WO2024251876A1 - Molécules protac et hyt-pd pour la dégradation ciblée de protéines de dcaf15 et leur utilisation dans le traitement de l'amylose - Google Patents

Molécules protac et hyt-pd pour la dégradation ciblée de protéines de dcaf15 et leur utilisation dans le traitement de l'amylose Download PDF

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WO2024251876A1
WO2024251876A1 PCT/EP2024/065601 EP2024065601W WO2024251876A1 WO 2024251876 A1 WO2024251876 A1 WO 2024251876A1 EP 2024065601 W EP2024065601 W EP 2024065601W WO 2024251876 A1 WO2024251876 A1 WO 2024251876A1
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dcaf15
ligand
hyt
linker
protac
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Sólveig Hlín BRYNJÓLFSDÓTTIR
Lisa Beth FRANKEL
Luca LARAIA
Joseph Franz Georg HOOCK
Maria Brandt SCHJØNNING
Matilde Lind Hartvig NIELSEN
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Kraeftens Bekaempelse
Danmarks Tekniske Universitet
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Kraeftens Bekaempelse
Danmarks Tekniske Universitet
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
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    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
    • C07D209/04—Indoles; Hydrogenated indoles
    • C07D209/30—Indoles; Hydrogenated indoles with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to carbon atoms of the hetero ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
    • C07D209/04—Indoles; Hydrogenated indoles
    • C07D209/30—Indoles; Hydrogenated indoles with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to carbon atoms of the hetero ring
    • C07D209/42—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
    • C07D413/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing three or more hetero rings

Definitions

  • the present invention relates to the finding of the role of DDB1 and CUL4 associated factor 15 (DCAF15) in protein aggregation in cells.
  • the present invention further relates to proteolysistargeting chimera (PROTACs) and hydrophobic tag-based protein degradation (HyT-PD) molecules for targeted protein degradation (TPD) of DCAF15 and their use in the treatment of amyloidosis, in particular in the treatment of synucleinopathies.
  • Proteins are essential for living organisms as they perform a vast array of functions, such as DNA replication and transcription, acting as enzymes for catalysing reactions, forming receptors for the cell to respond to stimuli and transporting molecules from one location to another.
  • proteins Once proteins have been synthesized in the cells, they fold into specific three-dimensional conformations (i.e. secondary and tertiary structures) that are thermodynamically favourable (i.e. their native state). This folding process is essential for the protein to acquire its proper structure and function and is driven by a tendency for hydrophobic portions of the protein to shield themselves from the hydrophilic environment of the cell by burying the hydrophobic portions into the interior of the protein.
  • the native state of the protein is stabilized by non-covalent interactions (e.g. hydrogen bonds, salt bridges, and van der Waals forces), as well as disulfide bonds between cysteine residues.
  • Amyloids are aggregates of proteins characterised by a fibrillar morphology, which have been associated with a group of diseases referred to as amyloidosis. About 60 amyloid proteins have been identified so far wherein at least 36 have been associated with human disease.
  • amyloidosis there is a group of neurodegenerative diseases referred to as synucleinopathies (also called o-synucleinopathies).
  • synucleinopathies also called o-synucleinopathies
  • alpha-synuclein deposits also referred to as Lewy bodies
  • Lewy bodies can be seen in neurons under a microscope.
  • Parkinson's disease dementia with Lewy bodies (DLB), and multiple system atrophy (MSA) are examples of synucleinopathies, wherein the cells are unable to effectively remove the highly cytotoxic alpha-synuclein deposits, damaging the cell and eventually leading to a loss of neurons (i.e. neurodegeneration).
  • the main pathological characteristics are cell death of dopamine secreting neurons (dopaminergic neurons) in the brain's basal ganglia leading to low dopamine levels.
  • the loss of dopaminergic neurons is accompanied by the death of astrocytes (star-shaped glial cells) and a significant increase in the number of microglia in the substantia nigra.
  • the symptomatic treatment in PD aims at restoring dopamine levels with e.g. the dopamine precursor levodopa (L-DOPA) or dopamine agonists and/or preventing the metabolism of dopamine with ocatechol-O-methyltransferase (COMT) inhibitors or monoamine oxidase-B (MAO-B) inhibitors.
  • L-DOPA dopamine precursor levodopa
  • COMP ocatechol-O-methyltransferase
  • MAO-B monoamine oxidase-B
  • the ubiquitin-proteasome system plays a critical role in maintaining intracellular protein homeostasis by eliminating misfolded, damaged, and worn-out proteins to prevent aggregation.
  • This process consists of a cascade of distinct steps, starting with ubiquitin activation by enzyme El.
  • Ubiquitin is then passed to the E2 or ubiquitin-conjugating enzyme by transthioesterification.
  • E3 ubiquitin ligases promote the transfer of ubiquitin onto a lysine of the substrate protein, after which it is targeted for proteasomal degradation.
  • the human genome includes two members of the El enzyme family, roughly 40 E2s, and more than 600 E3 ubiquitin ligases (Kleiger and Mayor, 2014).
  • the E3 ligases are categorized into three classes based on their mechanism of ubiquitin transfer, namely RING (really interesting new gene), HECT (homologous to E6AP C-terminus), and RBR (RING between RING).
  • RING really interesting new gene
  • HECT homologous to E6AP C-terminus
  • RBR RING between RING
  • the most abundant class RING is characterized by the direct transfer of ubiquitin from E2 to a substrate and includes approximately 600 RING E3 ligases.
  • Hijacking of the UPS forTPD has attracted substantial interest in the last decade owing to its potential to therapeutically modulate proteins that have previously been considered undruggable or proved difficult to target with conventional small molecules.
  • a major class of molecules that may enable such proteins to be modulated through TPD are known as proteolysis-targeting chimera (PROTAC) protein degraders. These are heterobifunctional small molecules consisting of two ligands joined by a linker: one ligand recruits and binds a protein of interest (POI) while the other recruits and binds an E3 ubiquitin ligase.
  • PROTACs As opposed to small molecule inhibitors which need to continuously occupy the active site of the POI (i.e. "occupancy driven") to exert a lasting inhibitory effect, PROTACs do not require extremely high affinity nor precise action sites to achieve the degradation effects due to its ability to degrade proteins in an "event-driven" manner. Compared to traditional small molecule inhibitors, PROTACs possess several advantages, including reduced drug resistance and the ability to mediate the degradation of what was previously considered undruggable proteins. However, the pharmacokinetic properties of PROTACs are often flawed due to their violation of Lipinski's rule of five.
  • Hydrophobic tag-based protein degradation also referred to as hydrophobic tagging (HyT) is another strategy for TPD.
  • Hydrophobic domains of native proteins are buried within the interior of the protein structure and only become exposed on the surface if the protein is misfolded. If hydrophobic domains are exposed on the protein surface, cells interpret them as unstable or misfolded proteins, leading to the degradation of these proteins through protein quality control (PQC) mechanisms.
  • PQC protein quality control
  • HyT-PD molecules (or hydrophobic tag tethering degraders (HyTTDs)) comprise a ligand for the POI linked to a highly hydrophobic group, such as adamantane, that serves as the hydrophobic tag (HyT).
  • HyT-PD molecules Upon binding of the HyT-PD molecule to the POI, the hydrophobic tag is exposed on the surface of the POI, leading to the degradation of the POI.
  • HyT-PD molecules have received relatively limited attention.
  • HyT- PD molecules may facilitate POI degradation via multiple pathways, such as the UPS pathway, the autophagy pathway, the unfolded protein response (UPR) pathway, and the ubiquitin-independent proteasome system (UIPS) pathway.
  • UPS UPS
  • UPR unfolded protein response
  • UIPS ubiquitin-independent proteasome system
  • HBDs/HBAs hydrogen bond donors/acceptors
  • HyT-PD molecules may present a compelling approach for enhancing druglike properties.
  • HyT-PD molecules are only affected by POI mutations, and the inactivation of a single protein quality control (PQC) pathway does not completely block the degradation of the POI.
  • PQC protein quality control
  • PROTACs require the simultaneous binding of E3 ubiquitin ligase and the POI, and thus any mutation in either related protein or failure to form the PROTAC-E3-POI complex may result in the inactivation of PROTACs and the development of drug resistance.
  • the present invention relates to the finding that knock-out cells lacking DCAF15 are effectively protected against protein aggregation compared to wild-type cells.
  • This finding may provide basis for a paradigm shift in the treatment of diseases caused by or associated with amyloid aggregates by targeted protein degradation (TPD) of DCAF15 using PROTACs or HyT-PD molecules based on ligands for DCAF15 known in the art.
  • TPD targeted protein degradation
  • indisulam an aryl sulfonamide drug
  • CQS chloroquinoxaline sulfonamide
  • SPLAMs - SPLicing inhibitor sulfonAMides have shown to act as ligands for DCAF15 (see e.g. Han et. al., 2017 and WO 2022/169755).
  • E3 ubiquitin ligase ligands see e.g. Bricelj et al., 2021
  • hydrophobic tags HyTs
  • HyT-PD molecules may also be effectively designed for targeted protein degradation of DCAF15.
  • the HyT-PD molecules have more druglike properties, such as lower molecular weight (MW) and less hydrogen bond donors/acceptors, which provides more drug-like properties making them suitable as orally active drugs.
  • the present invention relates to the finding of the pivotal role of DCAF15 in protein aggregation.
  • DCAF15 knock-out cells i.e. DCAF15 -/- cells
  • DCAF15 -/- cells were protected against the formation of stress-induced protein aggregates compared to wild-type cells (Examples 1, 2 and 3)
  • DCAF15 -/- cells showed improved cellular fitness in response to proteotoxic stress compared to wild-type cells (Example 4)
  • DCAF15 -/- neuroblastoma cells were protected against o-synuclein aggregation compared to wild-type cells (Example 5).
  • the present invention relates to PROTACs for targeted protein degradation of DCAF15 capable of preventing protein aggregation in cells.
  • the present invention relates to the medical use of said PROTACs.
  • the present invention relates to HyT-PD molecules for targeted protein degradation of DCAF15 capable of preventing aggregation in cells.
  • the present invention relates to the medical use of said HyT-PD molecules.
  • Fig. 1A shows HCT116 cells with the indicated genotypes treated with 5 pg/ml puromycin for 4h or left untreated (NT) before fixation. Cells were stained for ubiquitinated proteins and p62. Separate and merged channels from a representative experiment are shown (n>3).
  • Fig. IB shows the quantification of number of ubiquitin and p62 co-stained aggregates per cell from Fig. 1A.
  • Fig. 1C shows BE2C cells with the indicated genotypes treated with 5 pg/ml puromycin for 4h or left untreated (NT) before fixation. Cells were stained for ubiquitinated proteins and p62. Separate and merged channels from a representative experiment are shown (n>3).
  • Fig. ID shows the quantification of the number of ubiquitin and p62 co-stained aggregates per cell from Fig. 1C.
  • Fig. 2B shows quantifications of aggregates per cell from MG132 treatment in Fig. 2A for indicated three cell lines.
  • Fig. 3B shows quantification of double positive puncta per cell from Fig. 3A.
  • Fig. 4B shows HCT116 cells treated with 5 pg/ml puromycin for 4h before release into full media or left untreated for 7 days before fixation and crystal violet staining.
  • Fig. 4C shows quantification of Fig. 4B as integrated density per well.
  • PFF pre-formed fibrils
  • Fig. 5B shows quantification of o-synuclein inclusion number (>2 pm diameter) from Fig. 5A.
  • Fig. 5C shows quantification of total GFP levels from indicated cell lines/treatments (time point 1 in Fig. 5A).
  • Fig. 6A shows HCT116 DCAF15-/- cells stably expressing doxycycline-inducible GFP-DCAF15-3xF.
  • Cells were treated with doxycycline for 24h to induce GFP-DCAF15-3xF expression and treated with indicated PROTACS (0.5-2uM) for 24h.
  • Cells were lysed and analyzed for GFP-DCAF15 levels by western blot using anti-GFP antibody.
  • Fig. 6B shows BE2C WT cells treated with 0.5-2 uM of indicated PROTACs (or DMSO control) for 4h. 5ug/ml puromycin was added for 2h to induce aggregation and cells fixed and stained for ubiquitin and p62. "Aggregates per cell” represents the mean no. of double positive puncta (for ubiquitin and p62) per cell. Experiment was performed with technical duplicates analyzing > 110 cells per sample.
  • Fig. 6C shows BE2C WT cells treated with 1, 2 or 5 uM of indicated HyT-PD molecule (or DMSO control) for 24h.
  • Compound 24 was tested in HCT116 WT cells where cells were treated for 6h with the compound. 5ug/ml puromycin was added for the last 4h to induce aggregation and cells fixed and stained for ubiquitin and p62. "Aggregates per cell” represents the mean no. of double positive puncta (for ubiquitin and p62) per cell. Experiment was performed with technical triplicates analyzing > 120 cells per sample (see Fig. 6C).
  • Hi Bit levels were measured using the Hi Bit lytic detection system developed by Promega, following manufactures instructions. The obtained values were compared and plotted as the fold change vs the mean of the DMSO control sample (see Fig. 6D).
  • HiBit levels were measured using the HiBit lytic detection system developed by Promega, following manufactures instructions. The obtained values were compared and plotted as the fold change vs the mean of the DMSO control sample.
  • a linker comprising a PEG (polyethylene glycol) chain should be understood to include the motif -(O-CH2-CH2)n-O- in the linker, wherein n is an integer from 1-6.
  • a linker comprising an alkyl chain should be understood to include the motif -(CH2)n- (i.e. alkanediyl or alkylene) in the linker, wherein n is an interger from 1-16.
  • a linker comprising an extended glycol chain should be understood to include the motif, -(O-CH2-(CH2)n-CH2-O)-, in the linker, wherein n is an integer from 1-2.
  • a linker comrising a cyclic motif should be understood to include a(n) (un)saturated carbocycle or heterocycle as part of the linker to rigify the linker.
  • the cyclic motif of the linker comprises a cyclohexane ring system, a benzene ring system, a piperidine ring system, and/or a piperazine ring system of Fomula (L1-L4) as shown below:
  • a linker may comprise mixtures of PEG chains, extended glycol chains, or alkyl chain as exemplified herein. It should be understood that the linkers may be linear, branched or comprise one or more cyclic motifs. Preferably, the linkers are linear or rigidified with a cyclic motif. For the PROTACs, the linkers may have a total chain length of 6-24 atoms, preferably 6-22 atoms, more preferably 6-20 atoms, even more preferably 6-18 atoms, most preferably 8-16 atoms.
  • the linkers may have a chain length of 3-18 atoms, preferably 3-14 atoms, such as 3-10 atoms, more preferably 3-8 atoms, most preferably 3-6 atoms.
  • any functional groups used to connect the linker to the E3 ligase ligand or the hydrophobic tag (HyT) in one end and the linker to the DCAF15 ligand in the opposite end are included.
  • the linker of PROTAC 1 has a chain length of 9 atoms.
  • HyT-PD molecule 31 has a chain length of 4 atoms (see Table 1).
  • linkers comprising a PEG chain, an alkyl chain, and/or an extended glycol chain may be covalently attached to the DCAF15 ligand in one end and to the E3 ligase/HyT in the opposite via a variety of functional groups commonly used in PROTACs or HyT-PD molecules for linker attachment.
  • functional groups may include e.g.
  • the linker is covalently connected to the DCAF15 ligand with an amide or sulfonamide.
  • the PROTACs or HyT-PD molecules according to the invention may be in the form of a pharmaceutically acceptable salt and/or solvate thereof.
  • the salt may be acid addition salts or basic salts depending on whether acid or basic moieties are present in the DCAF15 ligand, the E3 ligase ligand, or the linker. Examples of pharmaceutically acceptable salts can be found in e.g. Handbook of Pharmaceutical Salts.
  • amyloidosis refers to a group of diseases in which abnormal accumulation of amyloid fibrils (also referred to as amyloids) occurs.
  • the amyloidosis is caused by amyloid fibrils of Tau, 13 amyloid (A
  • synucleinopathies also called o-synucleinopathies refers to a group of neurodegenerative diseases characterised by the abnormal accumulation of amyloid fibrils of o- synuclein. Examples of synucleinopathies are Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA).
  • DCAF15 knock-out cells were protected against protein aggregation compared to wild-type cells. This finding allows for the treatment of diseases caused by or associated with amyloid aggregates by targeted protein degradation (TPD) of DCAF15.
  • diseases include amyloidosis characterized by build-up of amyloid fibrils in tissue(s) or synucleinopathies characterised by the accumulation of alpha-synuclein aggregates in neurons, nerve fibres, or glial cells.
  • PROTACs for degradation of DCAF15 may be designed based on various known ligands for DCAF15 (POI) covalently linked to various known ligands for an E3 ubiquitin ligase other than the ones exemplified herein.
  • HyT-PD molecules for degradation of DCAF15 may be designed based on various known ligands for DCAF15 (POI) covalently linked to various known hydrophobic tags (HyT) other than the ones exemplified herein.
  • a number of ligands for DCAF15 are known in the art, such as the SPLicing inhibitor sulfonAMides indisulam, tasisulam, and chloroquinoxaline sulfonamide (CQS). Furthermore, various analogues of the indisulam scaffold have been synthesized and shown to bind to DCAF15, such as those described in e.g. WO 2022/169755. It should be appreciated that any of the known ligands for DCAF15 may be used as ligand in the PROTACs according to the invention.
  • the ligand for DCAF15 comprises a structure of Formula (DCAF15-I), (DCAF15-II), wherein the dotted lines denote a covalent connection to an E3 ligase ligand via a linker.
  • the DCAF15 ligand is selected from a compound of Formula (DCAF15-I), wherein R 1 is selected as F, Cl, Br, I, CN, Me, or CF3; R 2 is selected as H, CN, CF3 or Me; R 3 is selected as CN or H; and wherein the dotted line denotes a covalent connection to an E3 ligase ligand via a linker.
  • R 1 is selected as Cl or CN, most preferably Cl
  • R 2 is selected as H or Me, most preferably H
  • R 3 is selected as H.
  • the DCAF15 ligand is selected from a compound of Formula (DCAF15-Ia) or (DCAF15-Ib), wherein, R 1 is selected as F, Cl, Br, I, CN, Me, or CF3; R 2 is selected as H, CN, CF3 or Me; R 3 is selected as CN or H; and wherein the dotted lines denote a covalent connection to an E3 ubiquitin ligase ligand via a linker.
  • R 1 is selected as Cl or CN, most preferably Cl
  • R 2 is selected as H or Me, most preferably H
  • R 3 is selected as H.
  • the DCAF15 ligand is selected from a compound of Formula (DCAF15-Ia) wherein R 1 is selected as F, Cl, Br, I, CN, Me, or CF3; R 2 is selected as H, CN, CF3 or Me; R 3 is selected as CN or H; and wherein the dotted line denotes a covalent connection to an E3 ubiquitin ligase ligand via a linker.
  • R 1 is selected as Cl or CN, most preferably Cl;
  • R 2 is selected as H or Me, most preferably H; and
  • R 3 is selected as H.
  • ligands for E3 ubiquitin ligases are known in the art, which are either commercially available or may be synthesized using known procedures.
  • Such ligands include ligands for the cereblon (CRBN), von Hippel— Lindau (VHL), ring finger protein 114 (RNF114), DDB1- and CUL4- associated factor 11 (DCAF11), DDB1 and CUL4 associated factor 15 (DCAF15), or Mouse double minute 2 homolog (MDM2) E3 ubiquitin ligases as described in e.g. Bricelj et al., 2021.
  • E3 ubiquitin ligase ligands development of potent E3 ubiquitin ligase ligands has primarily targeted the von Hippel -Lindau (VHL) and cereblon (CRBN) E3 ubiquitin ligases, and the majority of recently reported PROTACs still utilize either VHL or CRBN as E3 ligases.
  • VHL von Hippel -Lindau
  • CRBN cereblon
  • any known E3 ubiquitin ligase ligand may be used in the PROTACs according to the invention.
  • the E3 ubiquitin ligase ligand is selected from a ligand for CRBN, VHL, RNF114, DCAF11, DCAF15, or MDM2 E3 ubiquitin ligases.
  • the E3 ubiquitin ligase ligand is selected from a CRBN or a VHL E3 ubiquitin ligase ligand.
  • CRBN ligands may be used in the PROTACs according to the invention. These ligands are based on thalidomide or analogues thereof and are commercially available or may be synthesized via known synthetic routes.
  • the E3 ubiquitin ligase ligand is a CRBN ligand selected from a compound of Formula (CRBN-I), (CRBN-II) wherein the dotted lines denote a covalent connection to an DCAF15 ligand via a linker.
  • the CRBN ligase ligand is a compound of Formula (CRBN- II).
  • the CRBN ligase ligand is a compound of Formula (CRBN-II)
  • VHL ligands may be used in the following way. It will be apparent to those skilled in the art that any of the known VHL ligands may be used in the
  • the E3 ligase ligand is a VHL ligand selected from a compound of Formula (VHL-I), (VHL- wherein the dotted lines denote a covalent connection to an DCAF15 ligand via a linker.
  • the VHL ligand consists of a compound of Formula (VHL-I).
  • the E3 ubiquitin ligase ligand is a RNF114 ligand of Formula (RNF114-I) or (RNF114- II), preferably (RNF114-I), / wherein the dotted lines denote a covalent connection to an DCAF15 ligand via a linker.
  • the E3 ubiquitin ligase ligand is a RNF114 ligand of Formula (RNF114-Ia), wherein the dotted line denotes a covalent connection to an DCAF15 ligand via a linker.
  • the E3 ligase ligand is a DCAF11 ligand having the structure of Formula (DCAF11-I- XXI)
  • the E3 ligase ligand is a DCAF11 ligand having the structure of Formula (DCAF11-I).
  • the protein DCAF15 forms in itself part of an E3 ligase complex (i.e. CUL4-DDB1-DDA1-DCAF15 E3 ubiquitin ligase complex). This allows for degradation of DCAF15 (POI) by recruiting the CUL4-DDB1- DDA1-DCAF15 E3 ubiquitin ligase complex.
  • the ligand for recruiting the E3 ubiquitin ligase may also be a ligand for DCAF15. It will be apparent to those skilled in the art that any of the known DCAF15 ligands may also be used as a ligand for recruiting the E3 ubiquitin ligase in the PROTACs according to the invention.
  • the PROTAC may comprise two ligands for DCAF15 covalently connected via a linker (one acting to bind the POI and the other acting to recruit the E3 ubiquitin ligase).
  • a linker one acting to bind the POI and the other acting to recruit the E3 ubiquitin ligase.
  • the ligand for recruiting the E3 ubiquitin ligase comprises a compound selected from Formula (DCAF15-I), (DCAF15-II), or (DCAF15-III) wherein the dotted lines denote a covalent connection to another DCAF15 ligand via a linker.
  • the E3 ligase ligand is a DCAF15 ligand (acting to recruit the E3 ligase) of Formula (DCAF15-I), wherein R 1 is selected as F, Cl, Br, I, CN, Me, or CF3; R 2 is selected as H, CN, CF3 or Me; R 3 is selected as CN or H; and wherein the dotted line denotes a covalent connection to another DCAF15 ligand via a linker.
  • R 1 is selected as Cl or CN, most preferably Cl
  • R 2 is selected as H or Me, most preferably H
  • R 3 is selected as H.
  • the E3 ligase ligand is a DCAF15 ligand (acting to recruit the E3 ligase) consisting of a compound of Formula (DCAF15-Ia), wherein R 1 is selected as F, Cl, Br, I, CN, Me, or CF3; R 2 is selected as H, CN, CF3 or Me; R 3 is selected as CN or H; and wherein the dotted line denotes a covalent connection to another DCAF15 ligand via a linker.
  • R 1 is selected as Cl or CN, most preferably Cl
  • R 2 is selected as H or Me, most preferably H
  • R 3 is selected as H.
  • the PROTACs may be either a heterobifunctional small molecule (e.g. a ligand of Formula (DCAF15-I) covalently linked to a ligand of Formula (DCAF15-II)) or a homobifunctional small molecule (e.g. a ligand of Formula (DCAF15-I) covalently linked to another ligand of Formula (DCAF15-I)).
  • the PROTAC is homobifunctional, and most preferably, the DCAF15 ligand comprises the structure of Formula (DCAF15-I), most preferably (DCAF15-Ia).
  • the E3 ubiquitin ligase ligand is a MDM2 ligand selected from a compound of Formula (MDM2-I), (MDM2-II), (MDM2-III), or Formula (MDM2-IV), wherein the dotted lines denote a covalent connection to an DCAF15 ligand via a linker.
  • linkers have been successfully employed in PROTACs in the prior art. Alkyl chains, PEG chains, and extended glycol chains are the most common linker motifs found in PROTACs. These linkers offer some key advantages, including their commercial availability, their synthetic accessibility, their flexibility, and their ability to easily tune their length and composition via a wide array of robust chemical methods. Furthermore, the development of PROTACs have shown that the binding affinity may be improved by lowering the number of rotational bonds in the linker by introducing ring systems, such as (un)saturated carbocycles or heterocycles to rigidity the linker, e.g.
  • the linker comprises a PEG chain, an extended glycol chain, an alkyl chain, and/or a chain comprising a cyclic motif selected from a disubstituted cyclohexane ring system, a disubstituted benzene ring system, a disubstituted piperidine ring system and/or a disubstituted piperazine ring system to rigidity the linker.
  • the linkers may be linear or branched or comprise one or more cyclic motif(s).
  • the linkers are linear or comprise one or more cyclic motifs.
  • the linkers comprising a PEG chain, an extended glycol chain, an alkyl chain, and/or a chain comprising a cyclic motif
  • the chain length is from 6-24 atoms, preferably 6-22 atoms, more preferably 6-20 atoms, even more preferably 6-18 atoms, most preferably 8-16 atoms.
  • the linkers comprising a PEG chain, an extended glycol chain, an alkyl chain, and/or a chain comprising a cyclic motif is covalently attached to the DCAF15 ligand in one end and to the E3 ubiquitin ligase ligand in the opposite end via common functional groups.
  • Such functional groups may include e.g. amides, sulfonamides, sulfinamides, sulfonates, sulfinates, carbamates, thiocarbamates, esters, ketones, ethers, thiol ethers, amines, alkynes, or tetrazoles depending on the E3 ligase ligand in the PROTAC.
  • the linker is covalently attached to the amine (denoted with the dotted line) in Formula (VHL-I) or (VHL-II).
  • the functional group for attachment of the linker to the ligand may be e.g. an amide, sulfonamide, sulfinamide, carbamate, or an amine, preferably an amide or amine commonly employed in PROTACs with VHL based ligands.
  • the E3 ubiquitin ligase ligand is selected from a scaffold of Formula (VHL-V), (VHL-VI) or (VHL-VII), the linker is attached to the hydroxy group (denoted with the dotted line) in Formula (VHL-V), (VHL-VI) or (VHL-VII).
  • the functional group for attachment of the linker to the E3 ligase ligand may be an e.g. a sulfonate, sulfinate, carbamate, ester, or ether, preferably an ether commonly employed in PROTACs with such VHL based ligands.
  • a sulfonate, sulfinate, carbamate, ester, or ether preferably an ether commonly employed in PROTACs with such VHL based ligands.
  • the E3 ligase ligand is selected from a scaffold of Formula (MDM2-I), (MDM2-II), (MDM2-III) or (MDM2-IV), the linker is covalently attached to the acyl group (denoted with the dotted line) in Formula (MDM2-I), (MDM2-II), (MDM2- III) or (MDM2-IV).
  • the functional group attachment of the linker to the ligand may be e.g. a ketone, amide, or an ester, preferably an amide.
  • the E3 ligase ligand is selected from a scaffold of Formula (CRBN-I) or (CRBN-II), the linker is covalently attached to the phenyl group (denoted with the dotted line) in Formula (CRBN-I) or (CRBN-II).
  • the functional group for attachment of the linker to the ligand may be e.g. an amide, sulfonamide, sulfinamide, sulfonate, sulfinate, carbamate, thiocarbamate, ester, ketone, ether, thiol ether, amine, alkyne, alkene, alkane, a triazole or a tetrazole.
  • the functional group for attachment of the linker to the CRBN ligands is an amine, ether, alkane, alkyne, or amide commonly employed in PROTACs with CRBN based ligands.
  • the linker comprises a structure selected from Formula L1-L8:
  • the linker is covalently connected to the DCAF15 ligand via a functional group selected from an amide, sulfonamide, or ether, more preferably an amide or sulfonamide, most preferably an amide as illustrated herein.
  • a functional group selected from an amide, sulfonamide, or ether, more preferably an amide or sulfonamide, most preferably an amide as illustrated herein.
  • the linker may be connected to the DCAF15 ligand and the E3 ligase ligand via different functional groups well known in the art.
  • the linker consists of a structure selected from the list consisting of:
  • the present invention relates to a proteolysis targeting chimera (PROTAC) or a pharmaceutically acceptable salt thereof, said PROTAC comprising a ligand for DCAF15 (i.e. protein of interest) covalently connected to an E3 ubiquitin ligase ligand via a linker, wherein
  • a proteolysis targeting chimera i.e. protein of interest
  • the ligand for DCAF15 is selected from a compound of Formula (DCAF15-I), (DCAF15-II), or wherein R 1 is selected as F, Cl, Br, I, CN, Me, or CF3; R 2 is selected as H, CN, CF3, or Me; R 3 is selected as CN or H; and wherein the dotted lines denote a covalent connection to the E3 ubiquitin ligase ligand via the linker;
  • the E3 ubiquitin ligase ligand is selected from a CRBN-ligand, a VHL-ligand, a RNF114-ligand, a DCAFll-ligand, or a MDM2-ligand.
  • the present invention relates to a proteolysis targeting chimera (PROTAC) or a pharmaceutically acceptable salt thereof, said PROTAC comprising a ligand for DCAF15 (i.e. protein of interest) covalently connected to an E3 ubiquitin ligase ligand via a linker, wherein
  • a proteolysis targeting chimera i.e. protein of interest
  • the linker comprises a PEG chain, an extended glycol chain, an alkyl chain, or a chain having a cyclic motif selected from a structure of Formula L1-L4,
  • the ligand for DCAF15 comprises a compound of Formula (DCAF15-I), (DCAF15-II), or wherein the dotted lines denote a covalent connection to the E3 ligase ligand via the linker;
  • the E3 ubiquitin ligase ligand is selected from a CRBN-ligand, a VHL-ligand, a RNF114-ligand, a DCAFll-ligand, a DCAF15-ligand, or a MDM2-ligand.
  • the ligand for DCAF15 is selected from a compound of Formula (DCAF15-I).
  • the present invention relates to a proteolysis targeting chimera (PROTAC) or a pharmaceutically acceptable salt thereof, said PROTAC comprising a ligand for DCAF15 (i.e. protein of interest) covalently connected to an E3 ubiquitin ligase ligand via a linker, wherein
  • a proteolysis targeting chimera i.e. protein of interest
  • the linker comprises a PEG chain, an extended glycol chain, an alkyl chain, or a chain having a cyclic motif selected from a structure of Formula L1-L4,
  • the ligand for DCAF15 comprises a compound of Formula (DCAF15-I), wherein the dotted line denotes a covalent connection to the E3 ubiquitin ligase ligand via the linker;
  • the E3 ubiquitin ligase ligand is selected from a CRBN-ligand, a VHL-ligand, a RNF114-ligand, a DCAFll-ligand, a DCAF15-ligand, or a MDM2-ligand.
  • the present invention relates to a proteolysis targeting chimera (PROTAC) or a pharmaceutically acceptable salt thereof, said PROTAC comprising a ligand for DCAF15 (i.e. protein of interest) covalently connected to an E3 ubiquitin ligase ligand via a linker, wherein
  • a proteolysis targeting chimera i.e. protein of interest
  • the linker comprises a PEG chain, an extended glycol chain, an alkyl chain, or a chain having a cyclic motif selected from a structure of Formula L1-L4,
  • the ligand for DCAF15 is selected from a compound of Formula (DCAF15-I), / wherein R 1 is selected as F, Cl, Br, I, CN, Me, or CF3; R 2 is selected as H, CN, CF3 or Me; R 3 is selected as CN or H; wherein the dotted line denotes a covalent connection to an E3 ubiquitin ligase ligand via the linker;
  • the E3 ligase ligand is selected from a CRBN-ligand, a VHL-ligand, a RNF114-ligand, a DCAFll-ligand, or a MDM2-ligand; preferably, the E3 ligase ligand is selected from a CRBN- ligand, a VHL-ligand, a RNF114-ligand, a DCAFll-ligand, or a MDM2-ligand; most preferably the E3 ligase ligand is selected from a CRBN-ligand, a VHL-ligand.
  • the CRBN-ligand, the VHL-ligand, the RNF114-ligand, the DCAF11- ligand, or the MDM2-ligand is preferably selected from a ligand illustrated herein under the examples of known CRBN E3 ubiquitin ligase ligands, the examples of known VHL E3 ubiquitin ligase ligands, the examples of known RNF114 E3 ubiquitin ligase ligands, the examples of known DCAF11 E3 ubiquitin ligase ligands, or the examples of known MDM2 E3 ubiquitin ligase ligands.
  • the CRBN-ligand, the VHL-ligand, the RNF114-ligand, the DCAF11- ligand, or the MDM2-ligand is preferably selected from the following structures:
  • the present invention relates to a proteolysis targeting chimera (PROTAC) or a pharmaceutically acceptable salt thereof, said PROTAC comprising a ligand for DCAF15 (i.e. protein of interest) covalently connected to an E3 ubiquitin ligase ligand via a linker, wherein
  • a proteolysis targeting chimera i.e. protein of interest
  • the linker comprises a PEG chain, an extended glycol chain, an alkyl chain, or a chain having a cyclic motif selected from a structure of Formula L1-L4,
  • the ligand for DCAF15 is selected from a compound of Formula (DCAF15-I), / wherein, R 1 is selected as F, Cl, Br, I, CN, Me, or CF3; R 2 is selected as H, CN, CF3 or Me; R 3 is selected as CN or H; wherein the dotted line denotes a covalent connection to an E3 ubiquitin ligase ligand via a linker;
  • the E3 ligase ligand is selected from a compound of Formula (VHL-I), (VHL-II), (VHL-III), (VHL-IV), (VHL-V), (VHL-VI), (VHL-VII), (DCAF15-I), (MDM2-I), (MDM2-II), (MDM2-III), (MDM2-IV), (DCAF11-I), (RNF114-I), (CRBN-I), or (CRBN-II); preferably, the E3 ligase ligand is selected from a compound of Formula (VHL-I), (VHL-II), (VHL-III), (VHL-IV), (VHL- V), (VHL-VI), (VHL-VII), (MDM2-I), (MDM2-III), (MDM2-IV), (DCAF11-I), (RNF114-I), (CRBN-I), or (CRBN-II); more preferably the E3 ligase ligand
  • the ligand for DCAF15 is selected from a compound of Formula (DCAF15a)
  • R 1 is selected as Cl or CN, most preferably Cl; that R 2 is selected as H or Me, most preferably H; and that R 3 is selected as H. It is further preferred, that the linker comprises a structure selected from Formula L1-L8:
  • the linker comprises a PEG chain, an extended glycol chain, or an alkyl chain. It is further highly preferred that the linker has a chain length of 6-24 atoms, preferably 6-22 atoms, more preferably 6-20 atoms, even more preferably 6-18 atoms, most preferably 8-16 atoms. In the above aspects and embodiments, the linker preferably consists of a structure selected from list consisting of:
  • the linker consists of a structure selected from list consisting of:
  • the linker consists of a structure selected from list consisting of:
  • the linker consists of a structure selected from list consisting of:
  • the linkers drawn above have a chain length of 6-24 atoms, preferably 6-22 atoms, more preferably 6-20 atoms, even more preferably 6-18 atoms, most preferably 8-16 atoms.
  • a proteolysis targeting chimera or a pharmaceutically acceptable salt thereof, said PROTAC comprising a ligand for DCAF15 (i.e. protein of interest) covalently connected to an E3 ubiquitin ligase ligand via a linker, wherein the ligand for DCAF15 is selected from a compound of Formula (DCAF15-I), wherein R 1 is selected as F, Cl, Br, I, CN, Me, or CF3; R 2 is selected as H, CN, CF3, or Me; R 3 is selected as CN or H; and wherein the dotted lines denote a covalent connection to the E3 ubiquitin ligase ligand via the linker; the E3 ubiquitin ligase ligand is selected from a compound of Formula (VHL-I), (VHL- II), (VHL-III), (VHL-IV), (VHL-V), (VHL-VI), (VHL-VII), (MDM2-I), (M
  • a PROTAC according to any one of the preceding items, wherein the E3 ubiquitin ligase ligand is selected from a compound of Formula (VHL-I), (VHL-II), (VHL-III), (VHL-IV), (VHL- V), (VHL-VI), (VHL-VII), (CRBN-I), or (CRBN-II).
  • a PROTAC according to any one of the preceding items, wherein the E3 ubiquitin ligase ligand is selected from a compound of Formula (VHL-I) or (CRBN-II).
  • linker has a chain length of 6-24 atoms, preferably 6-22 atoms, more preferably 6-20 atoms, even more preferably 6-18 atoms, most preferably 8-16 atoms.
  • PEG chain an extended glycol chain, an alkyl chain, or a chain having a cyclic motif selected from a structure of Formula L1-L4,
  • a PROTAC according to any one of the preceding items, wherein the linker comprises a PEG chain, an extended glycol chain, or an alkyl chain.
  • linker consists of a structure selected from the list consisting of:
  • the present invention relates to PROTACs, according to the first aspect and any one of its embodiments and items, for use as a medicament.
  • the PROTACs is for use in the treatment of amyloidosis; more preferably for use in the treatment of synucleinopathies; even more preferably for use in the treatment of Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA); most preferably, for use in the treatment of Parkinson's disease (PD).
  • PD Parkinson's disease
  • DLB dementia with Lewy bodies
  • MSA multiple system atrophy
  • a proteolysis targeting chimera or a pharmaceutically acceptable salt thereof for use in the treatment of amyloidosis, said PROTAC comprising a ligand for DCAF15 (i.e. protein of interest) covalently connected to an E3 ubiquitin ligase ligand via a linker, wherein the ligand for DCAF15 is selected from a compound of Formula (DCAF15-I), wherein, R 1 is selected as F, Cl, Br, I, CN, Me, or CF3; R 2 is selected as H, CN, CF3 or Me; R 3 is selected as CN or H; and wherein the dotted lines denote a covalent connection to the E3 ubiquitin ligase ligand via the linker; the E3 ligase ligand is selected from a CRBN-ligand, a VHL-ligand, a RNF114-ligand, a DCAFll-ligand, a DCAF15-ligand, or a
  • PROTAC for use according to any one of the preceding items, wherein the PROTACs is for use in the treatment of synucleinopathies.
  • a number of ligands for DCAF15 are known in the art, such as the SPLicing inhibitor sulfonAMides indisulam, tasisulam, and chloroquinoxaline sulfonamide (CQS). Furthermore, various analogues of the indisulam scaffold have been synthesized and shown to bind to DCAF15, such as those described in e.g. WO 2022/169755. It should be appreciated that any of the known ligands for DCAF15 may be used as ligand in the HyT-PD molecules according to the invention.
  • the ligand for DCAF15 comprises the structure of Formula (DCAF15-I), (DCAF15-II), (DCAF15-III), wherein the dotted lines denote a covalent connection to a hydrophobic tag (HyT) via a linker.
  • the DCAF15 ligand is selected from a compound of Formula wherein R 1 is selected as F, Cl, Br, I, CN, Me, or CF3; R 2 is selected as H, CN, CF3 or Me; R 3 is selected as CN or H; and wherein the dotted lines denote a covalent connection to a hydrophobic tag (HyT) via a linker.
  • R 1 is selected as Cl or CN, most preferably Cl
  • R 2 is selected as H or Me, most preferably H
  • R 3 is selected as H.
  • the DCAF15 ligand is selected from a compound of Formula (DCAF15-Ia) or (DCAF15-Ib) wherein R 1 is selected as F, Cl, Br, I, CN, Me, or CF3; R 2 is selected as H, CN, CF3 or Me; R 3 is selected as CN or H; and wherein the dotted lines denote a covalent connection to a hydrophobic tag (HyT) via a linker.
  • R 1 is selected as Cl or CN, most preferably Cl
  • R 2 is selected as H or Me, most preferably H
  • R 3 is selected as H.
  • the DCAF15 ligand consists of a compound of Formula (DCAF15- la) wherein R 1 is selected as F, Cl, Br, I, CN, Me, or CF3; R 2 is selected as H, CN, CF3 or Me; R 3 is selected as CN or H; and wherein the dotted lines denote a covalent connection to a hydrophobic tag (HyT) via a linker.
  • R 1 is selected as Cl or CN, most preferably Cl; R 2 is selected as H or Me, most preferably H; and R 3 is selected as H.
  • hydrophobic tags have been successfully employed in HyT-PD molecules in the art.
  • the hydrophobic tags are e.g. polycyclic aromatic hydrocarbons (PAH), a class of aromatic hydrocarbons that contain two or more fused benzene rings, such as naphthalene (Formula II), pyrene or p-naphthoflavone.
  • PAH polycyclic aromatic hydrocarbons
  • a hydrophobic tag is fluorene (Formula III) a rigid planar biphenyl compound.
  • adamantane has been widely employed as a hydrophobic tag in many HyT-PD molecules.
  • HyT-PD molecules with an adamantane tag have demonstrated superior protein degradation efficacy and some improved ADME properties.
  • hydrophobic tags include norbornene (Formula V), tri-Boc protected arginine (Formula VI), or menthoxyacetyl (Formula VII). It should be appreciated that any hydrophobic tag can be used in the synthesis of HyT-PD molecules according to the invention.
  • the hydrophobic tag (HyT) is selected from a compound of Formula (I)-(VII), wherein the dotted lines denote a covalent connection to the DCAF15 ligand via the linker; z is an integer of 0, 1, or 2; and R is/are independently selected from the list consisting of halogens, -0- (Ci-Cs-alkyl), -(Ci-Cs-alkyl), -(Ci-Cs-fluoroalkyl), -O-phenyl, sulfonamide (-SO2NH2), and alkyl sulfonamide (-SO2NH(Ci-Cs-alkyl) or -SO2N(Ci-Cs-alkyl)2).
  • hydrophobic tag (HyT) is selected from a compound for Formula (I)-
  • the hydrophobic tag (HyT) is selected from a compound for Formula (I)-(IV), wherein the dotted lines denote a covalent connection to the DCAF15 ligand via the linker; z is an integer of 0, 1, or 2; R is/are independently selected from the list consisting of halogens, -O-(Ci-Cs- alkyl), -(Ci-Cs-alkyl), -O-phenyl, sulfonamide (-SO2NH2).
  • linkers have been successfully employed in HyT-PD molecules in the art. Alkyl chains, PEG chains, and extended glycol chains are common linker motifs found in HyT-PD molecules. These linkers offer some key advantages, including their commercial availability, their synthetic accessibility, their flexibility, and their ability to easily tune their length and composition via a wide array of robust chemical methods.
  • the linker comprises a PEG chain, an extended glycol chain, an alkyl chain, and/or a chain comprising a cyclic motif selected from a disubstituted cyclohexane ring system, a disubstituted benzene ring system, a disubstituted piperidine ring system and/or a disubstituted piperazine ring system to rigidity the linker.
  • the linkers may be linear or branched or comprise one or more cyclic motif(s).
  • the linkers are linear or comprise one or more cyclic motifs.
  • the linkers may have a chain length of 3-18 atoms, preferably 3-14 atoms, more preferably 3-10 atoms, even more preferably 3-8 atoms, most preferably 3-6 atoms.
  • any functional groups used to connect the linker to the hydrophobic tag (HyT) in one end and the linker to the DCAF15 ligand in the opposite end are included.
  • the chain length is calculated as the number of atoms in a linear chain between the DCAF15 ligand and the hydrophobic tag (see Table 1 for examples).
  • HyT-PD molecule compound no. 30 has a chain length of 3 atoms
  • 25 has a chain length of 16 atoms.
  • a short linker length of e.g. 3 atoms was found to be sufficient for degradation of DCAF15. Shorter linkers have the benefit of lower molecular weight and improved drug-like properties.
  • linkers comprising a PEG chain, an alkyl chain, and/or an extended glycol chain may be covalently attached to the DCAF15 ligand in one end and to the HyT in the opposite end via a variety of functional groups commonly used in HyT-PD molecules for linker attachment.
  • Such functional groups may include e.g.
  • the linkers can be designed in variety of different ways. Most preferably, the linker is covalently connected to the DCAF15 ligand with an amide or sulfonamide, most preferably an amide as illustrated herein.
  • the linker preferably comprises a PEG chain, an extended glycol chain, an alkyl chain or a chain having a cyclic motif selected from a structure of Formula L1-L4,
  • the linker comprises a PEG chain, an extended glycol chain, an alkyl chain, most preferably, the linker comprises an alkyl chain.
  • the linker comprises a structural motif of Formula L1-L8:
  • the linker comprises a structural motif of Formula L1-L4 or L8: even more preferably the linker comprises a structural motif of Formula LI, L4 or L8: yet even more preferably the linker comprises a structural motif of Formula L4: even more preferably the linker comprises a structural motif of Formula L4:
  • the linker is covalently connected to the DCAF15 ligand via a functional group selected from an amide, sulfonamide, or ether, more preferably an amide or sulfonamide, most preferably an amide as illustrated herein.
  • the linker is covalently connected to the hydrophobic tag via an amine, amide, ketone or directly via a methylene (-CH2-) as illustrated herein.
  • the linker may be connected to the DCAF15 ligand and the hydrophobic tag via different functional groups well known in the art.
  • the linker consists of a structure selected from the list consisting of:
  • the linker consists of a structure selected from the list consisting of:
  • the linker consists of a structure selected from the list consisting of: wherein the dotted lines marked * denote the end of the linker covalently connected to the DCAF15 ligand and the dotted lines marked ** denote the end of the linker covalently connected to the hydrophobic tag (HyT); most preferably the linker consists of a structure selected from the list consisting of: wherein the dotted lines marked * denote the end of the linker covalently connected to the DCAF15 ligand and the dotted lines marked ** denote the end of the linker covalently connected to the hydrophobic tag (HyT).
  • the linkers drawn above in aspect 3 have a chain length of 3-18 atoms, preferably 3-14 atoms, such as 3-10 atoms, more preferably 3-8 atoms, most preferably 3-6 atoms.
  • the present invention relates to a hydrophobic tag-based protein degradation (HyT- PD) molecule or a pharmaceutically acceptable salt thereof, said HyT-PD molecule comprising a ligand for DCAF15 (i.e. protein of interest) covalently connected to a hydrophobic tag (HyT) via a linker, wherein
  • the ligand for DCAF15 is selected from a compound of Formula (DCAF15-I), (DCAF15-II), or wherein R 1 is selected as F, Cl, Br, I, CN, Me, or CF3; R 2 is selected as H, CN, CF3, or Me; R 3 is selected as CN or H; and wherein the dotted lines denote a covalent connection to the hydrophobic tag (HyT) via the linker;
  • hydrophobic tag (HyT) is selected from a compound of Formula (I)-(VII), wherein the dotted lines denote a covalent connection to the DCAF15 ligand via the linker; z is an integer of 0, 1, or 2;
  • R is/are independently selected from the list consisting of halogens, -O-(Ci-Cs-alkyl), -(Ci- Cs-alkyl), -O-(Ci-Cs-fluoroalkyl), -(Ci-Cs-fluoroalkyl), -O-phenyl, sulfonamide (-SO2NH2), and alkyl sulfonamide (-SO2NH(Ci-Cs-alkyl) or -SO2N(Ci-Cs-alkyl)2).
  • the ligand for DCAF15 is selected from a compound of Formula (DCAF15-I).
  • DCAF15-I hydrophobic tag-based protein degradation
  • the present invention relates to a hydrophobic tag-based protein degradation (HyT-PD) molecule or a pharmaceutically acceptable salt thereof, said HyT-PD molecule comprising a ligand for DCAF15 (i.e.
  • a hydrophobic tag HyT
  • the ligand for DCAF15 is selected from a compound of Formula (DCAF15-I), wherein R 1 is selected as F, Cl, Br, I, CN, Me, or CF3; R 2 is selected as H, CN, CF3, or Me; R 3 is selected as CN or H; and wherein the dotted line denotes a covalent connection to the hydrophobic tag (HyT) via a linker;
  • hydrophobic tag (HyT) is selected from a compound of Formula (I)-(VII), wherein the dotted lines denote a covalent connection to the DCAF15 ligand via the linker; z is an integer of 0, 1, or 2;
  • R is/are independently selected from the list consisting of halogens, -O-(Ci-Cs-alkyl), -(Ci- Cs-alkyl), -O-(Ci-Cs-fluoroalkyl), -(Ci-Cs-fluoroalkyl), -O-phenyl, sulfonamide (-SO2NH2), and alkyl sulfonamide (-SO2NH(Ci-Cs-alkyl) or -SO2N(Ci-Cs-alkyl)2).
  • the present invention relates to a hydrophobic tag-based protein degradation (HyT-PD) molecule or a pharmaceutically acceptable salt thereof, said HyT-PD molecule comprising a ligand for DCAF15 (i.e. protein of interest) covalently connected to a hydrophobic tag (HyT) via a linker, wherein
  • HyT-PD hydrophobic tag-based protein degradation
  • the ligand for DCAF15 is selected from a compound of Formula (DCAF15-I), wherein R 1 is selected as F, Cl, Br, I, CN, Me, or CF3; R 2 is selected as H, CN, CF3, or Me; R 3 is selected as CN or H; and wherein the dotted line denotes a covalent connection to the hydrophobic tag (HyT) via a linker;
  • the hydrophobic tag (HyT) is selected from a compound of Formula (I-IV), wherein the dotted lines denote a covalent connection to the DCAF15 ligand via the linker; z is an integer of 0, 1, or 2; R is/are independently selected from the list consisting of halogens, -O-(Ci-Cs-alkyl), -(Ci-Cs-alkyl), -O-(Ci-Cs-fluoroalkyl), -(Ci-Cs-fluoroalkyl), -0- phenyl, sulfonamide (-SO2NH2), and alkyl sulfonamide (-SChNH Ci-Cs-alkyl) or -SO2N(CI-C5- alkyl) 2 ).
  • the ligand for DCAF15 is selected from a compound of Formula (DCAF15a)
  • R 1 is selected as Cl or CN, most preferably Cl; that R 2 is selected as H or Me, most preferably H; and that R 3 is selected as H. It is preferred, that the linker comprises a structure selected from Formula L1-L8:
  • the linker comprises a PEG chain, an extended glycol chain, or an alkyl chain. It is further highly preferred that the linker has a chain length of 3-18 atoms, preferably 3-14 atoms, such as 3-10 atoms, more preferably 3-8 atoms, most preferably 3-6 atoms. Items of aspect 3
  • a hydrophobic tag-based protein degradation (HyT-PD) molecule or a pharmaceutically acceptable salt thereof said HyT-PD molecule comprising a ligand for DCAF15 (i.e. protein of interest) covalently connected to a hydrophobic tag (HyT) via a linker, wherein the ligand for DCAF15 is selected from a compound of Formula (DCAF15-I), wherein R 1 is selected as F, Cl, Br, I, CN, Me, or CF3; R 2 is selected as H, CN, CF3, or Me; R 3 is selected as CN or H; and wherein the dotted lines denote a covalent connection to the hydrophobic tag (HyT) via the linker;
  • DCAF15-I compound of Formula
  • hydrophobic tag (HyT) is selected from a compound of Formula (I)-(VII), wherein the dotted lines denote a covalent connection to the DCAF15 ligand via the linker; z is an integer of 0, 1, or 2;
  • R is/are independently selected from the list consisting of halogens, -O-(Ci-Cs-alkyl), -(Ci-Cs-alkyl), -O-(Ci-Cs-fluoroalkyl), -(Ci-Cs-fluoroalkyl), -O-phenyl, sulfonamide (- SO2NH2), and alkyl sulfonamide (-SO2NH(Ci-Cs-alkyl) or -SO2N(Ci-Cs-alkyl)2).
  • HyT-PD molecule according to item 1, wherein the ligand for DCAF15 is selected from a compound of Formula (DCAF15-I).
  • HyT-PD molecule according to any one of the preceding items, wherein the ligand for DCAF15 is selected from a compound of Formula (DCAF15-Ia) or (DCAF15-Ib):
  • HyT-PD molecule according to any one of the preceding items, wherein ligand for
  • DCAF15 is selected from a compound of Formula (DCAF15-Ia)
  • HyT-PD molecule according to any one of the preceding items, wherein R 1 is selected as Cl, and R 2 is selected as H.
  • HyT-PD molecule according to any one of the preceding items, wherein R 3 is selected as H.
  • HyT-PD molecule according to any one of the preceding items, wherein the hydrophobic tag (HyT) is selected from a compound of Formula (I)-(IV),
  • HyT-PD molecule according to any one of the preceding items, wherein linker has a chain length of 3-18 atoms, preferably 3-14 atoms, more preferably 3-10 atoms, even more preferably 3-8 atoms, most preferably 3-6 atoms.
  • the HyT-PD molecule according to any one of the preceding items, wherein the linker consists of a structural motif selected from the list consisting of:
  • HyT-PD molecule according to any one of the preceding items, wherein the linker consists of a structural motif selected from the list consisting of:
  • HyT-PD molecule according to any one of the preceding items, wherein the linker consists of a structural motif selected from the list consisting of:
  • HyT-PD molecule according to any one of the preceding items, wherein the HyT-PD molecule has the structure of compound nos. 24-41.
  • the present invention relates to a HyT-PD molecule, according to the third aspect and any one of its embodiments and items, for use as a medicament.
  • the HyT-PD molecules are for use in the treatment of amyloidosis; more preferably for use in the treatment of synucleinopathies; even more preferably for use in the treatment of Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA); most preferably, for use in the treatment of Parkinson's disease (PD).
  • the hydrophobic tag may also be selected as a Boc group.
  • the hydrophobic tag (HyT) is selected from a compound of Formula (I)-(VIII), EXEMPLIFIED PROTACs AND HyT-PD MOLECULES ACCORDING TO THE INVENTION
  • Table 1 exemplifies PROTACs and HyT-PD molecules according to the invention, using a ligand for DCAF15 covalently connected to different E3 ubiquitin ligase ligands or different hydrophobic tags (HyT) with a linker comprising a PEG chain, an extended glycol chain, an alkyl chain, or a chain comprising cyclic structures.
  • a linker comprising a PEG chain, an extended glycol chain, an alkyl chain, or a chain comprising cyclic structures.
  • Example 2 DCAF15 is required for protein aggregation following different types of proteotoxic stress
  • Examples 1 and 2 could theoretically be explained by an altered clearance of protein aggregates in DCAF15-/- cells.
  • TPD targeted protein degradation
  • Example 5 DCAF15 promotes aggregation of a-synuclein in neuroblastoma cells
  • PFF pre-formed fibrils
  • a cellular system to study a-synuclein aggregation in BE2C neuroblastoma cells was developed by stably expressing a GFP-tagged a-synuclein, containing a well-known A53T mutation that is especially prone to aggregation.
  • purified pre-formed a-synuclein fibrils (PFF) were transfected and formation of a-synuclein inclusions monitored by live imaging, a- synuclein inclusions were distinctly fewer (and smaller in size) in DCAF15 -/- cells relative to WT, while overall GFP intensity did not differ between the two cell lines.
  • the data suggest that DCAF15 facilitates PFF-induced a-synuclein aggregation/inclusion formation.
  • Example 6 Targeted protein degradation of DCAF15 via PROTACs or HyT-PD molecules inhibits aggregation
  • HCT116 DCAF15-/- cells stably expressing doxycycline-inducible GFP-DCAF15-3xF.
  • Cells were treated with doxycycline for 24h to induce GFP-DCAF15-3xF expression and treated with indicated PROTACS (2uM) for 24h.
  • Cells were lysed and analyzed for GFP-DCAF15 levels by western blot using anti-GFP antibody (see Fig. 6A).
  • Fig. 6B shows BE2C WT cells treated with 0.5-2 uM of indicated PROTACs (or DMSO control) for 4h. 5ug/ml puromycin was added for 2h to induce aggregation and cells fixed and stained for ubiquitin and p62.
  • “Aggregates per cell” represents the mean no. of double positive puncta (for ubiquitin and p62) per cell. Experiment was performed with technical duplicates analyzing > 110 cells per sample.
  • Fig. 6C shows BE2C WT cells treated with 1, 2 or 5 uM of indicated HyT-PD molecule (or DMSO control) for 24h.
  • Compound 24 was tested in HCT116 WT cells where cells were treated for 6h with the compound. 5ug/ml puromycin was added for the last 4h to induce aggregation and cells fixed and stained for ubiquitin and p62. "Aggregates per cell” represents the mean no. of double positive puncta (for ubiquitin and p62) per cell. Experiment was performed with technical triplicates analyzing > 120 cells per sample (see Fig. 6C).
  • HiBit levels were measured using the HiBit lytic detection system developed by Promega, following the manufactures instructions. The obtained values were compared and plotted as the fold change vs the mean of the DMSO control sample (see Fig. 6D and 6E).
  • the plates were either visualized under ultraviolet (UV)-light or stained by dipping in a developing agent followed by heating. Ninhydrin was used as developing agent. Flash column chromatography was performed using Merck Geduran® Si 60 (40-63 pm) silica gel. All purified compounds were characterized by Nuclear Magnetic Resonance Spectroscopy (NMR), and LRMS electrospray ionization (ESI) (by-products were not fully characterized). Melting point and optical rotation were measured when appropriate. Structural assignments were made, when possible, for new compounds using COSY, HSQC, HMBC spectra where appropriate.
  • NMR Nuclear Magnetic Resonance Spectroscopy
  • ESI electrospray ionization
  • a Bruker Avance III 400 spectrometer with a Bruker Ascend 400 magnet and a Prodigy CryoProbe (operating at 400 MHz for proton and 101 MHz for carbon) was used.
  • a Bruker Avance III 800 spectrometer with a Bruker Ascend 800 magnet and a 5 mm TCI CryoProbe (operating at 800 MHz for proton, 201 MHz for carbon) was used.
  • the chemical shifts (6) are reported in parts per million (ppm) and the coupling constants (J) in Hz.
  • Methyl 3-(N-(3-chloro-lH-indol-7-yl)sulfamoyl)benzoate (1) (2.19 g, 6.0 mmol, l.equiv.) was dissolved in THF/H2O (1:1, 50 ml). LiOH (1.44 g, 60.03 mmol, 10. equiv.) was added and the reaction mixture was stirred for 1-2 h until complete conversion of starting material. The mixture was acidified by addition of cone. HCI (until pH 3) and the product was extracted using DCM (3 x 50 ml). The combined organic phases were dried (Na 2 SO4) and put on rotary evaporator.
  • the mono-Boc protected linker (1.0 equiv.) was dissolved in dry DCM (10 ml) and kept under nitrogen atmosphere. DIPEA (4 equiv.) and pomalidomide (1.0 equiv.) was added and the reaction mixture was heated to 90°C for 6 h. The reaction mixture was cooled to room temperature and portioned between half sat. brine and EtOAc (3 x 50 mL). The combined organic phases were further washed with sat. NH4CI (50 ml), H 2 O (6 x 50 mL) and sat. brine (50 mL). The organic phase was dried (Na 2 SC>4), filtered and put on rotary evaporator. The crude product was purified by flash column chromatography to afford the CRBN ligand-linker conjugates. Genera! procedure for preparation of VHL ligand-linker conjugate (Intermediates for PROTAC 8-11, 16-17)
  • Boc-protected VH032 (1.0 equiv.) was dissolved in DCM (2 mL), followed by the addition of 4M HCI in dioxane (0.2 ml, ca.l mL/mmol). The reaction mixture was stirred at rt for 1 h. After complete deprotection was observed by TLC, the solvent was removed under reduced pressure and coevaporated with Et?O (3 x 10 mL). The residue was further dried in high vacuum for 2h.
  • Boc-protected carboxylic acid linker (1.0 equiv.) was dissolved in DCM (2 mL) followed by the addition of 3-(((ethylimino)methylene)amino)-N,N-dimethylpropan-l-amine hydrogen chloride (EDC x HCI, 2.0 equiv.) and lH-benzo[d][l,2,3]triazol-l-ol (HOBt x H2O, 1.2 equiv.) and activation of the carboxylic acid for 30 min.
  • EDC x HCI 3-(((ethylimino)methylene)amino)-N,N-dimethylpropan-l-amine hydrogen chloride
  • HBt x H2O lH-benzo[d][l,2,3]triazol-l-ol
  • reaction mixture was cooled to 0 °C and the VH032-amine (1.0 equiv.) dissolved in DCM (2 mL) and then /V,/V-Diisopropylethylamine (DIPEA, 2.0 equiv.) were sequentially charged into the reaction flask.
  • DIPEA /V,/V-Diisopropylethylamine
  • the reaction was warmed to rt and monitored by TLC analysis. After full conversion (typically lh), the solution was diluted with (DCM, 10 mL), washed with water and extracted with DCM (3 x 20 ml). The combined organic layers were washed with brine (2x50 mL), dried over Na2SC>4, filtered, and concentrated in vacuo.
  • the crude product was purified by column chromatography (DCM/MeOH 1 to 5%) to give a white solid.
  • the indisulam carboxylic acid derivative (1.0 equiv.) was dissolved in DMF (2 mL) followed by the addition of l-[Bis(dimethylamino)methylene]-l/7 L l,2,3-triazolo[4,5- ]pyridinium3-oxidhexafluoro- phosphate (HATU, 2.0 equiv.) and activation of the carboxylic acid for 30 min. Then, the reaction mixture was cooled to 0 °C and the respective mono-Boc protected diamine linker (0.5 equiv.) and then /V,/V-Diisopropylethylamine (DIPEA, 2.0 equiv.) were sequentially charged into the reaction flask.
  • DIPEA mono-Boc protected diamine linker
  • the Boc-protected linker-pomalidomide conjugate (1.1 equiv.) was dissolved in DCM (2 mL) and treated with TFA (0.75 mL). The reaction mixture was stirred at rt for 30 min to lh until complete deprotection and quenched by addition of sat. NaHCOs (15 mL). The aqueous solution was extracted with DCM (6 x 20 mL) until the organic phase is no longer fluorescent. The combined organic layers were dried using Na?SO4 and put on rotary evaporator. The residue was afterwards put on high vacuum.
  • the reaction is monitored by TLC and when reflecting zero change in spot intensities the reaction is quenched by diluting with water (10 mL) and extracting with DCM (3 x 10 mL). The combined organic phase was washed with brine (50 mL) dried using Na?SO4 and put on rotary evaporator. The crude product was purified by flash column chromatography yielding a yellow neon-like solid/oil.
  • the Boc-protected VHL-linker conjugate (1.0 equiv.) was dissolved in DCM (2 mL), followed by the addition of 4M HCI in dioxane (0.2 ml, ca.l mL/mmol). The reaction mixture was stirred at rt for 1 h. After complete deprotection was observed by TLC, the solvent was removed under reduced pressure and co-evaporated with EtzO (3 x 10 mL). The residue was further dried in high vacuum for 2h.
  • the indisulam carboxylic acid derivative (1.0 equiv.) was dissolved in DMF (2 mL) followed by the addition of l-[Bis(dimethylamino)methylene]-l/7 c l,2,3-triazolo[4,5- ]pyridinium3-oxidhexafluorophosphate (HATU, 2.0 equiv.) and activation of the carboxylic acid for 30 min.
  • HATU l-[Bis(dimethylamino)methylene]-l/7 c l,2,3-triazolo[4,5- ]pyridinium3-oxidhexafluorophosphate
  • reaction mixture was cooled to 0 °C and the respective VH032-linker conjugate (1.0 equiv.) dissolved in DCM (2 mL) and then /V,/V-Diisopropylethylamine (DIPEA, 2.0 equiv.) were sequentially charged into the reaction flask.
  • DIPEA /V,/V-Diisopropylethylamine
  • the reaction was warmed to rt and monitored by TLC analysis. After full conversion (typically lh) the solution was diluted with (DCM, 10 mL), washed with water and extracted with DCM (3 x 20 ml). The combined organic layers were washed with brine (2x50 mL), dried over NazSCU, filtered, and concentrated in vacuo.
  • the crude product was purified by column chromatography (DCM/MeOH 1 to 5%) to give a white solid.
  • the indisulam carboxylic acid derivative (1.0 equiv.) was dissolved in DMF (2 mL) followed by the addition of l-[Bis(dimethylamino)methylene]-l/7 L l,2,3-triazolo[4,5- ]pyridinium3-oxidhexafluoro- phosphate (HATU, 2.0 equiv.) and activation of the carboxylic acid for 30 min. Then, the reaction mixture was cooled to 0 °C and the respective diamine linker (0.5 equiv.) and then N,N- Diisopropylethylamine (DIPEA, 2.0 equiv.) were sequentially charged into the reaction flask.
  • DIPEA N,N- Diisopropylethylamine
  • RNF114 ligand as well as the DCAF15-RNF114 ligands were synthesized by closely following a literature procedure (Luo et al.). Shortly, the Boc-protected RNF114 ligand (1.0 equiv.) was dissolved in DCM (2 mL) and trifluoroacetic acid (TFA, 5 mL) added slowly over the course of 5 minutes. After stirring for an additional 20 minutes, deprotection was complete and the solvent was removed in vacuo. To remove residual TFA, the crude material was co-evaporated with toluene (3 x 10 mL) and then dried in high vacuum for 2h.
  • TFA trifluoroacetic acid
  • DCAF15-linker conjugate (1.0 equiv.) was Boc- deprotected by the addition of 4M HCI in dioxane (1 mL). After stirring for 30 min, the solvent was evaporated, and residues were co-evaporated with Et?O (3x10 mL). The crude was dried in high vacuum for 2 h and used directly for the next step.
  • the crude RNF114 acid was dissolved in DMF (1 mL) and HATU (2.0 equiv) was added, and the mixture was stirred for lh.
  • reaction mixture was cooled to 0 °C and the respective DCAF15-linker amine (1.0 equiv.), dissolved in DCM (2 mL) and then /V,/V-Diisopropylethylamine (DIPEA, 2.0 equiv.) were sequentially charged into the reaction flask.
  • the reaction was warmed to rt and monitored by TLC analysis. After full conversion (typically lh) the solution was diluted with (DCM, 10 mL), washed with water and extracted with DCM (3 x 20 ml). The combined organic layers were washed with brine (2x50 mL), dried over Na2SO4, filtered, and concentrated in vacuo.
  • the crude product was purified by column chromatography (DCM/MeOH 1 to 3%) to give an off-white solid.
  • the DCAFll-based PROTACs were synthesized by closely following a literature procedure (Zhang et. al.). Shortly, the DCAF15-Linker conjugate was dissolved in DCM (1 mL) and TFA (2 mL) was added slowly over the course of 5 minutes. After stirring for an additional 20 minutes, toluene was added, and the solvent was removed in vacuo. To generate the free amine, the resulting residue was dissolved in EtOAc (10 mL) and washed with sat. NaHCOs (2 x 5 mL). The organic layer was dried with Na2SC>4, filtered, and concentrated in vacuo or 2h.
  • PROTAC 1 3-(N-(3-chloro-lH-indol-7-yl)sulfamoyl)-N-(6-((2-(2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindolin-4- yl)amino)hexyl)benzamide (PROTAC 1): In the column chromatography an eluent of 98:2 DCM/MeOH was used. PROTAC (1) was afforded as a yellow solid (27.3 mg, 27%). Rf : 0.32 (95:5 DCM/MeOH).
  • PROTAC 2 3-(N-(3-chloro-lH-indol-7-yl)sulfamoyl)-N-(10-((2-(2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindolin-4- yl)amino)decyl)benzamide (PROTAC 2): In the column chromatography an eluent of DCM/MeOH 98:2 was used and later increased to DCM/MeOH 95:5. PROTAC 2 was afforded as a yellow solid (34.1 mg, 32%).
  • PROTAC 10 was afforded as a colourless solid (25.6 mg, 37%).
  • the indisulam carboxylic acid derivative (1.0 equiv.) was dissolved in DMF (2 mL) followed by the addition of l-[Bis(dimethylamino)methylene]-l/7 L l,2,3-triazolo[4,5- ]pyridinium3-oxidhexafluoro- phosphate (HATU, 2.0 equiv.) and activation of the carboxylic acid for 30 min. Then, the reaction mixture was cooled to 0 °C and the respective mono-Boc protected diamine linker (0.5 equiv.) and then /V,/V-Diisopropylethylamine (DIPEA, 2.0 equiv.) were sequentially charged into the reaction flask.
  • DIPEA mono-Boc protected diamine linker
  • Hyt molecule 28 l-(3-(N-(3-chloro-lH-indol-7-yl)sulfamoyl)phenyl)-l-oxo-6,9,12-trioxa-2-azapentadecan-15- aminium (intermediate for Hyt molecule 28): c> a solution of Hyt molecule 25 (25.00 mg, 0.04 mmol, 1.0 equiv.) in DCM (2 mL) TFA (0.5 mL) was added dropwise. The reaction mixture was stirred until completion was indicated by TLC analysis (2 h). At this point, the solution was evaporated by coevaporation with toluene (3 x 10 mL).
  • the indisulam carboxylic acid derivative (1.0 equiv., 0.09 mmol) was dissolved in DMF (2 mL) followed by the addition of l-[Bis(dimethylamino)methylene]-l/7 c l,2,3-triazolo[4,5- ]pyridinium3- oxidhexafluorophosphate (HATU, 1.5 equiv.) and activation of the carboxylic acid for 30 min. Then, the reaction mixture was cooled to 0 °C and the deprotected HyT 25 (1.1 equiv.) and then N,N- Diisopropylethylamine (DIPEA, 3.0 equiv.) were sequentially charged into the reaction flask.
  • DIPEA N,N- Diisopropylethylamine
  • the indisulam carboxylic acid derivative (1.0 equiv., 0.09 mmol) was dissolved in DMF (2 mL) followed by the addition of l-[Bis(dimethylamino)methylene]-l/7 L l,2,3-triazolo[4,5- ]pyridinium3- oxidhexafluorophosphate (HATU, 1.5 equiv.) and activation of the carboxylic acid for 30 min. Then, the reaction mixture was cooled to 0 °C and the respective amine (1.1 equiv.) and then N,N- Diisopropylethylamine (DIPEA, 3.0 equiv.) were sequentially charged into the reaction flask.
  • DIPEA N,N- Diisopropylethylamine
  • Hyt-iinker conjugate (Intermediates for Hyt 49-53) The corresponding carboxylic acid derivative (1.0 equiv.) was dissolved in DMF (2 mL) followed by the addition of l-[Bis(dimethylamino)methylene]-l/7 L l,2,3-triazolo[4,5- ]pyridinium3- oxid hexafluoro-phosphate (HATU, 1.1 equiv.) and activation of the carboxylic acid for 30 min.
  • HATU l-[Bis(dimethylamino)methylene]-l/7 L l,2,3-triazolo[4,5- ]pyridinium3- oxid hexafluoro-phosphate
  • reaction mixture was cooled to 0 °C and the respective mono-Boc protected diamine linker (0.5 equiv.) and then A(/V-Diisopropylethylamine (DIPEA, 2.0 equiv.) were sequentially charged into the reaction flask.
  • DIPEA A(/V-Diisopropylethylamine
  • the reaction was continued until full conversion was observed via TLC analysis (typically lh).
  • the solution was diluted with Dichloromethane (DCM, 10 mL), washed with water and extracted with DCM (3 x 20 ml). The combined organic layers were washed with brine (2x50 mL), dried over Na?SO4, filtered, and concentrated in vacuo.
  • the Boc-protected linker-Hyt conjugate (1.1 equiv.) was dissolved in DCM (2 mL) and treated with TFA (0.75 mL). The reaction mixture was stirred at rt for 30 min to lh until complete deprotection and quenched by addition of sat. NaHCOs (15 mL). The aqueous solution was extracted with DCM (6 x 20 mL) until the organic phase is no longer fluorescent. The combined organic layers were dried using Na?SO4 and put on rotary evaporator. The residue was afterwards put on high vacuum.
  • Anticancer sulfonamides target splicing by inducing RBM39 degradation via recruitment to DCAF15. SCIENCE, 16 Mar 2017, Vol 356, Issue 6336

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Abstract

La présente invention concerne la découverte du rôle de DCAF15 dans l'agrégation de protéines. La présente invention concerne en outre des molécules PROTAC et HyT-PD pour la dégradation ciblée de protéines (TPD) de DCAF15 ainsi que leur utilisation en tant que médicament pour le traitement de l'amylose, en particulier leur utilisation dans le traitement de synucléinopathies.
PCT/EP2024/065601 2023-06-07 2024-06-06 Molécules protac et hyt-pd pour la dégradation ciblée de protéines de dcaf15 et leur utilisation dans le traitement de l'amylose Ceased WO2024251876A1 (fr)

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