi
  • an heteroaryl is attached to the rest of the molecule via a ring carbon atom. In certain embodiments, an heteroaryl is attached to the rest of the molecule via a nitrogen atom (N-attached) or a carbon atom (C-attached) .
  • N-attached nitrogen atom
  • C-attached carbon atom
  • a group derived from pyrrole may be pyrrol-1-yl (N-attached) or pyrrol-3-yl (C-attached) .
  • a group derived from imidazole may be imidazol-1-yl (N-attached) or imidazol-3-yl (C-attached) .
  • heterocyclyl means a non-aromatic, monocyclic, bicyclic, tricyclic, or tetracyclic radical having a total of from 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 atoms in its ring system, and containing from 3 to 12 carbon atoms and from 1 to 4 heteroatoms each independently selected from O, S and N, and with the proviso that the ring of said group does not contain two adjacent O atoms or two adjacent S atoms.
  • a heterocyclyl group may include fused, bridged or spirocyclic ring systems.
  • a hetercyclyl group comprises 3 to 10 ring atoms (3-10 membered heterocyclyl) .
  • a hetercyclyl group comprises 3 to 8 ring atoms (3-8 membered heterocyclyl) . In certain embodiments, a hetercyclyl group comprises 4 to 8 ring atoms (4-8 membered heterocyclyl) . In certain embodiments, a hetercyclyl group comprises 3 to 6 ring atoms (3-6 membered heterocyclyl) .
  • a heterocyclyl group may contain an oxo substituent at any available atom that will result in a stable compound. For example, such a group may contain an oxo atom at an available carbon or nitrogen atom. Such a group may contain more than one oxo substituent if chemically feasible.
  • heterocyclyl group when such a heterocyclyl group contains a sulfur atom, said sulfur atom may be oxidized with one or two oxygen atoms to afford either a sulfoxide or sulfone.
  • An example of a 4 membered heterocyclyl group is azetidinyl (derived from azetidine) .
  • An example of a 5 membered cycloheteroalkyl group is pyrrolidinyl.
  • An example of a 6 membered cycloheteroalkyl group is piperidinyl.
  • An example of a 9 membered cycloheteroalkyl group is indolinyl.
  • An example of a 10 membered cycloheteroalkyl group is 4H-quinolizinyl.
  • Such heterocyclyl groups include, but are not limited to, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl, piperidino, morpholino, thiomorpholino, thioxanyl, piperazinyl, azetidinyl, oxetanyl, thietanyl, homopiperidinyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl, thiazepinyl, 1, 2, 3, 6-tetrahydropyridinyl, 2-pyrrolinyl, 3-pyrrolinyl, indolinyl, 2H-pyranyl, 4H-pyranyl, diox
  • a heteroaryl group may be attached to the rest of molecular via a carbon atom (C-attached) or a nitrogen atom (N-attached) .
  • a group derived from piperazine may be piperazin-1-yl (N-attached) or piperazin-2-yl (C-attached) .
  • cycloalkyl means a saturated, monocyclic, bicyclic, tricyclic, or tetracyclic radical having a total of from 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 carbon atoms in its ring system.
  • a cycloalkyl may be fused, bridged or spirocyclic.
  • a cycloalkyl comprises 3 to 8 carbon ring atoms (C 3 -C 8 cycloalkyl) .
  • a cycloalkyl comprises 3 to 6 carbon ring atoms (C 3 -C 6 cycloalkyl) .
  • Examples of such groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cycloheptyl, adamantyl, and the like.
  • cycloalkylene is a bidentate radical obtained by removing a hydrogen atom from a cycloalkyl ring as defined above.
  • groups include, but are not limited to, cyclopropylene, cyclobutylene, cyclopentylene, cyclopentenylene, cyclohexylene, cycloheptylene, and the like.
  • spirocyclic as used herein has its conventional meaning, that is, any ring system containing two or more rings wherein two of the rings have one ring carbon in common.
  • Each ring of the spirocyclic ring system independently comprises 3 to 20 ring atoms. Preferably, they have 3 to 10 ring atoms.
  • Non-limiting examples of a spirocyclic system include spiro [3.3] heptane, spiro [3.4] octane, and spiro [4.5] decane.
  • cyano refers to a -C ⁇ N group.
  • aldehyde refers to a –C (O) H group.
  • alkoxy refers to both an –O-alkyl, as defined herein.
  • alkoxycarbonyl refers to a -C (O) -alkoxy, as defined herein.
  • alkylaminoalkyl refers to an -alkyl-NR-alkyl group, as defined herein.
  • alkylsulfonyl refer to a -SO 2 alkyl, as defined herein.
  • amino refers to an optionally substituted -NH 2 .
  • aminoalkyl refers to an –alky-amino group, as defined herein.
  • aminocarbonyl refers to a -C (O) -amino, as defined herein.
  • arylalkyl refers to -alkylaryl, where alkyl and aryl are defined herein.
  • aryloxy refers to both an –O-aryl and an –O-heteroaryl group, as defined herein.
  • aryloxycarbonyl refers to -C (O) -aryloxy, as defined herein.
  • arylsulfonyl refers to a -SO 2 aryl, as defined herein.
  • carbonyl group refers to a -C (O) -group, as defined herein.
  • a “carboxylic acid” group refers to a –C (O) OH group.
  • cycloalkoxy refers to a –O-cycloalkyl group, as defined herein.
  • halo or halogen group refers to fluorine, chlorine, bromine or iodine.
  • haloalkyl group refers to an alkyl group substituted with one or more halogen atoms.
  • a "hydroxy” group refers to an -OH group.
  • a "nitro” group refers to a -NO 2 group.
  • trihalomethyl refers to a methyl substituted with three halogen atoms.
  • substituted means that the specified group or moiety bears one or more substituents independently selected from C 1 -C 4 alkyl, aryl, heteroaryl, aryl-C 1 -C 4 alkyl-, heteroaryl-C 1 -C 4 alkyl-, C 1 -C 4 haloalkyl, -OC 1 -C 4 alkyl, -OC 1 -C 4 alkylphenyl, -C 1 -C 4 alkyl-OH, -OC 1 -C 4 haloalkyl, halo, -OH, -NH 2 , -C 1 -C 4 alkyl-NH 2 , -N (C 1 -C 4 alkyl) (C 1 -C 4 alkyl) , -NH (C 1 -C 4 alkyl) , -N (C 1 -C 4 alkyl) (C 1 -C 4 alkylphenyl) , -NH (C 1 -C 4 alkyl
  • a C 6 aryl group also called “phenyl” herein
  • phenyl substituted with one additional substituent
  • one of ordinary skill in the art would understand that such a group has 4 open positions left on carbon atoms of the C 6 aryl ring (6 initial positions, minus one at which the remainder of the compound of the present invention is attached to and an additional substituent, remaining 4 positions open) .
  • the remaining 4 carbon atoms are each bound to one hydrogen atom to fill their valencies.
  • a C 6 aryl group in the present compounds is said to be “disubstituted, ” one of ordinary skill in the art would understand it to mean that the C 6 aryl has 3 carbon atoms remaining that are unsubstituted. Those three unsubstituted carbon atoms are each bound to one hydrogen atom to fill their valencies.
  • “Pharmaceutically acceptable salt” includes both acid and base addition salts.
  • a pharmaceutically acceptable salt of any one of the bivalent compounds described herein is intended to encompass any and all pharmaceutically suitable salt forms.
  • Preferred pharmaceutically acceptable salts of the compounds described herein are pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.
  • “Pharmaceutically acceptable acid addition salt” refers to those salts which retain the biological effectiveness and properties of the free bases, which are not biologically or otherwise undesirable, and which are formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, hydroiodic acid, hydrofluoric acid, phosphorous acid, and the like. Also included are salts that are formed with organic acids such as aliphatic mono-and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, alkanedioic acids, aromatic acids, aliphatic and. aromatic sulfonic acids, etc.
  • acetic acid trifluoroacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like.
  • Exemplary salts thus include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, nitrates, phosphates, monohydrogenphosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, trifluoroacetates, propionates, caprylates, isobutyrates, oxalates, malonates, succinate suberates, sebacates, fumarates, maleates, mandelates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, phthalates, benzenesulfonates, toluenesulfonates, phenylacetates, citrates, lactates, malates, tartrates, methanesulfonates, and the like.
  • salts of amino acids such as arginates, gluconates, and galacturonates (see, for example, Berge S. M. et al., “Pharmaceutical Salts, " Journal of Pharmaceutical Science, 66: 1-19 (1997) , which is hereby incorporated by reference in its entirety) .
  • Acid addition salts of basic compounds may be prepared by contacting the free base forms with a sufficient amount of the desired acid to produce the salt according to methods and techniques with which a skilled artisan is familiar.
  • “Pharmaceutically acceptable base addition salt” refers to those salts that retain the biological effectiveness and properties of the free acids, which are not biologically or otherwise undesirable. These salts are prepared from addition of an inorganic base or an organic base to the free acid. Pharmaceutically acceptable base addition salts may be formed with metals or amines, such as alkali and alkaline earth metals or organic amines. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like.
  • Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, for example, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, N, N-dibenzylethylenediamine, chloroprocaine, hydrabamine, choline, betaine, ethylenediamine, ethylenedianiline, N-methylglucamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like. See Berge et al
  • the present disclosure provides a bivalent compound including a target binding moiety (TBM) and a cereblon E3 ubiquitin ligase binding moiety (CLM) , or a pharmaceutically acceptable salt or analog thereof.
  • TBM target binding moiety
  • CLM cereblon E3 ubiquitin ligase binding moiety
  • the TBM may be connected to the CLM directly or via a linker moiety.
  • the TRK ligand may be connected to the degradation tag directly (TBM-CLM) .
  • the TRK ligand may be connected to the degradation tag via a linker moiety (TBM-L-CLM) .
  • TBM Target Binding Moiety
  • target binding moiety may refer to any molecules ranging from small molecules to large proteins that associate with or bind to a target protein to be degraded by the bivalent compounds disclosed herein, i.e. protein of interest ( “POI) .
  • the TBM can be, for example but not limited to, a small molecule compound (i.e., a molecule of molecular weight less than about 1.5 kilodaltons (kDa) ) , a peptide or polypeptide, nucleic acid or oligonucleotide, carbohydrate such as oligosaccharides, or an antibody or fragment thereof.
  • the TBM is capable of binding to an overexpressed protein associated with a disease or condition, a mutated protein associated with a disease or condition, a fusion protein associated with a disease or condition, or the like.
  • the first wave of clinical-stage protein degraders is aimed at classically drugged targets that have clinically validated roles in disease and readily available chemical matter. Success against these targets has begun to solidify PROTACs as a therapeutic modality and underscores the potential of these molecules to become best-in-class medicines by way of degrading a target instead of inhibiting it.
  • the true promise of the modality is reaching targets that are currently difficult to drug with existing modalities or have not yet been drugged at all.
  • the targets for PROTAC therapy can have several common characteristics, including: a change away from the natural state, via overexpression, mutation, aggregation, isoform expression or localization, that results in a disease-causing gain of function; a binding surface that is approachable by an E3 ligase; and an unstructured region to thread into the proteasome.
  • Proteins that have evolved resistance mutations to targeted therapies, proteins with scaffolding functions and proteins that are considered ‘undruggable’ with other modalities can also be highly suitable PROTAC targets.
  • the early PROTAC targets focused on POIs that had existing ligands, in the form of available inhibitors, but were still associated with clear unmet medical need.
  • PROTACable POIs do not necessarily need an enzyme active site, they do need a small-molecule binding site that is approachable by an E3 ligase. Using these sites does not require a high-affinity ligand if coupled to the right E3 ligand, but moderate affinity ( ⁇ 1–500 nM) is typically needed, and access to the POI surface near the binding site by a recruited E3 ligase is essential. Achieving such binding affinities can often be challenging and has promoted research into alternative degraders. Selection of the ligand-binding site is particularly important in the case of scaffolding proteins, where the POI may only be partially exposed within a given complex.
  • Androgen receptor a member of the nuclear hormone receptor family, is a transcriptional factor. Upon activation by androgen, AR translocate to the nucleus where it regulates nuclear gene transcription. AR is related to prostate cancer formation and progression, which is the second most prevalent cancer and the second-leading cause of cancer death in men. AR is a clinically fully validated target for the treatment of human prostate cancer, inhibition of AR has been extensively investigated to treat prostate cancer. Because AR protein plays a key role in metastatic castration-resistant prostate cancer (mCRPC) , AR degraders designed based upon the concept of PROTAC could be potentially very effective for the treatment of mCRPC when the disease becomes resistant to AR antagonists or to androgen synthesis inhibitors.
  • mCRPC metastatic castration-resistant prostate cancer
  • ER ⁇ + estrogen receptor-alpha-positive
  • the binding of estradiol to the ligand-binding domain activates ER ⁇ .
  • ER ⁇ can also be activated via the phosphorylation induced by growth factors.
  • Activated ER ⁇ functions as a transcriptional regulator with a pro-tumor activity in breast cancer cells.
  • ER degraders designed based upon the concept of PROTAC could be potentially very effective for the treatment of ER ⁇ + breast cancer.
  • BRD4 is a critical protein that is overexpressed in human cancer and promotes the growth and survival of cancer cells (Donati et al., 2018; Zhang F. et al., 2020) .
  • the present disclosure recognizes that development of the first CRBN-based PROTAC in 2015, with the structure of pomalidomide capturing CRBN and BRDs inhibitor JQ1 as TBM.
  • the resulting compound has been shown to induce highly selective CRBN-dependent BET protein degradation in vitro and in vivo and delay the progression of leukemia in mice, demonstrating the high efficiency and specificity of degradation of BRD family members, such as BRD2, BRD3, and BRD4, by using large-scale proteomic methods (Winter et al., 2015) .
  • TGF- ⁇ 1 is a pleiotropic cytokine and plays an important role in tumor progression (e.g., colorectal and prostate cancer) . Also, it is one of the key factors of tumor cell immune escape (Sun D. -Y. et al., 2019; Dai et al., 2019) . Feng’s team has developed a CRBN-based PROTAC DT-6 to degrade TGF- ⁇ 1.
  • the TGF- ⁇ 1 ligand is derived from its direct inhibitor P144, and CRBN is recruited by the widely used ligand thalidomide. It has been shown that DT-6 can effectively degrade TGF- ⁇ 1 in cells and reduce its secretion, which is of great significance for diseases that are correlated with the TGF- ⁇ 1 signaling (Feng et al., 2020) .
  • E3 ligases In light of the large effect of structure on degradation efficacy, Su’s team has designed a series of PROTACs with varying CDK6 targeting ligands, E3 ligases, and linkers. Considering that the terminal ligands of E3 ligase can also deeply affect the interaction angle between the target protein and the ligase, they have introduced flexible and rigid groups such as alkyl and alkyne into the ligand pomalidomide. To predict which ligase matches CDK6, they have also designed nutlin-3b, VH032, and Bestatin to recruit the E3 ligases MDM2, VHL, and cIAP, respectively.
  • CDK4/6 inhibitors palbociclib, ribociclib, and abemaciclib
  • CRBN-based PROTAC can degrade CDK6.
  • PROTACs with shorter linkers have shown a higher capacity in CDK6 degradation, suggesting that these shorter molecules have better CRBN recruitment ability on CDK6 (Su et al., 2019) .
  • PROTACs that have been designed with pomalidomide as the CRBN ligand to degrade various POIs, such as MCL-1/BCL-2, BCL-xL, HDAC6, and BTK (Myeku et al., 2016; Sun et al., 2018; Wang X. et al., 2019; Chi et al., 2019; Yang et al., 2019; Xue et al., 2020) .
  • PPI Protein-protein interaction
  • BTK a non-receptor cytoplasmic tyrosine kinase
  • BCR B cell receptor
  • CRBN as the E3 ligase
  • Crews’s team has found that MT802 can effectively degrade BTK. It has excellent degradation characteristics in vitro but shows a high clearance rate and short half-life in vivo. They have further replaced the CRBN ligand with the VHL ligand. Unfortunately, the resulting compound have shown low degradation efficiency.
  • the structure modification of the CRBN ligand has led to the identification of SJF620, with improved druggability compared with MT802 (Jaime-Figueroa et al., 2020) .
  • E3 ubiquitin ligases have been selected to degrade the target proteins.
  • Ibrutinib and PLS-123 two covalent inhibitors of BTK, have been chosen as the binding part of BTK due to the high affinity and different folding structures.
  • CRBN and VHL have been selected as the E3 ligase, which were recruited by pomalidomide and VH032, respectively.
  • Once irreversibly combined with target kinase an excellent degradation efficiency has been observed in living cells (Xue et al., 2020) .
  • CRBN and MDM2 have been selected as the E3 ligases in Rao’s study (Sun et al., 2018; Xue et al., 2020) .
  • RG-7112 has been designed as the ligand for MDM2 recruitment and ibrutinib and spebrutinib have been selected as the BTK ligands. It has been found that CRBN is generally more effective as E3 ligase than MDM2 (Sun et al., 2018) . Besides BTK, CRBN-and VHL-PROTAC can also effectively degrade EGFR, BRD4, PLK1, and CDK2 (Zhou F. et al., 2020; Zhang H. et al., 2020; Mu et al., 2020) .
  • Li et al. have developed a PROTAC that can degrade the cell cycle kinase Wee1 and provided a new direction for targeted cancer therapy (Jaeger and Winter, 2020; Li et al., 2020) .
  • Winzker et al. have described that PDE ⁇ -based PROTACs can effectively and selectively reduce the level of phosphodiesterase- ⁇ (PDE ⁇ ) in cells (Winzker et al., 2020) .
  • PDE ⁇ phosphodiesterase- ⁇
  • it has also increased the expression of various lipid-related enzymes and the level of cholesterol precursor.
  • the results have also shown that PDE ⁇ plays a role in the regulation of sterol synthesis (Winzker et al., 2020) .
  • STAT3 Signal transducer and activator of transcription 3 activation is beneficial to the survival, reproduction, metastasis, and immune escape of tumor cells (Furtek et al., 2016) .
  • STAT3 is closely related to the adverse prognosis of human cancer and has become a promising therapeutic target for cancer and other diseases.
  • Zhou et al. have developed SD-36 as a highly selective and potent PROTAC degrader of STAT3.
  • SD-36 can inhibit the growth of leukemia and lymphoma cell lines with highly phosphorylated STAT3 at low nanomolar concentrations in vitro. SD-36 can also completely and persistently regress the tumor growth in mice bearing the Molm-16 xenografts. SD-36 has been found to rapidly induce the degradation of STAT3 but has no significant effect on other STAT isoforms (Zhou et al., 2019) .
  • Bromodomain and Extra-Terminal domain (BET) family proteins are epigenetic regulatory factors related to the expression of multiple oncogenes (Stathis and Bertoni, 2018) .
  • BETd-260 is an effective PROTAC degradation agent synthesized on the basis of BET SMIs. The in vivo and in vitro experiments have shown that it can induce a large amount of apoptosis in osteosarcoma (OS) cells and OS xenograft tumor tissues and ultimately lead to the depth and sustained inhibition of tumor growth in both mouse OS cell line-derived xenograft and patient-derived xenograft (PDX) models (Shi et al., 2019) .
  • OS osteosarcoma
  • PDX patient-derived xenograft
  • the TBM is an antagonist of the POI. In some embodiments, the TBM is an agonist of the POI. In some embodiments, the TBM is a partial agonist of the POI. In some embodiments, the TBM is an inverse agonist of the POI.
  • the TBM is not selected from the group consisting of:
  • the TBM is not selected from the group consisting of:
  • A1 is selected from Cl, F, Br or CF3;
  • A2 is selected from O, NH, N-methyl or N-ethyl; and
  • A3, A4, A5 and A6 are each independently CH or N.
  • the TBM is not selected from the group consisting of:
  • Z1 is selected from the group consisting of an aryl group, a heteroaryl group, a bicyclic group, and a bi-heterocyclic group, each independently substituted by one or more substituents selected from the group consisting of a halo group, a hydroxyl group, a nitro group, CN, C ⁇ CH, an unsubstituted C1-6 alkyl group, a C1-6 alkyl group substituted by a C1-6 alkoxyl group or one or more halo groups, an unsubstituted C1-6 alkoxyl group, a C1-6 alkoxyl group substituted by one or more halo groups, an unsubstituted C2-6 alkenyl, a C2-6 alkenyl substituted by one or more halo groups, an unsubstituted C2-6 alkynyl, and a C3-6 alkynyl substituted by one or more halo groups;
  • Y1, Y2, Y6 are each independently NRY1, O or S;
  • M is a 3-to 6-membered ring with 0 to 4 heteroatoms, which is unsubstituted or substituted by 1 to 6 RM groups;
  • each RM group is independently selected from the group consisting of H, an unsubstituted C1-6 alkyl group, a C1-6 alkyl group substituted by a C1-6 alkoxyl group or one or more halo groups, halogen, and a C1-6 alkoxy group; or two RM groups are taken together with the atom they are attached to and form a 3-to 8-membered ring system containing 0 to 2 heteroatoms;
  • Ra, Rb, Rc, Rd, RY1, RY2 are each independently selected from the group consisting of H, an unsubstituted C1-6 alkyl group, a C1-6 alkyl group substituted by a C1-6 alkoxyl group or one or more halo groups, halogen, a C1-6 alkoxy group, a cyclic group, and a heterocyclic group; or Ra, Rb are taken together with the atom they are attached to and form a 3-to 8-membered ring system containing 0 to 2 heteroatoms;
  • Z2 is selected from the group consisting of a bond, a C1-6 alkyl group, a C1-6 heteroalkyl group, O, an aryl group, a heteroaryl group, an alicyclic group, a heterocyclic group, a biheterocyclic group, a biaryl group, and a biheteroaryl group, each of which is unsubstituted or substituted by 1 to 10 RZ2 groups;
  • each R Z2 group is independently selected from the group consisting of H, halo, an unsubstituted C1-6 alkyl group, a C1-6 alkyl group substituted by one or more F, -ORZ2A, a C3-6 cycloalkyl group, a C4-6 cycloheteroalkyl group, an unsubstituted C1-6 alkyl group, a C1-6 alkyl group substituted by a C1-3 alkyl group or a C1-6 alkoxyl group or one or more halo groups, an unsubstituted heterocyclic group, a heterocyclic group substituted by a C1-3 alkyl group or a C1-6 alkoxyl group or one or more halo groups, an unsubstituted aryl group, an aryl group substituted by a C1-3 alkyl group or a C1-6 alkoxyl group or one or more halo groups, an unsubstituted heteroaryl group, aryl group substituted by a C1-3
  • R Z2A is selected from the group consisting of H, a C1-6 alkyl group, and a C1-6 heteroalkyl group, each of which is unsubstituted or substituted by a cycloalkyl group, a cycloheteroalkyl group, an aryl group, a heterocyclic group, a heteroaryl group, halo, or a OC1-3 alkyl group.
  • CRBN a component of a cullin-RING ubiquitin ligase (CRL) complex
  • CRL4CRBN E3 ubiquitin ligase binding moiety refers to a compound, which associates with or binds to a ubiquitin ligase for recruitment of the corresponding ubiquitination machinery to the to-be-degraded target protein.
  • cereblon E3 ubiquitin ligase binding moiety represented by Formula (II) -1:
  • W1 and W2 are each independently selected from C, CRC2 and N;
  • G is selected from the group consisting of H, OH, CH2OH, RC3OCOORC4, RC3OCONRC4RC5, and 2- (trimethylsilyl) ethoxymethyl group;
  • Q1 to Q7 are each independently C, O, S, N, CRC2 or NRC2; at least one of W1, W2, Q1, Q2, Q3, Q4, Q5, Q6 and Q7 comprises a heteroatom;
  • K is selected from the group consisting of H, an unsubstituted alkyl group, an alkyl group substituted by RC2, an unsubstituted cycloalkyl group, and a cycloalkyl group substituted by RC2; K is bound to the 6-membered ring with a stereospecific bond or a non-stereospecific bond;
  • RC1 is selected from the group consisting of an unsubstituted alkyl group, an alkyl group substituted by RC2, an unsubstituted aryl group, an aryl group substituted by RC2, an unsubstituted alkyl-aryl group, an alkyl-aryl group substituted by RC2, an unsubstituted alkoxy group, and an alkoxy group substituted by RC2;
  • RC2 is selected from the group consisting of H, halo, CH2OH, CRC4, NRC4RC5, 2- (trimethylsilyl) ethoxymethyl, an alkoxyl group, an unsubstituted alkyl group, an alkyl group substituted by one or more halo groups, an unsubstituted cycloalkyl group, a cycloalkyl group substituted by one or more halo groups, an unsubstituted aryl group, an aryl group substituted by one or more halo groups, an unsubstituted heteroaryl group, a heteroaryl group substituted by one or more halo groups, an unsubstituted heterocyclyl group, and a heterocyclyl group substituted by one or more halo groups;
  • RC3 is selected from the group consisting of an unsubstituted alkylene group, and an alkylene group substituted by RC2;
  • RC4 and RC5 are independently selected from the group consisting of an unsubstituted alkyl group, an alkyl group substituted by RC2, an unsubstituted cycloalkyl group, a cycloalkyl group substituted by RC2, an unsubstituted heterocyclyl group, a heterocyclyl group substituted by RC2, an unsubstituted aryl group, an aryl group substituted by RC2, an unsubstituted heteroaryl group, and a heteroaryl group substituted by RC2; and n is 0, 1, 2, 3 or 4, or a pharmaceutically acceptable salt or analog thereof, .
  • Q1 is NRC2.
  • RC2 is an unsubstituted C1-6 alkyl group, a C1-6 alkyl group substituted by one or more halo groups, an unsubstituted C1-6 cycloalkyl group, a C1-6 cycloalkyl group substituted by one or more halo groups.
  • RC2 is an unsubstituted C1-6 alkyl group.
  • RC2 is an unsubstituted C1-4 alkyl group.
  • RC2 is methyl or ethyl.
  • W1 and W2 are each C and connected together through a double bond.
  • Q2, Q3, Q4, Q5, Q6 and Q7 together form a benzene or heterobenzen ring, optionally substituted with one or more RC2. In some embodiment, Q2, Q3, Q4, Q5, Q6 and Q7 together form an unsubstituted benzene or heterobenzen ring. In some embodiment, Q2, Q3, Q4, Q5, Q6 and Q7 together form an unsubstituted benzene ring.
  • K is H.
  • n is 0, 1, or 2. In some embodiment, n is 0 or 1. In some embodiment, n is 0.
  • G is selected from the group consisting of H, CH2OH, RC3OCOORC4, and RC3OCONRC4RC5. In some embodiment, G is selected from the group consisting of H, CH2OH, and RC3OCOORC4. In some embodiment, G is H. In some embodiment, G is CH2OH. In some embodiment, G is RC3OCOORC4. In some embodiment, RC3 is selected from the group consisting of an unsubstituted alkylene group. In some embodiment, RC3 is an unsubstituted C1-6 alkylene group. In some embodiment, RC3 is an unsubstituted C1-4 alkylene group. In some embodiment, RC3 is methylene or ethylene. In some embodiment, RC3 is methylene.
  • RC4 is an unsubstituted alkyl group or an alkyl group substituted by RC2. In some embodiment, RC4 is an unsubstituted alkyl group. In some embodiment, RC4 is an unsubstituted C1-6 alkyl group. In some embodiment, RC4 is an unsubstituted C1-4 alkyl group. In some embodiment, RC4 is methyl or ethyl. In some embodiment, RC4 is methyl.
  • the CLM is represented with Formula (II) -2:
  • Q1 is O, S or NRC2;
  • Q3 to Q5 are each independently C or CRC2;
  • G is selected from the group consisting of H, OH, CH2OH, RC3OCOORC4 and 2- (trimethylsilyl) ethoxymethyl group;
  • RC2 is selected from the group consisting of H, CH2OH, CRC4, 2- (trimethylsilyl) ethoxymethyl, an unsubstituted C1-6 alkyl group, a C1-6 alkyl group substituted by one or more halo groups, a unsubstituted C1-6 cycloalkyl group, a C1-6 cycloalkyl group substituted by one or more halo groups;
  • RC3 is selected from the group consisting of a methylene group and a methylene group substituted by RC2;
  • RC4 is selected from the group consisting of an unsubstituted alkyl group, an alkyl group substituted by RC2, an unsubstituted cycloalkyl group, a cycloalkyl group substituted by RC2, an unsubstituted heterocyclyl group, a heterocyclyl group substituted by RC2.
  • Q1 is NRC2.
  • RC2 is an unsubstituted C1-6 alkyl group, a C1-6 alkyl group substituted by one or more halo groups, an unsubstituted C1-6 cycloalkyl group, a C1-6 cycloalkyl group substituted by one or more halo groups.
  • RC2 is an unsubstituted C1-6 alkyl group.
  • RC2 is an unsubstituted C1-4 alkyl group.
  • RC2 is methyl or ethyl.
  • Q3-Q5 are each C.
  • G is selected from the group consisting of H, CH2OH, RC3OCOORC4, and RC3OCONRC4RC5. In some embodiment, G is selected from the group consisting of H, CH2OH, and RC3OCOORC4. In some embodiment, G is H. In some embodiment, G is CH2OH. In some embodiment, G is RC3OCOORC4. In some embodiment, RC3 is selected from the group consisting of an unsubstituted alkylene group. In some embodiment, RC3 is an unsubstituted C1-6 alkylene group. In some embodiment, RC3 is an unsubstituted C1-4 alkylene group. In some embodiment, RC3 is methylene or ethylene. In some embodiment, RC3 is methylene.
  • RC4 is an unsubstituted alkyl group or an alkyl group substituted by RC2. In some embodiment, RC4 is an unsubstituted alkyl group. In some embodiment, RC4 is an unsubstituted C1-6 alkyl group. In some embodiment, RC4 is an unsubstituted C1-4 alkyl group. In some embodiment, RC4 is methyl or ethyl. In some embodiment, RC4 is methyl.
  • the bivalent compound disclosed herein with the CLM including two carbonyls adjacent to the nitrogen connected to the 2, 6-dioxo-piperidine ring provides improved E3 ligase binding, protein ubiquitination, and/or target protein degradation over a control compound with the CLM including only one carbonyl adjacent to either side of the nitrogen connected to the 2, 6-dioxo-piperidine ring.
  • linker or “linker moiety” is a molecular structure capable of connecting two separate moieties to one another through covalent bonds. In some embodiments, linkers provide for desirable spacing of the two entities.
  • linker in some aspects refers to any agent or molecule that bridges the TBM to the CLM. The present disclosure recognizes suitable sites for attaching a linker, provided that the linker, once attached to the conjugate of the present disclosures, does not interfere with the function of the TBM, i.e., its ability to bind POI, or the function of the CLM, i.e., its ability to recruit a ubiquitin ligase.
  • the length of the linker of the bivalent compound can be adjusted to minimize the molecular weight of the bivalent compounds, avoid the clash of the TBM or targeting moiety with the ubiquitin ligase.
  • the linker comprises acyclic or cyclic saturated or unsaturated carbon, ethylene glycol, amide, amino, ether, urea, carbamate, aromatic, heteroaromatic, heterocyclic or carbonyl groups.
  • the length of the linker is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more atoms.
  • the TBM is connected to the CLM through a bond or a linker moiety (L) . In some embodiment, the TBM is connected to the CLM through a linker moiety (L) . In some embodiment, the TBM is connected to the CLM through Q5. In some embodiment, the TBM is connected to the CLM through Q4. In some embodiment, the TBM is connected to the CLM through Q3.
  • the linker moiety is of Formula (III) :
  • A, W, and B, at each occurrence, are independently selected from null, CO, CO 2 , C (O) NR 1 , C (S) NR 1 , O, S, SO, SO 2 , SO 2 NR 1 , NR 1 , NR 1 CO, NR 1 CONR 2 , NR 1 C (S) , optionally substituted C 1 -C 8 alkyl, optionally substituted C 1 -C 8 alkoxy, optionally substituted C 1 -C 8 alkoxyC 1 -C 8 alkyl, optionally substituted C 1 -C 8 haloalkyl, optionally substituted C 1 -C 8 hydroxyalkyl, optionally substituted C 2 -C 8 alkenyl, optionally substituted C 2 -C 8 alkynyl, optionally substituted C 3 -C 8 cycloalkyl, optionally substituted C 3 -C 8 cycloalkoxy, optionally substituted 3-8 membered heterocyclyl, optionally substituted
  • R 1 and R 2 are independently selected from hydrogen, optionally substituted C 1 -C 6 alkyl, optionally substituted C 3 -C 6 cycloalkyl, optionally substituted C 3 -C 6 cycloalkoxy, optionally substituted 3-6 membered heterocyclyl, optionally substituted C 1 -C 6 alkoxy, optionally substituted C 1 -C 6 alkoxyalkyl, optionally substituted C 1 -C 6 haloalkyl, optionally substituted C 1 -C 6 hydroxyalkyl, optionally substituted C 1 -C 6 alkylamino, and optionally substituted C 1 -C 6 alkylaminoC 1 -C 6 alkyl; and
  • m 0 to 15.
  • the linker moiety is of Formula (III) -1:
  • R 1 , R 2 , R 3 , and R 4 are independently selected from hydrogen, halogen, CN, OH, NH 2 , optionally substituted C 1 -C 6 alkyl, optionally substituted C 3 -C 6 cycloalkyl, optionally substituted C 3 -C 6 cycloalkoxy, optionally substituted 3-6 membered heterocyclyl, optionally substituted C 1 -C 6 alkoxy, optionally substituted C 1 -C 6 alkoxyalkyl, optionally substituted C 1 -C 6 haloalkyl, optionally substituted C 1 -C 6 hydroxyalkyl, optionally substituted C 1 -C 6 alkylamino, and optionally substituted C 1 -C 6 alkylaminoC 1 -C 6 alkyl;
  • A, W, and B, at each occurrence, are independently selected from null, CO, CO 2 , C (O) NR 5 , C (S) NR 5 , O, S, SO, SO 2 , SO 2 NR 5 , NR 5 , NR 5 CO, NR 5 CONR 6 , NR 5 C (S) , optionally substituted C 1 -C 8 alkyl, optionally substituted C 1 -C 8 alkoxy, optionally substituted C 1 -C 8 alkoxyC 1 -C 8 alkyl, optionally substituted C 1 -C 8 haloalkyl, optionally substituted C 1 -C 8 hydroxyalkyl, optionally substituted C 2 -C 8 alkenyl, optionally substituted C 2 -C 8 alkynyl, optionally substituted C 3 -C 8 cycloalkyl, optionally substituted C 3 -C 8 cycloalkoxy, optionally substituted 3-8 membered heterocyclyl, optionally substituted
  • R 5 and R 6 are independently selected from hydrogen, optionally substituted C 1 -C 6 alkyl, optionally substituted C 3 -C 6 cycloalkyl, optionally substituted C 3 -C 6 cycloalkoxy, optionally substituted 3-6 membered heterocyclyl, optionally substituted C 1 -C 6 alkoxy, optionally substituted C 1 -C 6 alkoxyalkyl, optionally substituted C 1 -C 6 haloalkyl, optionally substituted C 1 -C 6 hydroxyalkyl, optionally substituted C 1 -C 6 alkylamino, and optionally substituted C 1 -C 6 alkylaminoC 1 -C 6 alkyl;
  • n 0 to 15;
  • each n is 0 to 15;
  • o 0 to 15.
  • the linker moiety is of Formula (III) -2:
  • each R 1 , and each R 2 are independently selected from hydrogen, halogen, CN, OH, NH 2 , and optionally substituted C 1 -C 6 alkyl, optionally substituted C 3 -C 6 cycloalkyl, optionally substituted C 3 -C 6 cycloalkoxy, optionally substituted 3-6 membered heterocyclyl, optionally substituted C 1 -C 6 alkoxy, optionally substituted C 1 -C 6 alkoxyalkyl, optionally substituted C 1 -C 6 haloalkyl, optionally substituted C 1 -C 6 hydroxyalkyl, optionally substituted C 1 -C 6 alkylamino, or C 1 -C 6 alkylaminoC 1 -C 6 alkyl;
  • each A and each B are independently selected from null, CO, CO 2 , C (O) NR 3 , C (S) NR 3 , O, S, SO, SO 2 , SO 2 NR 3 , NR 3 , NR 3 CO, NR 3 CONR 4 , NR 3 C (S) , and optionally substituted C 1 -C 8 alkyl, optionally substituted C 1 -C 8 alkoxy, optionally substituted C 1 -C 8 alkoxyC 1 -C 8 alkyl, optionally substituted C 1 -C 8 haloalkyl, optionally substituted C 1 -C 8 hydroxyalkyl, optionally substituted C 2 -C 8 alkenyl, optionally substituted C 2 -C 8 alkynyl, optionally substituted C 3 -C 8 cycloalkyl, optionally substituted C 3 -C 8 cycloalkoxy, optionally substituted 3-8 membered heterocyclyl, optionally substituted aryl, optionally
  • R 3 and R 4 are independently selected from hydrogen, and optionally substituted C 1 -C 6 alkyl, optionally substituted C 3 -C 6 cycloalkyl, optionally substituted C 3 -C 6 cycloalkoxy, optionally substituted 3-6 membered heterocyclyl, optionally substituted C 1 -C 6 alkoxy, optionally substituted C 1 -C 6 alkoxyalkyl, optionally substituted C 1 -C 6 haloalkyl, optionally substituted C 1 -C 6 hydroxyalkyl, optionally substituted C 1 -C 6 alkylamino, or C 1 -C 6 alkylaminoC 1 -C 6 alkyl;
  • each m is 0 to 15;
  • n 0 to 15.
  • the linker moiety is of FORMULA (III) -3:
  • X is selected from O, NH, and NR 7 ;
  • R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 are independently selected from hydrogen, halogen, CN, OH, NH 2 , optionally substituted C 1 -C 6 alkyl, optionally substituted C 3 -C 6 cycloalkyl, optionally substituted C 3 -C 6 cycloalkoxy, optionally substituted 3-6 membered heterocyclyl, optionally substituted C 1 -C 6 alkoxy, optionally substituted C 1 -C 6 alkoxyalkyl, optionally substituted C 1 -C 6 haloalkyl, optionally substituted C 1 -C 6 hydroxyalkyl, optionally substituted C 1 -C 6 alkylamino, and optionally substituted C 1 -C 6 alkylaminoC 1 -C 6 alkyl;
  • a and B are independently selected from null, CO, CO 2 , C (O) NR 7 , C (S) NR 7 , O, S, SO, SO 2 , SO 2 NR 7 , NR 7 , NR 7 CO, NR 7 CONR 8 , NR 7 C (S) , optionally substituted C 1 -C 8 alkyl, optionally substituted C 1 -C 8 alkoxy, optionally substituted C 1 -C 8 alkoxyC 1 -C 8 alkyl, optionally substituted C 1 -C 8 haloalkyl, optionally substituted C 1 -C 8 hydroxyalkyl, optionally substituted C 2 -C 8 alkenyl, optionally substituted C 2 -C 8 alkynyl, optionally substituted C 3 -C 8 cycloalkyl, optionally substituted C 3 -C 8 cycloalkoxy, optionally substituted 3-8 membered heterocyclyl, optionally substituted aryl,
  • R 8 and R 8 are independently selected from hydrogen, optionally substituted C 1 -C 6 alkyl, optionally substituted C 3 -C 6 cycloalkyl, optionally substituted C 3 -C 6 cycloalkoxy, optionally substituted 3-6 membered heterocyclyl, optionally substituted C 1 -C 6 alkoxy, optionally substituted C 1 -C 6 alkoxyalkyl, optionally substituted C 1 -C 6 haloalkyl, optionally substituted C 1 -C 6 hydroxyalkyl, optionally substituted C 1 -C 6 alkylamino, and optionally substituted C 1 -C 6 alkylaminoC 1 -C 6 alkyl;
  • each m is 0 to 15;
  • each n is 0 to 15;
  • o 0 to 15;
  • p 0 to 15.
  • the linker moiety comprises a 3 to 13 membered ring, a 4 to 13 membered fused ring, a 5 to 13 membered bridged ring, and a 5 to13 membered spiro ring. In some embodiment, the linker moiety comprises a ring selected from the group consisting of Formula C1, C2, C3, C4 and C5:
  • the linker moiety is of Formula (IV) :
  • Z is selected from the group consisting of a 3-to 8-membered ring, a 5-to 12-membered bicyclic ring, an 8-to 15-membered tricyclic ring and a 6-to 12-membered spiro bicyclic ring, each independently having 0-4 heteroatoms;
  • RL1 is selected from the group consisting of H, an unsubstituted C1-6 alkyl group, a C1-6 alkyl group substituted by a C1-6 alkoxyl group or one or more halo groups, halogen, a C1-6 alkoxy group, a keto group, or an oxide group; wherein, two RL1 groups are optionally taken together to form a 3-8 membered ring system containing 0-2 heteroatoms;
  • RL2 is a bond or an ethynylene group
  • X1 is selected from the group consisting of a methylene group and an ethylene group
  • X3 is selected from the group consisting of an unsubstituted C1-8 alkylene group, a C1-8 alkylene group substituted by 1 to 6 RL1, an unsubstituted C1-8 heteroalkylene group, a C1-8 heteroalkylene group substituted by 1 to 6 RL1, an unsubstituted 3-to 8-membered arylene group, a 3-to 8-membered arylene group substituted by 1 to 6 RL1, an unsubstituted 3-to 8-membered heteroarylene group with 1 to 3 hetero atoms, and a 3-to 8-membered heteroarylene group with 1 to 3 hetero atoms and substituted by 1 to 6 RL1, an unsubstituted 3-to 7-membered cyclic alkylene group, a 3-to 7-membered cyclic alkylene group substituted by 1 to 6 RL1, an unsubstituted 3-to 7-membered heterocyclic alkylene group with 1 to 2 hetero atom
  • RL3 is hydrogen
  • RL4 and RL5 are independently selected from the group consisting of H, an unsubstituted C1-6 alkyl group, and a C1-6 alkyl group substituted by a C1-6 alkoxyl group or one or more halo groups;
  • n1 0, 1, 2, 3, 4, 5 or 6;
  • n2 is 0 or 1
  • n3 is 0 or 1;
  • n4 1;
  • m5 is 0 or 1.
  • heteroatoms in Formula (IV) are each independently selected N, O and S.
  • the Z ring comprises one or two heteroatoms selected from N, O and S.
  • Z is selected from the group consisting of a 3-to 8-membered ring, a 6-to 10-membered bicyclic ring and a 8-to 10-membered spiro bicyclic ring, each independently having 1-2 heteroatoms.
  • RL1 is selected from the group consisting of H, an unsubstituted linear C1-3 alkyl group, a keto group, or an oxide group;
  • RL2 is a bond or an ethynylene group.
  • X1 is selected from the group consisting of a methylene group and an ethylene.
  • X3 is selected from the group consisting of an unsubstituted C1-6 alkylene group, an unsubstituted 3-to 7-membered cyclic alkylene group, and an unsubstituted 3-to 7-heterocyclic alkylene group with 0 to 2 hetero atoms; or, X3 is a 10-to 12-membered spiro bicyclic ring having 0-2 heteroatoms.
  • X4 is selected from the group consisting of a methylene group, CONRL3 and O.
  • m1 is 0, 1, 2 or 3.
  • RL1 is selected from the group consisting of H, an unsubstituted C1-3 alkyl group, a C1-3 alkyl group substituted by a C1-3 alkoxyl group or one or more halo groups, halogen, a C1-3 alkoxy group, a keto group, or an oxide group; wherein, two RL1 groups are optionally taken together to form a 3-8 membered ring system containing 0-2 heteroatoms.
  • RL1 is an oxide group which is attached to one heteroatom N on the Z ring to form an N-oxide group (N + –O - ) .
  • RL1 is an alkyl selected from the group consisting of a linear C1-6 alkyl and a branched C1-6 alkyl, each of which is unsubstituted or substituted by a C1-6 alkoxyl group or one or more halo groups. In some embodiment, RL1 is an alkyl selected from the group consisting of a linear C1-3 alkyl and a branched C1-3 alkyl, each of which is unsubstituted or substituted by a C1-6 alkoxyl group or one or more halo groups.
  • X3 is selected from the group consisting of a bond, an unsubstituted C1-6 alkylene group, a C1-6 alkylene group substituted by 1 to 6 RL1, an unsubstituted C1-6 heteroalkylene group, a C1-6 heteroalkylene group substituted by 1 to 6 RL1. In some embodiment, X3 is selected from the group consisting of an unsubstituted C1-5 alkylene group.
  • RL4 and RL5 are independently an alkyl selected from the group consisting of a linear C1-6 alkyl and a branched C1-6 alkyl, each of which is unsubstituted or substituted by a C1-6 alkoxyl group or one or more halo groups. In some embodiment, RL4 and RL5 are independently an alkyl selected from the group consisting of a linear C1-3 alkyl and a branched C1-3 alkyl, each of which is unsubstituted or substituted by a C1-6 alkoxyl group or one or more halo groups.
  • linker moiety is selected from the group consisting of:
  • f is an integer of 0, 1, 2, 3 or 4; and g is an integer of 0, 1, 2 or 3.
  • the binding affinity of novel synthesized bivalent compounds can be assessed using standard biophysical assays known in the art (e.g., isothermal titration calorimetry (ITC) , surface plasmon resonance (SPR) ) . Cellular assays can then be used to assess the bivalent compound’s ability to induce target protein degradation. Besides evaluating a bivalent compound’s induced changes in the protein levels POI, POI mutants, or POI fusion proteins, enzymatic activity can also be assessed.
  • ITC isothermal titration calorimetry
  • SPR surface plasmon resonance
  • Assays suitable for use in any or all of these steps are known in the art, and include, e.g., western blotting, quantitative mass spectrometry (MS) analysis, flow cytometry, enzymatic activity assay, ITC, SPR, cell growth inhibition, xenograft, orthotopic, and patient-derived xenograft models.
  • Suitable mouse models for use in any or all of these steps are known in the art and include subcutaneous xenograft models, orthotopic models, patient-derived xenograft models, and patient-derived orthotopic models.
  • isotopic variations of the compounds disclosed herein are contemplated and can be synthesized using conventional methods known in the art or methods corresponding to those described in the Examples (substituting appropriate reagents with appropriate isotopic variations of those reagents) .
  • an isotopic variation is a compound in which at least one atom is replaced by an atom having the same atomic number, but an atomic mass different from the atomic mass usually found in nature.
  • Useful isotopes are known in the art and include, for example, isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine. Exemplary isotopes thus include, e.g., 2 H, 3 H, 13 C, 14 C, 15 N, 17 O, 18 O, 32 P, 35 S, 18 F, and 36 Cl.
  • Isotopic variations e.g., isotopic variations containing 2 H
  • certain isotopic variations can be used in drug or substrate tissue distribution studies.
  • the radioactive isotopes tritium ( 3 H) and carbon-14 ( 14 C) are particularly useful for this purpose in view of their ease of incorporation and ready means of detection.
  • solvates of the compounds disclosed herein are contemplated.
  • a solvate can be generated, e.g., by substituting a solvent used to crystallize a compound disclosed herein with an isotopic variation (e.g., D 2 O in place of H 2 O, d 6 -acetone in place of acetone, or d 6 -DMSO in place of DMSO) .
  • an isotopic variation e.g., D 2 O in place of H 2 O, d 6 -acetone in place of acetone, or d 6 -DMSO in place of DMSO
  • a fluorinated variation is a compound in which at least one hydrogen atom is replaced by a fluoro atom. Fluorinated variations can provide therapeutic advantages resulting from greater metabolic stability, e.g., increased in vivo half-life or reduced dosage requirements.
  • prodrugs of the compounds disclosed herein are contemplated and can be synthesized using conventional methods known in the art or methods corresponding to those described in the Examples (e.g., converting hydroxyl groups or carboxylic acid groups to ester groups) .
  • a prodrug refers to a compound that can be converted via some chemical or physiological process (e.g., enzymatic processes and metabolic hydrolysis) to a therapeutic agent.
  • prodrug also refers to a precursor of a biologically active compound that is pharmaceutically acceptable.
  • a prodrug may be inactive when administered to a subject, i.e.
  • prodrug compound often offers advantages of solubility, tissue compatibility or delayed release in an organism.
  • prodrug is also meant to include any covalently bonded carriers, which release the active compound in vivo when such prodrug is administered to a subject.
  • Prodrugs of an active compound may be prepared by modifying functional groups present in the active compound in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to the parent active compound.
  • Prodrugs include compounds wherein a hydroxy, amino or mercapto group is bonded to any group that, when the prodrug of the active compound is administered to a subject, cleaves to form a free hydroxy, free amino or free mercapto group, respectively.
  • Examples of prodrugs include, but are not limited to, acetate, formate and benzoate derivatives of an alcohol or acetamide, formamide and benzamide derivatives of an amine functional group in the active compound and the like.
  • PROTAC degraders work by involving more complicated three-body binding equilibria.
  • characterizing protein–protein interaction (PPI) characterizing protein–protein interaction (PPI) , ternary complex formation, ternary complex stability and cooperativity are also important in elucidating the SAR.
  • the SAR obtained from cell-free biochemical assays can provide valuable feedback to rational lead optimization.
  • ternary complex formation, ternary complex stability and cooperativity of PPI are more predictive of a PROTAC’s degradation activity than its binary interactions.
  • a ternary complex can be characterized by several different assays. Common biochemical and biophysical assays used to profile ternary complex formation, population, stability, binding affinities, cooperativity or kinetics are discussed below:
  • ⁇ is the rotation relaxation time, defined as the time taken by the fluorescent molecule to rotate 68.5°; V is molecular volume including hydration; ⁇ represents viscosity; R and T are the gas constant and thermodynamic temperatures, respectively.
  • Interaction of a large molecule (protein) with a fluorescent ligand (tracer) can change the effective molecular volume of the fluorescent ligand, and thus alter its rotational relaxation time and eventually its polarization that can be detected through plane-polarized light ( Figure 5) .
  • the binding affinity between a fluorescent ligand and a protein can be monitored using plane-polarized light in a nondestructive and separation-free manner.
  • the FP assay is an economical and homogeneous assay that can provide rapid screening for a large number of compounds. As such, the FP assay has been extensively used in high-throughput screening (HTS) programs in drug discovery. In the PROTAC field, the FP assay provides a powerful tool to determine binary binding affinities, ternary binding affinities and cooperativities. The FP assay is an accessible assays that can be easily set up with various protocols available. The fluorescent probe is the key for the FP assay, which can be synthesized by a wide range of commercial services and academic core facilities or can be prepared in-house by attaching a fluorophore to a ligand.
  • Fluorescent probes are preferred to be as small as possible to maximize the molecular weight difference between a fluorescent probe and its binding protein.
  • the binding affinity of a fluorescent probe to its target protein is also important, because the higher the binding affinity of a fluorescent probe, the wider the range of inhibitor potencies that can be resolved. However, the affinity of a fluorescent probe will be considered too high if the instrument cannot detect the fluorescent signal when it is lower than twice the Kd.
  • the application of the FP assay for characterizing the binary binding affinity of PROTACs to their target protein or E3 ligase is the same as that of a small-molecule ligand.
  • the FP assay requires saturating the PROTAC with one binding protein first, then titrating into the other protein as a binary complex. As such, a large amount of the proteins are required, making it infeasible for HTS.
  • Amplified luminescent proximity homogeneous assay (ALPHA) technology is a bead-based proximity assay that can be used to study interactions between molecules in a microplate format.
  • A one binding partner
  • B the other binding partner
  • the donor beads are coated with photosensitizer that can convert ambient O2 to singlet O2 (1O2) once illuminated at 680 nm.
  • the singlet O2 has a half-life of 4 ⁇ s, allowing it to travel about 200 nm in the solution.
  • acceptor beads are within this distance, thioxene derivatives coated on the acceptor beads will accept the energy from singlet O2 and emit light at 520–620 nm (ALPHAScreen) or at 615 nm (ALPHALISA) .
  • APHAScreen 520–620 nm
  • APHALISA 615 nm
  • the chance that acceptor beads are in proximity to donor beads would be increased significantly when binding partners A and B interact, and the tighter the binding, the higher the luminescence signal produced by the acceptor beads.
  • the interaction between A and B can be quantified by detecting the luminescence signal.
  • a competitor that competes with A in binding to B would decrease the luminescence signal in a concentration-dependent manner, providing a competitive assay for the screening of inhibitors.
  • ALPHA has made ALPHA very practical and easy by commercializing numerous ALPHA reagents. Meanwhile, the high signal-to-background ratio, high dynamic range, high sensitivity and wash-free procedure associated with the ALPHA makes this technology suitable for HTS application, allowing for the discovery of hits from a big library of compounds.
  • the ALPHA has also been utilized to quantify the concentrations of proteins, to capture PPI, and to characterize ternary complexes formed between a target protein, a PROTAC degrader and an E3 ligase. Detecting ternary complex formation is one of the most ueful applications of the ALPHA for PROTAC degraders.
  • a bell-shaped curve By titrating a PROTAC degrader to its target protein and E3 ligase, a bell-shaped curve can be produced when plotting ALPHA signals against the concentrations of the PROTAC.
  • the height of the bell-shaped curve reflects the relative population of the ternary complex, allowing scientists to rank PROTAC degraders according to their ability to form ternary complexes.
  • the time-resolved fluorescence energy transfer (TR-FRET) assay is also a proximity-based technology. It combines time-resolved fluorimetry with resonance energy transfer (FRET) , resulting in an assay that allows for very sensitive detection of binding/dissociation events in a homogeneous format. While the ALPHA uses a singlet oxygen transferred chemiluminescence signal to measure binding between two partners, the TR-FRET assay utilizes long-lived fluorophores combined with time-gated fluorescence intensity measurements to quantitate molecular association or dissociation events.
  • one binding partner is labeled with a donor fluorophore (usually a europium or terbium chelate/cryptate) ; the other binding partner is conjugated to a corresponding acceptor fluorophore.
  • a donor fluorophore usually a europium or terbium chelate/cryptate
  • Europium and terbium are both lanthanides that have a long fluorescence life-time, large Stokes shift and a narrow emission peak. With these properties, europium and terbium can emit the fluorescent signals after the delay time when background fluorescence has decayed.
  • the acceptor fluorophores used in the TR-FRET assay are chosen to have excitation wavelengths that are overlapped with the emission wavelengths of their paired donor fluorophores, allowing the energy transfer from donor fluorophores to acceptor fluorophores when they are in proximity.
  • the TR-FRET signal can be determined through a ratiometric or delta method, which is used to quantify the binding events of two binding partners that are conjugated to donor and acceptor fluorophores, respectively.
  • this assay can be applied to perform HTS on small-molecule libraries, to quantify protein concentrations, to screen PPI targeting small molecules, as well as to characterize the ternary complex for PROTACs. Similar to the ALPHA, the ternary complex formation assay performed with TR-FRET also results in a bell-shaped curve, whose peak height reflects the relative population of the ternary complex. Compared with the ALPHA that captures binding partner to beads, the TR-FRET assay labels binding partner with fluorescent molecules, which are much smaller than beads, to provide more entropic freedom.
  • ITC Isothermal titration calorimetry
  • ITC is superior to any other biophysical techniques in that it can determine all binding parameters including stoichiometry (n) , Kd. ⁇ H and ⁇ S in a single experiment.
  • ITC data can provide deeper insights into the mechanisms of binding, the binding driving force and the structure–function relationships [64] .
  • ITC is also a true label-free biophysical method that allows the binding affinities to be determined without the need to tag or immobilize the binding partner, which is advantageous as tagging or immobilizing an analyte is sometimes technically difficult and may even interfere with the binding.
  • ITC has been increasingly used in determining protein–protein, protein–nucleic acid and protein–small molecule interactions.
  • ITC has also been applied in profiling the thermodynamic parameters and cooperativities of PROTAC molecules, providing invaluable insights into the interplay between a PROTAC molecule and its target protein and E3 ligase.
  • ITC-200 from Malvern Instrument (previously MicroCal, Malvern, UK) and Nano-ITC from TA Instrument (DE, USA) .
  • Both SPR and BLI are biophysical techniques that detect binding events through a spectroscopic method; the resulting sensorgrams show the kinetics of binding in real-time.
  • the SPR assay is based on the changes in the refractive index of the medium directly in contact with the sensor chip surface, whereas BLI detects the changes in the interference pattern on the biosensor tip surface.
  • Both SPR and BLI are label-free biophysical methods for determining interactions between ligands and targets, although they both require immobilization of one binding partner on the sensor chips/tips.
  • the immobilization strategies for SPR and BLI are the same, but the availability of the corresponding modified sensor chips/tips is instrument dependent.
  • the two broad approaches for performing the immobilization are chemical coupling and capture. Chemical coupling strategy results in covalent bonds between the ligands and the sensor chips/tips surface, while the capture strategy takes advantage of the strong affinity between the chemistry of the sensor chips/tips and the tag of the ligand.
  • biosensor technology also finds its application in PROTACs, not only for binary binding determination but also for ternary complex characterization.
  • SPR is more advantageous than ITC and other assays, as it not only provides ternary binding affinity (Kd) but also kinetics of a ternary complex.
  • Nano-BRET is an optimized BRET technology using Nanoluc luciferase coupled with its substrate furimazine as the donor system [87] .
  • the optimum properties of the Nanoluc and furimazine combination such as high physical stability, high luminescence signal and small size, make Nano-BRET advantageous over previous BRET technologies and the combination of Nanoluc with various fluorescent protein acceptors greatly expands the application of BRET.
  • the recent introduction of the Halo-Tag system as an alternative to fluorescent proteins offers an opportunity for multiplexing as fluorophores can be chosen as needed as long as chloroalkane–fluorophore conjugates are available [91] .
  • one binding partner e.g., protein A
  • the other binding partner e.g., protein B
  • Halo-Tag The substrate of Nanoluc (furimazine) and ligand of Halo-Tag (chloroalkane–fluorophore conjugate) are added separately to the cells.
  • furimazine can be converted to furimamide by luciferase in the presence of oxygen and emit a luminescence signal at around 460 nm
  • the chloroalkane–fluorophore conjugate attaches to Halo-Tag covalently and can be excited at 460 nm and emit light at around 618 nm.
  • Nano-BRET technology can be applied to almost every step along the degradation pathway of PROTAC within live cells: from PROTAC target engagement, ternary complex formation and target ubiquitination to target degradation and target protein level detection. Nano-BRET can also be used to explain the mechanism of action of PROTAC molecules.
  • Nano-BRET is a relatively new technology developed and commercialized by Promega (WI, USA) . It offers several advantages over other methods in profiling PROTAC molecules: Nano-BRET assay is carried out within live cells, and thus the results are based on physiological conditions; Nano-BRET uses endogenous protein in live cells, thus avoiding protein expression and purification process, which is favorable for target proteins that are hard to obtain or are in a large protein complex; the Nano-BRET assay can kinetically monitor target engagement and ternary complex formation, as well as target ubiquitination and degradation in real-time, providing more insights into the mechanism of action of PROTAC molecules.
  • Assays other than the aforementioned that have also been used to characterize ternary complexes include size exclusive chromatography (SEC) , crystallography, co-immunoprecipitation (Co-IP) , mass spectrometry (MS) and the assay.
  • SEC size exclusive chromatography
  • Co-IP co-immunoprecipitation
  • MS mass spectrometry
  • the SEC ternary complex formation assay is based on the size difference between the binary complex and the ternary complex. By detecting the elution volume and elution time of proteins following PROTAC treatment, one can compare the relative ternary complex formation among different PROTAC molecules.
  • Crystallography is a challenging yet very useful biophysical technique.
  • the ternary complex crystal structures of PROTAC degraders can provide valuable insights into how they bind to their target protein and E3 ligase, which can provide better guidance on the rational design and optimization of new degraders.
  • Co-IP can be used to detect PPI in live cells by using target protein-specific antibodies to indirectly capture proteins that are bound to a specific target protein.
  • the enhanced capture in the presence of a PROTAC is an indication of ternary complex formation.
  • the advantage of co-IP over other ternary complex formation assays is that it is most related to physiological conditions.
  • MS is another label-free technique that can provide insights into PROTAC degrader-mediated PPI.
  • native MS can preferentially reveal the E3–PROTAC–POI ternary complex in competition experiments with multiple substrate proteins present, thereby suggesting that it is not only an ideal HTS strategy for the development of new PROTACs but also a valuable tool to dissect the mechanism of actions, selectivity and specificity of PROTACs.
  • NanoBiT is a protein-fragment complementation assay that can detect PPI and ternary complex formation in live cells.
  • one binding partner is fused to a LgBiT (18 kDa) subunit, while the other binding partner is fused to a small BiT (SmBiT; 11 amino acid peptide) .
  • SmBiT is a complementary peptide of LgBiT that is designed to have a very low binding affinity to LgBiT.
  • SmBiT and LgBiT interact, they form an active enzyme that can generate a bright luminescent signal in the presence of a substrate. Interaction between LgBiT-fused and SmBiT-fused binding partners brings these two complementary proteins together, resulting in a luminescent signal as a measurement of PPI between binding partner proteins.
  • the thermal shift assay also known as differential scanning calorimetry (DSF) , studies thermal stabilization of proteins upon ligand binding. Given its capability to screen ligands that occupy the nonactive sites of proteins, DSF assay could be the potential assay to offer nonfunctional binding ligands for PROTAC design.
  • TSA thermal shift assay
  • CETSA cellular TSA
  • the CETSA involves treatment of cells with a PROTAC of interest, heating to denature and precipitate proteins, cell lysis and the separation of cell debris and aggregates from the soluble protein fraction.
  • Poly-ubiquitination is often the defining step in triggering target protein degradation.
  • protein ubiquitination involves concerted actions of E1, E2 and E3 enzymes to attach ubiquitins to lysine residues of a target protein and subsequent ubiquitin chain elongation.
  • the ubiquitinated proteins can be recognized, recruited and degraded by the 26S proteasome, a very large multicatalytic protease complex that breaks down ubiquitinated proteins to small peptides.
  • Ubiquitination assays can be done both in a cell-free system and in live cells using either electrophoresis-based or nonelectrophoresis-based methods.
  • Substrate protein ubiquitination can also be detected in vitro with nonelectrophoresis-based methods such as FP, ALPHA and TR-FRET assays. Nevertheless, all these in vitro ubiquitination assays require additives such as ATP and E1, E2 and E3 enzymes, which is quite complicated in setting up the assay conditions and are not physiologically related.
  • Ubiquitination assays carried out in live cells allows for ubiquitination detection under native conditions by taking advantage of their own ubiquitination machinery.
  • Immunoprecipitation of substrate proteins followed by ubiquitin immunoblotting is the simplest and one of the most commonly used methods to detect protein ubiquitination and it is likely to capture the ubiquitinated target protein specifically.
  • the tagged versions of a target protein and ubiquitin can be expressed in cells to facilitate the detection.
  • NanoBRET technology was applied to monitor intracellular BET protein ubiquitination induced by PROTAC degraders.
  • HiBiT-BET protein, its complementary protein LgBiT, and the luminescence substrate furimazine were used as an energy donor system, while the Halo-Tag fused ubiquitin and haloalkane fluorophore were employed as the respective energy acceptor system.
  • a PROTAC degrader that can induce the ubiquitination of BET protein can bring HiBiT-BET protein in proximity to HaloTag-fused ubiquitin.
  • LgBiT, furimazine and haloalkane fluorophore an energy transfer from donor to acceptor can be expected, resulting in a fluorescence signal that reflects the intensity of ubiquitination.
  • MS is a powerful technology that can not only be used in detecting total protein ubiquitination but also allows identification of ubiquitination sites and ubiquitination types.
  • nanopore makes real-time detection of protein ubiquitination possible, yet it has not been applied to PROTAC degraders.
  • the nanopore is capable of monitoring the E1–E2–E3 ubiquitination cascade kinetically.
  • the TUBE-ALPHALISA and the TUBE-DELFIA are also two of the assays that can potentially be applied for quantifying ubiquitinated proteins in cell lysates following PROTAC treatment. The ease of operation and plate-based format of these two assays make high-throughput ubiquitination screening possible.
  • Protein degradation also called proteolysis, is a process that results in the hydrolysis of one or more peptide bonds in a protein.
  • the ubiquitin-proteasome pathway (UPP) and autophagy are two main pathways and machineries that mediate degradation of intracellular proteins, while extracellular proteins and some cell surface proteins are taken up by endocytosis and are degraded within lysosomes.
  • Protein degradation induced by PROTAC degraders is anticipated through UPP. Given the unpredictable nature of PROTAC degraders in inducing target protein degradation, it is of great importance to introduce reliable degradation assays to ultimately evaluate if the designed PROTAC is a target protein degrader.
  • Methods that have been used to detect intracellular protein levels post PROTAC treatment include western blot, capillary-based immunoassay, fluorescence or luminescence-based reporter assays and MS based-proteomics.
  • Western blot is the most frequently used method to measure the relative target protein levels in cells.
  • the western blot assay relies on specific and high-quality antibodies for protein detection and is not suitable to accurately quantify the protein levels.
  • the low sensitivity and multistep procedures of western blot could bring artifacts to the results.
  • a capillary electrophoresis immunoassay is simpler than western blot with less sample consumption, simpler procedures and a shorter analysis time. It has been applied to determine the levels of BTK and pirin protein after cells treated with respective PROTACs.
  • this method also depends on the specific interaction between an antibody and an antigen for protein detection, presenting as a semiquantitative technique for protein detection.
  • ELISA and ALPHA Other antibody-based methods that have been developed to quantitate protein levels include ELISA and ALPHA. Both ELISA and ALPHA provide highly sensitive protein level detection with the ALPHA being wash-free, having a larger dynamic range and allowing protein detection homogeneously. However, neither the ELISA nor the ALPHA have been applied to PROTAC degraders for protein degradation.
  • Fluorescence-or luminescence-based reporter assays represent a rapid and sensitive method to measure protein degradation in situ.
  • the development of a luciferase-based HiBiT tagging system offered another reporter assay to monitor protein degradation.
  • a target protein is fused with HiBiT using CRISPR/Cas9 editing in cell lines stably expressing LgBiT which complements with HiBiT to form the luminescent NanoBiT luciferase.
  • Post PROTAC treatment degradation of a target protein is reflected in the loss of luminescence signal.
  • One challenge in these fluorescence-and luminescence-based assays is background interference, which can potentially decrease the dynamic range and sensitivity of these assays.
  • these reporter assays can offer robust phenotype-based HTS methods for PROTACs.
  • MS analysis is a method that offers sensitive protein detection and quantification without relying on antibodies or tags.
  • Various proteomic approaches are available to understand the mechanism of action of PROTAC degraders.
  • the quantitative approach uses synthetic stable isotope-labeled proteins which can precisely mimic their endogenous counterparts as the internal standards to quantify the corresponding target protein.
  • a global proteomic analysis study is widely used to examine the abundance change of proteins post treatment of with PROTAC degraders, to validate the degradation selectivity of the PROTAC degrader, and to reveal any off-target effects. The activities of these off-targets are probably responsible for the observed molecular and phenotypic responses.
  • MS makes intact protein detection possible.
  • With its capability to investigate the causes and consequences of protein degradation in biological systems we anticipate its increasing application to the study of PROTAC degraders.
  • compositions and methods described herein include the manufacture and use of pharmaceutical compositions and medicaments that include one or more bivalent compounds as disclosed herein. Also included are the pharmaceutical compositions themselves.
  • the pH of the compositions disclosed herein can be adjusted with pharmaceutically acceptable acids, bases, or buffers to enhance the stability of the bivalent compound or its delivery form.
  • compositions typically include a pharmaceutically acceptable excipient, adjuvant, or vehicle.
  • pharmaceutically acceptable refers to molecular entities and compositions that are generally believed to be physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human.
  • a pharmaceutically acceptable excipient, adjuvant, or vehicle is a substance that can be administered to a patient, together with a compound of the invention, and which does not compromise the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the compound.
  • Exemplary conventional nontoxic pharmaceutically acceptable excipients, adjuvants, and vehicles include, but not limited to, saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • pharmaceutically acceptable excipients, adjuvants, and vehicles that can be used in the pharmaceutical compositions of this invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery systems (SEDDS) such as d- ⁇ -tocopherol polyethylene glycol 1000 succinate, surfactants used in pharmaceutical dosage forms such as Tweens or other similar polymeric delivery matrices, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxe
  • compositions may be used.
  • pharmaceutically acceptable excipients, adjuvants, and vehicles include lactose and corn starch.
  • Lubricating agents such as magnesium stearate, are also typically added.
  • useful diluents include lactose and dried corn starch.
  • the active ingredient may be suspended or dissolved in an oily phase is combined with emulsifying or suspending agents. If desired, certain sweetening, flavoring, or coloring agents can be added.
  • the bivalent compounds disclosed herein are defined to include pharmaceutically acceptable derivatives or prodrugs thereof.
  • a “pharmaceutically acceptable derivative” means any pharmaceutically acceptable salt, solvate, or prodrug, e.g., carbamate, ester, phosphate ester, salt of an ester, or other derivative of a compound or agent disclosed herein, which upon administration to a recipient is capable of providing (directly or indirectly) a compound described herein, or an active metabolite or residue thereof.
  • Particularly favored derivatives and prodrugs are those that increase the bioavailability of the compounds disclosed herein when such compounds are administered to a subject (e.g., by allowing an orally administered compound to be more readily absorbed into the blood) or which enhance delivery of the parent compound to a biological compartment (e.g., the brain or lymphatic system) relative to the parent species.
  • Preferred prodrugs include derivatives where a group that enhances aqueous solubility or active transport through the gut membrane is appended to the structure of formulae described herein. Such derivatives are recognizable to those skilled in the art without undue experimentation. Nevertheless, reference is made to the teaching of Burger’s Medicinal Chemistry and Drug Discovery, 5 th Edition, Vol. 1: Principles and Practice, which is incorporated herein by reference to the extent of teaching such derivatives.
  • the bivalent compounds disclosed herein include pure enantiomers, mixtures of enantiomers, pure diastereoisomers, mixtures of diastereoisomers, diastereoisomeric racemates, mixtures of diastereoisomeric racemates and the meso-form and pharmaceutically acceptable salts, solvent complexes, morphological forms, or deuterated derivatives thereof.
  • the pharmaceutical compositions disclosed herein can include an effective amount of one or more bivalent compounds.
  • effective amount and “effective to treat, ” as used herein, refer to an amount or a concentration of one or more compounds or a pharmaceutical composition described herein utilized for a period of time (including acute or chronic administration and periodic or continuous administration) that is effective within the context of its administration for causing an intended effect or physiological outcome (e.g., treatment or prevention of cell growth, cell proliferation, or cancer) .
  • compositions can further include one or more additional compounds, drugs, or agents used for the treatment of cancer (e.g., conventional chemotherapeutic agents) in amounts effective for causing an intended effect or physiological outcome (e.g., treatment or prevention of cell growth, cell proliferation, or cancer) .
  • additional compounds, drugs, or agents used for the treatment of cancer e.g., conventional chemotherapeutic agents
  • an intended effect or physiological outcome e.g., treatment or prevention of cell growth, cell proliferation, or cancer
  • compositions disclosed herein can be formulated or adapted for administration to a subject via any route, e.g., any route approved by the Food and Drug Administration (FDA) .
  • FDA Food and Drug Administration
  • Exemplary methods are described in the FDA Data Standards Manual (DSM) (available at http: //www. fda. gov/Drugs/DevelopmentApprovalProcess/FormsSubmissionRequirements/ElectronicSubmissions/DataStandardsManualmonographs) .
  • DSM Data Standards Manual
  • the pharmaceutical compositions can be formulated for and administered via oral, parenteral, or transdermal delivery.
  • parenteral includes subcutaneous, intracutaneous, intravenous, intramuscular, intraperitoneal, intra-articular, intra-arterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques.
  • compositions of this invention can be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, emulsions and aqueous suspensions, dispersions and solutions.
  • compositions of this invention can be administered by injection (e.g., as a solution or powder) .
  • Such compositions can be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as, for example, Tween 80) and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, e.g., as a solution in 1, 3-butanediol.
  • suitable vehicles and solvents that may be employed are mannitol, water, Ringer’s solution, and isotonic sodium chloride solution.
  • a bivalent compound described herein for treating or preventing one or more diseases or conditions disclosed herein in a subject in need thereof.
  • a bivalent compound in manufacture of a medicament for preventing or treating one or more diseases or conditions disclosed herein are provided herein.
  • the methods disclosed include the administration of a therapeutically effective amount of one or more of the compounds or compositions described herein to a subject (e.g., a mammalian subject, e.g., a human subject) .
  • the methods disclosed include selecting a subject and administering to the subject an effective amount of one or more of the compounds or compositions described herein, and optionally repeating administration as required for the prevention or treatment of a disease or condition such as cancer.
  • subject selection can include obtaining a sample from a subject (e.g., a candidate subject) and testing the sample for an indication that the subject is suitable for selection.
  • the subject can be confirmed or identified, e.g. by a health care professional, as having had, having an elevated risk to have, or having a condition or disease.
  • suitable subjects include, for example, subjects who have or had a condition or disease but that resolved the disease or an aspect thereof, present reduced symptoms of disease (e.g., relative to other subjects (e.g., the majority of subjects) with the same condition or disease) , or that survive for extended periods of time with the condition or disease (e.g., relative to other subjects (e.g., the majority of subjects) with the same condition or disease) , e.g., in an asymptomatic state (e.g., relative to other subjects (e.g., the majority of subjects) with the same condition or disease) .
  • exhibition of a positive immune response towards a condition or disease can be made from patient records, family history, or detecting an indication of a positive immune response.
  • multiple parties can be included in subject selection.
  • a first party can obtain a sample from a candidate subject and a second party can test the sample.
  • subjects can be selected or referred by a medical practitioner (e.g., a general practitioner) .
  • subject selection can include obtaining a sample from a selected subject and storing the sample or using the in the methods disclosed herein. Samples can include, e.g., cells or populations of cells.
  • methods of treatment can include a single administration, multiple administrations, and repeating administration of one or more compounds disclosed herein as required for the prevention or treatment of the disease or condition disclosed herein (e.g., a POI-associated disease) .
  • methods of treatment can include assessing a level of disease in the subject prior to treatment, during treatment, or after treatment. In some aspects, treatment can continue until a decrease in the level of disease in the subject is detected.
  • subject refers to any animal. In some instances, the subject is a mammal. In some instances, the term “subject, ” as used herein, refers to a human (e.g., a man, a woman, or a child) .
  • administer refers to implanting, ingesting, injecting, inhaling, or otherwise absorbing a compound or composition, regardless of form.
  • methods disclosed herein include administration of an effective amount of a compound or composition to achieve the desired or stated effect.
  • treat refers to partially or completely alleviating, inhibiting, ameliorating, or relieving the disease or condition from which the subject is suffering. This means any manner in which one or more of the symptoms of a disease or disorder (e.g., cancer) are ameliorated or otherwise beneficially altered.
  • amelioration of the symptoms of a particular disorder refers to any lessening, whether permanent or temporary, lasting or transient that can be attributed to or associated with treatment by the bivalent compounds, compositions and methods of the present invention.
  • prevent, ” and “prevention, ” as used herein, shall refer to a decrease in the occurrence of a disease or decrease in the risk of acquiring a disease or its associated symptoms in a subject.
  • the prevention may be complete, e.g., the total absence of disease or pathological cells in a subject.
  • the prevention may also be partial, such that the occurrence of the disease or pathological cells in a subject is less than, occurs later than, or develops more slowly than that which would have occurred without the present invention.
  • Specific dosage and treatment regimens for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health status, sex, diet, time of administration, rate of excretion, drug combination, the severity and course of the disease, condition or symptoms, the patient’s disposition to the disease, condition or symptoms, and the judgment of the treating physician.
  • An effective amount can be administered in one or more administrations, applications or dosages.
  • a therapeutically effective amount of a therapeutic compound depends on the therapeutic compounds selected.
  • treatment of a subject with a therapeutically effective amount of the compounds or compositions described herein can include a single treatment or a series of treatments.
  • effective amounts can be administered at least once.
  • the compositions can be administered from one or more times per day to one or more times per week; including once every other day. The skilled artisan will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health or age of the subject, and other diseases present.
  • the subject can be evaluated to detect, assess, or determine their level of disease.
  • treatment can continue until a change (e.g., reduction) in the level of disease in the subject is detected.
  • a maintenance dose of a compound, or composition disclosed herein can be administered, if necessary.
  • the dosage or frequency of administration, or both can be reduced, e.g., as a function of the symptoms, to a level at which the improved condition is retained.
  • Patients may, however, require intermittent treatment on a long-term basis upon any recurrence of disease symptoms.
  • a bivalent compound comprising a target binding moiety (TBM) , and a cereblon E3 ubiquitin ligase binding moiety (CLM) represented by Formula (II) -1:
  • W1 and W2 are each independently selected from C, CRC2 and N;
  • G is selected from the group consisting of H, OH, CH2OH, RC3OCOORC4, RC3OCONRC4RC5, and 2- (trimethylsilyl) ethoxymethyl group;
  • Q1 to Q7 are each independently C, O, S, N, CRC2 or NRC2; at least one of W1, W2, Q1, Q2, Q3, Q4, Q5, Q6 and Q7 comprises a heteroatom;
  • K is selected from the group consisting of H, an unsubstituted alkyl group, an alkyl group substituted by RC2, an unsubstituted cycloalkyl group, and a cycloalkyl group substituted by RC2; K is bound to the 6-membered ring with a stereospecific bond or a non-stereospecific bond;
  • RC1 is selected from the group consisting of an unsubstituted alkyl group, an alkyl group substituted by RC2, an unsubstituted aryl group, an aryl group substituted by RC2, an unsubstituted alkyl-aryl group, an alkyl-aryl group substituted by RC2, an unsubstituted alkoxy group, and an alkoxy group substituted by RC2;
  • RC2 is selected from the group consisting of H, halo, CH2OH, CRC4, NRC4RC5, 2- (trimethylsilyl) ethoxymethyl, an alkoxyl group, an unsubstituted alkyl group, an alkyl group substituted by one or more halo groups, an unsubstituted cycloalkyl group, a cycloalkyl group substituted by one or more halo groups, an unsubstituted aryl group, an aryl group substituted by one or more halo groups, an unsubstituted heteroaryl group, a heteroaryl group substituted by one or more halo groups, an unsubstituted heterocyclyl group, and a heterocyclyl group substituted by one or more halo groups;
  • RC3 is selected from the group consisting of an unsubstituted alkylene group, and an alkylene group substituted by RC2;
  • RC4 and RC5 are independently selected from the group consisting of an unsubstituted alkyl group, an alkyl group substituted by RC2, an unsubstituted cycloalkyl group, a cycloalkyl group substituted by RC2, an unsubstituted heterocyclyl group, a heterocyclyl group substituted by RC2, an unsubstituted aryl group, an aryl group substituted by RC2, an unsubstituted heteroaryl group, and a heteroaryl group substituted by RC2; and n is 0, 1, 2, 3 or 4, or a pharmaceutically acceptable salt or analog thereof, wherein the TBM is not selected from the group consisting of:
  • A1 is selected from Cl, F, Br or CF3;
  • A2 is selected from O, NH, N-methyl or N-ethyl; and
  • A3, A4, A5 and A6 are each independently CH or N.
  • Z1 is selected from the group consisting of an aryl group, a heteroaryl group, a bicyclic group, and a bi-heterocyclic group, each independently substituted by one or more substituents selected from the group consisting of a halo group, a hydroxyl group, a nitro group, CN, C ⁇ CH, an unsubstituted C1-6 alkyl group, a C1-6 alkyl group substituted by a C1-6 alkoxyl group or one or more halo groups, an unsubstituted C1-6 alkoxyl group, a C1-6 alkoxyl group substituted by one or more halo groups, an unsubstituted C2-6 alkenyl, a C2-6 alkenyl substituted by one or more halo groups, an unsubstituted C2-6 alkynyl, and a C3-6 alkynyl substituted by one or more halo groups;
  • Y1, Y2, Y6 are each independently NRY1, O or S;
  • M is a 3-to 6-membered ring with 0 to 4 heteroatoms, which is unsubstituted or substituted by 1 to 6 RM groups;
  • each RM group is independently selected from the group consisting of H, an unsubstituted C1-6 alkyl group, a C1-6 alkyl group substituted by a C1-6 alkoxyl group or one or more halo groups, halogen, and a C1-6 alkoxy group; or two RM groups are taken together with the atom they are attached to and form a 3-to 8-membered ring system containing 0 to 2 heteroatoms;
  • Ra, Rb, Rc, Rd, RY1, RY2 are each independently selected from the group consisting of H, an unsubstituted C1-6 alkyl group, a C1-6 alkyl group substituted by a C1-6 alkoxyl group or one or more halo groups, halogen, a C1-6 alkoxy group, a cyclic group, and a heterocyclic group; or Ra, Rb are taken together with the atom they are attached to and form a 3-to 8-membered ring system containing 0 to 2 heteroatoms;
  • Z2 is selected from the group consisting of a bond, a C1-6 alkyl group, a C1-6 heteroalkyl group, O, an aryl group, a heteroaryl group, an alicyclic group, a heterocyclic group, a biheterocyclic group, a biaryl group, and a biheteroaryl group, each of which is unsubstituted or substituted by 1 to 10 RZ2 groups;
  • each R Z2 group is independently selected from the group consisting of H, halo, an unsubstituted C1-6 alkyl group, a C1-6 alkyl group substituted by one or more F, -ORZ2A, a C3-6 cycloalkyl group, a C4-6 cycloheteroalkyl group, an unsubstituted C1-6 alkyl group, a C1-6 alkyl group substituted by a C1-3 alkyl group or a C1-6 alkoxyl group or one or more halo groups, an unsubstituted heterocyclic group, a heterocyclic group substituted by a C1-3 alkyl group or a C1-6 alkoxyl group or one or more halo groups, an unsubstituted aryl group, an aryl group substituted by a C1-3 alkyl group or a C1-6 alkoxyl group or one or more halo groups, an unsubstituted heteroaryl group, aryl group substituted by a C1-3
  • R Z2A is selected from the group consisting of H, a C1-6 alkyl group, and a C1-6 heteroalkyl group, each of which is unsubstituted or substituted by a cycloalkyl group, a cycloheteroalkyl group, an aryl group, a heterocyclic group, a heteroaryl group, halo, or a OC1-3 alkyl group.
  • Q1 is O, S or NRC2;
  • Q3 to Q5 are each independently C or CRC2;
  • G is selected from the group consisting of H, OH, CH2OH, RC3OCOORC4 and 2- (trimethylsilyl) ethoxymethyl group;
  • RC2 is selected from the group consisting of H, CH2OH, CRC4, 2- (trimethylsilyl) ethoxymethyl, an unsubstituted C1-6 alkyl group, a C1-6 alkyl group substituted by one or more halo groups, a unsubstituted C1-6 cycloalkyl group, a C1-6 cycloalkyl group substituted by one or more halo groups;
  • RC3 is selected from the group consisting of a methylene group and a methylene group substituted by RC2;
  • RC4 is selected from the group consisting of an unsubstituted alkyl group, an alkyl group substituted by RC2, an unsubstituted cycloalkyl group, a cycloalkyl group substituted by RC2, an unsubstituted heterocyclyl group, a heterocyclyl group substituted by RC2.
  • A, W, and B, at each occurrence, are independently selected from null, CO, CO 2 , C (O) NR 1 , C (S) NR 1 , O, S, SO, SO 2 , SO 2 NR 1 , NR 1 , NR 1 CO, NR 1 CONR 2 , NR 1 C (S) , optionally substituted C 1 -C 8 alkyl, optionally substituted C 1 -C 8 alkoxy, optionally substituted C 1 -C 8 alkoxyC 1 -C 8 alkyl, optionally substituted C 1 -C 8 haloalkyl, optionally substituted C 1 -C 8 hydroxyalkyl, optionally substituted C 2 -C 8 alkenyl, optionally substituted C 2 -C 8 alkynyl, optionally substituted C 3 -C 8 cycloalkyl, optionally substituted C 3 -C 8 cycloalkoxy, optionally substituted 3-8 membered heterocyclyl, optionally substituted
  • R 1 and R 2 are independently selected from hydrogen, optionally substituted C 1 -C 6 alkyl, optionally substituted C 3 -C 6 cycloalkyl, optionally substituted C 3 -C 6 cycloalkoxy, optionally substituted 3-6 membered heterocyclyl, optionally substituted C 1 -C 6 alkoxy, optionally substituted C 1 -C 6 alkoxyalkyl, optionally substituted C 1 -C 6 haloalkyl, optionally substituted C 1 -C 6 hydroxyalkyl, optionally substituted C 1 -C 6 alkylamino, and optionally substituted C 1 -C 6 alkylaminoC 1 -C 6 alkyl; and
  • m 0 to 15.
  • R 1 , R 2 , R 3 , and R 4 are independently selected from hydrogen, halogen, CN, OH, NH 2 , optionally substituted C 1 -C 6 alkyl, optionally substituted C 3 -C 6 cycloalkyl, optionally substituted C 3 -C 6 cycloalkoxy, optionally substituted 3-6 membered heterocyclyl, optionally substituted C 1 -C 6 alkoxy, optionally substituted C 1 -C 6 alkoxyalkyl, optionally substituted C 1 -C 6 haloalkyl, optionally substituted C 1 -C 6 hydroxyalkyl, optionally substituted C 1 -C 6 alkylamino, and optionally substituted C 1 -C 6 alkylaminoC 1 -C 6 alkyl;
  • A, W, and B, at each occurrence, are independently selected from null, CO, CO 2 , C (O) NR 5 , C(S) NR 5 , O, S, SO, SO 2 , SO 2 NR 5 , NR 5 , NR 5 CO, NR 5 CONR 6 , NR 5 C (S) , optionally substituted C 1 -C 8 alkyl, optionally substituted C 1 -C 8 alkoxy, optionally substituted C 1 -C 8 alkoxyC 1 -C 8 alkyl, optionally substituted C 1 -C 8 haloalkyl, optionally substituted C 1 -C 8 hydroxyalkyl, optionally substituted C 2 -C 8 alkenyl, optionally substituted C 2 -C 8 alkynyl, optionally substituted C 3 -C 8 cycloalkyl, optionally substituted C 3 -C 8 cycloalkoxy, optionally substituted 3-8 membered heterocyclyl, optionally substituted
  • R 5 and R 6 are independently selected from hydrogen, optionally substituted C 1 -C 6 alkyl, optionally substituted C 3 -C 6 cycloalkyl, optionally substituted C 3 -C 6 cycloalkoxy, optionally substituted 3-6 membered heterocyclyl, optionally substituted C 1 -C 6 alkoxy, optionally substituted C 1 -C 6 alkoxyalkyl, optionally substituted C 1 -C 6 haloalkyl, optionally substituted C 1 -C 6 hydroxyalkyl, optionally substituted C 1 -C 6 alkylamino, and optionally substituted C 1 -C 6 alkylaminoC 1 -C 6 alkyl;
  • n 0 to 15;
  • each n is 0 to 15;
  • o 0 to 15.
  • each R 1 , and each R 2 are independently selected from hydrogen, halogen, CN, OH, NH 2 , and optionally substituted C 1 -C 6 alkyl, optionally substituted C 3 -C 6 cycloalkyl, optionally substituted C 3 -C 6 cycloalkoxy, optionally substituted 3-6 membered heterocyclyl, optionally substituted C 1 -C 6 alkoxy, optionally substituted C 1 -C 6 alkoxyalkyl, optionally substituted C 1 -C 6 haloalkyl, optionally substituted C 1 -C 6 hydroxyalkyl, optionally substituted C 1 -C 6 alkylamino, or C 1 -C 6 alkylaminoC 1 -C 6 alkyl;
  • each A and each B are independently selected from null, CO, CO 2 , C (O) NR 3 , C (S) NR 3 , O, S, SO, SO 2 , SO 2 NR 3 , NR 3 , NR 3 CO, NR 3 CONR 4 , NR 3 C (S) , and optionally substituted C 1 -C 8 alkyl, optionally substituted C 1 -C 8 alkoxy, optionally substituted C 1 -C 8 alkoxyC 1 -C 8 alkyl, optionally substituted C 1 -C 8 haloalkyl, optionally substituted C 1 -C 8 hydroxyalkyl, optionally substituted C 2 -C 8 alkenyl, optionally substituted C 2 -C 8 alkynyl, optionally substituted C 3 -C 8 cycloalkyl, optionally substituted C 3 -C 8 cycloalkoxy, optionally substituted 3-8 membered heterocyclyl, optionally substituted aryl, optionally
  • R 3 and R 4 are independently selected from hydrogen, and optionally substituted C 1 -C 6 alkyl, optionally substituted C 3 -C 6 cycloalkyl, optionally substituted C 3 -C 6 cycloalkoxy, optionally substituted 3-6 membered heterocyclyl, optionally substituted C 1 -C 6 alkoxy, optionally substituted C 1 -C 6 alkoxyalkyl, optionally substituted C 1 -C 6 haloalkyl, optionally substituted C 1 -C 6 hydroxyalkyl, optionally substituted C 1 -C 6 alkylamino, or C 1 -C 6 alkylaminoC 1 -C 6 alkyl;
  • each m is 0 to 15;
  • n 0 to 15.
  • X is selected from O, NH, and NR 7 ;
  • R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 are independently selected from hydrogen, halogen, CN, OH, NH 2 , optionally substituted C 1 -C 6 alkyl, optionally substituted C 3 -C 6 cycloalkyl, optionally substituted C 3 -C 6 cycloalkoxy, optionally substituted 3-6 membered heterocyclyl, optionally substituted C 1 -C 6 alkoxy, optionally substituted C 1 -C 6 alkoxyalkyl, optionally substituted C 1 -C 6 haloalkyl, optionally substituted C 1 -C 6 hydroxyalkyl, optionally substituted C 1 -C 6 alkylamino, and optionally substituted C 1 -C 6 alkylaminoC 1 -C 6 alkyl;
  • a and B are independently selected from null, CO, CO 2 , C (O) NR 7 , C (S) NR 7 , O, S, SO, SO 2 , SO 2 NR 7 , NR 7 , NR 7 CO, NR 7 CONR 8 , NR 7 C (S) , optionally substituted C 1 -C 8 alkyl, optionally substituted C 1 -C 8 alkoxy, optionally substituted C 1 -C 8 alkoxyC 1 -C 8 alkyl, optionally substituted C 1 -C 8 haloalkyl, optionally substituted C 1 -C 8 hydroxyalkyl, optionally substituted C 2 -C 8 alkenyl, optionally substituted C 2 -C 8 alkynyl, optionally substituted C 3 -C 8 cycloalkyl, optionally substituted C 3 -C 8 cycloalkoxy, optionally substituted 3-8 membered heterocyclyl, optionally substituted aryl,
  • R 8 and R 8 are independently selected from hydrogen, optionally substituted C 1 -C 6 alkyl, optionally substituted C 3 -C 6 cycloalkyl, optionally substituted C 3 -C 6 cycloalkoxy, optionally substituted 3-6 membered heterocyclyl, optionally substituted C 1 -C 6 alkoxy, optionally substituted C 1 -C 6 alkoxyalkyl, optionally substituted C 1 -C 6 haloalkyl, optionally substituted C 1 -C 6 hydroxyalkyl, optionally substituted C 1 -C 6 alkylamino, and optionally substituted C 1 -C 6 alkylaminoC 1 -C 6 alkyl;
  • each m is 0 to 15;
  • each n is 0 to 15;
  • o 0 to 15;
  • p 0 to 15.
  • Z is selected from the group consisting of a 3-to 8-membered ring, a 5-to 12-membered bicyclic ring, an 8-to 15-membered tricyclic ring and a 6-to 12-membered spiro bicyclic ring, each independently having 0-4 heteroatoms;
  • RL1 is selected from the group consisting of H, an unsubstituted C1-6 alkyl group, a C1-6 alkyl group substituted by a C1-6 alkoxyl group or one or more halo groups, halogen, a C1-6 alkoxy group, a keto group, or an oxide group; wherein, two RL1 groups are optionally taken together to form a 3-8 membered ring system containing 0-2 heteroatoms;
  • RL2 is a bond or an ethynylene group
  • X1 is selected from the group consisting of a methylene group and an ethylene group
  • X3 is selected from the group consisting of an unsubstituted C1-8 alkylene group, a C1-8 alkylene group substituted by 1 to 6 RL1, an unsubstituted C1-8 heteroalkylene group, a C1-8 heteroalkylene group substituted by 1 to 6 RL1, an unsubstituted 3-to 8-membered arylene group, a 3-to 8-membered arylene group substituted by 1 to 6 RL1, an unsubstituted 3-to 8-membered heteroarylene group with 1 to 3 hetero atoms, and a 3-to 8-membered heteroarylene group with 1 to 3 hetero atoms and substituted by 1 to 6 RL1, an unsubstituted 3-to 7-membered cyclic alkylene group, a 3-to 7-membered cyclic alkylene group substituted by 1 to 6 RL1, an unsubstituted 3-to 7-membered heterocyclic alkylene group with 1 to 2 hetero atom
  • RL3 is hydrogen
  • RL4 and RL5 are independently selected from the group consisting of H, an unsubstituted C1-6 alkyl group, and a C1-6 alkyl group substituted by a C1-6 alkoxyl group or one or more halo groups;
  • n1 0, 1, 2, 3, 4, 5 or 6;
  • n2 is 0 or 1
  • n3 is 0 or 1;
  • n4 1;
  • m5 is 0 or 1.
  • X3 is selected from the group consisting of an unsubstituted C1-6 alkylene group, an unsubstituted 3-to 7-membered cyclic alkylene group, and an unsubstituted 3-to 7-heterocyclic alkylene group with 0 to 2 hetero atoms; or, X3 is a 10-to 12-membered spiro bicyclic ring having 0-2 heteroatoms.
  • RL1 is selected from the group consisting of H, an unsubstituted C1-3 alkyl group, a C1-3 alkyl group substituted by a C1-3 alkoxyl group or one or more halo groups, halogen, a C1-3 alkoxy group, a keto group, or an oxide group; wherein, two RL1 groups are optionally taken together to form a 3-8 membered ring system containing 0-2 heteroatoms.
  • RL1 is an alkyl selected from the group consisting of a linear C1-6 alkyl and a branched C1-6 alkyl, each of which is unsubstituted or substituted by a C1-6 alkoxyl group or one or more halo groups.
  • RL1 is an alkyl selected from the group consisting of a linear C1-3 alkyl and a branched C1-3 alkyl, each of which is unsubstituted or substituted by a C1-6 alkoxyl group or one or more halo groups.
  • RL4 and RL5 are independently an alkyl selected from the group consisting of a linear C1-6 alkyl and a branched C1-6 alkyl, each of which is unsubstituted or substituted by a C1-6 alkoxyl group or one or more halo groups.
  • RL4 and RL5 are independently an alkyl selected from the group consisting of a linear C1-3 alkyl and a branched C1-3 alkyl, each of which is unsubstituted or substituted by a C1-6 alkoxyl group or one or more halo groups.
  • f is an integer of 0, 1, 2, 3 or 4; and g is an integer of 0, 1, 2 or 3.
  • a bivalent compound selected from the group consisting of:
  • composition comprising a bivalent compound according to embodiment 1-88, and a pharmaceutically acceptable carrier.
  • the intermediate I-A was first prepared from commercially available 3-benzyloxy-phenylamine via the route shown below:
  • Pd(OAc) 2 (262 mg, 20 mol%) was added to a mixture of I-a2 (1.7 g, 6.99 mmol) , dimethyl acetylenedicarboxylate (0.71 mL, 5.8 mmol) , and dimethylacetamide (DMA) /pivalic acid (PivOH) (4: 1 v/v; 28 mL) in a 100 mL two-necks bottle, and the bottle then purged with air. The reaction mixture was gradually heated from room temperature to 120°C for 8 h.
  • the I-a5 (0.65 g, 1.51 mmol) was treated with a solution of NaOH (0.6 g, 15.1 mmol) in EtOH (30 mL) and heated to reflux. After 4 h, the reaction mixture was cooled to ambient temperature, removed excess EtOH, diluted with ethyl acetate (30 mL) and acidified with 1 M HCl to pH about 3. The aqueous layer was then extracted with ethyl acetate (30 mL) and the combined organic layers were washed with brine, dried (with MgSO 4 ) , filtered and concentrated to afford a white solid. Then the white solid and Ac 2 O (5 mL) were combined and heated to 140°C.
  • the aqueous layer was extracted with ethyl acetate (10 mL x 2) , and the organic layers were combined, washed with brine, and dried with MgSO 4 .
  • the solvent was removed to give a crude material, and the crude material was used in the next step without purification.
  • the above crude material was dissolved in THF (4 mL) , and carbonyldiimidazole (CDI) (0.11 g, 0.67 mmol, 2 eq) and 4-dimethylaminopyridine (DMAP) (4 mg, 0.1 eq) were added.
  • CDI carbonyldiimidazole
  • DMAP 4-dimethylaminopyridine
  • the intermediate I-B was prepared from 2, 6-Difluoro-pyridine via the route shown below:
  • the I-b4 (100 mg, 0.27 mmol) was dissolved in 1, 4-dioxane (5 mL) and the solution was added bis(pinacolato) diboron (B 2 Pin 2 ) (101 mg, 0.40 mmol) , potassium acetate (78 mg, 0.80 mmol) , and 1, 1’ -bis(diphenylphosphino) ferrocenedichloropalladium (II) (PdCl2 (dppf) (20 mg, 0.03 mmol) .
  • the reaction mixture was stirred at 90 °C for 2 h under the protection of nitrogen. After the reaction completed, the solvent was evaporated under reduced pressure and the resulting residue was purified by silica gel chromatography to obtain intermediate I-b5 (110 mg, 96%) .
  • Compound 1 was prepared via the route shown below:
  • the Intermediate I-C was first prepared from commercially available 4-Hydroxy-piperidine-1-carboxylic acid tert-butyl ester via the route shown below:
  • the I-c1 (800 mg, 1.79 mmol) was treated with a solution of NaOH (717 mg, 17.9 mmol) in EtOH (25 mL) and heated to reflux. After 4 h, the reaction mixture was cooled to ambient temperature, removed excess EtOH, diluted with ethyl acetate and acidified with 1 M HCl to pH 3. The aqueous layer was then extracted with ethyl acetate and the organics were washed with brine, dried (with MgSO 4 ) , filtered and concentrated to afford a white solid. Then the white solid and Ac 2 O (5 mL) were combined and heated to 140°C.
  • the aqueous layer was extracted with Ethyl acetate twice, and the organic layers were combined, washed with brine, and dried with MgSO 4 .
  • the solvent was removed to give a crude material, and the crude material was used in the next step without purification.
  • the above crude material was dissolved in THF (6 mL) , and CDI (324 mg, 2.0 mmol, 2 eq) and DMAP (12 mg, 0.1 eq) were added.
  • the reaction mixture was stirred at 50°C for 2 h. After cooling down, the reaction mixture was diluted with Ethyl acetate (20 mL) and water (20 mL) .
  • the intermediate I-D was first prepared from I-a1 via the route shown below:
  • the I-d5 (0.67 g, 1.51 mmol) was treated with a solution of NaOH (0.6 g, 15.1 mmol) in EtOH (30 mL) and heated to reflux. After 4 h, the reaction mixture was cooled to ambient temperature, removed excess EtOH, diluted with Ethyl acetate (30 mL) and acidified with 1 M HCl to pH about 3. The aqueous layer was then extracted with Ethyl acetate (30 mL) and the organic layer was washed with brine, dried (with MgSO 4 ) , filtered and concentrated to afford a solid. Then the solid and Ac 2 O (5 mL) were combined and heated to 140°C. After 2 h, After the reaction was completed, the reaction mixture was cooled to ambient temperature. Then excess Ac 2 O was removed, and then dried toluene was added and concentrated. The intermediate I-d6 was thus produced and then used without any further purification.
  • the aqueous layer was extracted with ethyl acetate (10 mL x 2) , and the organic layers were combined, washed with brine, and dried with MgSO 4 .
  • the solvent was removed to give a crude material, and the crude material was used in the next step without purification.
  • the above crude material was dissolved in THF (4 mL) , and CDI (0.11 g, 2 eq) and DMAP (4 mg, 0.1 eq) were added. The reaction mixture was stirred at 50°C for 2 h.
  • the intermediate I-E was first prepared from commercially available 3-Benzyloxy-propan-1-ol via the route shown below:
  • the intermediate I-F was first prepared from commercially available 2, 6-Difluoro-pyridine via the route shown below:
  • the intermediate I-G was prepared from I-a6 via the route shown below:
  • Synthetic Method A Synthetic schemes 1 to 3
  • Synthetic Method B Synthetic schemes 4 to 5
  • Synthetic Method C Synthetic schemes 6 to 7
  • Synthetic Method D Synthetic schemes 8 to 10
  • compound 5-16 were prepared by a method similar to Synthetic Method A , Synthetic Method B, Synthetic Method C, Synthetic Method D or Synthetic Method E with changing one or more starting materials to obtain the desired products.
  • FGC cells (Cat. 60088, Bioresource Collection and Research Center, 15 HsinChu City, Taiwan R. O. C. ) grown in RPMI 1640 (Cat. 31800022, Thermo Fisher Scientific, Waltham, Massachusetts, United States) medium supplemented with 10%FBS (Cat. 10437028, Thermo Fisher Scientific, Waltham, Massachusetts, United States) , 10mM HEPES (Cat. 15630080, Thermo Fisher Scientific, Waltham, Massachusetts, United States) and 1mM sodium pyruvate (Cat. 11360070, Thermo 20 Fisher Scientific, Waltham, Massachusetts, United States) were seeded at 2x105 cells per well in 24-well tissue culture plates.
  • Cells were incubated at 37°C, 5%CO2 for 24 hours (hr) , then treated with 100 nanomolar concentrations (nM) any of the compounds 1 to 148 and ARV-110 for 24hr. After the treatment, the cells were harvested, washed by PBS, and lysed with RIPA lysis and extraction buffer (Cat. 25 89900, Thermo Fisher Scientific, Waltham, Massachusetts, United States) supplemented with Halt Protease Inhibitor Cocktail (Cat. 78430, Thermo Fisher Scientific, Waltham, Massachusetts, United States) to collect protein samples. Cells not treated with any of the above-mentioned compounds are used as a negative control.
  • RIPA lysis and extraction buffer Cat. 25 89900, Thermo Fisher Scientific, Waltham, Massachusetts, United States
  • Halt Protease Inhibitor Cocktail Cat. 78430, Thermo Fisher Scientific, Waltham, Massachusetts, United States
  • the protein samples were separated by polyacrylamide gel electrophoresis, and then transferred to a piece of Immuno-Blot PVDF membrane (Cat. 1620177, Bio-Rad Laboratories, Hercules, California, United States) .
  • the presence of androgen receptor in the protein samples was detected by standard Western blotting procedure using an anti-AR antibody (1: 2000 dilution) (Cat. 5153, Cell Signaling Technology Inc., Danvers, Massachusetts, United States) and a goat anti-rabbit HRP-conjugated secondary antibody (1: 5000 dilution) (C04003, Crlinger Bioscience Co., Ltd., Taipei City, Taiwan R. O. C. ) .
  • the internal loading control GAPDH was detected using a mouse monoclonal antibody (1: 5000) (GTX627408, GeneTex International Corp., HsinChu City, Taiwan R.O.C. ) and a goat anti-mouse HRP-conjugated secondary antibody (1: 5000 dilution) (C04001, Crlinger Bioscience Co., Ltd., Taipei City, Taiwan R.O.C. ) .
  • Chemiluminescence signals were developed using Clarity Western ECL substrate (Cat. 1705061, Bio-Rad Laboratories, Hercules, California, United States) and detected with digital imager iBright FL1500 (Invitrogen Corp., Carlsbad, California, United States) .
  • Compounds 1-16 were selected and serial diluted (10x) with RPMI medium for treating LNCaP. FGC cells. A calibration curve was determined by the serial diluted samples for each compound, and the concentration needed for 50%AR degradation (AR DC50) for each compound was calculated. The results are shown in Table 2. “Cpd. ID” indicates the compound ID number in Table 1.

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