Compound and method

Abstract

The present invention relates to novel compounds and methods. 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. The compound is a hypoxia-activated proteolysis targeting chimera (hypoxia-activated PROTAC; HAP-TAC).

Description

[0001] COMPOUND AND METHOD Field of the Invention 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. Background of the Invention Proteolysis targeting chimeras (PROTACs) 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. Additionally, off-target toxicities might result from the presence of both the POI ligand and the E3 ubiquitin ligase ligand, meaning that both can act independently as occupancy- based inhibitors. For example, it has been previously shown that PROTACs based on immunomodulatory drugs (IMIDs) can maintain the ability to degrade the endogenous cereblon (CRBN) neosubstrates 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. To overcome these issues, strategies to develop pro-drugs of PROTACs which can be released selectively in the desired context or environment have been developed. One of the earliest approaches to achieve targeted protein degradation was the development of light-activated PROTACs. Attaching a photocleavable group to VHL or CRBN E3 ubiquitin ligase ligands allows spatial control of the release of the active degrader. However, this approach has clear limitations in therapeutic settings resulting from the poor tissue penetration of short wavelength light. Expanding on this concept, some efforts have been made to develop radiotherapy-triggered VHL-recruiting PROTACs, in which E3 ubiquitin ligase recruiter is unmasked upon X-ray irradiation of a tetrafluoro-phenyl azide group. Other strategies have harnessed components of the tumor microenvironment to achieve selective protein degradation, including folate-caged E3 ligase ubiquitin ligands and ROS-responsive PROTACs. Hypoxia-activated pro-drugs (HAPs) 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. Attempts have been made to develop distinct 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. However, this strategy did not exclude the unwanted release of the active PROTAC in normoxia. Summary of the Invention The inventors have devised a novel principle of general application which involves chemically modifying a PROTAC to increase its selectivity to hypoxic conditions. Advantageously, 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. An advantage to this approach is that the indolequinone group can be used to protect a number of different E3 ubiquitin ligase ligands, including the CRBN or VHL recruiting ligands, rendering them inactive in normoxia. Moreover, 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. reduced off-target effects and / or improved targeting, and, in therapeutic applications, may provide for reduced concentrations of therapeutic agents to be required in order to exert a desired therapeutic outcome, with benefits in terms of reduced toxicity, decreased costs, and / or improved patient compliance. Accordingly, provided herein is a compound which is a Hypoxia-Actived Proteolysis Targeting Chimera (HAP-TAC) of Formula A or a pharmaceutically acceptable salt thereof: [Formula A] in which: - 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; and - HAP is a hypoxia-activated moiety of formula (I): [Formula I] wherein: - M is -OR1or a 5- to 6-membered heterocyclyl ring which is unsubstituted or is substituted by 1 or 2 RYgroups; - each of R1, R2, R3and R4are independently C1-4alkyl which is unsubstituted or substituted by one, two or three R5groups; H; halo; or a group selected from C3-6cycloalkyl, 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 R6groups; - each R5is independently selected from C1-4alkoxy, halo, -OH, -CN and -N(RY)2; - each R6is independently selected from C1-4alkoxy, halo, -OH, -CN, -N(RY)2, and C1-4alkyl which is unsubstituted or substituted by one, two or three R5groups; - each RXis independently selected from H, methyl and 2-thienyl; - each RYis independently selected from H and C1-4alkyl; and - the point of attachment to U. In some embodiments, 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(O2)N(R’)-, -N(R’)S(O2)-, -(C1-6alkylene)-, -(C2-6alkenylene)-, 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 cycloalkylene, 6-membered arylene, 4- to 7-membered heterocyclylene and 4- to 7-membered heteroarylene; - each XLis independently selected from a single bond, -N(R’)-, -O-, -C(O)-, -C(O)N(R’)-, -N(R’)C(O)-, -S-, -SO-, -SO2-, -C(R’)=C(R’)- and -C≡C-; - each R’ is independently selected from H and C1-4alkyl; - each n is independently selected from 0 to 4; and - m is selected from 1 to 10. In some embodiments, U is a CRBN E3 ubiquitin ligase binding ligand of formula (U1): [Formula U1] wherein: - u is 1 or 2 - Q is N and LUis a single bond, or Q is CH and LUis selected from a single bond, - C(O)N(Rz)- and -N(Rz)C(O)-; wherein each Rzis independently H or C1-4 alkyl; - is the point of attachment to HAP; - RU1is 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 RU2group is a single bond to LINK and the other RU2groups when present are each independently selected from halogen, C1-4alkyl, NH2, NO2, OH, COOH, CN and CF3; - X is selected from -CH(RU3)-, -N(RU3)-, -O-, -C(O)-, -NH-CH(RU3)-, -N=C(RU3)-, and -N=N-; - RVis H or a group selected from C1-4alkyl, C3-6cycloalkyl, 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 C1-4alkoxy, halo, -OH, -CN, - N(RU3)2, and C1-4alkyl; and - RU3is H or C1-4alkyl; - the point of attachment to LU. In some embodiments, U is a VHL E3 ubiquitin ligase binding ligand of formula (U2): wherein: - RU4is 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 RU4being unsubstituted or substituted by C1-4alkyl; - RU5is H or C1-4 alkyl; - RU6is C1-4alkyl, phenyl or a 3- to 8-membered cycloalkyl ring; - RU7is a single bond to LINK; and - the point of attachment to HAP. In some embodiments, U is a DCAF15 E3 ubiquitin ligase binding ligand of formula (U3): wherein: - z is selected from 1 to 5; - one RU9group is a single bond to LINK and the other RU9groups when present are each independently selected from halogen, C1-4alkyl, NH2, NO2, OH, COOH, CN and CF3; - RU10is H or C1-4alkyl; and - attachment to HAP. In some embodiments, 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, the method 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. Also provided is a method of increasing the hypoxia selectivity of a PROTAC comprising a structure of formula: wherein POI, LINK and U are as described herein, the method comprising chemically modifying said structure to comprise a moiety of formula (I) as described herein. Brief Description of the Figures 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. (A) Enzymatic reduction of NI-pomalidomide with NADPH-CYP reductase. NI-pomalidomide was incubated with NADPH-CYP reductase and NADPH regenerating solutions A and B in phosphate buffer over 24 h in hypoxia at 37 °C. Aliquots were taken at T = 0, 3, 7 and 24 h, processed and analyzed by LCMS as described in the general procedure for the LCMS enzyme-based assay. One representative HPLC trace of three independent experiments is reported. Absorbance was recorded at 220 nm. For the sake of clarity, the injection peak was omitted and the chromatogram was reported after 2 min from the injection. FIGURE 4. Release of active PROTAC after 24 h of incubation with either NI- VHL (A), IQ-VHL (A), NI-CRBN (B) or IQ-CRBN (B) in normoxia (21% O2, red bars) and hypoxia (<0.1% O2, blue bars) with NADPH-CYP reductase or in hypoxia in the absence of the enzyme (control, light blue bars). Data are the mean of three independent experiments ± s.e.m. The release of active PROTAC was determined using a calibration curve. Time-course experiment in hypoxia (<0.1% O2, blue lines) in the presence of NADPH-CYP reductase for compounds NI-VHL (C), NI-CRBN (D) IQ-VHL (E), or IQ- CRBN (F) versus released of active PROTAC in normoxia (21% O2, red lines). Data are the mean of three independent experiments ± s.e.m. FIGURE 5. NI-VHL undergoes oxygen- and enzyme-dependent reduction to the corresponding active PROTAC MZ1. (A) Enzymatic reduction of NI-VHL using NADPH- CYP reductase. (B) NI-VHL was incubated with NADPH-CYP reductase over 24 h at 37 °C in normoxia and hypoxia. Additionally, the compound was incubated in hypoxia in the absence of the NADPH-CYP reductase (hypoxia control). Aliquots were taken at T = 0 and 24 h, processed and analyzed by HPLC, as described in the general procedure for the HPLC enzyme-based assay. One representative HPLC trace of three independent experiments is reported. Only the hypoxia T = 0 is shown in the figure. Absorbance was recorded at 254 nm. For the sake of clarity, the injection peak was omitted and the chromatogram was reported after 2 min from the injection. FIGURE 6. IQ-VHL undergoes oxygen- and enzyme-dependent reduction to the corresponding active PROTAC MZ1. (A) Enzymatic reduction of IQ-VHL using NADPH- CYP reductase. (B) IQ-VHL was incubated with NADPH-CYP over 24 h at 37 °C in normoxia and hypoxia. Additionally, the compound was incubated in hypoxia in the absence of the NADPH-CYP reductase (hypoxia control). Aliquots were taken at T = 0 and 24 h, processed and analyzed by HPLC, as described in the general procedure for the HPLC enzyme-based assay. One representative HPLC trace of three independent experiments is reported. Only the hypoxia T = 0 is shown in the figure. Absorbance was recorded at 254 nm. For the sake of clarity, the injection peak was omitted, and the chromatogram was reported after 2 min from the injection. FIGURE 7. 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). Aliquots were taken at T = 0 and 24 h, processed and analyzed by HPLC, as described in the general procedure for the HPLC enzyme-based assay. One representative HPLC trace of three independent experiments is reported. Only the hypoxia T = 0 is shown in the figure. Absorbance was recorded at 254 nm. For the sake of clarity, the injection peak was omitted, and the chromatogram was reported after 2 min from the injection. FIGURE 8. IQ-CRBN undergoes oxygen- and enzyme-dependent reduction to the corresponding active PROTAC PG-4c. (A) Enzymatic reduction of IQ-CRBN using NADPH-CYP reductase. (B) IQ-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). Aliquots were taken at T = 0 and 24 h, processed and analyzed by HPLC, as described in the general procedure for the HPLC enzyme-based assay. One representative HPLC trace of three independent experiments is reported. Only the hypoxia T = 0 is shown in the figure. Absorbance was recorded at 254 nm. For the sake of clarity, the injection peak was omitted, and the chromatogram was reported after 2 min from the injection. FIGURE 9. 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. 20 μM of compounds MZ1 (A), PG-4c (B), Bn-VHL (C) and Bn-CRBN (D) were incubated in hypoxia with (hypoxia) or without (hypoxia control) NADPH-CYP reductase over 24 h. Aliquots were taken at T = 0 and 24 h, processed and analyzed by HPLC, as described in the general procedure for the HPLC enzyme-based assay. One representative HPLC trace of three independent experiments is reported. For each compound, only the hypoxia T = 0 is shown in the figure. Absorbance was recorded at 254 nm. For the sake of clarity, the injection peak was omitted, and the chromatogram was reported after 2 min from the injection. 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% O2, red lines) or hypoxia (<0.1% O2, blue lines). Data are the mean of three independent experiments ± s.e.m. Representative western blot analysis of the data shown in panels A-D in normoxia (top, E) and hypoxia (bottom, F), with HIF-1α used as hypoxia biomarker and β-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 β-actin used as loading control. FIGURE 12. (A). Percentage of BRD4 levels compared to control, assessed using western blot analysis, in A549 cells after 24 h of incubation with IQ-VHL (0.25 µM, violet bars) or IQ-CRBN (3 µM, green bars) at the indicated levels of O2.Data are the mean of three independent experiments ± s.e.m. (B). Representative western blot analysis of the data shown in panel A, with HIF-1α used as hypoxia biomarker and β-actin used as a loading control. FIGURE 13. (A). 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 µM, violet bars) and IQ-CRBN (3 µM, 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-1α used as hypoxia biomarker and β-actin as loading control. (C). Representative western blot analysis of three independent experiments in A549 cells after 24 h of incubation with IQ-VHL (0.25 µM) and IQ-CRBN (3 µM) with or without the proteasome inhibitor carfilzomib (0.5 µM), or the neddylation inhibitor MLN-4924 (1 µM) in hypoxia (<0.1% O2). HIF-1α is used as hypoxia biomarker, β-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. Data are the mean of three independent experiments ± s.e.m. (B) Representative western blot analysis of the data showed in panel A with β-actin used as loading control. 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 µM) or IQ-CRBN (3 µM) for 24 h in normoxia (21% O2) 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-1α is used as hypoxia biomarker and β-actin is a loading control. FIGURE 16. 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% O2, red line) or hypoxia (<0.1% O2, 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 µM) in normoxia or IQ-VHL (0.25 µM) in normoxia or hypoxia over 6 days. Cell number is expressed as percentage related to DMSO control, respectively in normoxia or hypoxia. Data are the mean of three independent experiments ± s.e.m. (B). Growth over time of HCT116 cells after 24 h of incubation with PG4c (3 µM) in normoxia or IQ-CRBN (3 µM) in normoxia or hypoxia over 6 days. Cell number is expressed as percentage related to DMSO control, respectively in normoxia or hypoxia. Data are the mean of three independent experiments ± s.e.m. Detailed Description The present invention will be described with respect to particular embodiments and with reference to certain drawings but the disclosure is not limited thereto but only by the claims. Of course, it is to be understood that not necessarily all aspects or advantages may be achieved in accordance with any particular embodiment. Thus, for example those skilled in the art will recognize that the disclosed embodiments may be embodied or carried out in a manner that achieves or optimizes one advantage or group of advantages as taught herein without necessarily achieving other aspects or advantages as may be taught or suggested herein. The disclosure, both as to organization and method of operation, together with features and advantages thereof, may best be understood by reference to the following detailed description when read in conjunction with the accompanying drawings. The aspects and advantages of the invention will be apparent from and elucidated with reference to the embodiment(s) described hereinafter. Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one disclosed embodiment. Thus, appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Similarly, it should be appreciated that in the description of exemplary disclosed embodiments, various features are sometimes grouped together in a single embodiment, figure, or description thereof for the purpose of streamlining the disclosure and aiding in the understanding of one or more of the various inventive aspects. This method of disclosure, however, is not to be interpreted as reflecting an intention that the claimed invention requires more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive aspects lie in less than all features of a single foregoing disclosed embodiment. It should be appreciated that “embodiments” of the disclosure can be specifically combined together unless the context indicates otherwise. The specific combinations of all disclosed embodiments (unless implied otherwise by the context) are further disclosed embodiments of the claimed invention. In addition as used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a protein” includes two or more proteins, and the like. "About" as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ± 20 % or ± 10 %, more preferably ± 5 %, even more preferably ± 1 %, and still more preferably ± 0.1 % from the specified value, as such variations are appropriate to perform the disclosed methods. 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. The terms “hypoxia” and “hypoxic” are used to describe a state of insufficient oxygen concentration that can be present in tumors, normal tissues and wounds. Normal oxygen levels, described as “normoxia,” are defined as 21 % oxygen. Oxygenation in untreated tumors is significantly lower with median oxygen levels of <2% and typically a range of <0.1% to 4.2% oxygen. Thus, hypoxia may be defined as a tissue oxygen concentration of 4.2% or less, for instance 4% or less, or 3% or less. Preferably, 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. As used herein, a C1-4alkyl group is a linear or branched alkyl group containing from 1 to 4 carbon atoms. A C1-4alkyl group is often a C1-3alkyl group or a C1-2alkyl group. Examples of C1-4alkyl groups include methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, and tert-butyl. A C1-2alkyl group is methyl or ethyl, typically methyl. For the avoidance of doubt, where two alkyl groups are present, the alkyl groups may be the same or different. As used herein, a C1-4alkoxy group is typically a said C1-4alkyl group which is joined to the rest of the molecule via an oxygen atom. Typically, a C1-4alkoxy group is a C1-3 alkoxy group. Examples of C1-4 alkoxy groups include methoxy, ethoxy, propoxy and butoxy. Typically, a C1-3alkoxy group is a C1-2alkoxy group such as a methoxy or ethoxy group. For the avoidance of doubt, where two alkoxy groups are present, the alkoxy groups may be the same or different. As used herein, a C1-6alkylene group is a linear or branched divalent alkyl group that contains from 1 to 6 carbon atoms and is preferably linear. A C1-6alkylene group is typically a C1-4alkylene group or a C1-3alkylene group. Examples of C1-4alkylene groups include methylene, ethylene, n-propylene, iso-propylene, n-butylene, sec-butylene, and tert-butylene. A C1-3alkylene group is typically a C1-2alkylene group. A C1-2alkylene group is methylene or ethylene, typically methylene. For the avoidance of doubt, where multiple alkylene groups are present, the alkylene groups may be the same or different. As used herein, a C2-6alkenylene 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. Typically a C2-6alkenylene group is a C2-4alkenylene group. Examples of C2-4alkenylene groups include divalent ethenylene, propenylene and butenylene. For the avoidance of doubt, where multiple alkenylene groups are present, 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. As used herein, a halogen is typically chlorine, fluorine, bromine or iodine and is preferably chlorine, bromine or fluorine, especially chorine or fluorine, and most especially fluorine. Where a group or moiety is substituted with one or more halogen atoms, preferably it carries one, two, three or four halogen atoms, preferably one, two or three halogen atoms. Where a group or moiety is substituted with two or more halogen atoms, the halogen atoms may be the same or different. Typically, the halogen atoms are the same. As used herein 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. Examples include monocyclic rings such as phenyl and fused bicyclic rings such as naphthyl and indenyl. Phenyl (benzene) is preferred. As used herein, 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. Examples of 5- and 6- membered heteroaryl rings include pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyridine, pyridazine, pyrimidine, and pyrazine. As used herein, 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 C3-6cycloalkyl may be saturated or partially unsaturated, but is typically saturated. A partially unsaturated C3-6cycloalkyl is a cyclic hydrocarbon containing from 3 to 6 carbon atoms and containing 1 or 2, e.g. 1 double bond. A C3-6cycloalkyl is typically a C5-6cycloalkyl. Examples of saturated C5-6cycloalkyls include cyclopentyl and cyclohexyl groups. Examples of 5- to 6- membered saturated heterocyclic rings include piperazine, piperidine, morpholine, pyrrolidine, imidazolidine, and oxazolidine. Examples of 5- to 6- membered partially unsaturated heterocyclic rings include tetrahydropyrazine, tetrahydropyridine, dihydro-1,4-oxazine, tetrahydropyrimidine, dihydro-1,3-oxazine, dihydropyrrole, dihydroimidazole and dihydrooxazole. As used herein, 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. As used herein, 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 C3-6cycloalkylene is a divalent cyclic hydrocarbon containing from 3 to 6 carbon atoms. A C3-6cycloalkylene may be saturated or partially unsaturated, but is typically saturated. A partially unsaturated C3-6cycloalkylene is a divalent cyclic hydrocarbon containing from 3 to 6 carbon atoms and containing 1 or 2, e.g. 1 double bond. A C3-6cycloalkylene is typically a C5-6cycloalkylene. An aryl, heterocyclyl, heteroaryl, cycloalkyl, heterocyclylene, heteroarylene, or cycloalkylene ring may be unsubstituted or substituted as described herein. For example, said ring may be unsubstituted or substituted with 1, 2 or 3, typically 1 or 2 such as e.g. 1 substituent. Unless otherwise stated, 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. As used herein, the terms “monovalent” or “monovalent moiety” are used to describe a chemical group obtainable by removing a hydrogen atom from the corresponding compound. As used herein, the terms “divalent” or “divalent moiety” are used to describe a chemical group obtainable by removing a hydrogen atom from the corresponding monovalent moiety. Thus, as used herein, 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. All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety. Compounds The compounds provided herein comprise a hypoxia-activated moiety (HAP) of formula (I) that is covalently attached to the E3 ubiquitin ligase binding ligand part (U) of a PROTAC. 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). 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). 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. Thus, provided herein is a compound which is a Hypoxia-Actived Proteolysis Targeting Chimera (HAP-TAC) of Formula A or a pharmaceutically acceptable salt thereof: [Formula A] in which 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, and HAP is a hypoxia-activated moiety of formula (I) as defined herein. In the provided compounds, 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. As those skilled in the art will readily appreciate, methods for preparation of compounds (in which two discrete chemical entities contribute discrete biological functions to the overall compound) 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 (HAP) In the provided compounds, the hypoxia-activated moiety (HAP) is of formula (I) as defined herein. Typically, M is -OR1or a 5- to 6-membered heterocyclyl ring which is unsubstituted or is substituted by 1 or 2 RYgroups. When M is a 5- to 6-membered heterocyclyl ring, M is typically unsubstituted or is substituted with one methyl group. More typically, M is -OR1or a group selected from morpholino, N-methyl piperidinyl, and 4-methylpiperazin-1-yl. Preferably, M is -OR1. Each of R1, R2, R3and R4are independently C1-4alkyl which is unsubstituted or substituted by one, two or three R5groups; H; halo; or a group selected from C3-6cycloalkyl, 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 R6groups. Typically, each of R2and R4are independently C1-4alkyl which is unsubstituted or substituted by one, two or three R5groups; H; halo; or a group selected from C3-6cycloalkyl, 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 R6groups; and each of R1and R3are independently C1-4alkyl which is unsubstituted or substituted by one, two or three R5groups; H; or a group selected from C3-6cycloalkyl, 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 R6groups Typically, R1is C1-4alkyl which is unsubstituted or substituted by one or two R5groups; or a group selected from C5-6cycloalkyl, 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 R6groups. Preferably, R1is unsubstituted C1-4alkyl, for instance methyl. Typically, R3is C1-4alkyl which is unsubstituted or substituted by one or two R5groups; or a group selected from C5-6cycloalkyl, 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 R6groups. Preferably, R3is unsubstituted C1-4alkyl, for instance methyl. Typically, R2is H, halo or C1-4alkyl which is unsubstituted or substituted by one or two R5groups. Preferably, R2is H or unsubstituted C1-4alkyl. More preferably, R2is H. Typically, R4is H, halo or C1-4alkyl which is unsubstituted or substituted by one or two R5groups. Preferably, R4is H or unsubstituted C1-4alkyl. More preferably, R4is methyl. Typically, each R5is independently selected from C1-4alkoxy, halo, -OH, -CN and - N(RY)2. Preferably, each R5is independently selected from C1-4alkoxy, halo and -OH. Typically, each R6is independently selected from C1-4alkoxy, halo, -OH, -CN, -N(RY)2, and C1-4 alkyl which is unsubstituted or substituted by one, two or three, for instance one or two, R5groups. Preferably, each R6is independently selected from C1-4alkoxy, halo, -OH and unsubstituted C1-4 alkyl. Typically, each RXis independently selected from H and methyl. Preferably, each RXis independently H. Typically, each RYis independently selected from H and methyl. Preferably, each RYis independently H. Typically, therefore, in formula (I), M is -OR1or a group selected from morpholino, N-methyl piperidinyl, and 4-methylpiperazin-1-yl; R1and R3are each independently C1-4alkyl which is unsubstituted or substituted by one or two R5groups; or a group selected from C5-6cycloalkyl, 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 R6groups; R2is H, halo or C1-4 alkyl which is unsubstituted or substituted by one or two R5groups; R4is H, halo or C1-4alkyl which is unsubstituted or substituted by one or two R5groups; each R5is independently selected from C1-4alkoxy, halo, -OH, -CN and -N(RY)2; each R6is independently selected from C1-4alkoxy, halo, -OH, -CN, -N(RY)2, and C1-4alkyl which is unsubstituted or substituted by one, two or three, for instance one or two, R5groups; and each RXis independently selected from H and methyl; each RYis independently selected from H and methyl. Preferably, in formula (I), M is -OR1; R1and R3are each independently unsubstituted C1-4alkyl, for instance methyl; R2is H or unsubstituted C1-4alkyl, more preferably R2is H; R4is H or unsubstituted C1-4alkyl, more preferably R4is methyl; each R5is independently selected from C1-4 alkoxy, halo and -OH; each R6is independently selected from C1-4alkoxy, halo, -OH and unsubstituted C1-4alkyl; each RXis independently H; and each RYis independently H. In a particularly preferred embodiment, HAP is a hypoxia-activated moiety of Formula IA:

[0002] [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”. The term PROTAC is an acronym for proteolysis targeting chimera. In general, PROTACs are, as is known in the art, heterobifunctional molecules that comprise two active moieties attached covalently by a linker group. In a PROTAC, 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. Those skilled in the art will appreciate that routine methods known in the art can be used to determine whether a given substance binds to an E3 ubiquitin ligase (including but not limited to any of those disclosed specifically herein). For example, protein based assays such as those described in the examples can be used to identify E3 ubiquitin ligase binding ligands. In some embodiments, the moiety shows activity or binds to the E3 ubiquitin ligase with an IC50of 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 µM, less than 10 µM, or less than 1 µM. The IC50can be determined according to any method known in the art, e.g., a fluorescent polarization assay. Merely by way of example of the extensive disclosure in the field concerning PROTACs, and hence E3 ubiquitin ligase binding moieties, reference can be made to Gu et al. (BioEssays 2018, 40, 1700247), Sun et al. (Signal Transduction and Targeted Therapy 2019, 4:64), Diehl et al. (Chem. Soc. Rev., 2022, 51, 8216-8257), Ichikawa et al. (bioRxiv 2023.04.22537935), Krieger et al. (Chem Med Chem 2023, 18, e202200615), Li et al. (Sig Transduct Target Ther, 2020, 5, 129), WO 2020 / 041331, and WO 2019 / 140003, the contents of all of which are herein incorporated by reference in their entireties. Any of the E3 ubiquitin ligase binding ligands disclosed in these documents can be used as an E3 ubiquitin ligase binding ligand in the compounds of the present invention. For the avoidance of doubt, in certain documents such as WO 2020 / 041331 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). In WO 2019 / 140003 such ligands (labelled “B” in WO 2019 / 140003’s formula (I)) are referred to as a ubiquitin ligase ligand / binder. Further examples of documents disclosing suitable ligands that can be used in the compounds of the present invention include WO2013 / 106643, US2016 / 0045607, WO2014187777, US20140356322 and US 9,249, 153, US2016 / 0058872, US2015 / 0291562 and Winter et al (Science, June 19, 2015, p. 1376), the contents of all of which are herein incorporated by reference in their entireties. Thalidomide, lenalidomide, pomalidomide and analogs thereof are still further examples of suitable ligands. Examples of E3 ubiquitin ligases include von Hippel-Lindau (VHL), cereblon (CRBN) and DCAF15. Preferably 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. In one embodiment, U is a CRBN E3 ubiquitin ligase binding ligand. In another embodiment, U is a VHL E3 ubiquitin ligase binding ligand. In a further embodiment, U is a DCAF15 E3 ubiquitin ligase binding ligand. In some embodiments, 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 LUis a single bond, or Q is CH and LUis selected from a single bond, - C(O)N(Rz)- and -N(Rz)C(O)-; wherein each Rzis independently H or C1-4alkyl; - is the point of attachment to HAP; - RU1is selected from the following structures:

[0003] ; - ring A is selected from the following structures: - x is selected from 1 to 5; - y is selected from 1 to 4; - one RU2group is a single bond to LINK and the other RU2groups when present are each independently selected from halogen, C1-4alkyl, NH2, NO2, OH, COOH, CN and CF3; - X is selected from -CH(RU3)-, -N(RU3)-, -O-, -C(O)-, -NH-CH(RU3)-, -N=C(RU3)-, and -N=N-; - RVis H or a group selected from C1-4alkyl, C3-6cycloalkyl, 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 C1-4alkoxy, halo, -OH, -CN, - N(RU3)2, and C1-4 alkyl; - RU3is H or C1-4alkyl; and - the point of attachment to LU; B: a VHL E3 ubiquitin ligase binding ligand of formula (U2): wherein: - RU4is 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 RU4being unsubstituted or substituted by C1-4alkyl; - RU5is H or C1-4alkyl; - RU6is C1-4alkyl, phenyl or a 3- to 8-membered cycloalkyl ring; - RU7is a single bond to LINK; and the point of attachment to HAP; C: a DCAF15 E3 ubiquitin ligase binding ligand of formula (U3):

[0004] wherein: - z is selected from 1 to 5; - one RU9group is a single bond to LINK and the other RU9groups when present are each independently selected from halogen, C1-4alkyl, NH2, NO2, OH, COOH, CN and CF3; - RU10is H or C1-4alkyl; and - the point of attachment to HAP. In some embodiments, 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. Tyically, in formula (U1), Q is CH and LUis selected from a single bond, - C(O)N(Rz)- and -N(Rz)C(O)-. Preferably, Q is CH and LUis a single bond. Typically, in formula (U1), each Rzis independently H or methyl. Preferably, each Rzis independently H. Typically, in formula (U1), RU1is selected from the following structures:

[0005] . Preferably, RU1is the following structure: Ring A is selected from the following structures: . Typically, in formula (U1), x is selected from 1 to 5, for instance 1 to 4 or 1 to 3. Preferably, x is selected from 1 or 2, more preferably x is 1. Typically, in formula (U1), y is selected from 1 to 4, for instance 1 to 3. Preferably, y is selected from 1 or 2, more preferably y is 1. Typically, in formula (U1), one RU2group is a single bond to LINK and the other RU2groups when present are each independently selected from halogen, C1-4alkyl, NH2, NO2, OH, COOH, CN and CF3. Preferably, one RU2group is a single bond to LINK and the other RU2groups when present are each independently selected from halogen, C1-4alkyl and OH. Typically, in formula (U1), X is selected from -CH(RU3)-, -N(RU3)-, -O- and -C(O)- . Preferably, X is -CH(RU3)-. Typically, in formula (U1), RVis H or a group selected from C1-4alkyl, C3-6cycloalkyl, 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(RU3)2, C1-4alkyl, C3-6cycloalkyl, phenyl, a 5- to 6-membered heteroaryl ring and a 5- to 6-membered heterocyclyl ring. Preferably, RVis H or C1-4alkyl which is unsubstituted or substituted by one or two of C1-4alkoxy, halo, -OH, -CN, - N(RU3)2, C3-6cycloalkyl, phenyl, a 5- to 6-membered heteroaryl ring and a 5- to 6- membered heterocyclyl ring. In one preferred embodiment, RVis an amino acid side chain or derivative thereof. Typically, in formula (U1), each RU3is independently H or methyl. Preferably, each RU3is independently H. Typically, therefore, in formula (U1), u is 1 or 2; Q is CH and LUis selected from a single bond, -C(O)N(Rz)- and -N(Rz)C(O)-; each Rzis independently H or methyl; RU1is selected from the following structures:

[0006] ; x is selected from 1 to 5, for instance 1 to 4 or 1 to 3; y is selected from 1 to 4, for instance 1 to 3; one RU2group is a single bond to LINK and the other RU2groups when present are each independently selected from halogen, C1-4alkyl, NH2, NO2, OH, COOH, CN and CF3; X is selected from -CH(RU3)-, -N(RU3)-, -O- and -C(O)-; RVis H or a group selected from C1-4alkyl, C3-6cycloalkyl, 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 C1-4alkoxy, halo, -OH, -CN, -N(RU3)2, C1-4alkyl, C3-6cycloalkyl, phenyl, a 5- to 6- membered heteroaryl ring and a 5- to 6-membered heterocyclyl ring; and each RU3is independently H or methyl. Preferably, in formula (U1), u is 2; Q is CH and LUis a single bond; RU1is the following structure: x is selected from 1 or 2, more preferably x is 1; one RU2group is a single bond to LINK and the other RU2group when present is independently selected from halogen, C1-4alkyl and OH. In a particularly preferred embodiment, U is a CRBN E3 ubiquitin ligase binding ligand having the following structure: wherein is the point of attachment to LINK. In some embodiments, U is a VHL E3 ubiquitin ligase binding ligand of formula (U2). In some embodiments, 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. Typically, in formula (U2), RU4is a 5- to 6-membered heteroaryl ring, the group RU4being unsubstituted or substituted by C1-4 alkyl. Preferably, RU4is: wherein: - V is typically S or O, preferably S; - W is typically N or CH, preferably N; and - RU8is typically H or methyl, preferably methyl. Typically, in formula (U2), RU5is H or methyl. Preferably, RU5is H. Typically, in formula (U2), RU6is C1-4alkyl. Preferably, RU6is t-butyl. Typically, therefore, in formula (U2), RU4is a 5- to 6-membered heteroaryl ring, the group RU4being unsubstituted or substituted by C1-4alkyl; RU5is H or methyl; and RU6is C1-4alkyl. Preferably, in formula (U2), RU4is: V is typically S or O, preferably S; W is typically N or CH, preferably N; RU8is typically H or methyl, preferably methyl; RU5is H; and RU6is t-butyl. In some embodiments, U is a VHL E3 ubiquitin ligase binding ligand of formula (U2’).

[0007] In some embodiments, RU4, RU5, RU6and RU7are as described above for formula (U2). In a particularly preferred embodiment, U is a VHL E3 ubiquitin ligase binding . In some embodiments, U is a DCAF15 E3 ubiquitin ligase binding ligand of formula (U3). Typically, in formula (U3), z is selected from 1 to 4, for instance 1 to 3. Preferably, z is selected from 1 or 2, more preferably z is 1. Typically, in formula (U3), one RU9group is a single bond to LINK and the other RU9groups when present are each independently selected from halogen, C1-4alkyl, NH2, NO2, OH, COOH, CN and CF3. Preferably, one RU9group is a single bond to LINK and the other RU9groups when present are each independently selected from halogen, C1-4alkyl, and OH. Typically, in formula (U3), RU10is H or methyl. Preferably, RU10is methyl. Typically, therefore, in formula (U3), z is selected from 1 to 4, for instance 1 to 3; one RU9group is a single bond to LINK and the other RU9groups when present are each independently selected from halogen, C1-4alkyl, NH2, NO2, OH, COOH, CN and CF3; and RU10is H or methyl. Preferably, in formula (U3), z is selected from 1 or 2, more preferably z is 1; one RU9group is a single bond to LINK and the other RU9group when present is independently selected from halogen, C1-4alkyl, and OH; and RU10is methyl. In a particularly preferred embodiment, 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). 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. 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(O2)N(R’)-, -N(R’)S(O2)-, -(C1-6alkylene)-, -(C2-6alkenylene)-, 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 cycloalkylene, 6-membered arylene, 4- to 7-membered heterocyclylene and 4- to 7-membered heteroarylene; - each XLis independently selected from a single bond, -N(R’)-, -O-, -C(O)-, -C(O)N(R’)-, -N(R’)C(O)-, -S-, -SO-, -SO2-, -C(R’)=C(R’)- and -C≡C-; - each R’ is independently selected from H and C1-4alkyl; - each n is independently selected from 0 to 4; and - m is selected from 1 to 10. Typically, 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. When present, the 4- to 7-membered heterocyclylene ring of L1 typically contains at least one N atom, for instance one or two N atoms. Preferably, when 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. Preferably, 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’)-. Typically, 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. Preferably, 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. Preferably, 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. More preferably, L3 is selected from -C(O)N(H)-, -N(H)C(O)- and -O-. Typically, each L2 is independently a unit of formula described above. Typically, each XLis independently selected from a single bond, -O-, - NR’C(O)-, -C(O)NR’-, -NR’- or -S-, and n is selected from 1 or 2. In one embodiment, each L2 is the same. Alternatively, LINK may comprise two or more blocks, wherein in each block, each L2 is the same. For instance, (L2)mmay 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. Preferably, L2’, L2’’ and L2’’’ are selected from -CH2-, -CH2CH2O-, - OCH2CH2-, -CH2CH2S- and -SCH2CH2-. More preferably, L2’, L2’’ and L2’’’ are selected from -CH2-, -CH2CH2O- and -OCH2CH2-. p, q and r are integers of from 0 to 10, wherein p+q+r=m. Typically, m is selected from 1 to 10, for instance 2 to 8. Preferably, m is selected from 3 to 6, more preferably 3 to 4. Typically, in formula (L), each R’ is independently selected from H and methyl. Preferably, each R’ is H. In a preferred embodiment, LINK comprises or consists of one of the following structures:

[0008] preferably wherein 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. in vitro or in silico, against the structures of such proteins. Alternatively, compounds may be screened against a protein of interest in the absence of structural information in order to identify ligands for the protein of interest. Binding of ligands to proteins can be detected by suitable techniques such as size filtration, fluorescence techniques such as FRET, and the like. Such methods are routine in the art and are within the capacity of those skilled in the art. In some embodiments, the protein of interest targeting ligand (POI) 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. Typically, 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. As discussed above, those skilled in the art will appreciate that entirely routine laboratory methods can be used to determine whether a given ligand binds to a protein of interest (including but not limited to any of those disclosed specifically herein). Thus, those skilled in the art would have no difficulty in identifying protein of interest binding ligands, nor in establishing whether any existing chemical moiety falls within the bounds of this definition. In certain embodiments, the ligand shows activity or binds to the protein of interest with an IC50of 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 µM, less than 10 µM, or less than 1 µM. The IC50can 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:

[0009] 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. For example, POI may be attached to LINK by derivatisation of a carboxylic acid, ester, carbamide, amide, amine, or hydroxy group of POI. For instance, when the protein of interest targeting ligand is based on the structure of JQ1, 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. For the avoidance of doubt, those skilled in the art will readily appreciate that each of the definitions of moieties U, HAP, POI and LINK provided herein may be taken together in any combination to arrive at a definition of the compound of the present invention. As examples that are in no way limiting, 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, and 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. However, the skilled person would appreciate that a definition labelled “typically” of one moiety may be combined with a definition labelled “preferably” of another moiety to arrive at a definition of a compound of the present invention. Specific embodiments In one typical embodiment of formula (A): - HAP is a hypoxia-activated moiety of formula (I) in which M is -OR1or a group selected from morpholino, N-methyl piperidinyl, and 4-methylpiperazin-1-yl; R1and R3are each independently C1-4alkyl which is unsubstituted or substituted by one or two R5groups; or a group selected from C5-6cycloalkyl, 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 R6groups; R2is H, halo or C1-4alkyl which is unsubstituted or substituted by one or two R5groups; R4is H, halo or C1-4alkyl which is unsubstituted or substituted by one or two R5groups; each R5is independently selected from C1-4 alkoxy, halo, -OH, -CN and -N(RY)2; each R6is independently selected from C1-4alkoxy, halo, -OH, -CN, -N(RY)2, and C1-4alkyl which is unsubstituted or substituted by one, two or three, for instance one or two, R5groups; and each RXis independently selected from H and methyl; each RYis independently selected from H and methyl; - the E3 ubiquitin ligase binding moiety (U) is (i) a CRBN E3 ubiquitin ligase binding ligand of formula (U1) in which u is 1 or 2; Q is CH and LUis selected from a single bond, -C(O)N(Rz)- and -N(Rz)C(O)-; each Rzis independently H or methyl; RU1is selected from the following structures:

[0010] ; x is selected from 1 to 5, for instance 1 to 4 or 1 to 3; y is selected from 1 to 4, for instance 1 to 3; one RU2group is a single bond to LINK and the other RU2groups when present are each independently selected from halogen, C1-4alkyl, NH2, NO2, OH, COOH, CN and CF3; X is selected from -CH(RU3)-, -N(RU3)-, -O- and -C(O)-; RVis H or a group selected from C1-4alkyl, C3-6cycloalkyl, 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 C1-4alkoxy, halo, -OH, -CN, - N(RU3)2, C1-4alkyl, C3-6cycloalkyl, phenyl, a 5- to 6-membered heteroaryl ring and a 5- to 6-membered heterocyclyl ring; and each RU3is independently H or methyl; or (ii) a VHL E3 ubiquitin ligase binding ligand of formula (U2) in which RU4is a 5- to 6-membered heteroaryl ring, the group RU4being unsubstituted or substituted by C1-4 alkyl; RU5is H or methyl; and RU6is C1-4 alkyl; - LINK is a divalent chemical linker group in which 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; 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; each L2 is independently a unit of formula as described above, for instance wherein each XLis independently selected from a single bond, -O-, - NR’C(O)-, -C(O)NR’-, -NR’- or -S-, and n is selected from 1 or 2; m is selected from 1 to 10, for instance 2 to 8; each R’ is independently selected from H and methyl; and - POI is a ligand for a cancer-regulating protein. In one preferred embodiment of formula (A): - HAP is a hypoxia-activated moiety of formula (I) in which M is -OR1; R1and R3are each independently unsubstituted C1-4alkyl, for instance methyl; R2is H or unsubstituted C1-4alkyl, more preferably R2is H; R4is H or unsubstituted C1-4alkyl, more preferably R4is methyl; each R5is independently selected from C1-4alkoxy, halo and -OH; each R6is independently selected from C1-4alkoxy, halo, - OH and unsubstituted C1-4alkyl; each RXis independently H; and each RYis 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 LUis a single bond; RU1is the following structure: x is selected from 1 or 2, more preferably x is 1; one RU2group is a single bond to LINK and the other RU2group when present is independently selected from halogen, C1-4alkyl and OH; or (ii) a VHL E3 ubiquitin ligase binding ligand of formula (U2) in which RU4is: V is typically S or O, preferably S; W is typically N or CH, preferably N; RU8is typically H or methyl, preferably methyl; RU5is H; and RU6is t-butyl; - LINK is a divalent chemical linker group in which 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(H)- , -C(O)N(H)-, -O-, -N(H)C(O)-, and a divalent moiety of piperidine or diazinane, more preferably L3 is selected from -C(O)N(H)-, -N(H)C(O)- and -O-; each L2 is independently a unit of formula as described above, wherein each XLis independently selected from a single bond, -O-, -NR’C(O)-, - C(O)NR’-, -NR’- or -S-, and n is selected from 1 or 2; m is selected from 3 to 6, more preferably 3 to 4; each R’ is H;and - POI is a ligand for a cancer-regulating protein. In one particularly preferred embodiment of formula (A): - HAP is a hypoxia-activated moiety of Formula IA:

[0011] [Formula IA]; 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:

[0012] more preferably wherein 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. Synthesis Compounds of the invention can be prepared by the synthetic methods described in the Examples below, or by analogy with such methods using appropriate starting materials and methodologies familiar to the skilled chemist. Practitioners are directed to standard reference texts such as March’s Avanced Organic Chemistry (8thedition, Wiley) which describes suitable chemical transformations that can be used to synthesize compounds and components thereof provided herein. Pharmaceutical composition The invention provides a pharmaceutical composition comprising a compound as provided herein and a pharmaceutically acceptable excipient, carrier or diluent. Typically, 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. Alternatively, the carrier protein may be tetanus toxoid or diphtheria toxoid. Alternatively, 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. These 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. A thorough discussion of pharmaceutically acceptable excipients, vehicles and auxiliary substances is available in Remington’s Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991). Formulation of a suitable composition can be carried out using standard pharmaceutical formulation chemistries and methodologies all of which are readily available to the reasonably skilled artisan. Such compositions may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable compositions may be prepared, packaged, or sold in unit dosage form, such as in ampoules or in multi-dose containers optionally containing a preservative. Compositions include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. In one embodiment of a composition, 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. 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. 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. Alternatively, 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. Other particulate systems and polymers can also be used, for example, polymers such as polylysine, polyarginine, polyornithine, spermine, spermidine, as well as conjugates of these molecules. 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, 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 trophoblastic disease, hairy cell leukemia, hepatocellular cancer, histiocytosis, langerhans cell, hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumors, pancreatic neuroendocrine tumors, kaposi sarcoma, kidney (renal cell) cancer, langerhans cell histiocytosis, laryngeal cancer, leukemia, liver cancer, lung cancer (non- small cell, small cell, pleuropulmonary blastoma, and tracheobronchial tumor), lymphoma, malignant fibrous histiocytoma of bone and osteosarcoma, merkel cell carcinoma, mesothelioma, mouth cancer, multiple endocrine neoplasia syndromes, multiple myeloma / plasma cell neoplasms, mycosis fungoides, myelodysplastic syndromes, myelodysplastic / myeloproliferative neoplasms, myelogenous leukemia, neuroblastoma, non-hodgkin lymphoma, oropharyngeal cancer, osteosarcoma and undifferentiated pleomorphic sarcoma, pancreatic cancer, pancreatic neuroendocrine tumors (islet cell tumors), papillomatosis, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, prostate cancer, rectal cancer, retinoblastoma, rhabdomyosarcoma, t-cell lymphoma, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, tracheobronchial tumors and the like is particularly suitable to being treated with the compounds and compositions provided herein. 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). When used to treat a cancer, the compounds and compositions provided herein may be used in alleviating, ameliorating or preventing aggravation of the symptoms of the cancer. Typically, 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. Preferably, 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. As explained here, 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. Also provided is a method of treating a subject in need of such treatment, said method comprising administering to the subject a compound or composition provided herein. In some embodiments the subject suffers from or is at risk of suffering from one of the disorders disclosed herein. In some embodiments the subject suffers from a disorder characterised by a protein of interest as described herein. In one aspect, 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. In this embodiment, 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. In this embodiment, 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. Preferably, the agent may be administered parenterally. The agent is typically formulated for administration with a pharmaceutically acceptable carrier or diluent. For example, 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. Alternatively, the agent may be formulated for inhaled administration as a powdered drug, such formulations may be administered from a dry powder inhaler (DPI). When formulated for inhaled administration, the agent may be delivered in the form of particles which have a mass median aerodynamic diameter (MMAD) of from 1 to 100 µm, preferably from 1 to 50 µm, more preferably from 1 to 20 µm such as from 3 to 10 µm, e.g. from 4 to 6 µm. When the agent is delivered as a nebulized aerosol, the reference to particle diameters defines the MMAD of the droplets of the aerosol. The MMAD can be measured by any suitable technique such as laser diffraction. 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. Preferably, daily dosage levels are from 5 mg to 2 g. When the agent is administered to a subject in combination with another active substance (e.g. another medicament as described herein), 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. Preferably, 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. In some embodiments, 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. Typically, 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. In this context, in one embodiment 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. In one embodiment 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. In another embodiument, 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. In one embodiment 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. They do not however limit the invention in any way. In this regard, it is important to understand that the particular assay used in the Examples section is designed only to provide an indication of biological activity. There are many assays available to determine biological activity, and a negative result in any one particular assay is therefore not determinative. The invention is defined according to the claims. It is to be understood that although particular embodiments, specific configurations as well as materials and / or molecules, have been discussed herein for methods according to the present disclosure, various changes or modifications in form and detail may be made without departing from the scope and spirit of this invention. The preceding embodiment and following examples are provided for illustration only, and should not be considered limiting the application. The application is limited only by the claims Examples We selected MZ1 and dBet1, two BRD4-targeting PROTACs recruiting VHL and CRBN as E3 ligases, as the basis for our HAP-TACs. BRD4 was chosen as a non-limiting exemplary proof-of-concept target to illustrate the advantages of the claimed compounds and methods, but the approach and results discussed below are applicable to a wide range of target proteins of interest and are not limited to BRD4. To render HAP-TACs inactive in normoxic conditions, we identified positions that disrupt binding to the respective ligase to locate the bioreductive group: (i) the hydroxyl group on the prolyl ring of the ligand VH032, on which MZ1 is based; and (ii) the nitrogen on the piperidinedione group of the pomalidomide-based recruiter. Previous work by others has shown that functionalization at these positions results in disruption to E3 ligase binding, and consequent abrogation of PROTAC activity. To develop HAP-TACs based on VHL-recruiting PROTACs, we employed either 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. However, while this linker can improve release of the active drug (e.g. tenofovir disoproxil), it can also result in instability in a biological setting. In an attempt to prevent the premature release of the active PROTAC in non-hypoxic regions, we sought to synthesize more stable derivatives in which the bioreductive group was attached to the hydroxyproline. However, direct alkylation of the hydroxyl group with the 2-nitroimidazole was unsuccessful, and therefore we synthesized the carbonate derivative 1 (NI-VHL).. To our delight, however, we were able to obtain the indolequinone derivative (2, IQ-VHL, Scheme 3) directly attached to the VHL ligand in 32% yield. To provide a negative control for the VHL-recruiting HAP-TACs, a PROTAC alkylated with a benzyl group, which should not reduce or fragment in hypoxia, was also synthesized (3, Bn- VHL, Figure 1C). To develop the CRBN-recruiting HAP-TACs, we first functionalized an illustrative recruiter, pomalidomide (7, Figure 2A) with either a 4-nitrobenzyl (5, NB-POMA, Figure 2A) or a 2-nitroimidazol group (6, NI-POMA, Figure 2A) at the glutarimide nitrogen. While the 4-nitrobenzyl derivative 5 did not show any release of pomalidomide in initial studies using zinc to reduce the nitro group, the 2-nitroimidazole derivative 6 released significant amount of pomalidomide upon zinc reduction. Encouraged by this result, we next validated compound 6 in an enzyme-based assay using a NADPH-CYP bactosomal reductase (92 pmol / mL) in hypoxia. We have previously shown that pro-drug release in this assay corresponds well with pro-drug release in hypoxic mammalian cells.[1,2]The amount of remaining prodrug and degree of pomalidomide release over 24 h was monitored using LCMS analysis. While we observed pro-drug degradation over this time, we were surprised to detect no pomalidomide release (Figure 3B). We observed loss of pomalidomide 7 when it was incubated under the same conditions, and we were unable to determine the identity of the products formed (Figure 3C). These data are consistent with a previous report that investigated use of ROS-activated PROTACs. Here direct attachment of the ROS-activated moiety to the glutarimide nitrogen resulted in ring opening. It is also well established that IMiD-based PROTACs, including dBet1, often display most effective protein degradation after 6-8 h incubation, but typically protein levels recover after 24 h. This phenomenon likely results from metabolism of the glutarimide ring. To overcome this instability, we investigated the use of phenyl glutarimide-based CRBN ligands (Figure 2B), which were recently reported by Min et al.[3]as more stable alternatives to IMID-based compounds. We initially synthesized the nitroimidazole-based compound 8 (NI-CRBN, Figure 2B). However, we had previously shown that pro-drug fragmentation to release a nitrogen-based leaving group was inefficient, compared to release of an oxygen-based leaving group. Given our concerns over inefficient fragmentation, we included the indolequinone group as alternative bioreductive protecting group for HAP- TACs. Therefore, a HAP-TAC (9, IQ-CRBN, Figure 2B) incorporating this motif was synthesized, along with a benzyl-protected negative control (10, Bn-CRBN, Figure 2B). The 2-nitroimidazol (16 and 17) and indolequinone (24) precursors were synthesized following optimized protocols used in the group,[1,4]which are summarized in Scheme 1 and 2. Scheme 1. Synthesis of 5-(bromomethyl)-1-methyl-2-nitro-1H-imidazole 16 and (1-methyl- 2-nitro-1H-imidazol-5-yl)methyl (4-nitrophenyl) carbonate 17. Reagents and conditions: (a) (i) NaH (60% mineral oil), ethyl formate, dry THF, absolute EtOH, 0 ºC to rt, 18 h; (ii) HCl (37% aq.), EtOH, 78 °C, 2 h; (iii) NCNH2, EtOH, water, 100 °C, 2 h, 50–68% (over three steps), n = 3; (b) NaNO2, acetic acid, water, 0 °C to rt, 2 h, 42–53%, n = 3; (c) NaBH4, CH3OH, dry THF, 0 °C, 2 h, 35–40%, n = 2; (d) SOBr2, 0 °C to rt, 20 min, 80–81%, n = 2; (e) 4- Nitrobenzyl chloroformate, DMAP, dry pyridine, dry CH2Cl2, rt, 20 h, 68%.

[0013] Scheme 2. Synthesis of 3-(bromomethyl)-5-methoxy-1,2-dimethyl-1H-indole-4,7-dione 24. Reagents and conditions: (a) POCl3, dry DMF, −10 °C to 0 °C, 2 h, 68−70%, n = 3; (b) NaH (60% mineral oil), CH3I, dry DMF, 0 ºC to rt, 4 h; 41−90%, n = 3; (c) 1. Fuming nitric acid, acetic acid, 0 °C, 4 h, 53−81%, n = 3; 2. Sn, HCl (3M), EtOH, 80 °C, 1 h, 71−90%, n = 3; (d) Fremy’s salt, NaH2PO4 / Na2HPO4buffer, acetone, rt, 1 h, 50−89%, n = 3; (e) NaBH4, dry THF, dry CH3OH, 0 °C, 1 h, 69−88%, n = 3; (f) PBr3, dry CH2Cl2, 0 °C, 10 min, 90−99%, n = 3. To obtain the VHL ligand 31, a synthetic protocol reported by Han et al.[5]was followed (Scheme 3). Compound 31 was coupled to the commercially available PEG3linker to afford intermediate 32. Subsequent reduction of the azide (32), using Pd-catalyzed hydrogenation, gave the amine which was coupled with the commercially available carboxylic acid derivative of the BRD4 bromodomain ligand (+)-JQ1 ((+)-JQ1-COOH) to afford MZ1 (4). Reaction of 4 with (1-methyl-2-nitro-1H-imidazol-5-yl)methyl (4-nitrophenyl) carbonate 17, synthesized as described in Scheme 1, afforded compound 1 in a late-stage functionalization step, as previously reported by Shi et al.[6]Alternatively, alkylation of the hydroxyl group of compound 32, using either indolequinone bromide (24) or commercially available benzyl bromide in the presence of TBAI afforded compounds 33 and 34. A Staudinger reduction of these azides to give the corresponding amine, followed by (+)-JQ1- COOH coupling, afforded the final compounds 2 and 3.

[0014]

[0015]  Scheme 3. Synthesis of VHL-recruiting PROTAC 4 and HAP-TACs 1, 2 and 3. Reagents and conditions: (a) 1. HATU, DIPEA, dry DMF, rt, 16 h, 86%; 2. LiOH, H2O, THF, rt, 16 h, 83–100%, n = 2; (b) Boc2O, NaHCO3, H2O, EtOAc, 0 °C to rt, 2 h, 97%; (c) 4-methylthiazole, KOAc, Pd(OAc)2, dry DMA, 90 °C, 24 h, 48%; (d) 1. HCl (4M in dioxane), CH2Cl2, rt, 2 h; 2. HATU, DIPEA, dry DMF, rt, 16 h, 46% over two steps; (e) 1. HCl (4M in dioxane), CH2Cl2, rt, 2 h; 2.2-(2- (2-(2-Azidoethoxy)ethoxy)ethoxy)acetic acid, HATU, DIPEA, dry DMF, rt, 16 h, 41–63% over two steps, n = 4; (f) 1. Pd / C 10%, H2, CH3OH, rt, 4 h; 2. (+)-JQ1-COOH, HATU, DIPEA, dry DMF, rt, 16 h, 66% over two steps; (g) Compound 17, dry TEA, dry CH2Cl2, 40 °C, 40 h, 45%; (h) R2-Br, TBAI, NaOH, H2O, CH2Cl2, rt, 3 h, 42–56%, n = 3 for 33; 3 h, 81% for 34; (i) 1. PPh3, H2O, THF, rt, 16 h; 2. (+)-JQ1-COOH, HATU, DIPEA, dry DMF, rt, 16 h, 36–50% over two steps, n = 2 for 2; 16 h, 72% over two steps for 3. NB- (5) and NI-pomalidomide (6) were obtained as summarized in Scheme 4. From pomalidomide 7, a Mitsunobu reaction with polymer-supported PPh3(PS-PPh3) and DIAD in a 4:1 mixture of dry THF and dry DMF. Both compounds were obtained in 20% of yield. Scheme 4. Synthesis of pomalidomide 7 and pomalidomide derivatives 5 and 6. Reagents and conditions: (a) Sodium acetate, acetic acid, 120 ºC, 3–7 h, 70–88%, n = 3; (b) Pd / C 10%, H2, DMF, rt, 24 h, 91%; (c) R1-OH, PS-PPh3, DIAD, dry THF, dry DMF, 0 °C to rt for 5, 36 h, 20% and 0 °C to 60 °C for 6, 20 h, 20%. To synthesize the CRBN-recruiting HAP-TACs, we followed a previously reported route[3]to obtain compound 45 (Scheme 5). The Boc group of 45 was deprotected using HCl (4 M in dioxane) and a HATU-mediated amide coupling with (+)-JQ1-COOH afforded the active PROTAC PG-4c (11, Scheme 5). To obtain the protected PROTACs 8-10, alkylation of the glutarimide nitrogen with the 2-nitroimidazol bromide (16), indolequinone bromide (24), or benzyl bromide in the presence of Cs2CO3and TBAI in DMF gave compounds 46-48. Treatment of 48 with HCl (4 M in dioxane, 6 eq.), in an attempt to remove the Boc group from the terminal amine, revealed that the glutarimide ring is acid labile and opens under the strongly acidic conditions. When the intermediate was carried forward to an amide coupling to (+)-JQ1 carboxylic acid, the corresponding product was only formed in poor yield. To overcome this issue, we employed the milder combination of oxalyl chloride in methanol to remove the Boc group. Although the deprotection required 16 h, the reactions proceeded smoothy, forming one product, as judged by TLC analysis, and after evaporation and amide coupling with (+)-JQ1-COOH, the products 8-10 were obtained in yields 50–84%.

[0016] Scheme 5. Synthesis of CRBN-recruiting active PROTAC 11 and HAP-TACs 8-10. Reagents and conditions: (a) Benzyl alcohol, NaH (60% mineral oil), dry DMF, 0 °C to 80 °C, 16 h, 97–98%, n = 3; (b) NBS, CH3CN, 80 °C, 3 h, 64–79%, n = 3; (c) (4-Hydroxyphenyl)boronic acid, K3PO4, Pd(dppf)Cl2•CH2Cl2, dioxane, water, 100 °C, 16 h, 77–91%, n = 4; (d) Tert-butyl bromoacetate, K2CO3, dry DMF, rt, 3 h, 71–93%, n = 4; (e) Pd / C 10%, H2, DMF, rt, 16 h, 56–92%, n = 3; (f) TFA, CH2Cl2, rt, 16 h, 90–99%, n = 4; (g) N-Boc-cadaverine, HATU, DIPEA, dry DMF, rt, 16 h, 49–85%, n = 4; (h) 1. HCl (4M in dioxane), CH2Cl2, rt, 1 h; 2.(+)-JQ1-COOH, HATU, DIPEA, dry DMF, rt, 16 h, 86%; (i) R1-Br, Cs2CO3, TBAI, DMF, rt, 22 h, 39−46%, n = 2 for 46; 24 h, 23–32%, n = 3 for 47; 32 h, 40–41%, n = 2 for 48; (l) 1.Oxalyl chloride, MeOH, rt, 19 h; 2. (+)- JQ1-COOH, HATU, DIPEA, dry DMF, rt, 16 h, 36–50% over two steps, n = 2, for 8, 24 h, 47–84% over two steps, n = 2 for 9, 16 h, 66% over two steps for 10. We note that the protected intermediates 33-34 (Scheme 3) and 46-48 (Scheme 5) can potentially be applied to synthesize a wide variety of CRBN- or VHL-recruiting HAP-TACs by conjugation with the POI ligand of choice. 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. Compounds 1, 2, 8 and 9, the active PROTACs MZ1 (4) and PG-4c (11), and the two negative controls Bn-VHL (3) and Bn-CRBN (10) were incubated with NADPH-CYP reductase (CYP004) in hypoxic (<0.1% O2) conditions for 24 h.[1]Parallel incubations in hypoxia without the reductase enzyme were also performed to assess the hydrolytic stability of the compounds. The HAP-TACs were also incubated in normoxia (21% O2) for 24 h. The residual substrate of the tested compounds and the corresponding release of active PROTACs were monitored by HPLC analysis. We have previously shown that HAPs which are activated under these conditions tend to also be activated in hypoxic cells.[1,2]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). In contrast, 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). IQ-CRBN did not show any release of PG-4c in either normoxia or the hypoxic control experiment, with selective release of the active PROTAC (9%) observed only in hypoxia (Figure 4B and 8). Finally, the active PROTACs MZ1 and PG-4c, and the two negative controls Bn-VHL and Bn-CRBN, were stable after 24 h of incubation in hypoxia, with no substantial differences in stability in the presence or absence of the enzyme (Figure 9). To investigate the kinetics of HAP-TAC activation, a time-course was performed in hypoxia. This analysis showed that NI-VHL released 100% of MZ1 in less than 4 h (Figure 4C), while IQ-VHL plateaued at approximately 80% of MZ1 release after 24 h (Figure 4E), with a concomitant reduction of the parent pro-drug in both cases. The amount of PROTACs released from CRBN-recruiting compounds was significantly less over the 24 hours (Figure 5D and F), which is consistent with our previous observations that the pKaof the HAP cargo determines the rate of reduced pro-drug fragmentation to give the active component.31NI- CRBN formed multiple products, observed by HPLC analysis, after 2 h of incubation in hypoxia. This result is consistent with our previous observations, and we attribute to the electron-withdrawing nature of the nitroimidazole ring promoting hydrolysis of the glutarimide ring. Conversely, IQ-CRBN released 5% of the active PROTAC after 24 h, with no other products observed. Based on these data, IQ-VHL and IQ-CRBN showed release of the active PROTAC in hypoxia, with little or no release observed in normoxia, indicating a promising profile for use in cells. Without being bound by theory, we hypothesize that the indolequinone group may improve the stability and at the same time might give more efficient release of the active PROTAC as it lacks the withdrawing effect of the nitroimidazole. In-cell screening The four HAP-TACs (1, 2, 8 and 9) were next evaluated for their ability to induce BRD4 degradation in A549 cells in hypoxia (<0.1% O2) compared to normoxic (21% O2) 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 µM), or not substantially affecting BRD4 levels in either set of conditions at lower concentrations (17 ± 10% vs 30 ± 10% of degradation at 0.030 µM in normoxia and hypoxia, respectively) (Figure 10A). In contrast, IQ-VHL (2) showed little or no degradation of BRD4 in normoxia, but almost complete degradation of BRD4 in hypoxia. For example, at a concentration of 0.5 µM, IQ-VHL degraded 98 ± 2% of BRD4 in hypoxic conditions, but only modest degradation was observed in normoxia (19 ± 24%) (Figure 10B). NI-CRBN (8) had little effect on BRD4 levels in either hypoxia or normoxia (Figure 10C). This result is consistent with data from Cheng et al.[7]who reported an EGFR-targeted HAP-TAC, which comprised either the nitroimidazole or nitrobenzyl bioreductive group attached to the glutarimide nitrogen atom of a 4-hydroxylthalidomide-based PROTAC. The nitrobenzyl-based HAP-TACs showed modest EGFR degradation in both normoxia and hypoxia. Interestingly, 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.31However, the result is likely explained by the nitroimidazole group promoting the hydrolysis of the glutarimide ring. These data indicate that attachment of a nitroimidazole group to a CRBN-recruiting PROTAC is an ineffective strategy for developing HAP-TACs. Last, 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 µM, IQ-CRBN degraded 82 ± 16% of BRD4 in hypoxic conditions and only 16 ± 2% of BRD4 in normoxia (Figure 10D). To determine whether the (+)-JQ1-based component of the HAP-TACs affects BRD4 levels when bioreductive groups are attached to the E3 ligase ligand, we treated A549 cells with a range of concentrations of (+)-JQ1 (0.1-1-10 µM) in normoxia. In parallel, 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 µM) did not reduce BRD4 levels. Interestingly, 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. Based on the above results, we selected a concentration of 0.25 µM for IQ-VHL 2 (protein degradation: 9 ± 24% in normoxia vs 79% ± 8% in hypoxia) and 3 µM for IQ-CRBN 9 (2 ± 16% in normoxia vs 79 ± 6% in hypoxia) as optimum concentrations for further HAP- TAC evaluation. To determine how O2dependent the activation of IQ-VHL (2) or IQ-CRBN (9) is, we incubated A549 cells for 24 h in 2% or 0.5% O2(Figure 12A and B). Interestingly, no degradation was observed at 2% O2(BRD4 levels: 97 ± 8% and 120 ± 25% for 2 and 9, respectively), while at 0.5% O2, IQ-VHL significantly degraded BRD4 (59 ± 6%), while IQ- CRBN induced only a mild degradation (37 ± 13%) after 24 h. This indicates that both compounds would be activated in hypoxic tumors, but should not release substantial levels of PROTAC in tissue that is physiologically hypoxic, such as the bone marrow, the intestine, the heart or the retina. Having selected severe hypoxia as the most effective oxygen concentration for both compounds, to further investigate the hypoxia-dependent activity of HAP-TACs 2 and 9, we then performed a time-course in which we evaluated BRD4 levels after 2, 4, 16, or 24 h incubation. Approximately thirty percent of BRD4 was degraded by IQ-VHL (26 ± 8%) after 2 h incubation in hypoxia and further degradation was achieved after 16 h (51 ± 3%). Conversely, following treatment with IQ-CRBN, BRD4 levels remained constant until 16 h, when substantial degradation (37 ± 11%) was observed (Figure 13A and B). These data reflect the need for the bioreductive group to be reduced in hypoxia, and the reduced product to fragment, before the active PROTAC is released. As expected, given the instability of HIF-1α, its levels are higher after 2 h of hypoxic exposure, but they are reduced after 8 h, to reach the lowest amount after 24 h (Figure 13B). To ensure that the mechanism of BRD4 degradation was retained in hypoxic conditions, compounds 2 and 9 were incubated in A549 cells in the presence of carfilzomib, a proteasome inhibitor, or MLN-4629, an inhibitor of neddylation. Co-treatment of HAP- TACs with either one of the two inhibitors for 24 h reduced the amount of BRD4 degradation, confirming the dependency on the proteasome. In line with literature reports, HIF-1α levels were increased after treatment with the two inhibitors, resulting from further stabilization of this protein in hypoxia (Figure 13C). To demonstrate that the effect was not cell-line dependent, we used HCT116 cells as second model. Cells were initially treated with MZ1, PG4c and (+)-JQ1 at various concentrations for 24 h. As expected, while 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). In this second cell line, 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. To differentiate between the inhibitory activity related to the presence of (+)-JQ1 moiety and the degradation activity related to the release of the active PROTAC in hypoxia, a washout step was performed after 24 h of incubation with the compounds. Given the time required to re-synthesize BRD4 in cells, the washout would remove compounds from the media, but the effect related to the reduced protein levels would be retained. While active PROTACs show no significant difference in their cytotoxic profile in normoxia (21% O2) and hypoxia (<0.1% O2) (Figure 16A and C), both compounds 2 and 9 have different IC50values in the two conditions (29.05 vs 2.68 µM for 2 and 29.26 vs 2.46 µM for 9, respectively in normoxia and hypoxia, Figure 16B and D). Finally, we investigated the anti-proliferative effect of compounds 2 and 4. Cell growth over time was determined for HCT116 cells treated with IQ-VHL and IQ-CRBN in normoxia (21% O2) and hypoxia (<0.1% O2) and, in parallel, with MZ1 and PG4c in normoxia. Again, a washout step was performed after 24 h treatment. While IQ-VHL did not affect cell growth in normoxia, the cell number was significantly reduced over time in hypoxia, with a comparable trend to the anti-proliferative effect induced by MZ1 treatment (Figure 17A). On the other hand, treatment of IQ-CRBN in normoxia affected cell growth, an effect putatively related to the presence of (+)-JQ1 in the structure of the compound, which showed a marked cytotoxicity at 1 µM under the same assay conditions. Despite this, cell growth was significantly lower in hypoxia, similarly to the treatment with PG-4c in normoxia (Figure 17B). Conclusion Strategies to target the protein degradation activity of PROTACs to diseased tissue, while sparing healthy tissue will help to maximize the therapeutic window for this drug modality. This approach will increase the POIs that can be targeted clinically by reducing the risk of on-target toxicity. While a number of strategies have been applied to this end, hypoxia-activated pro-drugs (HAPs) have been proven to target the release of a cargo drug to hypoxic tumors, in vivo, in a therapeutically relevant manner. Although there have been previous reports of strategies to selectively activate PROTACs in hypoxia, compounds previously reported typically either contain unstable linkers between the bioreductive group and the E3 ligase ligand resulting in a small window of hypoxia / normoxia selectivity; or show very inefficient release of the active PROTAC, likely as a result of cellular instability conveyed by incorporation of the bioreductive group. By employing the indolequinone bioreductive group, we have overcome these issues to develop two HAP- TACs that can degrade target proteins (illustrated here for the proof-of-concept-target BRD4), via recruitment of either CRBN or VHL. These HAP-TACs show little effect on BRD4 in normoxia, but exhibit almost complete degradation of this protein in hypoxia. As the HAP-TACs incorporate the bioreductive group on the E3 ligase ligands, this strategy can, in principle, be applied to any protein that is degraded by an E3 ubiquitin ligase. Supplementary The oxygen-dependent enzymatic reduction assays were run in a RUSKINN InvivO2®400 hypoxic workstation at 37 °C with 0.1% oxygen and 99.9% nitrogen concentrations unless otherwise stated. Bactosomal human NADPH-CYP reductase (CYP004, Cypex) was used in combination with NADPH regenerating system (solution A, Cat. No 451220 and solution B, Cat. No 451200, Corning). Phosphate buffer, MilliQ water and absolute ethanol used in the assays were equilibrated under the conditions stated above in the hypoxic workstation overnight before use. LCMS or HPLC analysis were used as readout for reaction analysis. General procedure for hypoxia LCMS assay: To a 1.5 mL Eppendorf tube in the hypoxic workstation, MilliQ water (874.7 μL), phosphate buffer (0.5 M, pH 7.4, 240 μL), regenerating solution A (60 μL), regenerating solution B (12 μL), and a 10 mM DMSO solution of compound (2 μL, final concentration 20 μM) were added. An aliquot (200 μL) was collected from the reaction solution and quenched with 200 µL of methanol. This serves as T = 0 (where T = time). Then, NADPH-CYP reductase (11.3 µL, final concentration 92 pmol / mL) was added to the reaction mixture. At selected timepoints, aliquots were collected and quenched in the hypoxic workstation with 200 µL 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 CH2Cl2:methanol (4 × 200 µL), filtered using a syringe filter (Gilson Nylon Syringe Filter, size × porosity: 13 mm × 0.45 μm) and evaporated. The residue was solubilized in 200 µL of water with 0.5% of DMSO, and 100 µL were injected and analyzed using LCMS. General procedure for normoxia HPLC assay: To a 1.5 mL Eppendorf tube, MilliQ water (744.7 μL), phosphate buffer (0.5 M, pH 7.4, 240 μL), absolute ethanol (120 μL), regenerating solution A (60 μL), regenerating solution B (12 μL), and a 2 mM DMSO solution of compound (12 μL, final concentration 20 μM) were added. An aliquot (200 μL) was collected from the reaction solution and quenched with 200 µL of methanol. This serves as T = 0 timepoint. Then, NADPH-CYP reductase (11.3 μL, final concentration 92 pmol / mL) was added to the reaction mixture. At selected timepoints, aliquots were collected and quenched in the hypoxic workstation with 200 µL 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 μm), 100 µL were injected and analyzed using HPLC. General procedure for hypoxia HPLC assay: To a 1.5 mL Eppendorf tube in the hypoxic workstation, MilliQ water (744.7 μL), phosphate buffer (0.5 M, pH 7.4, 240 μL), absolute ethanol (120 μL), regenerating solution A (60 μL), regenerating solution B (12 μL), and a 2 mM DMSO solution of compound (12 μL, final concentration 20 μM) were added. An aliquot (200 μL) was collected from the reaction solution and quenched with 200 µL of methanol. This serves as T = 0 timepoint. Then, NADPH-CYP reductase (11.3 μL, final concentration 92 pmol / mL) was added to the reaction mixture. At selected timepoints, aliquots were collected and quenched in the hypoxic workstation with 200 µL 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 μm), 100 µL were injected and analyzed using HPLC. General procedure for hypoxia control HPLC assay: To a 1.5 mL Eppendorf tube in the hypoxic workstation, MilliQ water (756 μL), phosphate buffer (0.5 M, pH 7.4, 240 μL), absolute ethanol (120 μL), regenerating solution A (60 μL), regenerating solution B (12 μL), and a 2 mM DMSO solution of compound (12 μL, final concentration 20 μM) were added. The mixture was left in the hypoxic workstation and at selected timepoints aliquots (200 μL) were collected from the reaction solution and quenched with 200 µL 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 μm), 100 µL 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, GlutaMAX™ and pyruvate supplemented with 1% penstrep and 10% FBS. Cells were maintained in an incubator set at 37 °C and 5% CO2. 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% CO2for 24 h. Cells were harvest and lysed in UTB buffer (9M urea, 75 mM Tris-HCl, pH 7.5, 0.1M β-mercaptoethanol). Hypoxic Treatments Hypoxia treatments at <0.1% O2were carried out in a Bactron II anaerobic chamber (Shel Labs) while all oxygen concentrations from 0.5-2% O2were 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. For the proteasome / neddylation experiment, 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. After additional 24 hours in normoxia, cells were incubated with 0.5 mg / mL MTT reagent (Sigma Aldrich) in complete media for 3 hours at 37 °C protected from light. MTT was removed and formazan crystals were solubilized with 100 μL of DMSO for 15 minutes at 37 °C protected from light. Absorbance was read at 570 nm using a a FLUOstar Omega plate reader. Data are shown as percentage viability relative to DMSO control and are the result of three biological replicates with three technical replicates each ± s.e.m. Cell proliferation HCT116 cells were seeded at 8,300 cells / well in 24-well plates and allow 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 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 Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). Proteins were diluted in MilliQ water and 10% β- 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 Spectra™ Multicolor High Range Protein Ladder (Thermo Fisher Scientific). Semi-dry blotting was done on a Trans-Blot Turbo Transfer System (Bio-Rad) with the Trans-Blot Turbo RTA Mini 0.2 µm PVDF Transfer Kit (Bio-Rad) using the default high molecular weight mode. Membranes were blocked in 5% non-fat dry milk (Bio-Rad) in TBS-T for 1-2 h. Primary BRD4 (E2A7X, Cell Signaling Technology Europe), c-Myc (D84C12, Cell Signaling Technology Europe), HIF-1α (610958, BD Bioscience) and β-actin (sc-69879, Santa Cruz Biotechnologies) antibodies were used in concentrations recommended by the manufacturer, diluted in 5% non-fat dry milk in TBS- T. 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 β-actin bands, and reported as percentage of the DMSO control lane. General chemical methods Reagents and solvents were purchased from commercial sources (Sigma-Aldrich, Fluorochem, and Alfa Aesar) and were used without further purification, unless otherwise stated. Anhydrous solvents were taken from MBRAUN Solvent Purification System 5 and stored over 3 Å molecular sieves under an argon atmosphere. In vacuo refers to solvent removal under reduced pressure at 45 °C using a Buchi™ rotary evaporator. Petroleum ether refers to the fraction of light petroleum ether boiling in the range 40– 60 °C when distilled. 1H NMR spectra were recorded using a Bruker AVH400 (400 MHz) or Bruker NEO 600 spectrometers using the stated solvents as a reference for internal deuterium lock. The chemical shift data for each signal are given as δHin units of parts per million (ppm) relative to the tetramethyl silane (TMS) where δH(TMS) is 0.00 ppm. The multiplicity of 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. 13C 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 δCin parts per million (ppm) relative to the tetramethyl silane (TMS) where δH(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 F254aluminium-supported thin layer chromatography sheets. Visualisation was done by absorption of UV light (λmax 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 µm), 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. For the determination of compound purity, one of the following methods was used: (A) Poroshell 120 EC-C18 column 40 (4 μM, 4.6 × 100 mm) and (B) Poroshell 120 EC-C18 column 40 (4 μM, 4.6 × 150 mm); Gradient elution of 95% H2O / 5% MeCN + 0.1% formic acid modifier (1 min), 95% H2O / 5% MeCN to 5% H2O / 95% MeCN + 0.1% FA modifier (over 10 min), hold (5 min); flow rate 1 mL / min and detection at 220, 254, 280 and 365 nm. Semi-preparative high performance liquid chromatography (Semi Prep HPLC) was carried out on an Agilent 1260 Infinity II® system using one of the following methods: (A) 5 Prep C18 column (5 µm, 21.2 × 50 mm); Gradient elution: 95% H2O / 5% MeCN (1 min), 95% H2O / 5% MeCN to 5% H2O / 95% MeCN (over 9 min), hold (1 min); flow rate 25 mL / min; (B) Zorbax SB-C18 column (5 µm, 9.4 × 250 mm); Gradient elution: 95% H2O / 5% MeCN (1 min), 95% H2O / 5% MeCN to 5% H2O / 95% MeCN (over 10 min), hold (5 min); flow rate 4 mL / min. Synthetic procedures and compound data Ethyl 2-amino-1-methyl-1H-imidazole-5-carboxylate (13) Sarcosine ethyl ester hydrochloride (6.00 g, 39.06 mmol, 1.0 eq.) was suspended in a combination of dry THF (135 mL), absolute EtOH (13 mL), and ethyl formate (135 mL) and cooled to 0 °C under a stream of argon. NaH (60% mineral oil, 6.25 g, 156.24 mmol, 4.0 eq.) was added portionwise and, once gas evolution had ceased, the reaction mixture was warmed to rt and stirred for 18 h. The reaction was quenched by the addition of diethyl ether (200 mL) and evaporated in vacuo. The resulting solid was triturated with hexane (2 × 50 mL) and dried in vacuo. The resulting intermediate was then suspended in EtOH (130 mL) and concentrated HCl (26 mL) was slowly added to the suspension. The mixture was then stirred at reflux for 2 h then filtered in vacuo to remove the colourless salts. The resulting solution was concentrated in vacuo, then dissolved in EtOH (210 mL) and water (90 mL), and the pH adjusted to 3 with 6M aqueous NaOH solution (~50 mL). Cyanamide (3.27 g, 78.12 mmol, 2.0 eq.) was added to the solution and the reaction was heated at 100 °C for 2 h. The mixture was cooled to rt and concentrated in vacuo to reach 1 / 8 of the original volume, then solid K2CO3was added until pH 8-9. The formed precipitate was then filtered in vacuo, washed with saturated aqueous K2CO3solution (1 × 20 mL) and water (1 × 20 mL) and dried in vacuo to yield a yellow solid (4.52 g, 68%): Rf0.25 (CH2Cl2 / CH3OH 95:5); m.p. 145–147 °C (from water) [lit. 130-133 °C];1H NMR (400 MHz; CDCl3) δH 7.37 (1H, s, H-4), 4.38 (2H, br s, NH2), 4.19 (2H, q, J 7.1, H-8), 3.60 (3H, s, H-1'), 1.27 (3H, t, J 7.1, H-9); LRMS m / z (ESI+) 170 ([M+H]+, 100%). The spectroscopic data are in good agreement with the literature values. Ethyl 1-methyl-2-nitro-1H-imidazole-5-carboxylate (14) To a solution of NaNO2(18.40 g, 267.10 mmol, 10.0 eq.) in water (25 mL), intermediate 13 (4.52 g, 26.71 mmol, 1.0 eq.) in glacial acetic acid (47 mL) was added dropwise at 0 °C over 45 min. The solution was stirred at 0 °C for 1 h and then at rt for 2 h. The mixture was diluted with water (80 mL) and the product was extracted with CH2Cl2(6 × 80 mL). The combined organic layers were washed with a saturated aqueous Na2S2O3solution (100 mL), dried over sodium sulfate and evaporated. The residue was dissolved in CH2Cl2and filtered through a short pad of silica gel eluting with CH2Cl2to yield a colourless solid (2.65 g, 50%): Rf0.24 (CH2Cl2100%); m.p. 52–54 °C (from CH2Cl2) [lit. 56–58 °C];1H NMR (400 MHz; CDCl3) δH7.75 (1H, s, H-4), 4.42 (2H, q, J 7.1, H-8), 4.37 (3H, s, H-1'), 1.43 (3H, t, J 7.1, H-9); LRMS m / z (ESI+) 200 ([M+H]+, 100%). The spectroscopic data are in good agreement with the literature values. (1-Methyl-2-nitro-1H-imidazol-5-yl)methanol (15) Intermediate 14 (2.60 g, 13.05 mmol, 1.0 eq.) was solubilized in a mixture of dry THF (45 mL) and CH3OH (2.5 mL) and the reaction was cooled to 0 °C. NaBH4(1.48 g, 39.15 mmol, 3.0 eq.) was added portionwise and the resulting mixture was stirred at 0 °C for 2 h. The mixture was quenched by dropwise addition of a solution of diethyl ether and CH3OH (1:1, 100 mL in total) at 0 °C and stirred for 30 min. 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%): Rf0.47 (EtOAc 100%); m.p. 118–120 °C (from EtOAc) [lit. 141–143 °C];1H NMR (400 MHz; CDCl3) δH7.19 (1H, s, H-4), 4.66 (2H, s, H-6), 4.02 (3H, s, H-1'); LRMS m / z (ESI+) 158 ([M+H]+, 100%). The spectroscopic data are in good agreement with the literature values. 5-(Bromomethyl)-1-methyl-2-nitro-1H-imidazole (16) Under argon, intermediate 15 (200 mg, 1.27 mmol, 1.0 eq.) was solubilized in SOCl2(2.29 mL, 24.13 mmol, 19.0 eq.) at 0 °C and the resulting solution was warmed to rt and stirred for 20 min. The reaction was quenched by dropwise addition of ice-cold water (25 mL) and the product was extracted using EtOAc (3 × 25 mL). The combined organic layers were dried over sodium sulfate, filtered and concentrated in vacuo to yield the title compound as a dark yellow solid (227 mg, 81%): Rf0.61 (EtOAc 100%); m.p. 84–85 °C (from EtOAc);1H NMR (400 MHz; CDCl3) δH7.20 (1H, s, H-4), 4.47 (2H, s, H-6), 4.05 (3H, s, H-1'); LRMS m / z (ESI-) 220 ([79M+H]+, 22%), 222 ([81M+H]+, 22%). The spectroscopic data are in good agreement with the literature values. (1-Methyl-2-nitro-1H-imidazol-5-yl)methyl (4-nitrophenyl) carbonate (17) Under argon, intermediate 15 (100 mg, 0.64 mmol, 1.0 eq.) was solubilized in dry CH2Cl2(8 mL) and dry pyridine (154 µL, 1.92 mmol, 3.0 eq.) was added. The solution was cooled to 0 °C and a solution of 4-nitrophenyl chloroformate (192 mg, 0.96 mmol, 1.5 eq.) in dry CH2Cl2(2 mL) was added dropwise. The reaction mixture was warmed to rt and stirred for 4 h. An addition of 3.0 eq. of dry pyridine and 1.5 eq. of 4-nitrophenyl chloroformate was performed and the reaction was stirred at rt for 16 h. Water (50 mL) was added and the product was extracted with CH2Cl2 (1 × 50 mL). The organic layer was quickly washed with 1M aqueous HCl solution (3 × 50 mL), then dried over sodium sulfate, filtered and evaporated in vacuo. The crude material was purified using silica gel column chromatography, eluting with PE:EtOAc (gradient 25 to 60% EtOAc) to afford a pale pink solid (140 mg, 68%): Rf0.35 (PE / EtOAc 50:50); m.p. 118–120 °C (from PE / EtOAc); ῦmax(neat) / cm-12919 (C-H, w), 2850 (C-H, w), 1769 (C=O, s), 1524 (N-O, w), 1491 (w), 1336 (N-O, w), 1215 (C-C-O, s), 861 (C-H, w), 837 (C-H, w);1H NMR (600 MHz; DMSO-d6) δH8.33 (2H, d, J 9.2, H-3''), 7.58 (2H, d, J 9.2, H-2''), 7.36 (1H, s, H-4), 5.46 (2H, s, H-6), 3.98 (3H, s, H-1');13C NMR (151 MHz; DMSO-d6) δC155.6 (C-1''), 151.9 (C-8), 145.8 (2C, C-4'', C-5), 131.9 (C-2), 130.0 (C-4), 125.9 (C-3''), 123.0 (C-2''), 59.9 (C-6), 34.8 (C-1'); HRMS m / z (ESI+) [Found: 323.0619, C12H11N4O7 requires [M+H]+323.0622]; LRMS m / z (ESI+) 304 (100%), 323 ([M+H]+, 12%); HPLC Retention time 8.4 min, 94.2%. The spectroscopic data are in good agreement with the literature values. 5-Methoxy-2-methyl-1H-indole-3-carbaldehyde (19) POCl3(4.05 mL, 43.4 mmol, 1.4 eq.) was slowly added to dry DMF (10 mL) at 0 °C and the resulting solution was stirred at 0 °C for 10 min. The resulting mixture was then added dropwise to a solution of 5-methoxy-2-methyl-1H-indole (5.00 g, 31.00 mmol, 1.0 eq.) in dry DMF (10 mL) at −10 °C over a period of 45 min. The reaction mixture was then stirred at 0 °C for 1 h and then quenched by dropwise addition of an ice-cold 2M aqueous NaOH solution (50 mL). The compound was then extracted with CH2Cl2(5 × 50 mL). The combined organic layers were dried over sodium sulfate, filtered, and evaporated in vacuo. The resulting brown solid was then washed with cold EtOAc (2 × 50 mL) and filtration in vacuo yielded the product as a beige solid (3.99 g, 68%): Rf0.26 (PE / EtOAc 40:60); m.p. 180–182 °C (from EtOAc) [lit. 198–199 °C, lit.8191– 194 °C, lit.9120–122 °C];1H NMR (400 MHz; CDCl3) δH10.07 (1H, s, H-1'), 7.73 (1H, d, J 2.5, H-4), 7.23 (1H, d, J 8.8, H-7), 6.84 (1H, dd, J 8.8, 2.5, H-6), 3.86 (3H, s, H-2''), 2.70 (3H, s, H-1'''); LRMS m / z (ESI+) 190 ([M+H]+, 68%), 401 (100%). The spectroscopic data are in good agreement with the literature values. 5-Methoxy-1,2-dimethyl-1H-indole-3-carbaldehyde (20) Under argon, a solution of 5-methoxy-2-methyl-1H-indole-3-carbaldehyde 19 (1.32 g, 6.98 mmol, 1.0 eq.) was solubilized in dry DMF (9 mL) and NaH (60% mineral oil, 0.42 g, 10.47 mmol, 1.5 eq.) was added portionwise. Once gas evolution had ceased, the reaction mixture was warmed to rt and stirred for 2 h. To reaction mixture was then cooled to 0 °C, and iodomethane (0.52 mL, 8.38 mmol, 1.2 eq.) was added dropwise. The resulting mixture was warmed to rt and stirred for 2 h. The reaction was then quenched by slow addition of water (50 mL). The mixture was then extracted with CH2Cl2 (6 × 50 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 40 to 100% EtOAc) to afford compound 20 as a beige solid (1.27 g, 90%): Rf0.26 (PE / EtOAc 40:60); m.p. 124–126 °C (from PE / EtOAc) [lit. 132– 133 °C];1H NMR (400 MHz; CDCl3) δH9.82 (1H, s, H-1''), 7.73 (1H, d, J 2.5, H-4), 7.24 (1H, d, J 8.8, H-7), 6.95 (1H, dd, J 8.8, 2.5, H-6), 3.89 (3H, s, H-2'''), 3.74 (3H, s, H-1'), 2.69 (3H, s, H-1''''); LRMS m / z (ESI+) 204 ([M+H]+, 79%), 226 ([M+Na]+, 100%). The spectroscopic data are in good agreement with the literature values. 4-Amino-5-methoxy-1,2-dimethyl-1H-indole-3-carbaldehyde (21) 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. The reaction mixture was stirred for 3 h, then crushed ice-water (400 g) was added and the resulting suspension was stirred at rt for 1 h. The yellow precipitate was collected by vacuum filtration, washed with water (1 × 20 mL) and dried in vacuo to afford the crude material as a pale yellow solid (1.94 g, 63%), which was used in the next step without further purification. 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 NaHCO3. The aqueous layer was extracted with CHCl3(3 × 500 mL) and the combined organic phases were dried over sodium sulfate and concentrated in vacuo to yield the title compound as a brown-yellow solid (1.54 g, 90%): Rf0.60 (100% EtOAc); m.p. 155–157 °C (from CHCl3);1H NMR (400 MHz; CDCl3) δH9.83 (1H, s, H-1''), 6.88 (1H, d, J 8.6, H-7), 6.50 (1H, d, J 8.6, H-6), 3.89 (3H, s, H-2'''), 3.62 (3H, s, H-1'), 2.63 (3H, s, H-1'v); LRMS m / z (ESI+) 219 ([M+H]+, 100%). The spectroscopic data are in good agreement with the literature values. 5-Methoxy-1,2-dimethyl-4,7-dioxo-4,7-dihydro-1H-indole-3-carbaldehyde (22) 4-Amino-5-methoxy-1,2-dimethyl-1H-indole-3-carbaldehyde (1.53 g, 7.01 mmol, 1.0 eq.) was solubilized in acetone (450 mL) and a solution of Fremy’s salt (9.40 g, 35.05 mmol, 5.0 eq.) in NaH2PO4 / Na2HPO4buffer (450 mL, 0.4 M, pH 6.0) was added. The resulting mixture was stirred at rt for 1 h, then acetone was removed in vacuo and the compound was extracted with EtOAc (6 × 400 mL). The combined organic layers were dried over sodium sulfate, filtered, and evaporated in vacuo. Purification using silica gel column chromatography, eluting with acetone:CH2Cl2 (gradient 2 to 10% CH2Cl2) yielded the title compound as an orange-red solid (1.23 g, 75%): Rf0.65 (100% EtOAc); m.p. 236– 238 °C (from acetone / CH2Cl2) [lit. 239–242 °C];1H NMR (400 MHz; CDCl3) δH10.55 (1H, s, H-1''), 5.70 (1H, s, H-6), 3.94 (3H, s, H-1'), 3.85 (3H, s, H-2'''), 2.62 (3H, s, H-1'v); LRMS m / z (ESI+) 234 ([M+H]+, 100%), 256 ([M+Na]+, 75%). The spectroscopic data are in good agreement with the literature values. 3-(Hydroxymethyl)-5-methoxy-1,2-dimethyl-1H-indole-4,7-dione (23) Under argon, to a suspension of 5-methoxy-1,2-dimethyl-4,7-dioxo-4,7-dihydro- 1H-indole-3-carbaldehyde (1.22 g, 5.23 mmol, 1.0 eq.) in dry THF (100 mL) and dry CH3OH (100 mL) at 0 °C, NaBH4(0.99 g, 26.15 mmol, 5.0 eq.) was added portionwise. The mixture was stirred at 0 °C for 45 min and then water (100 mL) was slowly added. The compound was then quickly extracted with CH2Cl2(4 × 100 mL). The combined organic layers were dried over sodium sulfate, filtered, and concentrated in vacuo. Purification using silica gel column chromatography, eluting with PE:EtOAc (gradient 50 to 100% EtOAc) yielded the title compound as a red solid (1.08 g, 88%): Rf0.47 (100% EtOAc); m.p. 198–200 °C (from PE / EtOAc) [lit. 199–200 °C];1H NMR (400 MHz; CD3OD) δH5.61 (1H, s, H-6), 4.71 (2H, s, H-1''), 3.90 (3H, s, H-1'), 3.81 (3H, s, H-2'''), 2.29 (3H, s, H-1'v); LRMS m / z (ESI+) 258 ([M+Na]+, 100%). The spectroscopic data are in good agreement with the literature values. 3-(Bromomethyl)-5-methoxy-1,2-dimethyl-1H-indole-4,7-dione (24) 3-(Hydroxymethyl)-5-methoxy-1,2-dimethyl-1H-indole-4,7-dione (0.5 g, 2.12 mmol, 1.0 eq.) was solubilized in dry CH2Cl2(22 mL) under argon. The reaction mixture was cooled to 0 °C and PBr3(403 µL, 4.24 mmol, 2.0 eq.) was added dropwise. After 10 min, the reaction was concluded. It was quenched by slow addition of saturated aqueous NaHCO3solution (30 mL). The product was extracted with CH2Cl2(3 × 30 mL) and the collected organic phases were dried over sodium sulfate, filtered and evaporated in vacuo, yielding the title compound as a dark red solid (0.61 g, 97%): Rf0.25 (PE / EtOAc 60:40); m.p. 190–192 °C (from CH2Cl2);1H NMR (400 MHz; CDCl3) δH5.63 (1H, s, H- 6), 4.78 (2H, s, H-1''), 3.90 (3H, s, H-1'), 3.81 (3H, s, H-2'''), 2.26 (3H, s, H-1'v); LRMS m / z (ESI-) 296 ([79M-H]-, 24%), 298 ([81M-H]-, 24%). The spectroscopic data are in good agreement with the literature values. (2S,4R)-1-((S)-2-((Tert-butoxycarbonyl)amino)-3,3-dimethylbutanoyl)-4- hydroxypyrrolidine-2-carboxylic acid (27) L-4-Hydroxyproline methyl ester hydrochloride (3.45 g, 19.02 mmol, 1.1 eq.) was added to a solution of Boc-L-tert-leucine (4.00 g, 17.29 mmol, 1.0 eq.), HATU (7.89 g, 20.75 mmol, 1.2 eq.) and DIPEA (18 mL, 103.74 mmol, 6.0 eq.) in dry DMF (80 mL). The reaction was stirred at rt for 16 h, after which it was diluted with water (250 mL) and extracted with EtOAc (3 × 200 mL). The combined organic phases were washed with saturated aqueous NaHCO3solution (2 × 400 mL), 1M aqueous citric acid solution (3 × 400 mL), then dried over sodium sulfate, filtered, and concentrated in vacuo to afford (2S,4R)-methyl 1-((S)-2-((tert-butoxycarbonyl)amino)-3,3-dimethylbutanoyl)-4- hydroxypyrrolidine-2-carboxylate (5.85 g, 86%) which was used in the next step without further purifications. 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 H2O (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. The solid was collected by vacuum filtration, washed with H2O (1 × 20 mL) and dried in vacuo to yield a colourless solid (5.46 g, 100%): Rf0.15 (EtOAc / CH3OH 70:30); m.p. 84–86 °C (from water); (c 1.0, CH3OH); ῦmax(neat) / cm-13398 (O-H, s), 2976 (C-H, w), 1723 (C=O, s), 1689 (C=O, s), 1615 (C=O, s), 1254 (C-O, s), 1171 (s), 1058 (C-O, s), 690 (s);1H NMR (400 MHz; DMSO-d6) δH 6.46 (1H, d, J 9.4, NH-1''), 5.19 (1H, br s, OH), 4.34–4.30 (1H, m, H-4), 4.26 (1H, dd, Js8.3, H-2), 4.15 (1H, d, J 9.4, H-7), 3.64 (1H, dd, J 10.6, 4.6, H-5a), 3.57 (1H, dd, J 10.6, 4.6, H-5b), 2.09 (1H, ddd, J 13.2, 8.3, 4.6, H-3a), 1.88 (1H, ddd, J 13.2, 8.3, 4.6, H-3b), 1.38 (9H, s, H-5''), 0.93 (9H, s, H-9);13C NMR (101 MHz, DMSO-d6) δ 173.5 (C-1'), 170.0 (C-6), 155.4 (C-2''), 78.3 (C-4''), 68.8 (C-4), 58.3 (C-7), 57.9 (C-2), 56.1 (C-5), 37.4 (C-8), 35.5 (C-3), 28.3 (C-5''), 26.3 (C-9); HRMS m / z (ESI+) [Found: 367.1838, C16H28N2O6Na requires [M+Na]+367.1840]; LRMS m / z (ESI−) 343 ([M−H]−, 100%). The spectroscopic data are in good agreement with the literature values. Tert-butyl 4-bromobenzylcarbamate (29) To a solution of (4-bromophenyl)methanamine (3.39 mL, 26.87 mmol, 1.0 eq.) and NaHCO3(1.67 g, 19.88 mmol, 0.74 eq.) in water (50 mL) and EtOAc (50 mL), Boc2O (7.04 g, 32.24 mmol, 1.2 eq.) was added at 5 °C. The reaction mixture was stirred at rt for 2 h, then the aqueous layer was extracted with EtOAc (2 × 50 mL). The combined organic layers were dried over sodium sulfate, filtered and evaporated in vacuo to obtain a colourless solid. The solid was suspended in hexane (20 mL) and the resulting suspension was stirred at 0 °C for 30 min, then filtered in vacuo to yield the title compound as a colourless solid (7.47 g, 97%): Rf0.58 (PE / EtOAc 80:20); m.p. 80–82 °C (from PE / EtOAc) [lit. 86–88 °C]; ῦmax(neat) / cm-13316 (N-H, w), 2979 (C-H, w), 1679 (C=O, s), 1535 (N-C=O, s), 1281 (C-O, s), 1166 (s), 1059 (w), 681 (C-H, s);1H NMR (400 MHz; CDCl3) δH7.44 (2H, d, J 8.5, H-3'), 7.15 (2H, d, J 8.5, H-2'), 4.86 (1H, br s, NH-2), 4.26 (2H, d, J 6.0, H-1), 1.45 (9H, s, H-6);13C NMR (101 MHz; CDCl3) δC155.7 (C-3), 138.0 (C-1'), 131.7 (C-3'), 129.2 (C-2'), 121.1 (C-4'), 79.7 (C-5), 44.0 (C-1), 28.3 (C-6); LRMS m / z (ESI+) 307 ([79M+Na]+, 38%), 309 ([81M+Na]+, 38%); HPLC Retention time 10.2 min, 98.3%. The spectroscopic data are in good agreement with the literature values. Tert-butyl 4-(4-methylthiazol-5-yl)benzylcarbamate (30) 4-Methylthiazole (2.4 mL, 26.21 mmol, 1.5 eq.) was added to a solution of tert- butyl 4-bromobenzylcarbamate (5.00 g, 17.47 mmol, 1.0 eq.), KOAc (3.43 g, 34.94 mmol, 2.0 eq.) and Pd(OAc)2(39 mg, 0.17 mmol, 0.01 eq.) in dry DMA (12.5 mL) under argon. The reaction mixture was stirred for 24 h at 90 °C, cooled to rt and concentrated in vacuo. Water (100 mL) was added and the mixture was extracted with EtOAc (3 × 80 mL). The collected organic phases were washed with 0.5M aqueous LiCl solution (1 × 200 mL), dried over sodium sulfate, filtered, and concentrated in vacuo. Purification using silica gel column chromatography, eluting with PE:EtOAc (gradient 10 to 30% EtOAc) yielded the title compound as a yellow solid (2.58 g, 48%): Rf0.26 (PE / EtOAc 60:40); m.p. 102– 104 °C (from PE / EtOAc) [lit. 112–114 °C]; ῦmax(neat) / cm-13233 (N-H, w), 3053 (C-H, w), 2977 (C-H, w), 1703 (C=O, s), 1555 (N-C=O, s), 1270 (C-O, s), 1166 (s), 1050 (C-S, s), 936 (C-H, s), 722 (C-H, s);1H NMR (400 MHz; CDCl3) δH8.70 (1H, s, H-2), 7.41 (2H, d, J 8.8, H-2'), 7.35 (2H, d, J 8.8, H-3'), 4.92 (1H, br s, NH-6'), 4.36 (2H, d, J 6.0, H-5'), 2.53 (3H, s, H-1''), 1.47 (9H, s, H-10'); LRMS m / z (ESI+) 305 ([M+H]+, 100%). The spectroscopic data are in good agreement with the literature values. Tert-butyl ((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5- yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)carbamate (31) Step 1: Compound 30 (1.77 g, 5.80 mmol, 1.0 eq.) was solubilized in CH2Cl2(15 mL) and HCl (4M in dioxane, 8.70 mL, 34.80 mmol, 6.0 eq.) was added dropwise. The reaction was stirred for 2 h at rt. Then, the volatile was evaporated in vacuo to afford the crude material as a yellow solid, which was used in the next step without further purification. 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. Water (50 mL) was then added and the mixture was extracted with EtOAc (4 × 30 mL). The combined organic phases were washed with saturated aqueous NaHCO3solution (5 × 100 mL), 5% aqueous citric acid solution (1 × 100 mL), and 0.5M aqueous LiCl solution (2 × 100 mL), then dried over sodium sulfate, filtered, and concentrated in vacuo. The crude material was purified using silica gel column chromatography, eluting with PE:EtOAc (gradient 20 to 100% EtOAc) yielded the title compound as a colourless solid (1.43 g, 46%): Rf0.17 (EtOAc); m.p. 68–70 °C (from EtOAc / CH3OH) [lit. 82–83 °C]; ^ ^^ଶ^ହ= −29.5 (c 1.0, CH3OH); ῦmax(neat) / cm-13435 (O-H, s), 3313 (N-H, w), 2964 (C-H, w), 2937 (C-H, w), 1686 (C=O, s), 1633 (C=O, s), 1525 (N-C=O, s), 1435 (C-H, w), 1238 (C-O, s), 1167 (C-S, s), 804 (C-H, s);1H NMR (400 MHz; CDCl3) δH8.67 (1H, s, H-2'''), 7.51 (1H, br t, J 5.3, NH-2'), 7.34–7.30 (4H, m, H-2'', H-3''), 5.24 (1H, d, J 9.2, NH-1v), 4.71 (1H, dd, J 7.9, 8.0, H-2), 4.56–4.48 (2H, m, H-4, H-3'a), 4.30 (1H, dd, J 9.8, 5.3, H-3'b), 4.17 (1H, d, J 9.2, H- 7), 3.99 (1H, dd, J 11.3, 3.7, H-5a), 3.60 (1H, dd, J 11.3, 3.7, H-5b), 2.49–2.42 (4H, m, H- 3a, H-1'v), 2.10 (1H, ddd, J 11.8, 7.9, 5.5, H-3b), 1.39 (9H, s, H-5v), 0.90 (9H, s, H-9);13C NMR (101 MHz; CDCl3) δC172.5 (C-6), 171.0 (C-1'), 156.4 (C-2v), 150.5 (C-2'''), 148.5 (C-4'''), 138.2 (C-4''), 131.8 (C-1''), 130.9 (C-5'''), 129.6 (C-3''), 128.1 (C-2''), 80.5 (C-4v), 70.2 (C-4), 59.0 (C-7), 58.6 (C-2), 56.7 (C-5), 43.3 (C-3'), 36.1 (C-3), 35.2 (C-8), 28.4 (C- 5v), 26.4 (C-9), 16.1 (C-1'v); HRMS m / z (ESI+) [Found: 531.2627, C27H39N4O5S requires [M+H]+531.2636]; LRMS m / z (ESI+) 531 ([M+H]+, 100%); HPLC Retention time 8.4 min, 98.9%. The spectroscopic data are in good agreement with the literature values. (2S,4R)-1-((S)-14-Azido-2-(tert-butyl)-4-oxo-6,9,12-trioxa-3-azatetradecan-1- oyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (32) Step 1: Compound 31 (570 mg, 1.07 mmol, 1.0 eq.) was solubilized in CH2Cl2(5.6 mL) and HCl (4M in dioxane, 3.21 mL, 12.84 mmol, 12.0 eq.) was added. The mixture was stirred at rt for 2 h, then the volatiles were evaporated in vacuo to afford the crude material as a yellow solid, which was used in the next step without further purification. 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). The reaction was stirred at rt for 16 h, after which it was diluted with water (40 mL) and extracted with EtOAc (3 × 40 mL). The organic layers were combined, washed with 0.5M aqueous LiCl solution (1 × 100 mL), dried over sodium sulfate, filtered and concentrated in vacuo. The crude product was purified using silica gel column chromatography, eluting with EtOAc:CH3OH (gradient 0 to 10% CH3OH) to isolate the title compound as an amorphous yellow solid (433 mg, 63%): Rf0.27 (EtOAc / CH3OH 90:10);^^^ଶ^ହ= −15.8 (c 1.0, CH3OH); ῦmax(neat) / cm-13306 (O-H, s), 2918 (C-H, w), 2107 (N=N=N, s), 1665 (C=O, s), 1636 (C=O, s), 1525 (N-H, s), 1436 (N-H, s), 1112 (C- O, s) 850 (C-H, w);1H NMR (400 MHz, CDCl3) δH8.67 (1H, s, H-2'''), 7.39–7.31 (4H, m, H-2'', H-3''), 7.28 (1H, br s, NH-2'), 4.73 (1H, dd, Js7.8, H-2), 4.57–4.47 (3H, m, H-4, H-7, H-3'a), 4.33 (1H, ddd, J 15.4, 7.8, 5.3, H-3'b), 4.08 (1H, dd, J 11.5, 2.4, H-5a), 4.02 (1H, d, J 15.8, H-3va), 3.96 (1H, d, J 15.8, H-3vb), 3.68–3.59 (11H, m, H-5a, H-5v, H-6v, H-8v, H- 9v, H-11v), 3.36 (2H, t, J 5.1, H-12v), 3.26 (1H, br s, OH), 2.58–2.51 (4H, m, H-3a, H-1'v), 2.10 (1H, ddd, J 7.7, 5.4, 1.7, H-3b), 0.95 (9H, s, H-9);13C NMR (101 MHz; CDCl3) δC171.6 (C-6), 170.8 (C-1'), 170.7 (C-2v), 150.4 (C-2'''), 148.6 (C-4'''), 138.2 (C-4''), 131.7 (C-1''), 131.1 (C-5'''), 129.7 (C-3''), 128.3 (C-2''), 71.3 (C-5v), 70.8 (2C, C-8v, C-9v), 70.7 (C-6v), 70.5 (C-11v), 70.3 (C-3v), 70.2 (C-4), 58.5 (C-2), 57.3 (C-7), 56.7 (C-5), 50.8 (C- 12v), 43.4 (C-3'), 35.9 (C-8), 35.0 (C-3), 26.5 (C-9), 16.2 (C-1'v); HRMS m / z (ESI+) [Found: 646.3014, C30H44N7O7S requires [M+H]+646.3017]; LRMS m / z (ESI+) 646 ([M+H]+, 100%), 668 ([M+Na]+, 93%); HPLC Retention time 7.8 min, 93.4%.

[0017] (2S,4R)-1-((S)-2-(Tert-butyl)-17-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H- thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)-4,16-dioxo-6,9,12-trioxa-3,15- diazaheptadecan-1-oyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2- carboxamide (4, MZ1) Step 1: Compound 32 (100 mg, 0.15 mmol, 1.0 eq.) was added to a round-bottom flask containing Pd / C 10% (10 mg, 10% w / w) and CH3OH (15 mL). The reaction was flushed with hydrogen and the mixture was stirred at rt for 4 h. The mixture was then filtered over a pad of celite and washed with CH3OH (2 × 20 mL). The volatile was evaporated to afford the crude material, which was used in the next step without further purification. Step 2: The obtained intermediate was added to a stirring solution of DIPEA (104 µL, 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. Water (20 mL) was then added and the mixture was extracted with EtOAc (6 × 20 mL). The combined organic phases were dried over sodium sulfate, filtered, and evaporated in vacuo. The crude material was purified using silica gel column chromatography, eluting with EtOAc:CH3OH (gradient 0 to 15% CH3OH) yielding compound 4 as a colourless solid (99 mg, 66%): Rf 0.14 (EtOAc / CH3OH 80:20); m.p. 100–102 °C (from EtOAc / CH3OH);^^^ଶ^ହ= +7.7 (c 1.0, CH3OH); ῦmax(neat) / cm-13309 (O-H, s), 2923 (C-H, w), 1640 (C=O, s), 1532 (N-H, s), 1420 (N-H, s), 1370 (s), 1091 (C-O, s) 846 (C-H, w), 806 (C-H, w);1H NMR (400 MHz; CDCl3) δH 8.78 (1H, s, H-2'''), 8.03 (1H, br s, NH-13v), 7.45 (1H, br s, NH-1v), 7.41 (2H, d, J 8.2, H-3x), 7.35–7.31 (6H, m, H-2'', H-3'', H-2x), 4.83 (1H, dd, Js 7.9, H-2), 4.73–4.68 (2H, m, H-7, H-6v'), 4.57–4.47 (2H, m, H-4, H-3'a), 4.33– 4.27 (2H, m, H-3va, H-3'b), 4.15–4.11 (2H, m, H-3vb, H-5a), 3.72–3.54 (13H, m, H-5b, H-5v, H-6v, H-8v, H-9v, H-11v, H-12va, H-15va), 3.38–3.32 (2H, m, H-12vb, H-15vb), 2.66 (3H, s, H-1'x), 2.54 (3H, s, H-1'v), 2.45–2.39 (4H, m, H-1v'', H-3a), 2.19–2.14 (1H, m, H-3b), 1.66 (3H, s, H-1v'''), 0.99 (9H, s, H-9);13C NMR (101 MHz; CDCl3) δC171.5 (C-1'), 171.1 (C- 14v), 170.9 (C-2v), 170.7 (C-6), 163.9 (C-4v'), 155.9 (C-6av'), 150.3 (C-9v'), 149.9 (C-2'''), 148.4 (C-4'''), 138.4 (C-1''), 136.8 (C-1x), 136.6 (C-4x), 131.9 (C-10av'), 131.8 (C-3av'), 131.1 (C-5'''), 131.0 (C-4''), 130.8 (C-2v'), 130.7 (C-3v'), 130.0 (C-3x), 129.5 (C-2x), 128.8 (C-3''), 128.0 (C-2''), 71.4 (C-5v), 70.8 (C-9v), 70.5 (C-6v), 70.4 (2C, C-3v, C-8v), 70.3 (C- 11v), 70.0 (C-4), 59.0 (C-2), 57.2 (C-5), 56.8 (C-7), 54.2 (C-6v'), 43.1 (C-3'), 39.8 (C-12v), 38.2 (C-15v), 36.5 (C-3), 35.6 (C-8), 26.5 (C-9), 16.1 (C-1'v), 14.5 (C-1v'''), 13.2 (C-1v''), 11.8 (C-1'x); HRMS m / z (ESI+) [Found: 1002.3735, C49H61ClN9O8S2requires [M+H]+1002.3768]; LRMS mass not detected; HPLC Retention time 8.8 min, 94.6%; The spectroscopic data are in good agreement with the literature values. (3R,5S)-1-((S)-2-(Tert-butyl)-17-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H- thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)-4,16-dioxo-6,9,12-trioxa-3,15- diazaheptadecan-1-oyl)-5-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-3-yl ((1-methyl-2-nitro-1H-imidazol-5-yl)methyl) carbonate (1, NI-VHL) Compound 4 (90 mg, 0.09 mmol, 1.0 eq.) was solubilized in dry CH2Cl2(5 mL) and dry TEA (38 µL, 0.27 mmol, 3.0 eq.) and intermediate 17 (40 mg, 0.13 mmol, 1.4 eq.) were added in order. The solution was stirred at 40 °C for 16 h. After this time, the reaction was not concluded. An addition of 3 eq. of TEA and 1.4 eq. of intermediate 17 was performed and the reaction was stirred at 40 °C for further 8 h. A second addition of 0.5 eq. of intermediate 17 was performed and the reaction was stirred at 40 °C for further 16 h. The reaction was cooled to rt and water (5 mL) was added. The product was extracted with CH2Cl2(3 × 5 mL). The collected organic layers were dried over sodium sulfate, filtered and dried in vacuo. The crude material was purified using silica gel column chromatography, eluting with EtOAc:CH3OH (gradient 0 to 15% CH3OH) yielding compound 7 as a colourless solid (48 mg, 45%). The compound was further purified using semi prep HPLC, method A: Rf 0.26 (EtOAc / CH3OH 80:20); m.p. 93–95 °C (from EtOAc / CH3OH); ^ ^^ଶ^ହ= +18.9 (c 1.0, CH3OH); ῦmax(neat) / cm-13413 (N-H, w), 1750 (O- C(=O)-O, s), 1647 (C=O, s), 1538 (N-H, s), 1489 (N-H, s), 1433 (w), 1256 (C-O, s) 836 (C-H, w);1H NMR (600 MHz; CD3OD) δH 8.86 (1H, s, H-2'''), 7.43–7.37 (8H, m, H-2'', H- 3'', H-2x''', H-3x'''), 7.23 (1H, s, H-4v''), 5.33–5.26 (3H, m, H-4, H-3v'), 4.61–4.51 (4H, m, H-2, H-7, H-3'a, H-6'x), 4.35–4.27 (2H, m, H-5a, H-3'b), 4.03 (1H, d, J 7.8, H-3va), 4.00 (1H, d, J 7.8, H-3vb), 3.98 (3H, s, H-2v'''), 3.92 (1H, dd, J 12.2, 3.7, H-5b), 3.71–3.65 (9H, m, H-5v, H-6v, H-8v, H-9v, H-11va), 3.59–3.57 (2H, m, H-11vb, H-12va), 3.45–3.41 (2H, m, H-12va, H-15vb), 3.30–3.28 (1H, m, H-15va), 2.67 (3H, s, H-1x''), 2.47–2.45 (4H, m, H-3a, H-1'v), 2.44 (3H, s, H-1x), 2.26 (1H, ddd, J 14.2, 9.5, 4.6, H-3b), 1.69 (3H, s, H-1x'), 1.04 (9H, s, H-9);13C NMR (151 MHz; CD3OD) δC173.6 (C-1'), 172.9 (C-14v), 172.2 (C-2v), 171.8 (C-6), 166.1 (C-4'x), 157.0 (C-6a'x), 155.1 (C-1v'), 152.8 (C-2'''), 152.1 (C-9'x), 149.1 (C-4'''), 147.6 (C-2v''), 140.1 (C-1''), 138.1 (C-1x'''), 137.9 (C-4x'''), 133.6 (C-10a'x), 133.5 (C-5v''), 133.4 (C-5'''), 133.2 (C-4''), 132.0 (C-3'x), 131.9 (C-2'x), 131.6 (C-3a'x), 131.4 (C- 3x'''), 130.4 (C-2x'''), 130.1 (C-4v''), 129.8 (C-3''), 129.0 (C-2''), 78.9 (C-4), 72.2 (C-5v), 71.6 (C-9v), 71.5 (C-6v), 71.4 (C-8v), 71.1 (C-3v), 70.7 (C-11v), 60.5 (C-2), 59.4 (C-3v'), 58.4 (C-7), 55.3 (C-6'x), 55.2 (C-5), 43.8 (C-3'), 40.6 (C-12v), 38.7 (C-15v), 36.5 (C-8), 36.0 (C- 3), 35.0 (C-2v'''), 26.9 (C-9), 15.8 (C-1'v), 14.4 (C-1x'), 12.9 (C-1x), 11.6 (C-1x''); HRMS m / z (ESI+) [Found: 1185.4087, C55H66ClN12O12S2requires [M+H]+1185.4048]; LRMS m / z (ESI+) 1185 ([35M+H]+, 97%), 1187 ([37M+H]+, 83%), 1207 ([35M+Na]+, 100%), 1209 ([37M+Na]+, 90%); HPLC Retention time 9.4 min, 99.0%. (2S,4R)-1-((S)-14-Azido-2-(tert-butyl)-4-oxo-6,9,12-trioxa-3-azatetradecan-1- oyl)-4-((5-methoxy-1,2-dimethyl-4,7-dioxo-4,7-dihydro-1H-indol-3-yl)methoxy)-N-(4- (4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (33) To a solution of compound 32 (100 mg, 0.15 mmol, 1.0 eq.) and TBAI (18 mg, 0.05 mmol, 0.3 eq.) in CH2Cl2(5 mL), a 50% v / wt aqueous NaOH solution (1.25 mL) was added. After 15 min at rt, compound 24 (51 mg, 0.17 mmol, 1.1 eq.) was added. The mixture was stirred at rt protected from the dark for 1h, then an addition of 1.1 eq of compound 24 was performed and the reaction was stirred for a further 1 h at rt. A second addition of 0.4 eq. of compound 24 was then performed and the reaction was stirred for a further 1 h at rt. Water (5 mL) was added and the mixture was extracted with CH2Cl2(3 × 5 mL). The collected organic phases were dried over sodium sulfate, filtered, and concentrated in vacuo. Purification using silica gel column chromatography, eluting with EtOAc:CH3OH (gradient 0 to 10% CH3OH) yielded an orange solid, which was solubilized in EtOAc (15 mL) and the organic layer was washed with saturated aqueous NH4Cl solution (4 × 15 mL). The organic layer was dried over sodium sulfate, filtered and concentrated in vacuo to afford the title compound as an orange solid (47 mg, 36%): Rf0.28 (EtOAc / CH3OH 90:10); m.p. 42–44 °C (from EtOAc / CH3OH); ^ ^^ଶ^ହ= −2.5 (c 1.0, CH3OH); ῦmax(neat) / cm-13404 (N-H, w), 2980 (C-H, s), 2888 (C-H, w), 2106 (N=N=N, w), 1671 (C=O, s), 1635 (C=O, s), 1600 (C=O, s), 1507 (N-H, s), 1461 (N-H, s), 1225 (s), 1151 (C-O, s), 1088 (C-O, w), 1018 (C-O, w), 850 (C-H, w);1H NMR (600 MHz; CDCl3) δH8.67 (1H, s, H-2'''), 7.38–7.33 (4H, m, H-2'', H-3''), 7.23 (1H, br s, NH-1v), 7.08 (1H, br t, J 6.0, NH-2'), 5.59 (1H, s, H-6v''), 4.75 (1H, d, J 11.1, H-1v'b), 4.70 (1H, d, J 9.5, H-7), 4.64–4.55 (3H, m, H-2, H-3'a, H-1v'a), 4.44–4.39 (2H, m, H-4, H-3'b), 4.07 (1H, dd, J 10.7, 2.6, H-5a), 4.01 (1H, d, J 15.5, H-3va), 3.95 (1H, d, J 15.5, H-3vb), 3.86 (3H, s, H-1v'''), 3.78 (3H, s, H-2x), 3.71–3.61 (11H, m, H-5a, H-5v, H-6v, H-8v, H-9v, H-11v), 3.34 (2H, t, J 5.2, H-12v), 2.55–2.49 (4H, m, H-3a, H-1'v), 2.29–2.22 (4H, m, H-3b, H-1'x), 0.94 (9H, s, H-9);13C NMR (151 MHz; CDCl3) δC178.9 (C-4v''), 178.2 (C-7v''), 171.3 (C-6), 171.0 (C-1'), 169.4 (C-2v), 159.8 (C-5v''), 150.5 (C-2'''), 148.1 (C-4'''), 138.4 (C-1''), 138.0 (C-2v''), 131.7 (C-5'''), 130.7 (C-4''), 129.7 (C-3''), 129.0 (C-7av''), 128.4 (C-2''), 121.8 (C-3av''), 117.9 (C- 3v''), 106.9 (C-6v''), 76.9 (C-4), 71.3 (C-5v), 70.9 (2C, C-8v, C-9v), 70.7 (2C, C-3v, C-6v), 70.2 (C-11v), 60.7 (C-1v'), 59.2 (C-2), 56.6 (C-2x), 56.5 (C-7), 53.6 (C-5), 50.8 (C-12v), 43.4 (C-3'), 36.0 (C-8), 33.6 (C-3), 32.5 (C-1v'''), 26.5 (C-9), 16.1 (C-1'v), 9.7 (C-1'x); HRMS m / z (ESI+) [Found: 863.3756, C42H55N8O10S requires [M+H]+863.3735]; LRMS mass not detected; HPLC Retention time 9.1 min, 96.4%.

[0018] (2S,4R)-1-((S)-14-Azido-2-(tert-butyl)-4-oxo-6,9,12-trioxa-3-azatetradecan-1- oyl)-4-(benzyloxy)-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (34) Compound 34 was synthesized as previously described for compound 33, starting from compound 32 (100 mg, 0.15 mmol, 1.0 eq.) and benzyl bromide (20 µL, 0.17 mmol, 1.1 eq.). The mixture was stirred at rt protected from the dark for 1h, then an addition of 1.1 eq of benzyl bromide was performed and the reaction was stirred for a further 1 h at rt. A second addition of 0.4 eq. of benzyl bromide was then performed and the reaction was stirred for a further 1 h at rt. Water (5 mL) was added and the mixture was extracted with CH2Cl2(6 × 5 mL). The collected organic phases were dried over sodium sulfate, filtered, and concentrated in vacuo. Purification using silica gel column chromatography, eluting with PE:EtOAc (gradient 50 to 100% EtOAc) yielded the title compound as a yellow oil (90 mg, 81%): Rf0.14 (EtOAc 100%); ^ ^^ଶ^ହ= −2.4 (c 1.0, CH3OH); ῦmax(neat) / cm-12939 (C-H, s), 2869 (C-H, w), 2107 (N=N=N, s), 1678 (C=O, s), 1639 (C=O, s), 1520 (N-H, s), 1435 (N-H, s), 1112 (C-O, s), 1030 (C-O, w), 738 (C-H, w), 699 (C-H, w);1H NMR (400 MHz; CDCl3) δH8.67 (1H, s, H-2'''), 7.37–7.22 (11H, m, H-2'', H-3'', H-2v'', H-3v'', H-4v'', NH-2', NH-1v), 4.70–4.66 (2H, m, H-2, H-7), 4.57–4.45 (3H, m, H-3'a, H-1v'), 4.37–4.29 (2H, m, H-4, H-3'b), 4.07 (1H, dd, J 10.8, 3.3, H-5a), 4.02 (1H, d, J 15.5, H-3va), 3.92 (1H, d, J 15.5, H-3vb), 3.67–3.61 (11H, m, H-5a, H-5v, H-6v, H-8v, H-9v, H-11v), 3.34 (2H, t, J 5.2, H-12v), 2.59 (1H, ddd, J 11.6, 5.8, 1.6, H-3a), 2.51 (3H, s, H-1'v), 2.13 (1H, ddd, J 11.6, 8.1, 4.0, H-3b), 0.92 (9H, s, H-9);13C NMR (101 MHz; CDCl3) δC 171.5 (C-6), 170.7 (C-1'), 169.6 (C-2v), 150.4 (C-2'''), 148.5 (C-4'''), 138.1 (C-1''), 137.8 (C-1v''), 131.7 (C-5'''), 131.1 (C-4''), 129.6 (C-3''), 128.5 (C-2''), 128.3 (C-2v''), 127.9 (C-4v''), 127.8 (C-3v''), 77.0 (C-4), 71.2 (2C, C-5v, C-1v'), 70.8 (2C, C-8v, C-9v), 70.6 (C-3v), 70.5 (C-6v), 70.1 (C-11v), 58.7 (C-2), 56.3 (C-7), 53.0 (C-5), 50.7 (C-12v), 43.3 (C-3'), 35.7 (C-8), 33.5 (C-3), 26.4 (C-9), 16.1 (C-1'v); HRMS m / z (ESI+) [Found: 736.3466, C37H50N7O7S requires [M+H]+736.3487]; LRMS mass not detected; HPLC Retention time 9.8 min, 94.2%. (2S,4R)-1-((S)-2-(Tert-butyl)-17-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H- thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)-4,16-dioxo-6,9,12-trioxa-3,15- diazaheptadecan-1-oyl)-4-((5-methoxy-1,2-dimethyl-4,7-dioxo-4,7-dihydro-1H-indol- 3-yl)methoxy)-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (2, IQ- Compound 33 (42 mg, 0.05 mmol, 1.0 eq.) was solubilized in THF (1 mL) and PPh3(26 mg, 0.10 mmol, 2.0 eq.) was added. The reaction was stirred for 10 min at rt, then water (10 µL, 0.50 mmol, 10.0 eq.) was added. After 8 h at rt, 10 eq. of water were added and the reaction was stirred at rt overnight. The volatiles were evaporated to afford the crude material as an orange solid, which was used in the next step without further purification. Step 2: (+)-JQ1-COOH (18 mg, 0.05 mmol, 0.9 eq.) was solubilized in dry DMF (0.5 mL) and DIPEA (35 µL, 0.20 mmol, 4.0 eq.) and HATU (23 mg, 0.06 mmol, 1.2 eq.) were added in order under argon. After 10 min, the intermediate (0.05 mmol, 1.0 eq.) obtained in Step 1 in dry DMF (0.5 mL) was added and the reaction was stirred at rt overnight. Water (5 mL) was then added and the mixture was extracted with EtOAc (10 × 5 mL). The combined organic phases were dried over sodium sulfate, filtered, and evaporated in vacuo. The crude material was purified using silica gel column chromatography, eluting with EtOAc:CH3OH (gradient 0 to 15% CH3OH) to yield the title compound as an orange solid (22 mg, 36%). The compound was further purified using semi prep HPLC, method B: Rf0.15 (EtOAc / CH3OH 80:20); m.p. 112–114 °C (from EtOAc / CH3OH); ^ ^^ଶ^ହ= −5.3 (c 0.5, CH3OH); ῦmax(neat) / cm-13390 (N-H, w), 2924 (C-H, w), 2855 (C-H, w), 1669 (C=O, s), 1636 (C=O, s), 1599 (C=O, s), 1461 (N-H, s), 1437 (N-H, s), 1224 (s), 1090 (C-O, s), 1041 (C-O, w), 1015 (C-O, w), 842 (C-H, w), 804 (C-H, w);1H NMR (600 MHz; CD3OD) δH8.86 (1H, s, H-2'''), 7.45–7.35 (8H, m, H-2'', H-3'', H-2xv, H-3xv), 5.64 (1H, s, H-6v''), 4.74 (1H, s, H-7), 4.66 (1H, d, J 11.2, H-1v'a), 4.61–4.57 (2H, m, H-1v'b,H-6x'), 4.54–4.48 (2H, m, H-2,H-3'a), 4.34 (1H, d, J 15.4, H-3'b), 4.31–4.30 (1H, m, H-4), 4.14 (1H, dd, J 11.4, 4.4, H-5a), 4.07 (1H, d, J 15.5, H-3va), 4.03 (1H, d, J 15.5, H-3vb), 3.83 (3H, s, H- 1v'''), 3.78–3.76 (4H, m, H-5b, H-2x), 3.71–3.62 (8H, m, H-5v, H-6v, H-8v, H-9v), 3.57–3.55 (2H, m, H-11va, H-11vb), 3.46–3.40 (3H, m, H-12va, H-12vb, H-15va), 3.28 (1H, dd, J 15.1, 5.1, H-15vb), 2.68 (3H, s, H-1x'v), 2.50–2.46 (4H, m, H-3a, H-1'v), 2.43 (3H, s, H-1x''), 2.22 (3H, s, H-1'x), 2.06 (1H, ddd, J 13.6, 9.5, 4.4, H-3b), 1.68 (3H, s, H-1x'''), 1.04 (9H, s, H-9);13C NMR (151 MHz; CD3OD) δC 180.1 (C-4v''), 179.5 (C-7v''), 174.3 (C-1'), 172.9 (C-14v), 172.1 (C-2v), 171.7 (C-6), 166.0 (C-4x'), 161.1 (C-5v''), 157.1 (C-6ax'), 152.8 (C-2'''), 152.1 (C-9x'), 149.1 (C-4'''), 140.2 (C-2v''), 140.0 (C-1''), 138.1 (C-1xv), 137.9 (C-4xv), 133.5 (C- 10ax'), 133.4 (C-5'''), 133.2 (C-4''), 132.1 (C-3x'), 132.0 (C-2x'), 131.5 (C-3ax'), 131.3 (C- 3xv), 130.4 (C-2xv), 129.9 (C-7av''), 129.7 (C-3''), 129.0 (C-2''), 122.7 (C-3av''), 118.8 (C- 3v''), 107.6 (C-6v''), 78.4 (C-4), 72.2 (C-5v), 71.7 (C-9v), 71.6 (C-6v), 71.4 (C-8v), 71.2 (C- 3v), 70.7 (C-11v), 61.3 (C-1v'), 60.9 (C-2), 58.1 (C-7), 57.1 (C-2x), 55.2 (2C, C-5, C-6x'), 43.7 (C-3'), 40.6 (C-12v), 38.7 (C-15v), 37.1 (C-8), 36.3 (C-3), 32.8 (C-1v'''), 27.0 (C-9), 15.8 (C-1'v), 14.4 (C-1x'''), 12.9 (C-1x''), 11.6 (C-1x'v), 9.4 (C-1'x); HRMS m / z (ESI+) [Found: 1219.4509, C61H72ClN10O11S2 requires [M+H]+1219.4507]; LRMS m / z (ESI+) 1219 ([35M+H]+, 77%), 1221 ([37M+H]+, 40%), 1241 ([35M+Na]+, 78%), 1243 ([37M+Na]+, 43%); HPLC Retention time 9.7 min, 98.5%. (2S,4R)-4-(Benzyloxy)-1-((S)-2-(tert-butyl)-17-((S)-4-(4-chlorophenyl)-2,3,9- trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)-4,16-dioxo-6,9,12- trioxa-3,15-diazaheptadecan-1-oyl)-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2- carboxamide (3, Bn-VHL) Step 1: Compound 34 (78 mg, 0.11 mmol, 1.0 eq.) was solubilized in THF (1 mL) and PPh3(58 mg, 0.22 mmol, 2.0 eq.) was added. The reaction was stirred for 10 min at rt, then water (20 µL, 1.10 mmol, 10.0 eq.) was added. The reaction was stirred at rt overnight. The volatiles were evaporated to afford the crude material as a yellow solid, which was used in the next step without further purification. Step 2: (+)-JQ1-COOH (40 mg, 0.10 mmol, 0.9 eq.) was solubilized in dry DMF (0.5 mL) and DIPEA (77 µL, 0.44 mmol, 4.0 eq.) and HATU (50 mg, 0.13 mmol, 1.2 eq.) were added in order under argon. After 10 min, the intermediate (0.11 mmol, 1.0 eq.) obtained in Step 1 in dry DMF (0.5 mL) was added and the reaction was stirred at rt overnight. Water (5 mL) was added and the mixture was extracted with EtOAc (3 × 5 mL), then the aqueous phase was saturated with solid NaCl and the product was further extracted with EtOAc (3 × 5 mL). The combined organic phases were washed with 0.5M aqueous LiCl solution (1 × 5 mL), dried over sodium sulfate, filtered, and concentrated in vacuo. The crude material was purified using silica gel column chromatography, eluting with EtOAc:CH3OH (gradient 0 to 15% CH3OH) to yield the title compound as a colourless solid (87 mg, 72%). The compound was further purified using semi prep HPLC, method A: Rf0.17 (EtOAc / CH3OH 85:15); m.p. 90–92 °C (from EtOAc / CH3OH);^^^ଶ^ହ= +12.0 (c 1.0, CH3OH); ῦmax(neat) / cm-13316 (N-H, w), 2921 (C-H, w), 2870 (C-H, w), 1644 (C=O, s), 1487 (N-H, s), 1434 (N-H, s), 1417 (N-H, s), 1091 (C-O, s), 1042 (C-O, w), 1014 (C-O, w);1H NMR (600 MHz; CD3OD) δH8.86 (1H, s, H-2'''), 7.44–7.37 (8H, m, H-2'', H-3'', H- 2x'', H-3x''), 7.30–7.24 (5H, m, H-2v'', H-3v'', H-4v''), 4.74 (1H, s, H-7), 4.61–4.55 (3H, m, H-2, H-6v''', H-1v'a), 4.53–4.46 (2H, m, H-1v'b, H-3'a), 4.34 (1H, d, J 15.4, H-3'b), 4.33–4.29 (1H, m, H-4), 4.21 (1H, dd, J 11.4, 7.3, H-5a), 4.07 (1H, d, J 15.6, H-3va), 3.99 (1H, d, J 15.6, H-3vb), 3.75 (1H, dd, J 11.4, 7.3, H-5b), 3.69–3.62 (8H, m, H-5v, H-6v, H-8v, H-9v), 3.57 (2H, t, J 6.6, H-11v), 3.45–3.41 (3H, m, H-12v, H-15vb), 3.29–3.27 (1H, m, H-15va), 2.67 (3H, s, H-1x'), 2.46 (3H, s, H-1'v), 2.43–2.38 (4H, m, H-1'x, H-3a), 2.12–2.07 (1H, m, H-3b), 1.68 (3H, s, H-1x), 1.04 (9H, s, H-9);13C NMR (151 MHz; CD3OD) δC174.2 (C-1'), 172.9 (C-14v), 172.2 (C-2v), 171.8 (C-6), 166.1 (C-4v'''), 157.0 (C-6v'''), 152.8 (C-2'''), 152.1 (C-9v'''), 149.1 (C-4'''), 140.2 (C-1''), 139.4 (C-1v''), 138.1 (C-1x''), 137.9 (C-4x''), 133.5 (C-10av'''), 133.4 (C-5'''), 133.2 (C4''), 132.0 (C-3v'''), 132.0 (C-2v'''), 131.5 (C-3av'''), 131.3 (C-3x''), 130.4 (C-2x''), 129.8 (C-3''), 129.4 (C-2''), 129.0 (C-3v''), 129.0 (C-2v''), 128.7 (C-4v''), 78.5 (C-4), 72.2 (C-5v), 71.7 (2C, C-1v', C-9v), 71.5 (C-6v), 71.4 (C-8v), 71.1 (C-3v), 70.7 (C-11v), 60.8 (C-2), 58.1 (C-7), 55.2 (C-6v'''), 54.7 (C-5), 43.7 (C-3'), 40.6 (C- 12v), 38.7 (C-15v), 36.9 (C-8), 36.6 (C-3), 26.9 (C-9), 15.8 (C-1'v), 14.4 (C-1x), 12.9 (C- 1'x), 11.6 (C-1x'); HRMS m / z (ESI+) [Found: 1092.4244, C56H67ClN9O8S2 requires [M+H]+1092.4237]; LRMS m / z (ESI+) 1092 ([35M+H]+, 88%), 1094 ([37M+H]+, 48%), 1114 ([35M+Na]+, 100%), 1116 ([37M+Na]+, 45%); HPLC Retention time 10.2 min, 98.4%. 2-(2,6-Dioxopiperidin-3-yl)-4-nitroisoindoline-1,3-dione (37) 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. Cold water (20 mL) was added and the formed precipitated was filtered in vacuo and washed with cold water (2 × 5 mL). The resulting solid was dried in vacuo to yield a pale grey solid (4.19 g, 88%): Rf 0.44 (PE / EtOAc 40:60); m.p. 270– 272 °C (from water) [lit.256.5–257.5 °C];1H NMR (400 MHz; DMSO-d6) δH11.17 (1H, s, NH-1'), 8.36 (1H, d, J 7.9, H-5), 8.25 (1H, d, J 7.2, H-7), 8.13 (1H, dd, J 7.9, 7.2, H-6), 5.21 (1H, dd, J 12.9, 5.4, H-3'), 2.90 (1H, ddd, J 19.6, 12.9, 5.4, H-5'a), 2.64–2.49 (2H, m, H-5'b, H-4'a), 2.11–2.05 (1H, m, H-4'b); LRMS m / z (ESI-) 302 ([M-H]-, 100%). The spectroscopic data are in good agreement with the literature values. 4-Amino-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (7) Intermediate 37 (2.00 g, 6.60 mmol, 1.0 eq.) was added to a round-bottom flask containing Pd / C 10% (0.20 g, 10% w / w) and DMF (40 mL). The reaction was flushed with hydrogen and the mixture was stirred overnight at rt. The mixture was filtered over a pad of celite and washed with EtOAc (3 × 10 mL). The solvent was removed in vacuo and the dark green residue was then suspended in EtOAc and heated at reflux for 20 min, then left to cool to rt and further cooled to 0 °C. The solid precipitate was collected by vacuum filtration and further dried in vacuo to afford a yellow solid (1.64 g, 91%): Rf 0.45 (PE / EtOAc 40:60); m.p. >300 °C (from EtOAc) [lit. 315.5–317.5° C];1H NMR (400 MHz; DMSO-d6) δH11.08 (1H, s, NH-1'), 7.47 (1H, dd, J 7.0, 6.9, H-6), 7.03–6.99 (2H, m, H-5, H-7), 6.52 (2H, s, NH2), 5.05 (1H, dd, J 12.8, 5.2, H-3'), 2.89 (1H, ddd, J 15.6, 12.8, 5.2, H-5'a), 2.61–2.49 (2H, m, H-5'b, H-4'a), 2.06–2.00 (1H, m, H-4'b); LRMS m / z (ESI-) 272 ([M-H]-, 100%); HPLC Retention time 6.7 min, 99.9%. The spectroscopic data are in good agreement with the literature values. 4-Amino-2-(1-(4-nitrobenzyl)-2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (5) Compound 7 (0.30 g, 1.10 mmol, 1.0 eq.) was solubilized in dry THF (6 mL) and dry DMF (1 mL) and 4-nitrobenzyl alcohol (0.22 g, 1.43 mmol, 1.3 eq.) and PS-PPh3(0.37 g, 1.43 mmol, 1.3 eq.) were added. The mixture was cooled at 0 °C and DIAD (0.28 mL, 1.43 mmol, 1.3 eq.) was slowly added. After 1 h at 0 °C, the mixture was warmed to rt and stirred for further 16 h. The following day the reaction was not concluded and 0.3 eq. of PS-PPh3and 0.3 eq. of DIAD were added. After 20 h at rt, the reaction was filtered over a pad of celite, washed with EtOAc (2 × 5 mL) and the volatiles were removed in vacuo. Water (5 mL) was added and the aqueous phase was extracted with EtOAc (3 × 20 mL). The collected organic layers were dried over sodium sulfate, filtered and evaporated. The crude material was purified using silica gel column chromatography, eluting with PE:EtOAc (gradient 10 to 30% EtOAc) afforded the title compound as a yellow solid (88 mg, 20%): Rf 0.54 (PE / EtOAc 40:60); m.p. 170–172 °C (from PE / EtOAc); ῦmax(thin film) / cm-13449 (N-H, s), 3358 (N-H, s), 2921 (C-H, w), 1729 (C=O, s), 1682 (C=O, s), 1518 (N-O, s), 1371 (s), 1333 (N-O, s), 1168 (s), 770 (C-H, s);1H NMR (600 MHz; DMSO-d6) δH8.19 (2H, d, J 8.8, H-3''), 7.54 (2H, d, J 8.8, H-2''), 7.46 (1H, dd, J 8.5, 7.0, H-6), 7.03–7.00 (2H, m, H-5, H-7), 6.54 (2H, s, NH2), 5.29 (1H, dd, J 13.0, 5.4, H-3'), 5.02 (1H, d, J 15.7, H-5''a), 4.95 (1H, d, J 15.7, H-5''b), 3.09 (1H, ddd, J 17.2, 13.0, 5.4, H-5'a), 2.86–2.79 (1H, m, H-5'b), 2.62 (1H, ddt, J 17.2, 13.0, 5.4, H-4'a), 2.13–2.07 (1H, m, H-4'b);13C NMR (151 MHz; DMSO-d6) δC171.7 (C-6'), 170.0 (C-2'), 168.5 (C-3), 166.7 (C-1), 147.4 (C-4), 146.1 (C-4''), 144.9 (C-1''), 136.4 (C-6), 131.9 (C-7a), 128.2 (C-2''), 123.4 (C- 3''), 121.8 (C-5), 110.5 (C-7), 108.4 (C-3a), 48.6 (C-3'), 42.5 (C-5''), 31.1 (C-5'), 21.9 (C- 4'); HRMS m / z (ESI-) [Found: 407.0996, C20H15N4O6requires [M-H]- 407.0997]; LRMS m / z (ESI-) 407 ([M-H]-, 100%); HPLC Retention time 9.8 min, 99.4%. 4-Amino-2-(1-((1-methyl-2-nitro-1H-imidazol-5-yl)methyl)-2,6-dioxopiperidin- 3-yl)isoindoline-1,3-dione (6) Compound 6 was synthesized as previously described for compound 5, starting from pomalidomide 7 (0.20 g, 0.73 mmol, 1.0 eq.) and intermediate 15 (0.15 g, 0.95 mmol, 1.3 eq.). The reaction was heated at 60 °C for 20 h. Then, the reaction was filtered over a pad of celite, washed with EtOAc (2 × 5 mL) and the volatile was removed in vacuo. Water (10 mL) was added and the product was extracted with EtOAc (3 × 20 mL). The collected organic layers were dried over sodium sulfate, filtered and evaporated. The crude material was purified using neutral alumina column chromatography, eluting with toluene:acetone (gradient 10 to 20% acetone) to afford the title compound as a pale yellow solid (61 mg, 20%): Rf0.39 (toluene / acetone 80:20); m.p. 186–188 °C (from toluene / acetone); ῦmax(neat) / cm-13468 (N-H, s), 3364 (N-H, s), 2934 (C-H, w), 1731 (C=O, s), 1684 (C=O, s), 1533 (N-O, s), 1371 (s), 1328 (N-O, s), 1242 (s), 787 (C-H, s);1H NMR (600 MHz; DMSO-d6) δH7.53 (1H, dd, J 8.6, 6.9, H-6), 7.11 (1H, s, H-4''), 7.09– 7.06 (2H, m, H-5, H-7), 6.59 (2H, s, NH2), 5.33 (1H, dd, J 13.0, 5.4, H-3'), 5.03 (1H, d, J 14.7, H-6''a), 4.96 (1H, d, J 14.7, H-6''b), 4.00 (3H, s, H-1'''), 3.10 (1H, ddd, J 17.3, 13.0, 5.4, H-5'a), 2.91–2.85 (1H, m, H-5'b), 2.65 (1H, ddt, J 17.5, 13.0, 5.4, H-4'a), 2.17–2.12 (1H, m, H-4'b);13C NMR (151 MHz; DMSO-d6) δC171.5 (C-6'), 169.8 (C-2'), 168.5 (C-3), 167.3 (C-1), 146.8 (C-4), 145.2 (C-2''), 135.5 (C-6), 134.3 (C-5''), 131.9 (C-7a), 127.1 (C- 4''), 121.8 (C-5), 111.1 (C-7), 108.4 (C-3a), 49.0 (C-3'), 34.2 (C-1'''), 33.8 (C-6''), 31.1 (C- 5'), 21.2 (C-4'); HRMS m / z (ESI+) [Found: 413.1204, C18H17N6O6requires [M+H]+413.1204]; LRMS m / z (ESI+) 304, (100%), 413 ([M+H]+, 36%), 435 ([M+Na]+, 20%); HPLC Retention time 8.2 min, 96.9%. 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. The reaction was cooled to rt and quenched by addition of water (30 mL). The precipitate formed was filtered in vacuo, washed with water (1 × 20 mL) and dried in vacuo to yield a yellow solid (1.92 g, 98%): Rf0.54 (PE / EtOAc 98:2); m.p. 55–57 °C (from water); ῦmax(neat) / cm-12921 (C-H, w), 2887 (C-H, w), 1616 (w), 1591 (w), 1453 (C-O, s), 1224 (s), 1078 (w), 788 (C-H, s) 732 (C-H, s);1H NMR (400 MHz; CDCl3) δH7.54 (1H, t, J 7.9, H- 4), 7.50–7.47 (4H, m, H-4'), 7.43–7.36 (6H, m, H-5', H-6'), 6.44 (2H, d, J 7.9, H-3), 5.40 (4H, s, H-2');13C NMR (101 MHz; CDCl3) δC162.4 (C-2), 141.2 (C-4), 137.7 (C-3'), 128.5 (C-5'), 127.9 (C-4'), 127.8 (C-6'), 102.1 (C-3), 67.7 (C-2'); HRMS m / z (ESI+) [Found: 292.1330, C19H18NO2requires [M+H]+292.1332]; LRMS m / z (ESI+) 292 ([M+H]+, 28%), 380 (100%); HPLC Retention time 11.9 min, 97.0%. The spectroscopic data are in good agreement with the literature values. 2,6-Bis(benzyloxy)-3-bromopyridine (40) To a stirred solution of 2,6-bis(benzyloxy)pyridine (1.00 g, 3.43 mmol, 1.0 eq.) in acetonitrile (10 mL), N-bromosuccinimide (0.49 g, 2.74 mmol, 0.8 eq.) was added portionwise and the reaction mixture was heated at 80 °C for 3 h. The reaction was cooled to rt and the volatile was evaporated in vacuo. EtOAc (25 mL) was added and the organic phase was washed with water (1 × 25 mL). The organic phase was dried over sodium sulfate, filtered, and concentrated in vacuo. Purification using silica gel column chromatography, eluting with PE:EtOAc (gradient 0 to 5% EtOAc) yielded compound 40 as a colourless solid (820 mg, 64%): Rf0.54 (PE / EtOAc 98:2); m.p. 45–47 °C (from PE / EtOAc); ῦmax(neat) / cm-12942 (C-H, w), 1604 (w), 1441 (C-O, s), 1378 (s), 1120 (w), 825 (w) 760 (C-H, s);1H NMR (400 MHz; CDCl3) δH7.68 (1H, d, J 8.3, H-4), 7.46–7.31 (10H, m, H-4', H-5', H-6', H-4'', H-5'', H-6''), 6.32 (1H, d, J 8.3, H-5), 5.43 (2H, s, H-2'), 5.31 (2H, s, H-2'');13C NMR (101 MHz; CDCl3) δC161.3 (C-2), 157.8 (C-6), 144.1 (C-4), 137.3 (C-3''), 137.2 (C-3'), 128.6 (2C, C-5', C-5''), 127.9 (C-4''), 128.0 (C-6''), 127.8 (C-6'), 127.4 (C-4'), 103.9 (C-5), 96.9 (C-3), 68.4 (C-2''), 68.3 (C-2'); HRMS m / z (ESI+) [Found: 370.0437 and 372.0416, C19H17BrNO2 requires [M+H]+370.0437 and 372.0417]; LRMS m / z (ESI+) 370 ([79M+H]+, 93%), 372 ([81M+H]+, 93%), 789 (100%); HPLC Retention time 12.6 min, 93.0%. The spectroscopic data are in good agreement with the literature values. 4-(2,6-Bis(benzyloxy)pyridin-3-yl)phenol (41) 2,6-Bis(benzyloxy)-3-bromopyridine (1.00 g, 2.70 mmol, 1.0 eq.), (4- hydroxyphenyl)boronic acid (0.74 g, 5.40 mmol, 2.0 eq.) and potassium phosphate tribasic (1.20 g, 5.67 mmol, 2.1 eq.) in a 6:1 mixture of dioxane (30 mL) and water (5 mL) were degassed under argon and 1,1'- bis(diphenylphosphino)ferrocene-palladium(II)dichloride dichloromethane complex (220 mg, 0.27 mmol, 0.1 eq.) was then added. The reaction mixture was stirred at 100 °C for 16 h, cooled to rt, filtered over a pad of celite, and washed with EtOAc (2 × 50 mL). The filtrate was then diluted with water (150 mL) and the product was extracted with EtOAc (1 × 200 mL). The combined organic phases were dried over sodium sulfate, filtered, and concentrated in vacuo. Purification using silica gel column chromatography, eluting with PE:EtOAc (gradient 10 to 20% EtOAc) yielded compound 41 as a pale pink solid (910 mg, 88%): Rf 0.58 (PE / EtOAc 70:30); m.p. 113– 115 °C (from PE / EtOAc); ῦmax(neat) / cm-13263 (O-H, w), 1598 (C-O, s), 1470 (C-O, s), 1356 (w), 1242 (s), 1005 (w), 841 (C-H, w), 697 (C-H, w);1H NMR (400 MHz; CDCl3) δH7.57 (1H, d, J 8.1, H-4), 7.46–7.42 (4H, m, H-3', H-6'', H-6'''), 7.39–7.33 (4H, m, H-4'', H- 4'''), 7.32–7.27 (4H, m, H-5'', H-5'''), 6.85 (2H, d, J 8.6, H-2'), 6.36 (1H, d, J 8.1, H-5), 5.42 (2H, s, H-2'''), 5.36 (2H, s, H-2''), 4.74 (1H, br s, OH);13C NMR (101 MHz; CDCl3) δC161.0 (C-6), 158.2 (C-4'), 154.4 (C-2), 141.5 (C-4), 137.9 (C-3'''), 137.6 (C-3''), 130.3 (C- 2'), 129.5 (C-1'), 128.5 (C-5'''), 128.3 (C-5''), 127.8 (2C, C-6'', C-6'''), 127.4 (C-4''), 127.2 (C-4'''), 115.7 (C-3), 115.1 (C-3'), 102.4 (C-5), 67.8 (C-2''), 67.6 (C-2'''); HRMS m / z (ESI+) [Found: 384.1597, C25H22NO3requires [M+H]+384.1594]; LRMS m / z (ESI+) 384 ([M+H]+, 100%), 406 ([M+Na]+, 34%); HPLC Retention time 11.7 min, 93.7%. The spectroscopic data are in good agreement with the literature values. Tert-butyl 2-(4-(2,6-bis(benzyloxy)pyridin-3-yl)phenoxy)acetate (42) 4-(2,6-Bis(benzyloxy)pyridin-3-yl)phenol (790 mg, 2.06 mmol, 1.0 eq.) was solubilized in dry DMF (19 mL) and K2CO3(427 mg, 3.09 mmol, 1.5 eq.) was added. The mixture was stirred at rt for 10 min, and then tert-butyl bromoacetate (319 µL, 2.16 mmol, 1.05 eq.) was added dropwise. The reaction mixture was stirred at rt for 3 h, then diluted with water (80 mL) and the product was extracted with EtOAc (2 × 100 mL). The combined organic phases were dried over sodium sulfate, filtered, and concentrated in vacuo. Purification using silica gel column chromatography, eluting with PE:EtOAc (gradient 2 to 10% EtOAc) yielded compound 5 as a colourless solid (866 mg, 84%): Rf0.39 (PE / EtOAc 90:10); m.p. 65–67 °C (from PE / EtOAc); ῦmax(neat) / cm-12918 (C-H, w), 1752 (C=O, s), 1470 (C-O, s), 1452 (C-O, s), 1240 (C-O, s), 1223 (C-O, s), 1165 (w), 1047 (w), 825 (w);1H NMR (400 MHz; CDCl3) δH7.46 (1H, d, J 8.0, H-4), 7.39 (2H, d, J 8.8, H-3'), 7.34–7.32 (2H, m, H-6'', H-6'''), 7.28–7.18 (8H, m, H-4'', H-5'', H-4''', H-5'''), 6.82 (2H, d, J 8.8, H-2'), 6.36 (1H, d, J 8.0, H-5), 5.31 (2H, s, H-2''), 5.26 (2H, s, H-2'''), 4.43 (2H, s, H-6'), 1.39 (9H, s, H-10');13C NMR (101 MHz; CDCl3) δC168.2 (C-7'), 161.1 (C- 6), 158.3 (C-4'), 157.0 (C-2), 141.6 (C-4), 138.0 (C-3'''), 137.7 (C-3''), 130.2 (C-2'), 130.1 (C-1'), 128.6 (C-5'''), 128.5 (C-5''), 127.9 (2C, C-6'', C-6'''), 127.5 (C-4'''), 127.3 (C-4''), 115.7 (C-3), 114.4 (C-3'), 102.5 (C-5), 82.4 (C-9'), 67.9 (C-2'''), 67.7 (C-2''), 65.9 (C-6'), 28.2 (C-10'); HRMS m / z (ESI+) [Found: 498.2271, C31H32NO5requires [M+H]+498.2275]; LRMS m / z (ESI+) 498 ([M+H]+, 79%), 520 ([M+Na]+, 90%); HPLC Retention time 13.3 min, 98.6%. The spectroscopic data are in good agreement with the literature values. Tert-butyl 2-(4-(2,6-dioxopiperidin-3-yl)phenoxy)acetate (43) Tert-butyl 2-(4-(2,6-bis(benzyloxy)pyridin-3-yl)phenoxy)acetate 42 (855 mg, 1.72 mmol, 1.0 eq.) was added to a round-bottom flask containing Pd / C 10% (86 mg, 10% w / w) and DMF (12 mL). The reaction was flushed with hydrogen and the mixture was stirred overnight at rt. The mixture was filtered over a pad of celite and washed with EtOAc (2 × 20 mL). The solvent was removed in vacuo and the crude material was purified using silica gel column chromatography, eluting with PE:EtOAc (gradient 30 to 60% EtOAc) yielding compound 43 as a colourless solid (504 mg, 92%): Rf0.43 (PE / EtOAc 40:60); m.p. 60– 62 °C (from PE / EtOAc); ῦmax(neat) / cm-13194 (N-H, w), 2979 (C-H, w), 1722 (C=O, s), 1712 (C=O, s), 1701 (C=O, s), 1513 (C-O, s), 1368 (w), 1154 (C-O, s), 831 (w);1H NMR (400 MHz; CDCl3) δH8.36 (1H, br s, NH), 7.11 (2H, d, J 8.7, H-2'), 6.88 (2H, d, J 8.7, H- 3'), 4.49 (2H, s, H-6'), 3.72 (1H, dd, J 9.6, 5.2, H-3), 2.67 (1H, ddd, J 17.7, 12.4, 5.2, H-5a), 2.62 (1H, ddd, J 17.7, 12.4, 5.2, H-5b), 2.28–2.13 (2H, m, H-4a, H-4b), 1.48 (9H, s, H-10');13C NMR (101 MHz; CDCl3) δC173.4 (C-2), 172.5 (C-6), 167.9 (C-7'), 157.5 (C-4'), 130.0 (C-1'), 129.2 (C-2'), 115.1 (C-3'), 82.5 (C-9'), 65.8 (C-6'), 47.1 (C-3), 30.8 (C-5), 28.1 (C- 10'), 26.4 (C-4); HRMS m / z (ESI+) [Found: 342.1317, C17H21NNaO5requires [M+Na]+ 342.131]; LRMS m / z (ESI+) 342 ([M+Na]+, 100%); HPLC Retention time 8.3 min, 91.6%. The spectroscopic data are in good agreement with the literature values. 2-(4-(2,6-Dioxopiperidin-3-yl)phenoxy)acetic acid (44) Tert-butyl 2-(4-(2,6-dioxopiperidin-3-yl)phenoxy)acetate 43 (288 mg, 0.90 mmol, 1.0 eq.) was solubilized in CH2Cl2(9 mL) and trifluoroacetic acid (2.7 mL) was added. The mixture was stirred at rt overnight, then concentrated in vacuo to afford the title compound as a pale pink solid (229 mg, 97%): Rf0.13 (EtOAc / CH3OH 80:20); m.p. 190– 192 °C (from CH2Cl2); ῦmax(neat) / cm-13197 (N-H, w), 3086 (O-H, w), 2918 (C-H, w), 1765 (C=O, s), 1706 (C=O, s), 1665 (C=O, s), 1515 (C-O, s), 1280 (w), 1195 (C-O, s), 1182 (C-O, s), 1084 (w), 835 (s);1H NMR (400 MHz; CD3OD) δH7.18 (2H, d, J 8.7, H- 2'), 6.93 (2H, d, J 8.7, H-3'), 4.66 (2H, s, H-6'), 3.81 (1H, dd, J 9.8, 5.9, H-3), 2.74–2.58 (2H, m, H-5a, H-5b), 2.27–2.15 (2H, m, H-4a, H-4b);13C NMR (101 MHz; CD3OD) δC175.2 (C-2), 174.3 (C-6), 171.2 (C-7'), 157.3 (C-4'), 131.3 (C-1'), 129.2 (C-2'), 114.4 (C- 3'), 62.7 (C-6'), 47.0 (C-3), 30.7 (C-5), 26.4 (C-4); HRMS m / z (ESI−) [Found: 262.0719, C13H12NO5requires [M−H]−262.0721]; LRMS m / z (ESI−) 262 ([M−H]−, 61%), 525 (100%); HPLC Retention time 5.2 min, 89.3%. The spectroscopic data are in good agreement with the literature values. Tert-butyl (5-(2-(4-(2,6-dioxopiperidin-3- yl)phenoxy)acetamido)pentyl)carbamate (45) Compound 44 (150 mg, 0.57 mmol, 1.0 eq.) was solubilized in dry DMF (3 mL) and DIPEA (496 µL, 2.85 mmol, 5.0 eq.), HATU (433 mg, 1.14 mmol, 2.0 eq.) and N- Boc-cadaverine (142 µL, 0.68 mmol, 1.2 eq.) were added in order under argon. The reaction was stirred at rt overnight. Water (20 mL) was then added and the mixture was extracted with CH2Cl2(2 × 20 mL). The combined organic phases were dried over sodium sulfate, filtered, and concentrated in vacuo. The solvent was removed in vacuo and the crude material was purified using silica gel column chromatography, eluting with CH2Cl2:CH3OH (gradient 0 to 10% CH3OH) yielding compound 45 as a colourless solid (192 mg, 75%): Rf0.24 (100% EtOAc); m.p. 90–92 °C (from PE / EtOAc); ῦmax(neat) / cm-13351 (N-H, w), 3078 (N-H, w), 2932 (C-H, w), 2853 (C-H, w), 1721 (C=O, s), 1699 (C=O, s), 1683 (C=O, s), 1661 (C=O, s), 1518 (C-O, s), 1364 (w), 1234 (C-O, s), 1175 (C-O, s), 845 (s);1H NMR (400 MHz; CD3OD) δH 8.11 (1H, br s, NH-14'), 7.20 (2H, d, J 8.6, H-2'), 6.97 (2H, d, J 8.6, H-3'), 4.49 (2H, s, H-6'), 3.81 (1H, dd, J 10.3, 5.6, H-3), 3.27 (2H, q, J 7.0, H-13'), 3.00 (2H, t, J 7.0, H-9'), 2.67 (1H, ddd, J 17.6, 10.3, 5.6, H-5a), 2.62 (1H, ddd, J 17.6, 10.3, 5.6, H-5b), 2.27–2.14 (2H, m, H-4a, H-4b), 1.57–1.45 (4H, m, H-10', H-12'), 1.42 (9H, s, 18'), 1.34–1.26 (2H, m, H-11');13C NMR (101 MHz; CD3OD) δC176.6 (C-2), 175.7 (C-6), 171.0 (C-7'), 158.6 (C-15'), 158.4 (C-4'), 133.1 (C-1'), 130.8 (C-2'), 116.0 (C- 3'), 79.8 (C-17'), 68.3 (C-6'), 47.0 (C-3), 41.2 (C-13'), 40.0 (C-9'), 32.2 (C-10'), 30.6 (C-5), 30.1 (C-12'), 28.8 (C-18'), 27.8 (C-4), 25.1 (C-11'); HRMS m / z (ESI−) [Found: 446.2296, C23H32N3O6requires [M−H]−446.2297]; LRMS m / z (ESI−) 446 ([M−H]−, 100%); HPLC Retention time 7.7 min, 97.9%. The spectroscopic data are in good agreement with the literature values.

[0019] 2-((S)-4-(4-Chlorophenyl)- 2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)-N-(5-(2- (4-(2,6-dioxopiperidin-3-yl)phenoxy)acetamido)pentyl)acetamide (11, PG4c) Step 1: Compound 45 (51 mg, 0.11 mmol, 1.0 eq.) was solubilized in CH2Cl2(0.5 mL) and HCl (4M in dioxane, 172 µL, 0.67 mmol 6.0 eq.) was added. The reaction was stirred for 1 h at rt. Then, the volatile was evaporated to afford the crude material as a colourless solid, which was used in the next step without further purification. Step 2: The obtained intermediate (0.11 mmol, 1.0 eq.) was solubilized in dry DMF (1.2 mL) and DIPEA (100 µL, 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. Water (5 mL) was then added and the mixture was extracted with EtOAc (6 × 5 mL). The combined organic phases were dried over sodium sulfate, filtered, and evaporated in vacuo. The crude material was purified using silica gel column chromatography, eluting with EtOAc:CH3OH (gradient 0 to 10% CH3OH) yielding compound 11 as a colourless solid (69 mg, 86%). The compound was further purified using semi prep HPLC, method A: Rf0.41 (EtOAc / CH3OH / NH385:10:0.5); m.p. 117– 119 °C (from EtOAc / CH3OH);^^^ଶ^ହ= +11.0 (c 1.0, CH3OH); ῦmax(neat) / cm-13335 (N-H, w), 3075 (N-H, w), 2928 (C-H, w), 2859 (C-H, w), 1702 (C=O, s), 1662 (C=O, s), 1536 (C-H, s), 1488 (C-H, s), 1230 (w), 1184 (C-O, s), 894 (C-H, w), 838 (C-H, w);1H NMR (600 MHz; CD3OD) δH7.43 (2H, d, J 8.5, H-2'v), 7.39 (2H, d, J 8.5, H-3'v), 7.15 (2H, d, J 8.8, H-2'), 6.91 (2H, d, J 8.8, H-3'), 4.63 (1H, dd, J 8.9, 5.5, H-6''), 4.48 (2H, s, H-6'), 3.79 (1H, dd, J 10.4, 5.4, H-3), 3.41 (1H, dd, J 14.8, 8.9 H-16'a), 3.30–3.20 (5H, m, H-13', H-9', H-16'b), 2.70–2.64 (4H, m, H-1v, H-5a), 2.61–2.56 (1H, m, H-5b), 2.44 (3H, s, H-1'''), 2.20– 2.12 (2H, m, H-4a, H-4b), 1.69 (3H, s, H-1'v), 1.61–1.55 (4H, m, H-10', H-12'), 1.40–1.36 (2H, m, H-11');13C NMR (151 MHz; CD3OD) δC176.5 (C-2), 175.7 (C-6), 172.7 (C-15'), 171.1 (C-7'), 166.2 (C-4''), 158.3 (C-4'), 157.0 (C-6a''), 152.2 (C-9''), 138.1 (C-4v'), 138.0 (C-1v'), 133.5 (C-10a''), 133.2 (C-3a''), 133.1 (C-1'), 132.0 (C-3''), 132.0 (C-2''), 131.3 (C- 2v'), 130.7 (C-2'), 129.8 (C-3v'), 115.9 (C-3'), 68.3 (C-6'), 55.3 (C-6''), 48.4 (C-3), 40.3 (C- 9'), 39.8 (C-13'), 38.9 (C-16'), 32.2 (C-5), 30.0 (C-12'), 29.9 (C-10'), 27.8 (C-4), 25.0 (C- 11'), 14.4 (C-1'v), 12.9 (C-1'''), 11.6 (C-1v); HRMS m / z (ESI+) [Found: 730.2567, C37H41ClN7O5S requires [M+H]+730.2573]; LRMS m / z (ESI+) 730 ([35M+H]+, 100%), 732 ([37M+H]+, 86%), 752 ([35M+Na]+, 72%), 754 ([37M+Na]+, 45%); HPLC Retention time 8.3 min, 97.0%; The spectroscopic data are in good agreement with the literature values. Tert-butyl (5-(2-(4-(1-((1-methyl-2-nitro-1H-imidazol-5-yl)methyl)-2,6- dioxopiperidin-3-yl)phenoxy)acetamido)pentyl)carbamate (46) Compound 45 (72 mg, 0.16 mmol, 1.0 eq.) was solubilized in dry DMF (2 mL) and Cs2CO3(63 mg, 0.19 mmol, 1.2 eq.) and TBAI (12 mg, 0.03 mmol, 0.2 eq.) were added under an argon atmosphere. After 10 min, 2-nitroimidazol bromide 16 (39 mg, 0.18 mmol, 1.1 eq.) was added. The reaction was stirred at rt for 6 h. After 6 h at rt, the reaction was not concluded. 0.5 eq. of Cs2CO3and 0.5 eq. of compound 16 were then added and the reaction was stirred at rt for additional 16 h. Water (5 mL) was then added and the mixture was extracted with EtOAc (6 × 5 mL). The combined organic phases were washed with 0.5M aqueous LiCl solution (1 × 5 mL), dried over sodium sulfate, filtered, and concentrated in vacuo. The solvent was removed in vacuo and the crude material was purified using silica gel column chromatography, eluting with PE:EtOAc (gradient 60 to 90% EtOAc) yielding compound 46 as a yellow solid (37 mg, 39%): Rf0.47 (EtOAc 100%); m.p. 46–47 °C (from PE / EtOAc); ῦmax (neat) / cm-13322 (N-H, w), 2929 (C-H, w), 2858 (C-H, w), 1679 (C=O, s), 1531 (N-H, w), 1512 (N-H, w), 1487 (C-O, s), 1365 (s), 1273 (w), 1246 (C-O, s), 1156 (C-O, s), 731 (s);1H NMR (600 MHz; CDCl3) δH7.18 (1H, s, H-4'''), 7.10 (2H, d, J 8.6, H-3'), 6.93 (2H, d, J 8.6, H-2'), 6.54 (1H, br t, J 6.9, NH-14'), 5.04 (1H, d, J 15.1, H-1''b), 5.00 (1H, d, J 15.1, H-1''a), 4.54 (1H, br s, NH-8'), 4.47 (2H, s, H-6'), 4.11 (3H, s, H1'v), 3.80 (1H, dd, J 10.3, 5.2, H-3), 3.34 (2H, q, J 7.0, H-13'), 3.08 (2H, q, J 7.0, H-9'), 2.89 (1H, dt, J 13.0, 5.2, H-5a), 2.76 (1H, ddd, J 13.0, 10.3, 5.2, H-5b), 2.28–2.18 (2H, m, H-4a, H-4b), 1.56 (2H, quint, J 7.0, H-12'), 1.49 (2H, quint, J 7.0, H- 10'), 1.43 (9H, s, 18'), 1.32 (2H, quint, J 7.0, H-11');13C NMR (151 MHz; CDCl3) δC173.5 (C-2), 172.0 (C-6), 168.0 (C-7'), 157.0 (C-4'), 156.2 (C-15'), 145.5 (C-2'''), 133.8 (C-5'''), 130.9 (C-1'), 130.0 (C-4'''), 129.6 (C-3'), 115.4 (C-2'), 79.3 (C-17'), 67.6 (C-6'), 48.2 (C-3), 40.5 (C-9'), 39.0 (C-13'), 34.5 (C-1'v), 32.8 (C-1''), 32.0 (C-5), 29.8 (C-10'), 29.4 (C-12'), 28.6 (C-18'), 25.1 (C-4), 24.1 (C-11'); HRMS m / z (ESI−) [Found: 585.2666, C28H37N6O8requires [M−H]−585.2678]; LRMS m / z (ESI−) 585 ([M−H]−, 100%); HPLC Retention time 9.4 min, 88.9%. Tert-butyl (5-(2-(4-(1-((5-methoxy-1,2-dimethyl-4,7-dioxo-4,7-dihydro-1H- indol-3-yl)methyl)-2,6-dioxopiperidin-3-yl)phenoxy)acetamido)pentyl)carbamate (47) Compound 47 was synthesized as previously described for compound 46, starting from compound 45 (62 mg, 0.14 mmol, 1.0 eq.) and indolequinone bromide 24 (45 mg, 0.15 mmol, 1.1 eq.). The reaction was stirred at rt for 16 h, then an addition of 0.4 eq. of Cs2CO3, 0.3 eq. of indolequinone bromide 24 and 0.1 eq. of TBAI was performed and the reaction was stirred at rt for additional 8 h. Water (5 mL) was added and the mixture was extracted with EtOAc (6 × 5 mL). The combined organic phases were dried over sodium sulfate, filtered, and concentrated in vacuo. The solvent was removed in vacuo and the crude material was purified using silica gel column chromatography, eluting with PE:EtOAc (gradient 60 to 100% EtOAc) yielding compound 47 as an orange solid (21 mg, 23%): Rf0.41 (EtOAc / CH3OH 90:10); m.p. 55–56 °C (from PE / EtOAc); ῦmax(neat) / cm-13339 (N-H, w), 2928 (C-H, w), 2860 (C-H, w), 1674 (C=O, s), 1636 (C=O, s), 1511 (N-H, w), 1244 (C-O, s), 1221 (C-O, s), 1175 (C-O, s), 837 (s);1H NMR (600 MHz; CDCl3) δH7.12 (2H, d, J 8.8, H-3'), 6.87 (2H, d, J 8.8, H-2'), 6.53 (1H, br t, J 6.7, NH-14'), 5.59 (1H, s, H-6'''), 5.06 (1H, d, J 14.7, H-1''b), 4.98 (1H, d, J 14.7, H-1''a), 4.45 (2H, s, H-6'), 3.99 (1H, br s, NH-8'), 3.87 (3H, s, H-1'v), 3.87 (4H, m, H-3, H-2v'), 3.34 (2H, q, J 6.7, H-13'), 3.07 (2H, q, J 6.7, H-9'), 2.86 (1H, dt, J 17.6, 5.1, H-5a), 2.78 (1H, ddd, J 17.6, 9.6, 5.1, H- 5b), 2.27 (3H, s, H-1v), 2.19–2.11 (2H, m, H-4a, H-4b), 1.55 (2H, quint, J 6.7, H-12'), 1.48 (2H, quint, J 6.7, H-10'), 1.43 (9H, s, 18'), 1.32 (2H, quint, J 6.7, H-11');13C NMR (151 MHz; CDCl3) δC178.8 (C-4'''), 177.6 (C-7'''), 173.5 (C-2), 172.4 (C-6), 168.2 (C-7'), 159.8 (C-5'''), 156.6 (C-4'), 156.2 (C-15'), 137.0 (C-2'''), 132.2 (C-1'), 129.8 (C-3'), 129.0 (C- 7a'''), 121.6 (C-3a'''), 117.7 (C-3'''), 115.1 (C-2'), 106.7 (C-6'''), 79.3 (C-17'), 67.6 (C-6'), 56.6 (C-2v'), 48.3 (C-3), 40.6 (C-9'), 39.0 (C-13'), 35.3 (C-1''), 32.6 (C-1'v), 32.0 (C-5), 29.8 (C-10'), 29.4 (C-12'), 28.6 (C-18'), 25.6 (C-4), 24.1 (C-11'), 9.8 (C-1v); HRMS m / z (ESI+) [Found: 665.3179, C35H45N4O9requires [M+H]+665.3181]; LRMS m / z (ESI+) 665 ([M+H]+, 29%), 687 ([M+Na]+, 100%); HPLC Retention time 8.9 min, 77.7%. Tert-butyl (5-(2-(4-(1-benzyl-2,6-dioxopiperidin-3- yl)phenoxy)acetamido)pentyl)carbamate (48) Compound 48 was synthesized as previously described for compound 46, starting from compound 45 (92 mg, 0.20 mmol, 1.0 eq.) and benzyl bromide (26 µL, 0.22 mmol, 1.1 eq.). The reaction was stirred at rt for 16 h, then an addition of 0.5 eq. of Cs2CO3and 0.5 eq. of benzyl bromide was performed and the reaction was stirred at rt for additional 16 h. Water (5 mL) was added and the mixture was extracted with EtOAc (1 × 5 mL). The combined organic phases were dried over sodium sulfate, filtered, and concentrated in vacuo. The solvent was removed in vacuo and the crude material was purified using silica gel column chromatography, eluting with PE:EtOAc (gradient 60 to 100% EtOAc) yielding compound 48 as a colourless oil (43 mg, 40%): Rf0.47 (EtOAc 100%); ῦmax(neat) / cm-13335 (N-H, w), 2927 (C-H, w), 2857 (C-H, w), 1673 (C=O, s), 1512 (N-H, w), 1364 (w), 1247 (w), 1165 (C-O, s), 702 (C-H, w);1H NMR (400 MHz; CDCl3) δH7.40– 7.38 (2H, m, H-3'''), 7.31–7.24 (3H, m, H-2''', H-4'''), 7.07 (2H, d, J 8.6, H-3'), 6.87 (2H, d, J 8.6, H-2'), 6.54 (1H, br t, J 6.9, NH-14'), 5.02 (1H, d, J 13.4, H-1''b), 4.98 (1H, d, J 13.4, H-1''a), 4.57 (1H, br s, NH-8'), 4.46 (2H, s, H-6'), 3.78 (1H, dd, J 9.8, 5.2, H-3), 3.33 (2H, q, J 6.9, H-13'), 3.08 (2H, q, J 6.9, H-9'), 2.81 (1H, dt, J 17.6, 5.2, H-5a), 2.71 (1H, ddd, J 17.6, 9.8, 5.2, H-5b), 2.26–2.11 (2H, m, H-4a, H-4b), 1.55 (2H, quint, J 6.9, H-12'), 1.47 (2H, quint, J 6.9, H-10'), 1.43 (9H, s, 18'), 1.31 (2H, quint, J 6.9, H-11');13C NMR (101 MHz; CDCl3) δC173.4 (C-2), 172.2 (C-6), 168.1 (C-7'), 156.7 (C-4'), 156.1 (C-15'), 137.3 (C-1'''), 131.8 (C-1'), 129.6 (C-3'), 129.2 (C-3'''), 128.5 (C-2'''), 127.7 (C-4'''), 115.1 (C-2'), 79.1 (C-17'), 67.6 (C-6'), 48.1 (C-3), 43.4 (C-1''), 40.5 (C-9'), 39.0 (C-13'), 31.9 (C-5), 29.8 (C-10'), 29.3 (C-12'), 28.6 (C-18'), 25.4 (C-4), 24.1 (C-11'); HRMS m / z (ESI−) [Found: 536.2767, C30H38N3O6requires [M−H]−536.2766]; LRMS m / z (ESI−) 536 ([M−H]−, 100%); HPLC Retention time 9.7 min, 96.9%.

[0020] 2-((S)-4-(4-Chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3- a][1,4]diazepin-6-yl)-N-(5-(2-(4-(1-((1-methyl-2-nitro-1H-imidazol-5-yl)methyl)-2,6- dioxopiperidin-3-yl)phenoxy)acetamido)pentyl)acetamide (8, NI-CRBN) Step 1: Compound 46 (33 mg, 0.06 mmol, 1.0 eq.) was solubilized in dry CH3OH (1 mL) and oxalyl chloride (15 µL, 0.18 mmol, 3.0 eq.) was added while maintaining an argon atmosphere. After 3 h at rt, the reaction was not concluded. A further addition of 3.0 eq. of oxalyl chloride was performed and the reaction was stirred at rt for 16 h. Then, the volatile was evaporated to afford the crude material as a yellow oil, which was used in the next step without further purification. 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 µL, 0.36 mmol, 6.0 eq.) and HATU (46 mg, 0.12 mmol, 2.0 eq.) in dry DMF (1 mL) under argon. The reaction was stirred at rt for 16 h, then diluted with water (15 mL) and extracted with EtOAc (3 × 15 mL). The combined organic layers were washed with 0.5M aqueous LiCl solution (1 × 30 mL), dried over sodium sulfate, filtered, and evaporated in vacuo. The crude material was purified using silica gel column chromatography, eluting with EtOAc:CH3OH (0 to 15% CH3OH) to afford compound 8 as a yellowish solid solid (26 mg, 50%). The compound was further purified using semi prep HPLC, method A: Rf0.18 (EtOAc / CH3OH 85:15); m.p. 94–96 °C (from EtOAc / CH3OH); ^ ^^ଶ^ହ= +7.6 (c 1.0, CH3OH); ῦmax(neat) / cm-13307 (N-H, w), 2928 (C-H, w), 2857 (C-H, w), 1730 (C=O, s), 1534 (N-H, s), 1511 (N-H, w), 1487 (C-O, s), 1420 (s), 1371 (s), 1244 (w), 1191 (C-O, s), 1155 (w), 834 (C-H, s);1H NMR (400 MHz; CD3OD) δH7.44–7.37 (4H, m, H-2'x, H-3'x), 7.11 (2H, d, J 8.8, H-3'), 7.06 (1H, s, H-4'''), 6.90 (2H, d, J 8.8, H-2'), 5.03 (2H, s, H-1''), 4.62 (1H, dd, J 5.8, 3.0, H-6v), 4.48 (2H, s, H- 6'), 4.06 (3H, s, H-1'v), 3.91 (1H, dd, J 11.3, 4.9, H-3), 3.40 (1H, dd, J 5.8, 2.6, H-16'a), 3.29–3.19 (5H, m, H-9', H-13', H-16'b), 2.79 (1H, ddd, J 17.5, 7.0, 4.8, H-5a), 2.70–2.67 (4H, m, H-5b, H-1v'''), 2.44 (3H, s, H-1v'), 2.29–2.10 (2H, m, H-4a, H-4b), 1.68 (3H, s, H- 1v''), 1.61–1.54 (4H, m, H-10', H-12'), 1.36 (2H, quint, J 6.2, H-11');13C NMR (151 MHz; CD3OD) δC175.6 (C-2), 175.1 (C-6), 174.2 (C-2'''), 172.7 (C-15'), 171.0 (C-7'), 166.2 (C- 4v), 158.5 (C-4'), 157.0 (C-6av), 152.2 (C-9v), 138.1 (C-4'x), 137.9 (C-1'x), 137.0 (C-5'''), 133.9 (C-1'), 133.5 (C-10av), 133.2 (C-3av), 132.0 (C-2v), 131.3 (C-3v), 131.2 (C-2'x), 130.0 (C-4'''), 129.8 (C-3'), 127.7 (C-3'x), 116.0 (C-2'), 68.3 (C-6'), 55.3 (C-6v), 51.7 (C-3), 40.3 (C-9'), 39.9 (C-13'), 38.9 (C-16'), 34.6 (C-1'v), 34.2 (C-1''), 32.6 (C-5), 30.0 (C-10'), 29.9 (C-12'), 29.3 (C-4), 25.0 (C-11'), 14.4 (C-1v''), 12.9 (C-1v'''), 11.6 (C-1v'); HRMS m / z (ESI+) [Found: 869.2959, C42H46ClN10O7S requires [M+H]+869.2955]; LRMS m / z (ESI+) 383 (100%), 428 (90%), 869 ([35M+H]+, 34%), 871 ([37M+H]+, 17%); HPLC Retention time 8.9 min, 96.1%.

[0021] 2-((S)-4-(4-Chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3- a][1,4]diazepin-6-yl)-N-(5-(2-(4-(1-((5-methoxy-1,2-dimethyl-4,7-dioxo-4,7-dihydro- 1H-indol-3-yl)methyl)-2,6-dioxopiperidin-3-yl)phenoxy)acetamido)pentyl)acetamide (9, IQ-CRBN) Step 1: Compound 47 (30 mg, 0.05 mmol, 1.0 eq.) was solubilized in dry CH3OH (1 mL) and oxalyl chloride (13 µL, 0.15 mmol, 3.0 eq.) was added while maintaining an argon atmosphere. After 3 h at rt, the reaction was not concluded. A further addition of 3.0 eq. of oxalyl chloride was performed and the reaction was stirred at rt for 16 h. Then, the volatile was evaporated to afford the crude material as an orange solid, which was used in the next step without further purification. 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 µL, 0.30 mmol, 6.0 eq.) and HATU (38 mg, 0.1 mmol, 2.0 eq.) in dry DMF (1 mL) under argon. The reaction was stirred at rt for 24 h, then diluted with water (5 mL) and extracted with EtOAc (3 × 5 mL). The combined organic layers were dried over sodium sulfate, filtered, and evaporated in vacuo. The crude material was purified using silica gel column chromatography, eluting with EtOAc:CH3OH (0 to 10% CH3OH) to afford compound 9 as an orange solid (22 mg, 47%). The compound was further purified using semi prep HPLC, method B: Rf 0.16 (EtOAc / CH3OH 85:15); m.p. 113–115 °C (from EtOAc / CH3OH);^^^ଶ^ହ= +8.2 (c 0.5, CH3OH); ῦmax(neat) / cm-13311 (N-H, w), 2926 (C-H, s), 2855 (C-H, w), 1731 (C=O, s), 1670 (C=O, s), 1600 (C=O, s), 1538 (N-H, s), 1509 (N-H, w), 1222 (C-O, s), 1176 (C-O, s), 840 (s);1H NMR (600 MHz; CD3OD) δH7.43–7.35 (4H, m, H-2x', H-3x'), 7.08 (2H, d, J 8.6, H-3'), 6.85 (2H, d, J 8.6, H-2'), 5.65 (1H, s, H-6'''), 5.06 (1H, d, J 14.8, H-1''a), 4.95 (1H, d, J 14.8, H-1''b), 4.61 (1H, dd, J 3.8, 1.7, H-6v''), 4.46 (2H, s, H-6'), 3.86 (3H, s, H- 1'v), 3.84–3.80 (1H, m, H-3), 3.79 (3H, s, H-2v'), 3.43–3.36 (1H, m, H-16'a), 3.28–3.18 (5H, m, H-9', H-13', H-16'b), 2.76–2.73 (2H, m, H-5a, H-5b), 2.68 (3H, s, H-1x), 2.43 (3H, s, H-1v'''), 2.23 (3H, s, H-1v), 2.12–2.06 (2H, m, H-4a, H-4b), 1.67 (3H, s, H-1'x), 1.58–1.54 (4H, m, H-10', H-12'), 1.38–1.33 (2H, m, H-11');13C NMR (151 MHz; CD3OD) δC180.1 (C-4'''), 179.0 (C-7'''), 175.7 (C-2), 174.5 (C-6), 172.7 (C-15'), 171.1 (C-7'), 166.2 (C-4v''), 161.1 (C-5'''), 158.3 (C-4'), 157.0 (C-6av''), 152.2 (C-9v''), 139.0 (C-3'''), 138.1 (C-4x'), 137.9 (C-1x'), 133.5 (2C, C-3av'', C-10av''), 133.2 (C-1'), 132.0 (2C, C-2v'', C-3v''), 131.3 (C-2x'), 130.7 (C-3'), 129.8 (C-7a'''), 129.8 (C-3x'), 122.6 (C-3a'''), 118.9 (C-2), 115.8 (C- 2'), 107.4 (C-6'''), 68.3 (C-6'), 57.1 (C-2v'), 55.3 (C-6v''), 49.6 (C-3), 40.3 (C-9'), 39.8 (C- 13'), 38.9 (C-16'), 35.8 (C-1''), 32.9 (C-5), 32.7 (C-1'v), 29.9 (C-12'), 29.8 (C-10'), 26.6 (C- 4), 24.9 (C-11'), 14.4 (C-1'x), 12.9 (C-1v), 11.6 (C-1x), 9.6 (C-1v'''); HRMS m / z (ESI+) [Found: 947.3326, C49H52ClN8O8S requires [M+H]+947.3312]; LRMS m / z (ESI+) 304 (100%), 428 (93%), 947 ([35M+H]+, 88%), 949 ([37M+H]+, 43%), 969 ([35M+Na]+, 49%), 971 ([37M+H]+, 15%); HPLC Retention time 9.2 min, 96.8%.

[0022] 2-(4-(1-Benzyl-2,6-dioxopiperidin-3-yl)phenoxy)-N-(5-(2-((S)-4-(4- chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6- yl)acetamido)pentyl)acetamide (10, Bn-CRBN) Step 1: Compound 48 (37 mg, 0.07 mmol, 1.0 eq.) was solubilized in dry CH3OH (1 mL) and oxalyl chloride (18 µL, 0.21 mmol, 3.0 eq.) was added while maintaining an argon atmosphere. After 3 h at rt, the reaction was not concluded. A further addition of 3.0 eq. of oxalyl chloride was performed and the reaction was stirred at rt for 16 h. Then, the volatile was evaporated to afford the crude material as a yellow oil, which was used in the next step without further purification. 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 µL, 0.42 mmol, 6.0 eq.) and HATU (53 mg, 0.14 mmol, 2.0 eq.) in dry DMF (1 mL) under argon. The reaction was stirred at rt for 16 h, then diluted with water (5 mL) and extracted with EtOAc (2 × 5 mL). The combined organic layers were washed with 0.5M aqueous LiCl solution (1 × 10 mL), dried over sodium sulfate, filtered, and evaporated in vacuo. The crude material was purified using silica gel column chromatography, eluting with EtOAc:CH3OH (gradient 0 to 5% CH3OH) to afford the title compound as a colourless solid (38 mg, 66%). The compound was further purified using semi prep HPLC, method A: Rf0.19 (EtOAc / CH3OH 85:15); m.p. 68–70 °C (from EtOAc / CH3OH);^^^ଶ^ହ= +12.7 (c 1.0, CH3OH); ῦmax(neat) / cm-13309 (N-H, w), 2927 (C-H, w), 2860 (C-H, w), 1725 (C=O, s), 1673 (C=O, s), 1534 (N-H, s), 1511 (N-H, w), 1420 (w), 1226 (w), 1164 (C-O, s), 1013 (w), 839 (C-H, w), 730 (C-H, s);1H NMR (600 MHz; CD3OD) δH7.43–7.37 (4H, m, H-2v''', H-3v'''), 7.30–7.20 (5H, m, H-2''', H-3''', H-4'''), 7.06 (2H, d, J 8.7, H-3'), 6.87 (2H, d, J 8.7, H-2'), 4.95 (2H, s, H-1''), 4.61 (1H, dd, J 3.6, 2.6, H-6'v), 4.46 (2H, s, H-6'), 3.88 (1H, dd, J 10.6, 5.3, H-3), 3.40 (1H, dd, J 8.9, 2.6 H-16'a), 3.29–3.19 (5H, m, H-9', H-13', H-16'b), 2.82–2.67 (5H, m, H-5a, H-5b, H-1v''), 2.43 (3H, s, H-1v), 2.21–2.11 (2H, m, H-4a, H-4b), 1.68 (3H, s, H-1v'), 1.62–1.53 (4H, m, H-10', H-12'), 1.40–1.35 (2H, m, H-11');13C NMR (151 MHz; CD3OD) δC175.8 (C-2), 174.5 (C-6), 172.7 (C-15'), 171.1 (C-7'), 166.2 (C-4'v), 158.3 (C-4'), 157.0 (C-6a'v), 152.2 (C-9'v), 138.9 (C-1'''), 138.1 (C-4v'''), 137.9 (C-1v'''), 133.5 (C-10a'v), 133.4 (C-3a'v), 133.2 (C-1'), 132.0 (C-3'v), 132.0 (C-2'v), 131.3 (C-2v'''), 130.6 (C-3'), 129.8 (C-3v'''), 129.4 (C- 2'''), 129.3 (C-3'''), 128.3 (C-4'''), 115.9 (C-2'), 68.3 (C-6'), 55.3 (C-6'v), 49.6 (C-3), 44.0 (C-1''), 40.3 (C-13'), 39.8 (C-9'), 38.9 (C-16'), 32.8 (C-5), 29.9 (C-12') ,29.8 (C-10'), 26.5 (C-4), 25.0 (C-11'), 14.4 (C-1v'), 12.9 (C-1v), 11.6 (C-1v'); HRMS m / z (ESI+) [Found: 820.3067, C44H47ClN7O5S requires [M+H]+820.3042]; LRMS m / z (ESI+) 820 ([35M+H]+, 100%), 822 ([37M+H]+, 71%), 842 ([35M+Na]+, 83%), 844 ([37M+Na]+, 53%); HPLC Retention time 9.9 min, 99.3%. Abbreviations 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; (+)-JQ1, (S)-(+)-tert-butyl 2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin- 6-yl)acetate; KOAc, potassium acetate; LCMS, liquid chromatography–mass spectrometry; LRMS, low-resolution mass spectrometry; MTT, (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl tetrazolium bromide); NBS, N-bromosuccinimide; NB, 4-nitrobenzyl; NI, 2- nitroimidazol; Pd(OAc)2, palladium(II) acetate; PE, petroleum ether; PG, phenyl glutarimide; PROTAC, PROteolysis TArgeting Chimera; PS, polymer-supported; TBAI, tetra-n-butylammonium iodide; TBS-T, Tris-buffered saline + Tween20; TEA, triethylamine; TFA, trifluoroacetic acid; THF, tetrahydrofuran; TLC, thin layer chromatography; UV / vis, ultraviolet–visible; VHL, von Hippel- Lindau. References [1] O’Connor, L. J.; Cazares-Körner, C.; Saha, J.; Evans, C. N. G.; Stratford, M. R. L.; Hammond, E. M.; Conway, S. J. Design, Synthesis and Evaluation of Molecularly Targeted Hypoxia-Activated Prodrugs. Nat. Protoc. 2016, 11, 781. [2] Skwarska, A.; Calder, E. D. D.; Sneddon, D.; Bolland, H.; Odyniec, M. L.; Mistry, I. N.; Martin, J.; Folkes, L. K.; Conway, S. J.; Hammond, E. M. Development and pre- clinical testing of a novel hypoxia-activated KDAC inhibitor. Cell Chem. Biol. 2021, 28, 1258. [3] Min, J.; Mayasundari, A.; Keramatnia, F.; Jonchere, B.; Yang, S. W.; Jarusiewicz, J.; Actis, M.; Das, S.; Young, B.; Slavish, J.; Yang, L.; Li, Y.; Fu, X.; Garrett, S. H.; Yun, M. K.; Li, Z.; Nithianantham, S.; Chai, S.; Chen, T.; Shelat, A.; Lee, R. E.; Nishiguchi, G.; White, S. W.; Roussel, M. F.; Potts, P. R.; Fischer, M.; Rankovic, Z. Phenyl-Glutarimides: Alternative Cereblon Binders for the Design of PROTACs. Angew. Chem. Int. Ed. Engl. 2021, 60, 26663. [4] Wallabregue, A. L. D.; Bolland, H.; Faulkner, S.; Hammond, E. M.; Conway, S. J. Two Color Imaging of Different Hypoxia Levels in Cancer Cells. J. Am. Chem. Soc.2023, 145, 2572. [5] Han, X.; Wang, C.; Qin, C.; Xiang, W.; Fernandez-Salas, E.; Yang, C. Y.; Wang, M.; Zhao, L.; Xu, T.; Chinnaswamy, K.; Delproposto, J.; Stuckey, J.; Wang, S. Discovery of ARD-69 as a Highly Potent Proteolysis Targeting Chimera (PROTAC) Degrader of Androgen Receptor (AR) for the Treatment of Prostate Cancer. J. Med. Chem. 2019, 62, 941. [6] Shi, S.; Du, Y.; Zou, Y.; Niu, J.; Cai, Z.; Wang, X.; Qiu, F.; Ding, Y.; Yang, G.; Wu, Y.; Xu, Y.; Zhu, Q. Rational Design for Nitroreductase (NTR)-Responsive Proteolysis Targeting Chimeras (PROTACs) Selectively Targeting Tumor Tissues. J. Med. Chem. 2022, 65, 5057. [7] Cheng, W.; Li, S.; Han, S.; Miao, R.; Wang, S.; Liu, C.; Wei, H.; Tian, X.; Zhang, X. Design, Synthesis and Biological Evaluation of the Tumor Hypoxia-Activated PROTACs Bearing Caged CRBN E3 Ligase Ligands. Bioorg. Med. Chem. 2023, 82, 117237.

Claims

CLAIMS 1. A compound which is a Hypoxia-Actived Proteolysis Targeting Chimera (HAP- TAC) of Formula A or a pharmaceutically acceptable salt thereof: [Formula A] in which: - 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; and - HAP is a hypoxia-activated moiety of formula (I):[Formula I] wherein: - M is -OR1or a 5- to 6-membered heterocyclyl ring which is unsubstituted or is substituted by 1 or 2 RYgroups; - each of R1, R2, R3and R4are independently C1-4alkyl which is unsubstituted or substituted by one, two or three R5groups; H; halo; or a group selected from C3-6cycloalkyl, a 6- to 10-membered aryl ring, a 5- to 10-membered heteroaryl ring anda 5- to 10-membered heterocyclyl ring, each of which is unsubstituted or substituted by one, two or three R6groups; - each R5is independently selected from C1-4alkoxy, halo, -OH, -CN and -N(RY)2; - each R6is independently selected from C1-4alkoxy, halo, -OH, -CN, -N(RY)2, and C1-4alkyl which is unsubstituted or substituted by one, two or three R5groups; - each RXis independently selected from H, methyl and 2-thienyl; - each RYis independently selected from H and C1-4alkyl; and -the point of attachment to U.

2. A compound according to claim 1, wherein: - M is -OR1or a group selected from morpholino, N-methyl piperidinyl, and 4- methylpiperazin-1-yl, wherein preferably M is -OR1; - R1and R3are each independently C1-4alkyl which is unsubstituted or substituted by one or two R5groups; or a group selected from C5-6cycloalkyl, 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 R6groups; - R2and R4are each independently H, halo or C1-4alkyl which is unsubstituted or substituted by one or two R5groups.

3. A compound according to claim 1 or 2, wherein R1is unsubstituted C1-4alkyl.

4. A compound according to any one of claims 1 to 3, wherein R3is unsubstituted C1-4alkyl.

5. A compound according to any one of claims 1 to 4, wherein R2and R4are each independently H.

6. A compound according to any one of claims 1 to 5, wherein each RXis independently H.

7. A compound according to any one of claims 1 to 6, wherein HAP is a hypoxia- activated moiety of Formula IA:[Formula IA] 8. A compound according to any one of claims 1 to 7, wherein 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(O2)N(R’)-, -N(R’)S(O2)-, -(C1-6alkylene)-, -(C2-6alkenylene)-, 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 cycloalkylene, 6-membered arylene, 4- to 7-membered heterocyclylene and 4- to 7-membered heteroarylene; - each XLis independently selected from a single bond, -N(R’)-, -O-, -C(O)-, -C(O)N(R’)-, -N(R’)C(O)-, -S-, -SO-, -SO2-, -C(R’)=C(R’)- and -C≡C-; - each R’ is independently selected from H and C1-4alkyl; - each n is independently selected from 0 to 4; and - m is selected from 1 to 10.

9. A compound according to claim 8, wherein each L2 is independently a unit of formulawherein: - each XLis independently selected from a single bond, -O-, -NR’C(O)-, -C(O)NR’-, -NR’- or -S-; - each n is independently selected from 1 or 2; and - m is an integer from 3 to 6.

10. A compound according to claim 8 or 9, wherein LINK comprises or consists of one of the following structures:preferably wherein LINK comprises or consists of one of the following structures:.

11. A compound according to any one of claims 1 to 10, wherein U is a CRBN, VHL or DCAF15 E3 ubiquitin ligase binding ligand; preferably wherein U is a CRBN E3 ubiquitin ligase binding ligand or a VHL E3 ubiquitin ligase binding ligand.

12. A compound according to any one of claims 1 to 11, wherein 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 LUis a single bond, or Q is CH and LUis selected from a single bond, - C(O)N(Rz)- and -N(Rz)C(O)-; wherein each Rzis independently H or C1-4alkyl; - is the point of attachment to HAP;- RU1is 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 RU2group is a single bond to LINK and the other RU2groups when present are each independently selected from halogen, C1-4alkyl, NH2, NO2, OH, COOH, CN and CF3; - X is selected from -CH(RU3)-, -N(RU3)-, -O-, -C(O)-, -NH-CH(RU3)-, -N=C(RU3)-, and -N=N-;- RVis H or a group selected from C1-4alkyl, C3-6cycloalkyl, 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 C1-4alkoxy, halo, -OH, -CN, - N(RU3)2, C1-4alkyl, , C3-6cycloalkyl, phenyl, a 5- to 6-membered heteroaryl ring and a 5- to 6-membered heterocyclyl ring; - each RU3is independently H or C1-4alkyl; and -the point of attachment to LU; B: a VHL E3 ubiquitin ligase binding ligand of formula (U2):wherein: - RU4is 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 RU4being unsubstituted or substituted by C1-4alkyl; - RU5is H or C1-4alkyl; - RU6is C1-4 alkyl, phenyl or a 3- to 8-membered cycloalkyl ring; - RU7is a single bond to LINK; and -the point of attachment to HAP;or C: a DCAF15 E3 ubiquitin ligase binding ligand of formula (U3):wherein: - z is selected from 1 to 5; - one RU9group is a single bond to LINK and the other RU9groups when present are each independently selected from halogen, C1-4alkyl, NH2, NO2, OH, COOH, CN and CF3; - RU10is H or C1-4 alkyl; and -the point of attachment to HAP.

13. A compound according to claim 12, wherein U is a CRBN E3 ubiquitin ligase binding ligand of formula (U1), Q is CH, LUis a single bond and RU1is:.

14. A compound according to claim 13, wherein U is a CRBN E3 ubiquitin ligase binding ligand having the following structure:wherein is the point of attachment to LINK.

15. A compound according to claim 12, wherein U is a VHL E3 ubiquitin ligase binding ligand of formula (U2), and RU4is:wherein: - V is S or O; - W is N or CH; and - RU8is H or C1-4alkyl.

16. A compound according to any one of claims 12 or 15, wherein U is a VHL E3 ubiquitin ligase binding ligand having the following structure:.

17. A compound according to claim 12, wherein U is a DCAF15 E3 ubiquitin ligase binding ligand having the following structure:.

18. A compound according to any one of claims 1 to 17, wherein POI is a ligand for a cancer-regulating protein.

19. A pharmaceutical composition comprising a compound as defined in any one of claims 1 to 18 and one or more pharmaceutically acceptable excipient, carrier or diluent.

20. A compound according to any one of claims 1 to 18, or a pharmaceutical composition according to claim 19, for use in medicine.

21. A compound according to any one of claims 1 to 18, or a pharmaceutical composition according to claim 19, for use in the treatment of cancer.

22. A compound according to any one of claims 1 to 18, or a pharmaceutical composition according to claim 19, for use in degrading under hypoxic conditions a therapeutic target protein of interest.

23. A compound according to any one of claims 1 to 18, or a pharmaceutical composition according to claim 19, for use in degrading a hypoxic cell comprising a therapeutic target protein of interest.

24. A method for degrading a target protein of interest in a hypoxic cell, the method comprising: a) introducing a compound as defined in any one of claims 1 to 18 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.

25. A method of increasing the hypoxia selectivity of a PROTAC comprising a structure of formula: wherein POI, LINK and U are as defined in any one of claims 8 to 18, the method comprising chemically modifying said structure to comprise a moiety of formula (I) as defined in any one of claims 1 to 7.

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