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WO2025056913A1 - Composé et procédé - Google Patents

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Publication number
WO2025056913A1
WO2025056913A1 PCT/GB2024/052383 GB2024052383W WO2025056913A1 WO 2025056913 A1 WO2025056913 A1 WO 2025056913A1 GB 2024052383 W GB2024052383 W GB 2024052383W WO 2025056913 A1 WO2025056913 A1 WO 2025056913A1
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alkyl
formula
hypoxia
compound
group
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Stuart Conway
Marta Serafini
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Oxford University Innovation Ltd
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Oxford University Innovation Ltd
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    • 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, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
    • 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
    • A61K47/545—Heterocyclic compounds
    • 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
    • C07D495/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms
    • C07D495/12—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 three hetero rings
    • C07D495/14—Ortho-condensed systems

Definitions

  • the present invention relates to novel compounds and methods.
  • the compounds provided herein are hypoxia-targeting agents. Accordingly, as described herein, the invention particularly provides a compound that finds use in the treatment of cancer by selectively degrading under hypoxic conditions a therapeutic target protein of interest.
  • Proteolysis targeting chimeras are heterobifunctional molecules comprising a ligand for a protein of interest (POI) attached to a ligand for an E3 ubiquitin ligase by a linker.
  • PROTACs function by inducing proximity between the POI and the E3 ubiquitin ligase leading to polyubiquitination of the POI, and subsequent proteasome- mediated degradation. This mode of action gives PROTACs advantages over traditional occupancy-based drugs, such as enabling the degradation of multidomain proteins with the consequent cellular ablation of undruggable domains. Consequently, the interest in PROTAC field has been intense and a number of PROTACs have progressed in clinical trials. However, some intrinsic limitations are emerging for this modality, especially in the translation from preclinical to clinical settings. One issue relates to the effects that degrading a complete protein from both normal and diseased tissues may have, potentially causing dose-limiting on-target toxicities.
  • IMIDs immunomodulatory drugs
  • CRBN endogenous cereblon
  • IKZF1 and IKZF3 proteins i.e. the natural substrates of cereblon responsible for the side effects of thalidomide and analogues when used as immunomodulating drugs (IMiDs). This activity can result in inhibition of cell proliferation and stimulation of the immune system.
  • hypoxia-activated pro-drugs are an effective strategy to target therapeutic agents to tumors in vivo. Hypoxia involves a reduced concentration of oxygen, and while mild hypoxia occurs physiologically in some contexts, more extreme hypoxia is a hallmark of cancer.
  • hypoxia results from the rapid growth of tumor cells, leading to a higher metabolic demand and a disorganized vasculature.
  • This hypoxic tumor microenvironment leads to aggressive phenotypes and resistance to chemo- and radiotherapy.
  • HAPs employ bioresponsive chemistry to take advantage of the reducing hypoxic environment: they are inactive prodrugs in normoxic cells but undergo enzyme-catalyzed reduction in hypoxia, resulting in the release of the active drugs.
  • This strategy has been previously applied to the development of HDAC inhibitors, Chk1 inhibitor, DNA-PK inhibitor, the pan-HER inhibitor tarloxotinib, and to the nitrogen mustard alkylating agent TH032, which reached Phase III.
  • hypoxia-activated PROTACs HAP- TACs
  • One approach that has been considered involved a pomalidomide-based EGFR- degrading PROTAC in which a nitroaryl group was attached to the POI ligand. This resulted in POI engagement only being observed in hypoxia, but the CRBN binder remained active in both normoxia and hypoxia.
  • Another approach involved a second HAP-TAC for EGFR degradation in which a 2-nitroimidazole group was attached to a VHL ligand via a carbonate group, to protect the hydroxyl group of the VHL ligand.
  • this strategy did not exclude the unwanted release of the active PROTAC in normoxia.
  • the inventors have devised a novel principle of general application which involves chemically modifying a PROTAC to increase its selectivity to hypoxic conditions.
  • this modification results in a HAP-TAC with improved specificity relative to known HAP-TACs.
  • the invention provides an indolequinone bioreductive group that provides an effective mechanism for releasing PROTACs in a range of hypoxic conditions.
  • the indolequinone bioreductive group has been employed to develop structurally new PROTACs that expand the design of HAP-TACs and can be applied to, for example, VHL and cereblon (CRBN) ligands.
  • HAP-TACs prepared by the inventors have been shown to have little effect on a target protein of interest in normoxia, but exhibit almost complete degradation of the target protein of interest in hypoxia.
  • the HAP-TACS provided herein thus exhibit reduced or elimintated interactions with their endogenous targets in normoxia and therefore minimise unwanted side effects.
  • the compounds provided herein may therefore provide for i.a.
  • HAP-TAC Hypoxia-Actived Proteolysis Targeting Chimera
  • - POI is a protein of interest targeting ligand
  • - LINK is a linking moiety that covalently links POI and U
  • - U is an E3 ubiquitin ligase binding ligand
  • - HAP is a hypoxia-activated moiety of formula (I): [Formula I] wherein: - M is -OR 1 or a 5- to 6-membered heterocyclyl ring which is unsubstituted or is substituted by 1 or 2 R Y groups; - each of R 1 , R 2
  • LINK is (a) a chemical linker group represented by formula (L); or (b) a single bond: [Formula L] wherein: - LINK is attached to POI via L1; - LINK is attached to U via L3; - L1 and L3 are each independently selected from a single bond, -N(R’)-, -C(O)N(R’)-, -N(R’)C(O)-, -O-, -C(O)-, -OC(O)-, -C(O)O-, -S(O 2 )N(R’)-, -N(R’)S(O 2 )-, -(C 1-6 alkylene)-, -(C 2-6 alkenylene)-, a 3- to 6-membered cycloalkylene ring and a 4- to 7-membered heterocyclylene ring; - each L2 is independently selected from a unit of formula: 3- to 6-membered cycl
  • U is a CRBN E3 ubiquitin ligase binding ligand of formula (U1): [Formula U1] wherein: - u is 1 or 2 - Q is N and L U is a single bond, or Q is CH and L U is selected from a single bond, - C(O)N(R z )- and -N(R z )C(O)-; wherein each R z is independently H or C1-4 alkyl; - is the point of attachment to HAP; - R U1 is selected from the following structures: ; - ring A is selected from the following structures: - x is selected from 1 to 5; - y is selected from 1 to 4; - one R U2 group is a single bond to LINK and the other R U2 groups when present are each independently selected from halogen, C 1-4 alkyl, NH 2 , NO 2 , OH, COOH, CN and CF 3 ; - X is selected from
  • U is a VHL E3 ubiquitin ligase binding ligand of formula (U2): wherein: - R U4 is a group selected from a 5- to 6-membered heteroaryl ring, phenyl, a 5- to 6- membered heterocyclyl ring, and a 5- to 6-membered cycloalkyl ring, the group R U4 being unsubstituted or substituted by C 1-4 alkyl; - R U5 is H or C1-4 alkyl; - R U6 is C 1-4 alkyl, phenyl or a 3- to 8-membered cycloalkyl ring; - R U7 is a single bond to LINK; and - the point of attachment to HAP.
  • U2 VHL E3 ubiquitin ligase binding ligand of formula (U2): wherein: - R U4 is a group selected from a 5- to 6-membered heteroaryl ring, phenyl, a 5- to 6-
  • U is a DCAF15 E3 ubiquitin ligase binding ligand of formula (U3): wherein: - z is selected from 1 to 5; - one R U9 group is a single bond to LINK and the other R U9 groups when present are each independently selected from halogen, C 1-4 alkyl, NH 2 , NO 2 , OH, COOH, CN and CF 3 ; - R U10 is H or C 1-4 alkyl; and - attachment to HAP.
  • POI is a ligand for a cancer-regulating protein. Also provided is a compound as provided herein for use in medicine.
  • Also provided is a method for degrading a target protein of interest in a hypoxic cell comprising: a) introducing a compound as provided herein into the hypoxic cell, thereby cleaving the hypoxia-activated moiety HAP from the rest of the compound to form an activated PROTAC; b) contacting the protein of interest with the activated PROTAC within the hypoxic cell; thereby inducing the degradation of the target protein of interest; preferably wherein the hypoxic cell is a cancer cell.
  • FIGURE 1 Design of VHL-recruiting HAP-TACs.
  • FIGURE 2. Initial attempts to synthesize hypoxia-activated CRBN ligands and subsequent strategy to overcome the instability and increase the release of the active recruiter.
  • FIGURE 3. NI-pomalidomide is degraded over time in the presence of NADPH- CYP reductase under hypoxia.
  • IQ-VHL undergoes oxygen- and enzyme-dependent reduction to the corresponding active PROTAC MZ1.
  • A Enzymatic reduction of IQ-VHL using NADPH- CYP reductase.
  • NI-CRBN undergoes oxygen- and enzyme-dependent reduction to the corresponding active PROTAC PG-4c.
  • A Enzymatic reduction of NI-CRBN using NADPH-CYP reductase.
  • B NI-CRBN was incubated with NADPH-CYP reductase over 24 h in normoxia and hypoxia. Additionally, the compound was incubated in hypoxia in the absence of the NADPH-CYP reductase (hypoxia control).
  • the active PROTACs, MZ1 and PG-4c, and the negative controls, Bn- VHL and Bn-CRBN, are stable in the presence of the NADPH-CYP reductase.
  • FIGURE 10 Percentage of BRD4 levels compared to control, assessed using western blot analysis, in A549 cells after 24 h of incubation with the indicated concentrations of NI- VHL (A), IQ-VHL (B), NI-CRBN (C) or IQ-CRBN (D) in normoxia (21% O 2 , red lines) or hypoxia ( ⁇ 0.1% O 2 , blue lines). Data are the mean of three independent experiments ⁇ s.e.m.
  • FIGURE 11 Representative western blot analysis of the data shown in panels A-D in normoxia (top, E) and hypoxia (bottom, F), with HIF-1 ⁇ used as hypoxia biomarker and ⁇ -actin used as a loading control.
  • FIGURE 11. (A). Percentage of BRD4 levels compared to control, assessed using western blot analysis, after 24 h of incubation in A549 cells with the indicated concentrations of MZ1, PG4c or (+)-JQ1. Data are the mean of three independent experiments ⁇ s.e.m.
  • B Representative western blot analysis of the data showed in panel A with ⁇ -actin used as loading control.
  • FIGURE 12. (A).
  • BRD4 Percentage of BRD4 levels, compared to control, assessed using western blot analysis in A549 cells after incubation in hypoxia ( ⁇ 0.1% O2) with IQ-VHL (0.25 ⁇ M, violet bars) and IQ-CRBN (3 ⁇ M, green bars) for the time points shown. Data are the mean of three independent experiments ⁇ s.e.m.
  • B Representative western blot analysis of the time-course experiment shown in panel A, with HIF-1 ⁇ used as hypoxia biomarker and ⁇ -actin as loading control.
  • C Representative western blot analysis of the time-course experiment shown in panel A, with HIF-1 ⁇ used as hypoxia biomarker and ⁇ -actin as loading control.
  • FIGURE 14 Representative western blot analysis of three independent experiments in A549 cells after 24 h of incubation with IQ-VHL (0.25 ⁇ M) and IQ-CRBN (3 ⁇ M) with or without the proteasome inhibitor carfilzomib (0.5 ⁇ M), or the neddylation inhibitor MLN-4924 (1 ⁇ M) in hypoxia ( ⁇ 0.1% O 2 ).
  • HIF-1 ⁇ is used as hypoxia biomarker
  • ⁇ -actin is a loading control.
  • FIGURE 14 (A). Percentage of BRD4 levels compared to control, assessed using western blot analysis, after 24 h of incubation in HCT116 cells with the indicated concentrations of MZ1, PG4c or (+)-JQ1.
  • FIGURE 15. (A). Percentage of BRD4 levels compared to control, assessed using western blot analysis, after 24 h of incubation in HCT116 cells with IQ-VHL (0.25 ⁇ M) or IQ-CRBN (3 ⁇ M) for 24 h in normoxia (21% O 2 ) or hypoxia ( ⁇ 0.1%). Data are the mean of three independent experiments ⁇ s.e.m. (B). Representative western blot analysis of the data shown in panel A. HIF-1 ⁇ is used as hypoxia biomarker and ⁇ -actin is a loading control. FIGURE 16.
  • FIGURE 17 Concentration-response curves of MTT assay in HCT116 cells treated with compounds MZ1 (A), IQ-VHL (B), PG-4c (C) and IQ-CRBN (D) in normoxia (21% O 2 , red line) or hypoxia ( ⁇ 0.1% O 2 , blue line). Data are the mean of three independent experiments ⁇ s.e.m. FIGURE 17.
  • A Growth over time of HCT116 cells after 24 h of incubation with MZ1 (0.25 ⁇ M) in normoxia or IQ-VHL (0.25 ⁇ M) in normoxia or hypoxia over 6 days. Cell number is expressed as percentage related to DMSO control, respectively in normoxia or hypoxia.
  • the term “protein” is used to describe a folded polypeptide having a secondary or tertiary structure.
  • the protein may be composed of a single polypeptide, or may comprise multiple polypeptides that are assembled to form a multimer.
  • the multimer may be a homooligomer, or a heterooligmer.
  • the protein may be a naturally occurring, or wild type protein, or a modified, or non-naturally, occurring protein.
  • the protein may, for example, differ from a wild type protein by the addition, substitution or deletion of one or more amino acids.
  • hyperoxia and “hypoxic” are used to describe a state of insufficient oxygen concentration that can be present in tumors, normal tissues and wounds.
  • hypoxia may be defined as a tissue oxygen concentration of 4.2% or less, for instance 4% or less, or 3% or less.
  • hypoxia is defined as a tissue oxygen concentration of 2 % or less, for instance 1 % or less. More preferably, hypoxia is defined as a tissue oxygen concentration of 0.1 % or less.
  • a C 1-4 alkyl group is a linear or branched alkyl group containing from 1 to 4 carbon atoms.
  • a C 1-4 alkyl group is often a C 1-3 alkyl group or a C 1-2 alkyl group.
  • Examples of C 1-4 alkyl groups include methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, and tert-butyl.
  • a C 1-2 alkyl group is methyl or ethyl, typically methyl.
  • the alkyl groups may be the same or different.
  • a C 1-4 alkoxy group is typically a said C 1-4 alkyl group which is joined to the rest of the molecule via an oxygen atom.
  • a C 1-4 alkoxy group is a C1-3 alkoxy group.
  • Examples of C1-4 alkoxy groups include methoxy, ethoxy, propoxy and butoxy.
  • a C 1-3 alkoxy group is a C 1-2 alkoxy group such as a methoxy or ethoxy group.
  • the alkoxy groups may be the same or different.
  • a C 1-6 alkylene group is a linear or branched divalent alkyl group that contains from 1 to 6 carbon atoms and is preferably linear.
  • a C 1-6 alkylene group is typically a C 1-4 alkylene group or a C 1-3 alkylene group.
  • C 1-4 alkylene groups include methylene, ethylene, n-propylene, iso-propylene, n-butylene, sec-butylene, and tert-butylene.
  • a C 1-3 alkylene group is typically a C 1-2 alkylene group.
  • a C 1-2 alkylene group is methylene or ethylene, typically methylene.
  • the alkylene groups may be the same or different.
  • a C 2-6 alkenylene group is a linear or branched divalent alkenyl group containing from 2 to 6 carbon atoms and having one or more, e.g. one or two, typically one double bonds.
  • a C 2-6 alkenylene group is a C 2-4 alkenylene group.
  • Examples of C 2-4 alkenylene groups include divalent ethenylene, propenylene and butenylene.
  • the alkenylene groups may be the same or different.
  • An alkyl, alkoxy, alkylene or alkenylene group as used herein may be unsubstituted or substituted. Unless otherwise stated, substituted alkyl, alkoxy, alkylene or alkenylene groups typically carry one or more, e.g. 1, 2, 3 or 4, such as one, two or three e.g. one or two substituents.
  • Preferred substituents include aryl, heteroaryl, heterocyclyl, alkoxy, amino and halogen substituents.
  • the substituents on a substituted alkyl, alkoxy, alkylene or alkenylene group are typically themselves unsubstituted. Where more than one substituent is present, these may be the same or different.
  • a halogen is typically chlorine, fluorine, bromine or iodine and is preferably chlorine, bromine or fluorine, especially chorine or fluorine, and most especially fluorine.
  • halogen atoms preferably it carries one, two, three or four halogen atoms, preferably one, two or three halogen atoms.
  • the halogen atoms may be the same or different.
  • the halogen atoms are the same.
  • a 6- to 10-membered aryl ring is a substituted or unsubstituted, monocyclic or fused polycyclic aromatic ring containing from 6 to 10 carbon atoms in the ring portion.
  • a 5- to 10-membered heteroaryl ring is a substituted or unsubstituted, monocyclic or fused polycyclic aromatic ring containing from 5 to 10 atoms in the ring portion, including at least one heteroatom, for example 1, 2 or 3 heteroatoms, typically selected from O, S and N; and is typically a 5- to 6- membered heteroaryl ring which is a substituted or unsubstituted, monocyclic aromatic ring containing 5 or 6 atoms in the ring portion, including at least one heteroatom, for example 1, 2 or 3 heteroatoms, typically selected from O, S and N.
  • 5- and 6- membered heteroaryl rings include pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyridine, pyridazine, pyrimidine, and pyrazine.
  • a 5- to 10- membered heterocyclyl ring is a cyclic ring containing from 5 to 10 atoms selected from C, O, N and S in the ring, including at least one heteroatom, and typically one or two heteroatoms; and is typically a 5- to 8- membered heterocyclyl ring, typically a 5- to 6- membered heterocyclyl ring which is a cyclic ring containing from 5 to 6 atoms selected from C, O, N and S in the ring, including at least one heteroatom, and typically one or two heteroatoms.
  • the heteroatom or heteroatoms are typically selected from O, N, and S.
  • a heterocyclic ring may be saturated or partially unsaturated.
  • a 5- to 6- membered partially unsaturated heterocyclic ring is a cyclic ring containing from 5 to 6 atoms selected from C, O, N and S in the ring and containing 1 or 2, e.g. 1 double bond.
  • a C3-6 cycloalkyl is a cyclic hydrocarbon containing from 3 to 6 carbon atoms.
  • a C 3-6 cycloalkyl may be saturated or partially unsaturated, but is typically saturated.
  • a partially unsaturated C 3-6 cycloalkyl is a cyclic hydrocarbon containing from 3 to 6 carbon atoms and containing 1 or 2, e.g. 1 double bond.
  • a C 3-6 cycloalkyl is typically a C 5-6 cycloalkyl.
  • saturated C 5-6 cycloalkyls include cyclopentyl and cyclohexyl groups.
  • 5- to 6- membered saturated heterocyclic rings include piperazine, piperidine, morpholine, pyrrolidine, imidazolidine, and oxazolidine.
  • 5- to 6- membered partially unsaturated heterocyclic rings include tetrahydropyrazine, tetrahydropyridine, dihydro-1,4-oxazine, tetrahydropyrimidine, dihydro-1,3-oxazine, dihydropyrrole, dihydroimidazole and dihydrooxazole.
  • a 4- to 7-membered heteroarylene ring is a substituted or unsubstituted, monocyclic or fused polycyclic divalent aromatic ring containing from 4 to 7 atoms in the ring portion, including at least one heteroatom, for example 1, 2 or 3 heteroatoms, typically selected from O, S and N; and is typically a 5- to 6- membered heteroarylene ring which is a substituted or unsubstituted, monocyclic divalent aromatic ring containing 5 or 6 atoms in the ring portion, including at least one heteroatom, for example 1, 2 or 3 heteroatoms, typically selected from O, S and N.
  • a 4- to 7- membered heterocyclylene ring is a cyclic ring containing from 4 to 7 atoms selected from C, O, N and S in the ring, including at least one heteroatom, and typically one or two heteroatoms; and is typically a 5- to 6- membered heterocyclylene ring which is a divalent cyclic ring containing from 5 to 6 atoms selected from C, O, N and S in the ring, including at least one heteroatom, and typically one or two heteroatoms.
  • the heteroatom or heteroatoms are typically selected from O, N, and S.
  • a heterocyclylene ring may be saturated or partially unsaturated.
  • a 5- to 6- membered partially unsaturated heterocyclylene ring is a divalent cyclic ring containing from 5 to 6 atoms selected from C, O, N and S in the ring and containing 1 or 2, e.g. 1 double bond.
  • a C 3-6 cycloalkylene is a divalent cyclic hydrocarbon containing from 3 to 6 carbon atoms.
  • a C 3-6 cycloalkylene may be saturated or partially unsaturated, but is typically saturated.
  • a partially unsaturated C 3-6 cycloalkylene is a divalent cyclic hydrocarbon containing from 3 to 6 carbon atoms and containing 1 or 2, e.g. 1 double bond.
  • a C 3-6 cycloalkylene is typically a C 5-6 cycloalkylene.
  • An aryl, heterocyclyl, heteroaryl, cycloalkyl, heterocyclylene, heteroarylene, or cycloalkylene ring may be unsubstituted or substituted as described herein.
  • said ring may be unsubstituted or substituted with 1, 2 or 3, typically 1 or 2 such as e.g. 1 substituent.
  • suitable substituents include alkyl group, alkoxyl groups, alkenyl groups, alkynyl groups, halogen, OH, and amino groups, which are typically themselves unsubstituted or substituted with one or more halogen atoms.
  • the terms “monovalent” or “monovalent moiety” are used to describe a chemical group obtainable by removing a hydrogen atom from the corresponding compound.
  • the terms “divalent” or “divalent moiety” are used to describe a chemical group obtainable by removing a hydrogen atom from the corresponding monovalent moiety.
  • the terms “divalent” and “divalent moiety” are used to describe a chemical group obtainable by removing two hydrogen atoms from the corresponding compound.
  • the compounds of the present invention may exist in unsolvated as well as solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like, and it is intended that the invention embrace both solvated and unsolvated forms.
  • a compound of the present invention can be converted into a pharmaceutically acceptable salt thereof, and a salt can be converted into the free compound, by conventional methods. For instance, a compound of the present invention can be contacted with a pharmaceutically acceptable acid to form a pharmaceutically acceptable salt.
  • a pharmaceutically acceptable salt is a salt with a pharmaceutically acceptable acid or base.
  • Pharmaceutically acceptable acids include both inorganic acids such as hydrochloric, sulphuric, phosphoric, diphosphoric, hydrobromic or nitric acid and organic acids such as oxalic, citric, fumaric, maleic, malic, ascorbic, succinic, tartaric, benzoic, acetic, methanesulphonic, ethanesulphonic, benzenesulphonic or p-toluenesulphonic acid.
  • Pharmaceutically acceptable bases include alkali metal (e.g. sodium or potassium) and alkaline earth metal (e.g. calcium or magnesium) hydroxides and organic bases such as alkyl amines, aralkyl amines and heterocyclic amines.
  • Preferred pharmaceutically acceptable salts are salts formed with pharmaceutically acceptable bases, in particular quaternary ammonium salts e.g. tetrabutylammonium salts, or alkali metal salts, e.g. sodium or potassium salts, most preferably sodium salts.
  • quaternary ammonium salts e.g. tetrabutylammonium salts
  • alkali metal salts e.g. sodium or potassium salts, most preferably sodium salts.
  • Said PROTAC comprises a protein of interest targeting ligand (POI) that is covalently attached via a linking moiety (LINK) to the E3 ubiquitin ligase binding ligand (U).
  • POI protein of interest targeting ligand
  • LINK linking moiety
  • HAP-TACs Hypoxia- Actived Proteolysis Targeting Chimeras
  • the inventors have surpisingly discovered that such compounds (which are Hypoxia- Actived Proteolysis Targeting Chimeras (HAP-TACs)) may have substantially enhanced selectivity for hypoxic conditions compared with a corresponding PROTAC that is not attached to a hypoxia-activated moiety of formula (I).
  • HAP-TACs Hypoxia- Actived Proteolysis Targeting Chimeras
  • the inventors have also surpisingly discovered that such compounds may have substantially enhanced selectivity for hypoxic conditions compared with a corresponding PROTAC that is attached to a hypoxia- activated moiety of the prior art
  • HAP-TAC Hypoxia-Actived Proteolysis Targeting Chimera
  • LINK is a linking moiety that covalently links POI and U
  • U is an E3 ubiquitin ligase binding ligand
  • HAP is a hypoxia-activated moiety of formula (I) as defined herein.
  • the protein of interest targeting ligand is covalently attached to the E3 ubiquitin ligase binding ligand via the moiety labelled LINK.
  • LINK is either a single bond or a chemical group that covalently attaches the protein of interest targeting ligand to the E3 ubiquitin ligase binding ligand.
  • methods for preparation of compounds are very well known in the art.
  • a hugely diverse range of techniques for covalently attaching the respective chemical entities, via a vast number of chemical linker moieties, is well established.
  • Such is the ubiquity of these methodologies, that standard text books devoted entirely to this topic have long been available.
  • One such textbook is “Bioconjugate Techniques” (Greg T. Hermanson, Academic Press Inc., 1996), the content of which is herein incorporated by reference in its entirety.
  • hypoxia-activated moiety In the provided compounds, the hypoxia-activated moiety (HAP) is of formula (I) as defined herein.
  • M is -OR 1 or a 5- to 6-membered heterocyclyl ring which is unsubstituted or is substituted by 1 or 2 R Y groups.
  • M is typically unsubstituted or is substituted with one methyl group.
  • M is -OR 1 or a group selected from morpholino, N-methyl piperidinyl, and 4-methylpiperazin-1-yl.
  • M is -OR 1 .
  • R 1 , R 2 , R 3 and R 4 are independently C 1-4 alkyl which is unsubstituted or substituted by one, two or three R 5 groups; H; halo; or a group selected from C 3-6 cycloalkyl, a 6- to 10-membered aryl ring, a 5- to 10-membered heteroaryl ring and a 5- to 10-membered heterocyclyl ring, each of which is unsubstituted or substituted by one, two or three R 6 groups.
  • each of R 2 and R 4 are independently C 1-4 alkyl which is unsubstituted or substituted by one, two or three R 5 groups; H; halo; or a group selected from C 3-6 cycloalkyl, a 6- to 10-membered aryl ring, a 5- to 10-membered heteroaryl ring and a 5- to 10-membered heterocyclyl ring, each of which is unsubstituted or substituted by one, two or three R 6 groups; and each of R 1 and R 3 are independently C 1-4 alkyl which is unsubstituted or substituted by one, two or three R 5 groups; H; or a group selected from C3- 6 cycloalkyl, a 6- to 10-membered aryl ring, a 5- to 10-membered heteroaryl ring and a 5- to 10-membered heterocyclyl ring, each of which is unsubstituted or substituted by one, two or three R 6 groups
  • R 1 is C
  • R 1 is unsubstituted C 1-4 alkyl, for instance methyl.
  • R 3 is C 1-4 alkyl which is unsubstituted or substituted by one or two R 5 groups; or a group selected from C 5-6 cycloalkyl, phenyl, a 5- to 6-membered heteroaryl ring and a 5- to 6-membered heterocyclyl ring, the group being unsubstituted or substituted by one or two R 6 groups.
  • R 3 is unsubstituted C 1-4 alkyl, for instance methyl.
  • R 2 is H, halo or C 1-4 alkyl which is unsubstituted or substituted by one or two R 5 groups.
  • R 2 is H or unsubstituted C 1-4 alkyl. More preferably, R 2 is H.
  • R 4 is H, halo or C 1-4 alkyl which is unsubstituted or substituted by one or two R 5 groups.
  • R 4 is H or unsubstituted C 1-4 alkyl. More preferably, R 4 is methyl.
  • each R 5 is independently selected from C 1-4 alkoxy, halo, -OH, -CN and - N(R Y ) 2 .
  • each R 5 is independently selected from C 1-4 alkoxy, halo and -OH.
  • each R 6 is independently selected from C 1-4 alkoxy, halo, -OH, -CN, -N(R Y )2, and C1-4 alkyl which is unsubstituted or substituted by one, two or three, for instance one or two, R 5 groups.
  • each R 6 is independently selected from C 1-4 alkoxy, halo, -OH and unsubstituted C1-4 alkyl.
  • each R X is independently selected from H and methyl.
  • each R X is independently H.
  • each R Y is independently selected from H and methyl.
  • each R Y is independently H.
  • M is -OR 1 or a group selected from morpholino, N-methyl piperidinyl, and 4-methylpiperazin-1-yl;
  • R 1 and R 3 are each independently C 1-4 alkyl which is unsubstituted or substituted by one or two R 5 groups; or a group selected from C 5-6 cycloalkyl, phenyl, a 5- to 6-membered heteroaryl ring and a 5- to 6-membered heterocyclyl ring, the group being unsubstituted or substituted by one or two R 6 groups;
  • R 2 is H, halo or C1-4 alkyl which is unsubstituted or substituted by one or two R 5 groups;
  • R 4 is H, halo or C 1-4 alkyl which is unsubstituted or substituted by one or two R 5 groups;
  • each R 5 is independently selected from C 1-4 alkoxy, halo, -OH, -CN and -N(R Y ) 2 ;
  • M is -OR 1 ;
  • R 1 and R 3 are each independently unsubstituted C 1-4 alkyl, for instance methyl;
  • R 2 is H or unsubstituted C 1-4 alkyl, more preferably R 2 is H;
  • R 4 is H or unsubstituted C 1-4 alkyl, more preferably R 4 is methyl;
  • each R 5 is independently selected from C1-4 alkoxy, halo and -OH;
  • each R 6 is independently selected from C 1-4 alkoxy, halo, -OH and unsubstituted C 1-4 alkyl;
  • HAP is a hypoxia-activated moiety of Formula IA:
  • E3 ubiquitin ligase binding moieties The presence of the E3 ubiquitin ligase binding ligand U in the compound of the invention means that the compound functions as a “PROTAC”.
  • PROTAC is an acronym for proteolysis targeting chimera.
  • PROTACs are, as is known in the art, heterobifunctional molecules that comprise two active moieties attached covalently by a linker group.
  • the first active moiety (the protein of interest targeting ligand, POI in the compounds provided herein) binds to a target protein that is intended for degradation (for example, the compounds provided herein are particularly suited for targeting target proteins which are cancer- regulating proteins).
  • the second active moiety (U in the compounds provided herein) is capable of binding to an E3 ubiquitin ligase, thereby inducing selective intracellular proteolysis. Recruitment of the E3 ligase to the target protein results in ubiquitination and subsequent degradation of the target protein by the proteasome.
  • E3 ubiquitin ligase binding ligands are known in the art. Any suitable E3 ligase binding ligand can be used in the compounds provided herein.
  • the E3 ligase binding ligtand is capable of binding to an E3 ubiquitin ligase.
  • E3 ubiquitin ligase including but not limited to any of those disclosed specifically herein.
  • protein based assays such as those described in the examples can be used to identify E3 ubiquitin ligase binding ligands.
  • the moiety shows activity or binds to the E3 ubiquitin ligase with an IC 50 of less than about 200 mM, e.g. less than about 100 mM, such as less than about 10 mM, less than 1 mM, less than 100 ⁇ M, less than 10 ⁇ M, or less than 1 ⁇ M.
  • the IC 50 can be determined according to any method known in the art, e.g., a fluorescent polarization assay.
  • a fluorescent polarization assay e.g., a fluorescent polarization assay.
  • such ligands may be referred to as a “ULM” or (small molecule) E3 ubiquitin ligase binding moiety (that binds an E3 ubiquitin ligase) (wherein the term “ULM” includes each of “ILM”, “CLM”, “VLM” and “MLM”, any of which can be used in the present compounds).
  • ULM small molecule E3 ubiquitin ligase binding moiety
  • MLM E3 ubiquitin ligase binding moiety
  • E3 ubiquitin ligases include von Hippel-Lindau (VHL), cereblon (CRBN) and DCAF15.
  • the E3 ubiquitin ligase binding ligand (U) is capable of binding to CRBN, VHL or DCAF15. More preferably the E3 ubiquitin ligase binding ligand (U) is capable of binding to CRBN or VHL. Such moieties are referred to herein as CRBN or VHL E3 ubiquitin ligase binding ligands.
  • U is a CRBN E3 ubiquitin ligase binding ligand.
  • U is a VHL E3 ubiquitin ligase binding ligand.
  • U is a DCAF15 E3 ubiquitin ligase binding ligand.
  • the E3 ubiquitin ligase binding moiety (U) is: A: a CRBN E3 ubiquitin ligase binding ligand of formula (U1): [Formula U1] wherein: - u is 1 or 2 - Q is N and L U is a single bond, or Q is CH and L U is selected from a single bond, - C(O)N(R z )- and -N(R z )C(O)-; wherein each R z is independently H or C 1-4 alkyl; - is the point of attachment to HAP; - R U1 is selected from the following structures:
  • - z is selected from 1 to 5; - one R U9 group is a single bond to LINK and the other R U9 groups when present are each independently selected from halogen, C 1-4 alkyl, NH 2 , NO 2 , OH, COOH, CN and CF 3 ; - R U10 is H or C 1-4 alkyl; and - the point of attachment to HAP.
  • the E3 ubiquitin ligase binding moiety (U) is a CRBN E3 ubiquitin ligase binding ligand of formula (U1). Tyically, in formula (U1), u is 1 or 2. Preferably, u is 2.
  • Q is CH and L U is selected from a single bond, - C(O)N(R z )- and -N(R z )C(O)-.
  • Q is CH and L U is a single bond.
  • each R z is independently H or methyl.
  • each R z is independently H.
  • R U1 is selected from the following structures:
  • R U1 is the following structure: Ring A is selected from the following structures: .
  • x is selected from 1 to 5, for instance 1 to 4 or 1 to 3.
  • x is selected from 1 or 2, more preferably x is 1.
  • y is selected from 1 to 4, for instance 1 to 3.
  • y is selected from 1 or 2, more preferably y is 1.
  • one R U2 group is a single bond to LINK and the other R U2 groups when present are each independently selected from halogen, C 1-4 alkyl, NH 2 , NO 2 , OH, COOH, CN and CF 3 .
  • one R U2 group is a single bond to LINK and the other R U2 groups when present are each independently selected from halogen, C 1-4 alkyl and OH.
  • X is selected from -CH(R U3 )-, -N(R U3 )-, -O- and -C(O)- .
  • X is -CH(R U3 )-.
  • R V is H or a group selected from C 1-4 alkyl, C 3-6 cycloalkyl, phenyl, a 5- to 6-membered heteroaryl ring and a 5- to 6-membered heterocyclyl ring, the group being unsubstituted or substituted by one or two of C1-4 alkoxy, halo, -OH, -CN, -N(R U3 ) 2 , C 1-4 alkyl, C 3-6 cycloalkyl, phenyl, a 5- to 6-membered heteroaryl ring and a 5- to 6-membered heterocyclyl ring.
  • R V is H or C 1-4 alkyl which is unsubstituted or substituted by one or two of C 1-4 alkoxy, halo, -OH, -CN, - N(R U3 ) 2 , C 3-6 cycloalkyl, phenyl, a 5- to 6-membered heteroaryl ring and a 5- to 6- membered heterocyclyl ring.
  • R V is an amino acid side chain or derivative thereof.
  • each R U3 is independently H or methyl.
  • each R U3 is independently H.
  • u is 1 or 2;
  • Q is CH and L U is selected from a single bond, -C(O)N(R z )- and -N(R z )C(O)-; each R z is independently H or methyl;
  • R U1 is selected from the following structures:
  • R U2 group is a single bond to LINK and the other R U2 groups when present are each independently selected from halogen, C 1-4 alkyl, NH 2 , NO 2 , OH, COOH, CN and CF 3 ;
  • X is selected from -CH(R U3 )-, -N(R U3 )-, -O- and -C(O)-;
  • R V is H or a group selected from C 1-4 alkyl, C 3-6 cycloalkyl, phenyl, a 5- to 6-membered heteroaryl ring and a 5- to 6- membered heterocyclyl ring, the group being unsubstituted or substituted by one or two of C 1-4 alkoxy, halo, -OH, -CN, -N(R U3 ) 2 , C 1-4 alkyl, C 3-6
  • U is a CRBN E3 ubiquitin ligase binding ligand having the following structure: wherein is the point of attachment to LINK.
  • U is a VHL E3 ubiquitin ligase binding ligand of formula (U2).
  • the phenyl ring moiety and/or the pyrrolidine ring moiety of formula U2 may be substituted by one or more substituents as described herein. More typically, the phenyl ring moiety and the pyrrolidine ring moiety are unsubstituted as depicted in formula U2 above.
  • R U4 is a 5- to 6-membered heteroaryl ring, the group R U4 being unsubstituted or substituted by C1-4 alkyl.
  • R U4 is: wherein: - V is typically S or O, preferably S; - W is typically N or CH, preferably N; and - R U8 is typically H or methyl, preferably methyl.
  • R U5 is H or methyl.
  • R U5 is H.
  • R U6 is C 1-4 alkyl.
  • R U6 is t-butyl.
  • R U4 is a 5- to 6-membered heteroaryl ring, the group R U4 being unsubstituted or substituted by C 1-4 alkyl;
  • R U5 is H or methyl; and
  • R U6 is C 1-4 alkyl.
  • R U4 is: V is typically S or O, preferably S; W is typically N or CH, preferably N; R U8 is typically H or methyl, preferably methyl; R U5 is H; and R U6 is t-butyl.
  • U is a VHL E3 ubiquitin ligase binding ligand of formula (U2’).
  • R U4 , R U5 , R U6 and R U7 are as described above for formula (U2).
  • U is a VHL E3 ubiquitin ligase binding .
  • U is a DCAF15 E3 ubiquitin ligase binding ligand of formula (U3).
  • z is selected from 1 to 4, for instance 1 to 3.
  • z is selected from 1 or 2, more preferably z is 1.
  • one R U9 group is a single bond to LINK and the other R U9 groups when present are each independently selected from halogen, C 1-4 alkyl, NH 2 , NO 2 , OH, COOH, CN and CF 3 .
  • one R U9 group is a single bond to LINK and the other R U9 groups when present are each independently selected from halogen, C 1-4 alkyl, and OH.
  • R U10 is H or methyl.
  • R U10 is methyl.
  • z is selected from 1 to 4, for instance 1 to 3; one R U9 group is a single bond to LINK and the other R U9 groups when present are each independently selected from halogen, C 1-4 alkyl, NH 2 , NO 2 , OH, COOH, CN and CF 3 ; and R U10 is H or methyl.
  • z is selected from 1 or 2, more preferably z is 1; one R U9 group is a single bond to LINK and the other R U9 group when present is independently selected from halogen, C 1-4 alkyl, and OH; and R U10 is methyl.
  • U is a DCAF15 E3 ubiquitin ligase binding ligand having the following structure: .
  • Linkers As discussed herein, in the compounds provided herein, the protein of interest targeting ligand (POI) is covalently attached to an E3 ubiquitin ligase binding ligand (U). This covalent attachment is labelled LINK and may be a direct single bond or a divalent chemical linking moiety that forms covalent bonds both to the protein of interest targeting ligand (POI) and the E3 ubiquitin ligase binding ligand (U).
  • LINK there is no particular limitation on the nature of LINK in the compounds of the present invention (beyond that the respective moieties, e.g., U and POI, are able to exert their desired function and LINK is capable of covalently attaching them together). Accordingly, any suitable linker group can be used. Those skilled in the art will appreciate that chemical linker groups are routinely used in the construction of bifunctional molecules and are able routinely to provide appropriate chemical linker groups for attaching particular U and POI ligands together. Typically, a chemical linker group for use in the present invention is an organic group.
  • LINK When LINK is a divalent chemical linker group, LINK may be represented by formula (L): [Formula L] wherein: - LINK is attached to POI via L1; - LINK is attached to U via L3; - L1 and L3 are each independently selected from a single bond, -N(R’)-, -C(O)N(R’)-, -N(R’)C(O)-, -O-, -C(O)-, -OC(O)-, -C(O)O-, -S(O 2 )N(R’)-, -N(R’)S(O 2 )-, -(C 1-6 alkylene)-, -(C 2-6 alkenylene)-, a 3- to 6-membered cycloalkylene ring and a 4- to 7-membered heterocyclylene ring; - each L2 is independently selected from a unit of formula: 3- to 6-membered cyclo
  • L1 is selected from a single bond, -N(R’)-, -C(O)N(R’)-, -N(R’)C(O)-, - O-, and a 4- to 7-membered heterocyclylene ring.
  • the 4- to 7-membered heterocyclylene ring of L1 typically contains at least one N atom, for instance one or two N atoms.
  • L1 is a 4- to 7-membered heterocyclylene ring, it is a 5- to 6- membered heterocyclylene ring, more preferably a saturated 5- to 6-membered heterocyclylene ring, for instance a divalent moiety of piperidine or diazinane.
  • L1 is selected from a single bond, -N(R’)-, -C(O)N(H)-, -N(H)C(O)-, and a divalent moiety of piperidine or diazinane. More preferably, L1 is -N(R’)-.
  • L3 is selected from a single bond, -N(R’)-, -C(O)N(R’)-, -N(R’)C(O)-, - O-, and a 4- to 7-membered heterocyclylene ring. When present, the 4- to 7-membered heterocyclylene ring of L3 typically contains at least one N atom, for instance one or two N atoms.
  • L3 when L3 is a 4- to 7-membered heterocyclylene ring, it is a 5- to 6- membered heterocyclylene ring, more preferably a saturated 5- to 6-membered heterocyclylene ring, for instance a divalent moiety of piperidine or diazinane, preferably 1,4-diazinane.
  • L3 is selected from a single bond, -N(H)-, -C(O)N(H)-, -O-, - N(H)C(O)-, and a divalent moiety of piperidine or diazinane.
  • L3 is selected from -C(O)N(H)-, -N(H)C(O)- and -O-.
  • each L2 is independently a unit of formula described above.
  • each X L is independently selected from a single bond, -O-, - NR’C(O)-, -C(O)NR’-, -NR’- or -S-, and n is selected from 1 or 2.
  • each L2 is the same.
  • LINK may comprise two or more blocks, wherein in each block, each L2 is the same.
  • (L2) m may be represented by the formula -(L2’)p-(L2’’)q-(L2’’’)r-, wherein L2’, L2’’ and L2’’’ are selected from those moieties described above for L2, and wherein each L2’ is the same, each L2’’ is the same and each L2’’’ is the same.
  • L2’, L2’’ and L2’’’ are selected from -CH 2 -, -CH 2 CH 2 O-, - OCH 2 CH 2 -, -CH 2 CH 2 S- and -SCH 2 CH 2 -.
  • L2’, L2’’ and L2’’ are selected from -CH 2 -, -CH 2 CH 2 O- and -OCH 2 CH 2 -.
  • m is selected from 1 to 10, for instance 2 to 8.
  • m is selected from 3 to 6, more preferably 3 to 4.
  • each R’ is independently selected from H and methyl.
  • each R’ is H.
  • LINK comprises or consists of one of the following structures:
  • LINK comprises or consists of one of the following structures: .
  • Protein of interest targeting ligand (POI) The compounds provided herein may be used to selectively target any desired protein of interest. It is within the skill of those in the art to provide ligands for a desired protein of interest. Any suitable method may be used to identify or design ligands for a desired protein of interest. For example, structure-determination methods such as NMR or X-ray crystallography may be used to identify binding sites in a protein of interest. Structures may also be accurately predicted in silico, for example using tools such as AlphaFold (DeepMind) which may be used to identify ligand binding sites. Compounds may be screened, e.g.
  • the protein of interest targeting ligand may be a small molecule ligand that has a molecular weight of less than 1000 Da, for instance less than 900 Da or less than 800 Da.
  • the target protein may be any protein of interest, particularly proteins that are expressed in the cell and are associated with a particular disease.
  • POI is a ligand for a cancer-regulating protein.
  • the protein may, for example, be a bromodomain- containing protein, a histone methyltransferase, a kinase, a phosphorylase, a cytosolic signaling protein, a nuclear protein, a histone deacetylase, a lysine methyltransferase, a protein regulating angiogenesis, a protein regulating immune response, an aryl hydrocarbon receptor, a hormone receptor, or a transcription factor.
  • Protein of interest targeting ligands are known in the art. Substantially any such ligand can be used. The sole limitation on the ligand is that it be capable of binding to a protein of interest.
  • the ligand shows activity or binds to the protein of interest with an IC 50 of less than about 200 mM, e.g. less than about 100 mM, such as less than about 10 mM, less than 1 mM, less than 100 ⁇ M, less than 10 ⁇ M, or less than 1 ⁇ M.
  • the IC 50 can be determined according to any method known in the art, e.g., a fluorescent polarization assay.
  • An illustrative and non-limiting example of the protein of interest targeting ligand may be based on the structure of JQ1, which inhibits the BET bromodomain BRD4.
  • the structure of JQ1 is:
  • the protein of interest targeting ligand may be attached to the linker group LINK at any suitable position.
  • Those skilled in the art are readily able to identify suitable positions on a ligand compound that are amenable to attachment to a linker.
  • POI may be attached to LINK by derivatisation of a carboxylic acid, ester, carbamide, amide, amine, or hydroxy group of POI.
  • POI may have the following structure in which POI is attached to LINK by derivatisation of an ester group on POI: wherein is the point of attachment to LINK.
  • moieties U, HAP, POI and LINK may be taken together in any combination to arrive at a definition of the compound of the present invention.
  • definitions of moieties U, HAP, POI and LINK labelled “typically” may be combined to arrive at a definition of a typical compound of the present invention
  • definitions of moieties U, HAP, POI and LINK labelled “preferably” may be combined to arrive at a definition of a preferred compound of the present invention.
  • - HAP is a hypoxia-activated moiety of formula (I) in which M is -OR 1 or a group selected from morpholino, N-methyl piperidinyl, and 4-methylpiperazin-1-yl; R 1 and R 3 are each independently C 1-4 alkyl which is unsubstituted or substituted by one or two R 5 groups; or a group selected from C 5-6 cycloalkyl, phenyl, a 5- to 6- membered heteroaryl ring and a 5- to 6-membered heterocyclyl ring, the group being unsubstituted or substituted by one or two R 6 groups; R 2 is H, halo or C 1-4 alkyl which is unsubstituted or substituted by one or
  • R U2 group is a single bond to LINK and the other R U2 groups when present are each independently selected from halogen, C 1-4 alkyl, NH 2 , NO 2 , OH, COOH, CN and CF 3 ;
  • X is selected from -CH(R U3 )-, -N(R U3 )-, -O- and -C(O)-;
  • R V is H or a group selected from C 1-4 alkyl, C 3-6 cycloalkyl, phenyl, a 5- to 6- membered heteroaryl ring and a 5- to 6-membered heterocyclyl ring, the group being unsubstituted or substituted by one or two of C 1-4 alkoxy, halo, -OH, -CN, - N(R U3 ) 2 , C 1-4 alkyl, C 3-6
  • - HAP is a hypoxia-activated moiety of formula (I) in which M is -OR 1 ; R 1 and R 3 are each independently unsubstituted C 1-4 alkyl, for instance methyl; R 2 is H or unsubstituted C 1-4 alkyl, more preferably R 2 is H; R 4 is H or unsubstituted C 1-4 alkyl, more preferably R 4 is methyl; each R 5 is independently selected from C 1-4 alkoxy, halo and -OH; each R 6 is independently selected from C 1-4 alkoxy, halo, - OH and unsubstituted C 1-4 alkyl; each R X is independently H; and each R Y is independently H; - the E3 ubiquitin ligase binding moiety (U) is (i) a CRBN E3 ubiquitin ligase binding ligand of formula (U1) in which u is 2; Q is CH and L U is
  • the E3 ubiquitin ligase binding moiety (U) is (i) a CRBN E3 ubiquitin ligase binding ligand having the following structure: wherein is the point of attachment to LINK; or (ii) a VHL E3 ubiquitin ligase binding ligand having the following structure: - LINK comprises or consists of one of the following structures:
  • LINK comprises or consists of one of the following structures: - - POI is a ligand for a cancer-regulating protein, for instance a bromodomain- containing protein, a histone methyltransferase, a kinase, a phosphorylase, a cytosolic signaling protein, a nuclear protein, a histone deacetylase, a lysine methyltransferase, a protein regulating angiogenesis, a protein regulating immune response, an aryl hydrocarbon receptor, a hormone receptor, or a transcription factor.
  • a cancer-regulating protein for instance a bromodomain- containing protein, a histone methyltransferase, a kinase, a phosphorylase, a cytosolic signaling protein, a nuclear protein, a histone deacetylase, a lysine methyltransferase, a protein regulating angiogenesis, a protein regulating immune response, an
  • compositions comprising a compound as provided herein and a pharmaceutically acceptable excipient, carrier or diluent.
  • the composition contains up to 85 wt% of a compound of the invention. More typically, it contains up to 50 wt% of a compound of the invention.
  • Preferred pharmaceutical compositions are sterile and pyrogen free.
  • the carrier may be any suitable carrier known to a person skilled in the art.
  • Carrier proteins include keyhole limpet hemocyanin, serum proteins such as transferrin, bovine serum albumin, human serum albumin, thyroglobulin or ovalbumin, immunoglobulins, or hormones, such as insulin or palmitic acid.
  • the carrier protein may be tetanus toxoid or diphtheria toxoid.
  • the carrier may be a dextran such as sepharose.
  • Auxiliary substances, such as wetting or emulsifying agents, pH buffering substances and the like, may be present in the excipient.
  • excipients and auxiliary substances are generally pharmaceutical agents that do not induce an immune response in the individual receiving the composition, and which may be administered without undue toxicity.
  • Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, polyethyleneglycol, hyaluronic acid, glycerol and ethanol.
  • Pharmaceutically acceptable salts can also be included therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like.
  • compositions include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations.
  • the active ingredient is provided in dry (for e.g., a powder or granules) form for reconstitution with a suitable vehicle (e. g., sterile pyrogen-free water) prior to administration of the reconstituted composition.
  • a suitable vehicle e. g., sterile pyrogen-free water
  • the composition may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution.
  • This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the adjuvants, excipients and auxiliary substances described herein.
  • Such sterile injectable formulations may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1,3-butane diol, for example.
  • a non-toxic parenterally-acceptable diluent or solvent such as water or 1,3-butane diol, for example.
  • Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono-or di-glycerides.
  • Other compositions which are useful include those which comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer systems.
  • compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.
  • the active ingredients of the composition may be encapsulated, adsorbed to, or associated with, particulate carriers.
  • suitable particulate carriers include those derived from polymethyl methacrylate polymers, as well as PLG microparticles derived from poly(lactides) and poly(lactide-co-glycolides). See, e.g., Jeffery et al. (1993) Pharm. Res. 10:362-368.
  • the compounds of the present invention are therapeutically useful. Accordingly, the invention provides a compound as described herein, or a pharmaceutical composition comprising said compound, for use in medicine. Also provided is a compound as described herein, or a pharmaceutical composition comprising said compound for use in treating the human or animal body. As explained above, the compounds provided herein are useful in treating or preventing various disorders, particularly in subjects suffering from a disorder characterised by one or more of the proteins of interest described herein. Cancer, e.g.
  • acute lymphoblastic leukemia acute myeloid leukemia, adrenocortical carcinoma, aids-related lymphoma, primary CNS lymphoma, anal cancer, astrocytomas, brain cancer, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer (e.g.
  • ewing sarcoma ewing sarcoma, osteosarcoma and malignant fibrous histiocytoma
  • breast cancer bronchial tumors, medulloblastoma and other CNS embryonal tumors
  • cervical cancer chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative neoplasms, colorectal cancer, craniopharyngioma, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, ewing sarcoma, extragonadal germ cell tumor, intraocular melanoma, retinoblastoma, fallopian tube cancer, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumors (gist), germ cell tumors, extragonadal germ cell tumors, ovarian germ cell tumors, testicular cancer, gestational trophoblast
  • the compounds and compositions provided herein may be used as standalone therapeutic agents. Alternatively, they may be used in combination with other active agents such as chemotherapeutic agents. For example, they may be used in combination with an EGFR inhibitor (for instance erlotinib, gefitinib, lapatinib or cetuximab), an immunotherapy (for instance pembrolizumab or nivolumab), a tumour-agnostic therapy (for instance larotrectinib) or a chemotherapy (for instance 5-fluorouracil, cisplatin or docetaxel).
  • an EGFR inhibitor for instance erlotinib, gefitinib, lapatinib or cetuximab
  • an immunotherapy for instance pembrolizumab or nivolumab
  • a tumour-agnostic therapy for instance larotrectinib
  • a chemotherapy for instance 5-fluorouracil, cisplatin or docetaxel.
  • the compounds and compositions provided herein
  • treating a cancer may comprise reducing progression of the cancer, e.g. increasing progression free survival. Treating a cancer may comprise preventing or inhibiting growth of a tumour associated with the cancer. Treating a cancer may comprise preventing metastasis of the cancer. Preferably, treating a cancer may comprise reducing the size of a tumour associated with the cancer. As such, the treatment may cause tumour regression in the cancer. Treating a cancer may comprise reducing the number of tumours or lesions present in the patient. When the treatment reduces the size of a tumour associated with the cancer, the size of the tumour is typically reduced from base line by at least 10%. Base line is the size of the tumour at the date treatment with the compound is first started.
  • the size of the tumour is typically as measured in accordance with version 1.1 of the RECIST criteria (for instance as described in Eisenhauer et al., European Journal of Cancer 45 (2009) 228-247).
  • the response to the treatment may be complete response, partial response or stable disease, in accordance with version 1.1 of the RECIST criteria.
  • the response is partial response or complete response.
  • the treatment may achieve progression free survival for at least 60 days, at least 120 days or at least 180 days.
  • the reduction in tumour size may be greater 20%, greater than 30% or greater than 50% reduction relative to base line.
  • the reduction in tumour size may be observed after 30 days of treatment or after 60 days of treatment.
  • the compounds and compositions provided herein are useful in treating or preventing various disorders.
  • the present invention therefore provides compounds and compositions as provided herein for use in medicine.
  • the invention also provides the use of compounds and compositions as provided herein in the manufacture of a medicament.
  • a method of treating a subject in need of such treatment comprising administering to the subject a compound or composition provided herein.
  • the subject suffers from or is at risk of suffering from one of the disorders disclosed herein.
  • the subject suffers from a disorder characterised by a protein of interest as described herein.
  • the subject is a mammal, in particular a human. However, it may be non-human.
  • Preferred non-human animals include, but are not limited to, primates, such as marmosets or monkeys, commercially farmed animals, such as horses, cows, sheep or pigs, and pets, such as dogs, cats, mice, rats, guinea pigs, ferrets, gerbils or hamsters.
  • a subject is typically a human patient. The patient may be male or female. The age of the patient is typically at least 18 years, for instance from 30 to 70 years or from 40 to 60 years.
  • An agent or formulation comprising a compound, or composition of the invention can be administered to the subject in order to prevent the onset or reoccurrence of one or more symptoms of the disorder. This is prophylaxis.
  • the subject can be asymptomatic.
  • a prophylactically effective amount of the agent or formulation may be administered to such a subject.
  • a prophylactically effective amount is an amount which prevents the onset of one or more symptoms of the disorder.
  • An agent or formulation of the invention can be administered to the subject in order to treat one or more symptoms of the disorder.
  • the subject is typically symptomatic.
  • a therapeutically effective amount of the agent or formulation may be administered to such a subject.
  • a therapeutically effective amount is an amount effective to ameliorate one or more symptoms of the disorder.
  • the agent may be administered in a variety of dosage forms. Thus, it can be administered orally, for example as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules.
  • the agent may also be administered parenterally, whether subcutaneously, intravenously, intramuscularly, intrasternally, transdermally or by infusion techniques.
  • the agent may also be administered as a suppository.
  • the agent may be administered parenterally.
  • the agent is typically formulated for administration with a pharmaceutically acceptable carrier or diluent.
  • solid oral forms may contain, together with the active compound, diluents, e.g. lactose, dextrose, saccharose, cellulose, corn starch or potato starch; lubricants, e.g. silica, talc, stearic acid, magnesium or calcium stearate, and/or polyethylene glycols; binding agents; e.g.
  • starches arabic gums, gelatin, methylcellulose, carboxymethylcellulose or polyvinyl pyrrolidone; disaggregating agents, e.g. starch, alginic acid, alginates or sodium starch glycolate; effervescing mixtures; dyestuffs; sweeteners; wetting agents, such as lecithin, polysorbates, laurylsulphates; and, in general, non-toxic and pharmacologically inactive substances used in pharmaceutical formulations.
  • Such pharmaceutical preparations may be manufactured in known manner, for example, by means of mixing, granulating, tableting, sugar coating, or film coating processes.
  • the agent may be formulated for inhaled (aerosolised) administration as a solution or suspension.
  • the agent may be administered by a metered dose inhaler (MDI) or a nebulizer such as an electronic or jet nebulizer.
  • MDI metered dose inhaler
  • a nebulizer such as an electronic or jet nebulizer
  • the agent may be formulated for inhaled administration as a powdered drug, such formulations may be administered from a dry powder inhaler (DPI).
  • DPI dry powder inhaler
  • the agent may be delivered in the form of particles which have a mass median aerodynamic diameter (MMAD) of from 1 to 100 ⁇ m, preferably from 1 to 50 ⁇ m, more preferably from 1 to 20 ⁇ m such as from 3 to 10 ⁇ m, e.g. from 4 to 6 ⁇ m.
  • MMAD mass median aerodynamic diameter
  • Liquid dispersions for oral administration may be syrups, emulsions and suspensions.
  • the syrups may contain as carriers, for example, saccharose or saccharose with glycerine and/or mannitol and/or sorbitol.
  • Suspensions and emulsions may contain as carrier, for example a natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose, or polyvinyl alcohol.
  • the suspension or solutions for intramuscular injections or inhalation may contain, together with the active compound, a pharmaceutically acceptable carrier, e.g. sterile water, olive oil, ethyl oleate, glycols, e.g. propylene glycol, and if desired, a suitable amount of lidocaine hydrochloride.
  • Solutions for inhalation, injection or infusion may contain as carrier, for example, sterile water or preferably they may be in the form of sterile, aqueous, isotonic saline solutions.
  • Pharmaceutical compositions suitable for delivery by needleless injection, for example, transdermally, may also be used. A therapeutically or prophylactically effective amount of the agent is administered to a subject.
  • the dose may be determined according to various parameters, especially according to the compound used; the age, weight and condition of the subject to be treated; the route of administration; and the required regimen. Again, a physician will be able to determine the required route of administration and dosage for any particular subject.
  • a typical daily dose is from about 0.01 to 100 mg per kg, preferably from about 0.1 mg/kg to 50 mg/kg, e.g. from about 1 to 10 mg/kg of body weight, according to the activity of the specific inhibitor, the age, weight and conditions of the subject to be treated, the type and severity of the disease and the frequency and route of administration.
  • daily dosage levels are from 5 mg to 2 g.
  • the dose of the other active substance can be determined as described above.
  • the dose may be determined according to various parameters, especially according to the agent used; the age, weight and condition of the subject to be treated; the route of administration; and the required regimen. Again, a physician will be able to determine the required route of administration and dosage for any particular subject.
  • a typical daily dose is from about 0.01 to 100 mg per kg, preferably from about 0.1 mg/kg to 50 mg/kg, e.g. from about 1 to 10 mg/kg of body weight, according to the activity of the specific agent, the age, weight and conditions of the subject to be treated, the type and severity of the disease and the frequency and route of administration.
  • daily dosage levels are from 5 mg to 2 g.
  • the invention also provides a pharmaceutical composition according to the invention, for use in degrading a therapeutic target protein or a cell comprising a therapeutic target protein in a subject.
  • Systems and methods The invention also provides a method for degrading a target protein of interest in a hypoxic cell, the method comprising: a) introducing a compound as defined herein into the hypoxic cell, thereby cleaving the hypoxia-activated moiety HAP from the rest of the compound to form an activated PROTAC; b) contacting the protein of interest with the activated PROTAC within the hypoxic cell; thereby inducing the degradation of the target protein of interest.
  • the hypoxic cell is a cancer cell.
  • a hypoxic cell is a cell in which the intracellular environment is under hypoxic conditions as defined herein.
  • the compound may be any of the compounds described herein.
  • the target protein of interest is as provided herein.
  • the invention further provides a method of increasing the hypoxia selectivity of a PROTAC comprising a structure of formula: wherein POI, LINK and U are as defined herein, the method comprising chemically modifying said structure to comprise a moiety of formula (I) as defined herein.
  • increasing hypoxia selectivity of a PROTAC comprises reducing the activity of the PROTAC under normoxic conditions while maintaining the activity of the PROTAC under hypoxic conditions.
  • increasing hypoxyia sensitivity comprises reducing the activity of the PROTAC under hypoxic conditions to a lesser extent than the reduction of activity of the PROTAC under normoxic conditions.
  • increasing hypoxia selectivity of a PROTAC comprises maintaining the activity of the PROTAC under normoxic conditions while increasing the activity of the PROTAC under hypoxic conditions.
  • increasing hypoxia sensitivity of a PROTAC comprises increasing the activity of the PROTAC under hypoxic conditions to a greater extent than the increase of activity of the PROTAC under normoxic conditions.
  • the invention further provides a system comprising: (i) a compound as provided herein; (ii) a target protein of interest as defined herein; (iii) an E3 ubiquitin ligase as defined herein; wherein the target protein of interest binds to the protein of interest targeting ligand within the compound, and the E3 ubiquitin ligase binds to the E3 ubiquitin ligase binding ligand within the compound.
  • the invention further provides a kit comprising a compound as provided herein.
  • the kit provides the components required to implement the system of the invention. As a consequence, the kit may allow a user to selectively target under hypoxic conditions a protein for degradation.
  • the kit may comprise instructions for use. The following examples illustrate the invention.
  • HAP-TACs based on VHL-recruiting PROTACs
  • a 2-nitroimidazole bioreductive group attached to hydroxyproline through a carbonate linker (1, NI-VHL, Figure 1A), or an indolequinone group (2, IQ-VHL, Figure 1B) attached directly to the hydroxyproline the VHL ligand VH032.
  • Indolequinone-modified synthetic dyes have been described.
  • Carbonate groups have been used in prodrug design to give an improved PK profile and assist oral delivery.
  • this linker can improve release of the active drug (e.g. tenofovir disoproxil), it can also result in instability in a biological setting.
  • R 2 -Br, TBAI, NaOH, H2O, CH2Cl2, rt, 3 h, 42–56%, n 3 for 33; 3 h, 81% for 34;
  • Enzyme-based assay With the four HAP-TACs (1, 2, 8 and 9) in hand, we next evaluated their hydrolytic stability and their ability to release the active PROTACs in a hypoxia-dependent manner using an enzyme-based assay.
  • Parallel incubations in hypoxia without the reductase enzyme were also performed to assess the hydrolytic stability of the compounds.
  • HAP-TACs were also incubated in normoxia (21% O 2 ) for 24 h.
  • the residual substrate of the tested compounds and the corresponding release of active PROTACs were monitored by HPLC analysis.
  • HAPs which are activated under these conditions tend to also be activated in hypoxic cells.
  • NI-VHL released 82% of MZ1 in normoxia and 100% of MZ1 in hypoxic conditions.
  • MZ1 release (31%) from NI-VHL also occurred in the absence of enzyme, i.e. in the hypoxic control experiment, suggesting that this compound is unstable in the buffer conditions employed ( Figure 4A and 5).
  • IQ-VHL was very stable in normoxic conditions, releasing only 8% of MZ1, while 41% of MZ1 was released in hypoxia.
  • This compound was also stable in the hypoxic control incubation without enzyme present, where no release of MZ1 was observed ( Figure 4A and 6).
  • PG-4c release from NI-CRBN was comparable between the normoxic and hypoxic incubations (10% vs 2%, Figure 4B). Additionally, incubation with the enzyme in either normoxia or hypoxia resulted in multiple peaks observed during HPLC analysis, with little of the parent compound present after 24 h ( Figure 7).
  • HAP-TACs (1, 2, 8 and 9) were next evaluated for their ability to induce BRD4 degradation in A549 cells in hypoxia ( ⁇ 0.1% O 2 ) compared to normoxic (21% O 2 ) conditions.
  • Concentrations of the HAP-TACs were chosen based on the results from the enzyme-based assay (above), and therefore the CRBN-based PROTACs were used at higher concentrations to give a sufficient amount of PROTAC release.
  • NI-VHL (1) had a very narrow window of selectivity at higher concentrations (e.g., 94 ⁇ 3% of BRD4 degradation in normoxia and 95 ⁇ 6% in hypoxia at 1 ⁇ M), or not substantially affecting BRD4 levels in either set of conditions at lower concentrations (17 ⁇ 10% vs 30 ⁇ 10% of degradation at 0.030 ⁇ M in normoxia and hypoxia, respectively) (Figure 10A).
  • IQ-VHL (2) showed little or no degradation of BRD4 in normoxia, but almost complete degradation of BRD4 in hypoxia.
  • nitrobenzyl-based HAP-TACs showed modest EGFR degradation in both normoxia and hypoxia.
  • the nitroimidazole based HAP-TAC showed no EGFR degradation in normoxia, but was less effective at degrading BRD4 in hypoxia than the nitrobenzyl-based HAP-TAC.
  • This result initially seems surprising given that it is well established the nitroimidazole group is more effectively bioreduced in cells than the nitrobenzyl group. 31 However, the result is likely explained by the nitroimidazole group promoting the hydrolysis of the glutarimide ring.
  • IQ-CRBN showed a good selectivity window of concentrations where the HAP-TAC had little or no effect on BRD4 levels in normoxia, but almost completely degraded BRD4 in hypoxia. For example, at 5 ⁇ M, IQ-CRBN degraded 82 ⁇ 16% of BRD4 in hypoxic conditions and only 16 ⁇ 2% of BRD4 in normoxia (Figure 10D).
  • (+)-JQ1-based component of the HAP-TACs affects BRD4 levels when bioreductive groups are attached to the E3 ligase ligand.
  • the two active PROTACs were tested in A549 ( Figure 11). While MZ1 and PG-4c degraded BRD4 (values ranging from 62-100% and 68-91%, respectively), the treatment with (+)-JQ1 alone for 24 h (up to 10 ⁇ M) did not reduce BRD4 levels.
  • BRD4 levels were increased in A549 cells after treatment with (+)-JQ1 at all the tested concentrations (174- 154% of protein levels after 24 h of treatment, Figure 11A and B). This effect might explain the upregulation of BRD4 seen at lower non-degrading concentrations and is consistent with previous reports of BRD4 bromodomain inhibition leading to higher detectable levels of BRD4, perhaps as a result of displacement from chromatin. Given these data we concluded that the modest degradation exhibited by the compounds in normoxia results from a small amount of active PROTAC release. The catalytic nature of some PROTACs means that even a very small amount of PROTAC release can result in some protein degradation, providing an extra challenge compared to the development of HAPs for occupancy-based drugs.
  • HIF-1 ⁇ levels were increased after treatment with the two inhibitors, resulting from further stabilization of this protein in hypoxia (Figure 13C).
  • HCT116 cells were initially treated with MZ1, PG4c and (+)-JQ1 at various concentrations for 24 h.
  • the active PROTACs showed high BRD4 degradation (62-100% for MZ1 and 68-92% for PG-4c of BRD4 degraded), treatment with (+)-JQ1 didn’t affect BRD4 levels (91-95% of remaining protein, Figure 14).
  • the two HAP-TACs showed low degradation of BRD4 in normoxia (19 ⁇ 4% for IQ-VHL and 19 ⁇ 16% for IQ-CRBN) and substantial degradation in hypoxia (79 ⁇ 3% for IQ-VHL and 72 ⁇ 5% for IQ-CRBN, Figure 15A and B), a pattern comparable to the activity showed in A549 cells.
  • Compound cytotoxicity was then determined by MTT assay in HCT116 cells.
  • a washout step was performed after 24 h of incubation with the compounds.
  • hypoxia-activated pro-drugs have been proven to target the release of a cargo drug to hypoxic tumors, in vivo, in a therapeutically relevant manner.
  • HAPs hypoxia-activated pro-drugs
  • HAP-TACs that can degrade target proteins (illustrated here for the proof-of-concept-target BRD4), via recruitment of either CRBN or VHL.
  • HAP-TACs show little effect on BRD4 in normoxia, but exhibit almost complete degradation of this protein in hypoxia.
  • this strategy can, in principle, be applied to any protein that is degraded by an E3 ubiquitin ligase.
  • aliquots were collected and quenched in the hypoxic workstation with 200 ⁇ L of methanol.
  • the samples were removed from the hypoxic workstation and placed on ice before being centrifuged at 13,000 g for 10 min at 4 °C.
  • the supernatants were extracted with a 90:10 mixture of CH 2 Cl 2 :methanol (4 ⁇ 200 ⁇ L), filtered using a syringe filter (Gilson Nylon Syringe Filter, size ⁇ porosity: 13 mm ⁇ 0.45 ⁇ m) and evaporated.
  • the residue was solubilized in 200 ⁇ L of water with 0.5% of DMSO, and 100 ⁇ L were injected and analyzed using LCMS.
  • aliquots were collected and quenched in the hypoxic workstation with 200 ⁇ L of methanol.
  • the samples were removed from the hypoxic workstation and placed on ice before being centrifuged at 13,000 g for 10 min at 4 °C.
  • the supernatants were filtered using a syringe filter (Gilson Nylon Syringe Filter, size ⁇ porosity: 13 mm ⁇ 0.45 ⁇ m), 100 ⁇ L were injected and analyzed using HPLC.
  • NADPH-CYP reductase (11.3 ⁇ L, final concentration 92 pmol/mL) was added to the reaction mixture.
  • aliquots were collected and quenched in the hypoxic workstation with 200 ⁇ L of methanol.
  • the samples were removed from the hypoxic workstation and placed on ice before being centrifuged at 13,000 g for 10 min at 4 °C.
  • the supernatants were filtered using a syringe filter (Gilson Nylon Syringe Filter, size ⁇ porosity: 13 mm ⁇ 0.45 ⁇ m), 100 ⁇ L were injected and analyzed using HPLC.
  • hypoxia control HPLC assay To a 1.5 mL Eppendorf tube in the hypoxic workstation, MilliQ water (756 ⁇ L), phosphate buffer (0.5 M, pH 7.4, 240 ⁇ L), absolute ethanol (120 ⁇ L), regenerating solution A (60 ⁇ L), regenerating solution B (12 ⁇ L), and a 2 mM DMSO solution of compound (12 ⁇ L, final concentration 20 ⁇ M) were added. The mixture was left in the hypoxic workstation and at selected timepoints aliquots (200 ⁇ L) were collected from the reaction solution and quenched with 200 ⁇ L of methanol.
  • the samples were removed from the hypoxic workstation and placed on ice before being centrifuged at 13,000 g for 10 min at 4 °C.
  • the supernatants were filtered using a syringe filter (Gilson Nylon Syringe Filter, size ⁇ porosity: 13 mm ⁇ 0.45 ⁇ m), 100 ⁇ L were injected and analyzed using HPLC.
  • Cell Lines and Reagents Colorectal HCT116 and lung adenocarcinoma A549 cells were grown in Dulbecco's Modified Eagle Medium (DMEM) with high glucose, GlutaMAXTM and pyruvate supplemented with 1% penstrep and 10% FBS.
  • DMEM Dulbecco's Modified Eagle Medium
  • Cells were maintained in an incubator set at 37 °C and 5% CO 2 . All cell lines were routinely tested for mycoplasma using MycoAlert Detection Kit (Lonza). Normoxic Treatments Cells were seeded on plastic dishes and allowed to settle overnight at 37 °C and 5% CO2. Cells were treated with the tested compounds in 1% DMSO at the indicated concentrations and incubated at 37 °C and 5% CO 2 for 24 h. Cells were harvest and lysed in UTB buffer (9M urea, 75 mM Tris-HCl, pH 7.5, 0.1M ⁇ -mercaptoethanol).
  • Hypoxic Treatments Hypoxia treatments at ⁇ 0.1% O 2 were carried out in a Bactron II anaerobic chamber (Shel Labs) while all oxygen concentrations from 0.5-2% O 2 were carried out in a M35 variable atmosphere workstation (Don Whitley Scientific). Oxygen concentrations were periodically validated using anaerobic oxygen indicator strips (ThermoFisher). Cells were seeded on glass dishes and allowed to settle overnight. Then, they were placed inside the chamber with equilibrated solutions for 4 h, before treatment with the tested compounds in 1% DMSO at the indicated concentrations.
  • cells were pre-treated with either the proteasome or the neddylation inhibitor for 4 h inside the chamber, then treated with the tested compounds in 1% DMSO at the indicated concentrations. After the time required for the experiment, cells were harvest and lysed in UTB buffer while maintained inside the chamber. MTT assay HCT116 cells were seeded at 6,000 cells/well in 96-well plates and allowed to adhere overnight. Cells were treated with the tested compounds in 1% DMSO at the indicated concentration for 24 hours in hypoxia or normoxia, then media was removed and replaced with fresh, compound-free, media.
  • Cells were treated with the tested compounds in 1% DMSO at the indicated concentration for 24 hours in hypoxia or normoxia, then media was removed and replaced with fresh, compound-free, media. Cells were then incubated in normoxia. At endpoints, cells were suspended and mixed with trypan blue at 1:1 before proceeding to cell count using a haemocytometer. Means from three technical replicates of cell counts were calculated and three biological replicates were plotted with s.e.m. Immunoblotting Cells lysates were sonicated (3 ⁇ 10 sec bursts, 10 sec pause on ice) on a FB-505 sonic dismembrator with a 1/16” tapered microtip at 40% maximum power.
  • the lysates were centrifuged (4 °C, 16,000 ⁇ g, 10 min) and the supernatants were transferred to a fresh tube. Protein concentrations were measured using a PierceTM BCA Protein Assay Kit (Thermo Fisher Scientific). Proteins were diluted in MilliQ water and 10% ⁇ - mercaptoethanol in Laemmli sample buffer (Bio-Rad), and denatured at 100 °C for 5 min, then separated by SDS-PAGE (Mini-PROTEAN TGX gels, Bio-Rad, 8-20%) and compared to the SpectraTM Multicolor High Range Protein Ladder (Thermo Fisher Scientific).
  • Membranes were incubated with primary antibodies overnight at 4 °C, washed with TBS-T (3 ⁇ ), incubated with HRP-conjugated secondary antibodies (Promega UK) for 1 h at rt, washed with TBS-T (3 ⁇ ) and imaged with Clarity western ECL substrate (Bio-Rad). Membranes were scanned with a Bio-Rad ChemiDoc XRS+ Imaging System and signal intensities were quantified using Image Lab software (Bio-Rad). Band densitometry was assessed, normalized to ⁇ -actin bands, and reported as percentage of the DMSO control lane.
  • each signal is indicated by s (singlet), br s (broad singlet), d (doublet), t (triplet), br t (broad triplet), q (quartet), quint (quintet), dd (doublet of doublets), dt (doublet of triplets), m (multiplet).
  • the number of protons, n, for a given resonance signal is indicated by nH.
  • Coupling constants (J) are expressed in Hz and are recorded to the nearest 0.1 Hz. Identical proton coupling constants (J) are averaged in each spectrum and reported to the nearest 0.1 Hz. MestReNova and TopSpin software were used for NMR analysis.
  • Spectra were assigned using COSY, HSQC, and HMBC experiments as necessary.
  • Bruker TopSpin software was used to plot the spectra.
  • 1 3 C NMR spectra were recorded using a Bruker AVH400 (101 MHz) or Bruker NEO600 (151 MHz) spectrometers in the stated solvents, with broadband proton decoupling using the stated solvent as a reference for internal deuterium lock.
  • the chemical shift data for each signal are quoted as ⁇ C in parts per million (ppm) relative to the tetramethyl silane (TMS) where ⁇ H (TMS) is 0.00 ppm.
  • the shift values of resonances are quoted to 1 decimal place and were determined using MestReNova and TopSpin software.
  • Spectra were assigned using COSY, HSQC, and HMBC experiments as necessary.
  • Bruker TopSpin software was used to plot the spectra. Mass spectra were recorded using either an Agilent 6120 (low resolution) or a Bruker microToF (high resolution) spectrometer using electrospray ionisation (ESI). Samples were submitted as solutions in either methanol or acetonitrile. m/z values are given in Daltons (Da) and followed by their percentage abundance in parentheses. Melting points were obtained using a Griffin capillary tube melting apparatus and are uncorrected. The crystallisation solvent is given in parentheses. Infrared (IR) spectra were obtained from neat samples.
  • the spectra were recorded using a Bruker Tensor 27 spectrometer with a diamond ATR module. Absorption maxima are given in wavenumbers (cm ⁇ 1 ) and reported as s (strong), m (medium), or w (weak). Analytical thin layer chromatography (TLC) was carried out on normal phase Merck silica gel 60 F 254 aluminium-supported thin layer chromatography sheets. Visualisation was done by absorption of UV light ( ⁇ max 254 nm), or thermal development after staining in an aqueous solution of potassium permanganate. UV light was provided by a LF – 206 LS 230V – 50 Hz from UVltec Limited.
  • Flash column chromatography was carried out using Geduran® silica gel 60 (40– 63 ⁇ m), eluting using solvents as supplied under a positive pressure of nitrogen.
  • Analytical HPLC was carried out on an Agilent 1260 Infinity II® system with a quaternary LC pump and UV/vis LC detector.
  • the mixture was gradually acidified to pH 5 with 2M aqueous HCl solution (10 mL) and concentrated in vacuo to give a mostly aqueous solution.
  • the product was extracted with ethyl acetate (6 ⁇ 20 mL), then the aqueous phase was saturated with solid NaCl and the product extracted with EtOAc (6 ⁇ 20 mL).
  • the combined organic layers were dried over sodium sulfate, filtered and concentrated in vacuo.
  • the crude material was purified using silica gel column chromatography, eluting with PE:EtOAc (gradient 30 to 100% EtOAc) to afford a pale yellow solid (824 mg, 40%): R f 0.47 (EtOAc 100%); m.p.
  • Step 1 A solution of fuming nitric acid (11.3 mL, 279.90 mmol, 22.5 eq.) in acetic acid (46 mL) was added dropwise over 1 h to a solution of 5-methoxy-1,2-dimethyl-1H-indole- 3-carbaldehyde 20 (2.53 g, 12.44 mmol, 1.0 eq.) in acetic acid (290 mL) at 0 °C.
  • Step 2 The intermediate obtained in Step 1 (1.94 g, 7.82 mmol, 1.0 eq.) was solubilized in ethanol (146 mL), and powdered tin (4.87 g, 41.05 mmol, 5.25 eq.) and 3M aqueous HCl solution (60 mL) were added in order. The reaction mixture was heated to 80 °C for 1 h. Then, water (400 mL) was added, and the pH adjusted to 8 with solid NaHCO 3 .
  • Step 2 The obtained intermediate (5.69 g, 15.87 mmol, 1.0 eq.) was solubilized in THF (80 mL) and a solution of LiOH (3.81 g, 158.75 mmol, 10.0 eq.) in H 2 O (70 mL) was slowly added. The mixture was stirred at rt for 16 h, then THF was removed in vacuo and ice (50 g) was added. The pH was adjusted to 2-3 with 6M aqueous HCl solution (10 mL) and a colourless precipitate was formed.
  • Step 2 Compound 27 (2.00 g, 5.80 mmol, 1.0 eq.) was solubilized in dry DMF (20 mL) and DIPEA (6.06 mL, 34.80 mmol, 6.0 eq.) and HATU (2.65 g, 6.96 mmol, 1.2 eq.) were added under nitrogen. After 10 min under stirring at rt, the intermediate (5.80 mmol, 1.0 eq.) obtained in Step 1 was added. The reaction was stirred at rt overnight.
  • Step 2 The intermediate obtained in Step 1 (1.07 mmol, 1.0 eq.) was added to a stirring solution of 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)acetic acid (249 mg, 1.07 mmol, 1.0 eq.), HATU (813 mg, 2.14 mmol, 2.0 eq.) and DIPEA (1.12 mL, 6.42 mmol, 6.0 eq.) in dry DMF (7.8 mL).
  • Step 2 The obtained intermediate was added to a stirring solution of DIPEA (104 ⁇ L, 0.60 mmol, 4.0 eq.), HATU (68 mg, 0.18 mmol, 1.2 eq.) and (+)-JQ1-COOH (54 mg, 0.14 mmol, 0.9 eq.) in dry DMF (2 mL) under argon. The reaction was stirred at rt overnight.
  • Step 2 (+)-JQ1-COOH (18 mg, 0.05 mmol, 0.9 eq.) was solubilized in dry DMF (0.5 mL) and DIPEA (35 ⁇ L, 0.20 mmol, 4.0 eq.) and HATU (23 mg, 0.06 mmol, 1.2 eq.) were added in order under argon.
  • the crude material was purified using silica gel column chromatography, eluting with EtOAc:CH 3 OH (gradient 0 to 15% CH 3 OH) to yield the title compound as a colourless solid (87 mg, 72%).
  • the compound was further purified using semi prep HPLC, method A: R f 0.17 (EtOAc/CH 3 OH 85:15); m.p.
  • 3-Nitrophthalic anhydride (3.66 g, 18.96 mmol, 1.2 eq.) was solubilized in acetic acid (50 mL) and sodium acetate (1.55 g, 18.96 mmol, 1.2 eq.) and 3-aminopiperidine-2,6- dione hydrogen chloride (2.60 g, 15.80 mmol, 1.0 eq.) were added sequentially. The resulting mixture was heated at 120 °C for 7 h. The reaction mixture was then cooled to rt and concentrated in vacuo.
  • 2,6-Bis(benzyloxy)pyridine (39) Benzyl alcohol (2.11 mL, 20.28 mmol, 3.0 eq.) was solubilized in dry DMF (27 mL) and the solution was cooled to 0 °C under a stream of argon. NaH (60% mineral oil, 1.08 g, 27.04 mmol, 4.0 eq.) was added portionwise and, once gas evolution had ceased, the reaction mixture was warmed to rt and stirred for 30 min. 2,6-Dichloropyridine (1.00 g, 6.76 mmol, 1.0 eq.) was then added and the reaction mixture was heated at 80 °C for 16 h.
  • Step 2 The obtained intermediate (0.11 mmol, 1.0 eq.) was solubilized in dry DMF (1.2 mL) and DIPEA (100 ⁇ L, 0.57 mmol, 5.0 eq.), HATU (87 mg, 0.23 mmol, 2.0 eq.) and (+)-JQ1-COOH (55 mg, 0.14 mmol, 1.2 eq.) were added in order under argon. The reaction was stirred at rt overnight.
  • Step 2 The obtained intermediate (0.06 mmol, 1.0 eq.) was added to a solution of (+)-JQ1- COOH (26 mg, 0.07 mmol, 1.1 eq.), DIPEA (63 ⁇ L, 0.36 mmol, 6.0 eq.) and HATU (46 mg, 0.12 mmol, 2.0 eq.) in dry DMF (1 mL) under argon.
  • Step 2 The obtained intermediate (0.05 mmol, 1.0 eq.) was added to a solution of (+)-JQ1- COOH (20 mg, 0.05 mmol, 1.1 eq.), DIPEA (52 ⁇ L, 0.30 mmol, 6.0 eq.) and HATU (38 mg, 0.1 mmol, 2.0 eq.) in dry DMF (1 mL) under argon.
  • Step 2 The obtained intermediate (0.07 mmol, 1.0 eq.) was added to a solution of (+)-JQ1- COOH (31 mg, 0.08 mmol, 1.1 eq.), DIPEA (73 ⁇ L, 0.42 mmol, 6.0 eq.) and HATU (53 mg, 0.14 mmol, 2.0 eq.) in dry DMF (1 mL) under argon.
  • Bn benzyl; CRBN, cereblon; DIPEA, N,N-diisopropylethylamine; DIAD, diisopropyl azodicarboxylate; DMAP, 4-dimethylaminopyridine; DMA, dimethylacetamide; DMEM, Dulbecco's Modified Eagle Medium; DMF, dimethylformamide; DMSO, dimethyl sulfoxide; EtOAc, ethyl acetate; EtOH, ethanol; FBS, fetal bovine serum; HAP-TAC, hypoxia-activated PROTAC; HATU, 1- [bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate; HPLC, high-performance liquid chromatography; HRMS, high resolution mass spectrometry; IQ, indolequinone; IR, infrared; (+)-J

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Abstract

La présente invention concerne de nouveaux composés et procédés. L'invention concerne en particulier un composé qui trouve une utilisation dans le traitement du cancer par dégradation sélective dans des conditions hypoxiques d'une protéine cible thérapeutique d'intérêt. Le composé est une chimère ciblant la protéolyse activée par l'hypoxie (PROTAC activé par l'hypoxie ; HAP-TAC).
PCT/GB2024/052383 2023-09-15 2024-09-13 Composé et procédé Pending WO2025056913A1 (fr)

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