WO2021140343A1

WO2021140343A1 - Ligand drug conjugates and modified bet inhibitors

Info

Publication number: WO2021140343A1
Application number: PCT/GB2021/050058
Authority: WO
Inventors: Rui Miguel Petrucci Albuquerque TRAQUETE; Gonçalo José Lopes Bernardes
Original assignee: Hovione Scientia Limited; Turner, Craig
Priority date: 2020-01-09
Filing date: 2021-01-11
Publication date: 2021-07-15
Also published as: PT116050B; PT116050A

Abstract

A ligand drug conjugate comprising a) a targeting ligand; b) a cleavable bridge; and a bromodomain and extraterminal (BET) inhibitor is provided. The targeting ligand may comprise a cancer targeting ligand. The BET inhibitor may comprise I-BET762, (+)-JQ1, MS417, OXT015, (2), RVX-208, (3), OXFBD02, OXFBD03, I-BET151, (4), PFI-1, I-BET726, MS436, XD14 or a modified BET inhibitor. Modified BET inhibitors are also provided, including RT48 ((S) -2- (6-(4-chlorophenyl)-8-methoxy-1-methyl-4H-benzo[f] [1,2,4] triazolo [4,3-a] [1,4] diazepin-4-yl)-1-(4-(dimethylamino) piperidin-1-yl) ethan-1-one) and RT53 ((S) -2- (6- (4-chlorophenyl) -8-methoxy-1-methyl-4H-benzo[f] [1,2,4] triazolo [4,3-a] [1,4] diazepin-4-yl) -N-(4- (4- (dimethylamino) piperidin-1-yl) phenyl) acetamide). A pro-drug of a BET inhibitor is also provided. A method of preparing a ligand drug conjugate according to the invention is also provided. The method comprises i) functionalising a BET inhibitor with a tertiary amine to form a modified BET inhibitor according to the invention; ii) bonding the modified BET inhibitor formed in step i) to a cleavable bridge; and iii) bonding the cleavable bridge bound to the modified BET inhibitor formed in step ii) to a targeting ligand. A ligand drug conjugate according to the invention for use as a medicament is also provided.

Description

Ligand drug conjugates and modified BET inhibitors

The present invention relates to ligand drug conjugates and modified BET inhibitors for use in treating cancer.

Epigenome-targeting drugs are an emerging class of therapeutic agents against a variety of human cancers. This results from the increasingly clear understanding of histone post- translational modifications and their role in tumorigenesis and tumor progression. Proteins of the bromodomain and extraterminal (BET) family, in particular, are epigenetic regulators of great interest as biological targets. These family of proteins is comprised of BRD2, BRD3, BRD4 and BRDT, which bind to chromatin through recognition of acetylated lysine residues (KAc) on histone tails, leading to the recruitment and co-activation of master regulatory transcription factors including the oncogene MYC. BET inhibitors are compounds that reversibly bind the bromodomains of BET proteins and prevent protein-protein interaction between BET proteins and acetylated histones and transcription factors. Preclinical modulation of BET protein function in malignant models through small molecule inhibition has therefore resulted in marked phenotypic changes and promising therapeutic benefits, driving multiple effectors into clinical evaluation. However, early clinical trials have had modest results with only a few, short-lived responses in both hematologic and solid tumors. Relevant toxicities were observed in many patients, including severe thrombocytopenia, fatigue, gastrointestinal (Gl)-side effects, nausea and vomiting, which significantly limited compliance to treatment.

Targeted drug delivery has been applied to many chemotherapeutic agents in clinical use and consists of an established chemotherapeutic agent being reversibly bound to a targeting ligand that can deliver the chemotherapeutic agent selectively to a cell to be treated. This strategy shows great promise to maximise the safety and efficacy of a given chemotherapeutic agent, as their selective delivery into target cells avoids the nonspecific uptake and associated toxicities to healthy cells (Srinivasarao, M. & Low, P. S. Ligand-Targeted Drug Delivery Chemical reviews 117 , 12133-12164, 2017) that can result in higher maximum tolerated doses (MTD). Most anticancer drugs need to be used near their MTD to achieve a clinically meaningful therapeutic effect, which significantly impairs the therapeutic window of these compounds (Chari Ravi, V. J., Miller Michael, L. & Widdison Wayne, C. Antibody-Drug Conjugates: An Emerging Concept in Cancer Therapy Angewandte Chemie International Edition 53, 3796-3827, 2014). Targeted drugs should be able to achieve the same clinical effect at lower doses than their non-targeted counterparts since receptor binding and internalisation within the target cells enables a higher concentration of the drugs within receptor-positive pathologic cells (Srinivasarao, M., Galliford, C. V. & Low, P. S. Principles in the design of ligand-targeted cancer therapeutics and imaging agents. Nature reviews. Drug discovery 14, 203-219, 2015).

The present invention provides ligand drug conjugates that enable the targeted delivery of BET inhibitors to cancer cells such that their overall efficacy is increased by the targeted release of the BET inhibitor within cancer cells whilst sparing healthy cells and the associated collateral toxicity. The present invention also provides modified BET inhibitors and pro-drugs of BET inhibitors, which pro-drugs of BET inhibitors help accomplish the selective delivery of BET inhibitors.

The present invention provides a ligand drug conjugate comprising: a) a targeting ligand; b) a cleavable bridge; and c) a BET inhibitor.

The present invention also provides a modified BET inhibitor, a pro drug of a BET inhibitor, a method of preparing a ligand drug conjugate, the ligand drug conjugate for use as a medicament, and a method of medical treatment using the ligand drug conjugate.

A major determinant for the selective delivery of drugs lies in the differential expression of a targeted receptor in diseased versus normal tissues. Examples of often exploited tumour- enriched antigens that are expressed in vast excess include folate receptor, prostate-specific membrane antigen (PSMA) and glucose transporter 1 (Srinivasarao, M., Galliford, C. V. & Low, P. S. Principles in the design of ligand-targeted cancer therapeutics and imaging agents. Nature reviews. Drug discovery 14, 203-219, 2015). The cell surface enzyme PSMA is up to 1000-fold overexpressed in prostate cancer cells and minimally detected in normal cells 257,258 (Srinivasarao, M. & Low, P. S. Ligand-Targeted Drug Delivery Chemical reviews 117, 12133-12164, 2017 and Wustemann, T., Haberkorn, U., Babich, J. & Mier, W. Targeting prostate cancer: Prostatespecific membrane antigen based diagnosis and therapy Medicinal research reviews, 39(1), 40-69, 2018).

Once the criteria for the identification of a disease-specific receptor are established, a targeting-ligand that is also suitable to be a drug delivery vehicle must be found. All targeting ligands currently in clinical development are characterised by a high affinity and high specificity towards their receptor. Ligands having a target affinity in the low nanomolar range are usually preferred (KD < 10 nM, Srinivasarao, M. & Low, P. S. Ligand-Targeted Drug Delivery Chemical reviews 117, 12133-12164, 2017). Targeting ligands should enable the direct or indirect attachment of a cleavable bridge whilst preserving their ability to target a disease-specific receptor. The most used functionalities to tether ligands to spacers or cleavable bridges include carboxylic acid, amine, alcohol, thiol and aldehyde functionalities (Srinivasarao, M. & Low, P. S. Ligand-Targeted Drug Delivery Chemical reviews 117, 12133-12164, 2017).

The targeting ligand in the ligand drug conjugate of the present invention may comprise a cancer targeting ligand. The cancer targeting ligand may comprise a prostate cancer targeting ligand, a brain cancer targeting ligand, a breast cancer targeting ligand, a colon cancer targeting ligand, a pancreatic cancer targeting ligand, a liver cancer targeting ligand, a lung cancer targeting ligand, an ovarian cancer targeting ligand, a blood cancer targeting ligand such as a multiple myeloma targeting ligand, a Burkitt’s lymphoma targeting ligand, an acute myeloblastic leukemia (AML) targeting ligand, a mixed-lineage leukemia (MLL) targeting ligand or an acute lymphoblastic leukemia (ALL) targeting ligand, or a bone marrow cancer targeting ligand.

Suitable prostate cancer targeting ligands include PSMA targeting ligands, transferrin- receptor-expressing prostate cancer targeting ligands, folate-receptor-expressing prostate cancer targeting ligands, oncogenic glucose-regulated proteins (GRPs) in prostate cancer (PC-3) targeting ligands, or LHRH-receptor targeting ligands. Therefore, the ligand drug conjugate of the present invention may comprise a PSMA targeting ligand, a transferrin- receptor-expressing prostate cancer targeting ligand, a folate-receptor-expressing prostate cancer targeting ligand, an oncogenic glucose-regulated proteins (GRPs) in prostate cancer (PC-3) targeting ligand, or an LHRH-receptor targeting ligand.

Suitable brain cancer targeting ligands include epidermal growth factor receptor (EGFR) glioblastoma targeting ligands. Therefore, the ligand drug conjugate of the present invention may comprise an EGFR glioblastoma targeting ligand.

Suitable breast cancer targeting ligands include LHRH-receptor targeting ligands, N- acetylgalactosamine and b-D galactose targeting ligands, HER-2-expressing breast cancer targeting ligands, and MCF-7 breast cancer targeting ligands. Therefore, the ligand drug conjugate of the present invention may comprise an LHRH-receptor targeting ligand, a N- acetyl-galactosamine and b-D galactose targeting ligand, an HER-2-expressing breast cancer targeting ligand, or an MCF-7 breast cancer targeting ligand. Suitable colon cancer targeting ligands include EGFR colorectal cancer targeting ligands and mucin 1 -glycoprotein-expressing colon cancer (C26) targeting ligands. Therefore, the ligand drug conjugate of the present invention may comprise an EGFR colorectal cancer targeting ligand or a mucinl -glycoprotein-expressing colon cancer (C26) targeting ligand.

Suitable lung cancer targeting ligands include mucin 1 -transmembrane protein targeting ligands and H69AR (human small cell lung carcinoma) targeting ligands. Therefore, the ligand drug conjugate of the present invention may comprise a mucin 1 -transmembrane protein targeting ligand or an H69AR targeting ligand.

Suitable ovarian cancer targeting ligands include CD44+ targeting ligands. Therefore, the ligand drug conjugate of the present invention may comprise a CD44+ targeting ligand.

Suitable blood cancer targeting ligands include folate-expressing leukemia targeting ligands. Therefore, the ligand drug conjugate of the present invention may comprise a folate expressing leukemia targeting ligand.

Suitable PSMA targeting ligands include small molecules (e.g. 2-[3-(1,3-dicarboxypropyl)- ureidojpentanedioic acid (DU PA), glycosylphosphatidylinositol (GPI), peptides (e.g. Lys- NHCONH-Glu) and aptamers (e.g. WQPDTAHHWATL)) and monoclonal antibodies (e.g. CYT-356 and J591). Therefore, the ligand drug conjugate of the present invention may comprise DUPA, GPI, Lys-NHCONH-Glu, WQPDTAHHWATL, CYT-356, or J591.

Suitable ligands for all folate-expressing cancers include folic acid.

Suitable ligands for transferrin-receptor-targeting ligands include transferrin, HAIYPRH (T7) peptide, and monoclonal antibodies (e.g. 8D3 and RI7-217).

Suitable ligands for folate-receptor-targeting ligands include folic acid.

Suitable oncogenic GRPs in PC-3 targeting ligands include siRNAs.

Suitable LHRH-receptor targeting ligands include LHRH and (D-Lys⁶)- LHRH.

Suitable EGFR cancer targeting ligands include EGFR and EGFRvlll antibodies. Suitable N-acetyl-galactosamine and b-D galactose targeting ligands include soybean agglutinin.

Suitable HER-2-expressing breast cancer targeting ligands include HER-2 antibodies. Suitable MCF-7 breast cancer targeting ligands include folic acid.

Suitable C26 targeting ligands include aptamers (e.g. a 5TR1 aptamer).

Suitable mucin 1 -transmembrane protein targeting ligands include aptamers.

Suitable H69AR targeting ligands include peptides (e.g. JB434).

Suitable CD44+ targeting ligands include hyaluronic acid.

Whilst the purpose of a targeting ligand is to ensure that a ligand drug conjugate is delivered to targeted cells, the purpose of a cleavable bridge is to ensure a drug is securely attached to the targeting ligand when in circulation, but readily released when it is delivered to the targeted cells. The release of the drug typically occurs when a specific triggering event in a target cell leads to a series of cascading reactions and ultimately the release of the drug in its active form.

Cleavable bridges may comprise a linker, optionally in combination with a self-immolative moiety. Alternatively, the linker itself may be self-immolative.

Linkers may be released from the targeting ligand by proteolytic cleavage within lysosomes or by reductive cleavage within cytosol. Proteolytic cleavage exploits the differential expression of hydrolytic enzymes inside targeted cells in comparison with healthy cells, which effectively creates a controlled release mechanism. The dipeptide valine-citruline (VC) has been widely used as one such release mechanism since it is well known to cleave on exposure to lysosomal proteases and particularly to cathepsin B, a cysteine protease highly upregulated in a wide variety of cancers (Mohamed, M. M. & Sloane, B. F. Cysteine cathepsins: multifunctional enzymes in cancer Nature reviews. Cancer6, 764-775, 2006). Other cathepsin B sensitive dipeptides include phenylalanine-arginine, phenylalanine-lysine and valine- alanine. Alternative types of linkers include disulphide linkers, hydrazone linkers (such as hydrazone acetyl butyrate), and glucosidase sensitive linkers (which may be activated by an enzyme such as a b-galactosidase). The structure p-aminobenzylcarbamate (PABC) has been extensively used in antibody drug conjugates, but presents some disadvantages due to its hydrophobic nature. Quaternary ammonium groups have been explored as a means to enhance solubility and decrease aggregation of small molecule-peptide or protein constructs. The combination of this knowledge led to the development of a self-immolative p-aminobenzyl quaternary (PABQ) ammonium salt.

Thus, the cleavable bridge in the ligand drug conjugate of the present invention may comprise PABC and a VC linker or PABQ and a VC linker.

Additionally, the ligand drug conjugate of the present invention may comprise a spacer. Spacers are used to physically separate the ligand from the cleavable bridge, which reduces stearic hindrance that might make it difficult for an enzyme to reach an active site on a linker hence making it easier for a drug to be released when it is delivered to the targeted cells. Suitable spacers include alkyl chains such as a propyl chain or a pentyl chain, or polyethylene glycol (PEG) chains such as a 5-atom PEG unit or an 8-atom PEG unit.

Several BET inhibitors are in clinical development in phase I or II studies for patients with haematological malignancies and solid tumours. Although the results available so far are preliminary, many of the compounds appear to show potential in treating a range of cancers including I-BET762 (CAS: 1260907-17-2), (+)-JQ1 (CAS: 1268524-70-4), MS417 (CAS: 916489-36-6), OXT015, (2), RVX-208, (3), OXFBD02, OXFBD03, I-BET151 (CAS: 1300031- 49-5), (4), PFI-1 (CAS: 1403764-72-6), I-BET726 (CAS: 2010159-45-0), MS436 (CAS: 1395084-25-9) or XD14.

The BET inhibitor in the ligand drug conjugate of the present invention may comprise one of the listed BET inhibitors or a modified version of one of the listed BET inhibitors, optionally a modified version of I-BET762. I-BET762 shows a high affinity to target proteins, high solubility in physiological media, low plasma protein binding, good passive permeability, excellent metabolic stability and lack of immunogenicity (Nicodeme, E. et al. Suppression of inflammation by a synthetic histone mimic Nature 468, 1119-1123, 2010 and Zhao, Y., Yang, C. Y. & Wang, S. The making of I-BET762, a BET bromodomain inhibitor now in clinical development Journal of medicinal chemistry 56, 7498-7500, 2013).

To enable the attachment of cleavable bridges to a drug, the drug may be functionalised to include a functional group such as an hydroxyl, carboxyl, amine, carbonyl or thiol. Thus the modification of a known BET inhibitor may comprise the functionalisation of the BET inhibitor. The modified BET inhibitor of the present invention comprises a BET inhibitor and a terminal tertiary amine. The terminal tertiary amine may comprise a piperidine ring bound to the terminal tertiary amine or a piperazine ring bound to the terminal tertiary amine. The subsequent formation of a quaternary amine at the terminal tertiary amine reduces the lability of the BET inhibitor, thus forming a pro-drug. Hence, the pro-drug of the present invention comprises a BET inhibitor and a quaternary amine. The quaternary amine may comprise a quaternary alkylamine or a quaternary aniline.

The modified BET inhibitor of the present invention may comprise derivatives of I-BET762. Hence, the modified BET inhibitor of the present invention may comprise RT48 ((S) -2- (6- (4-chlorophenyl) -8-methoxy-1-methyl-4H -benzo[f] [1,2,4] triazolo [4,3-a] [1,4] diazepin- 4-yl)-1-(4-(dimethylamino) piperidin-1-yl) ethan-1-one) or RT53 ((S) -2- (6- (4-chlorophenyl) - 8-methoxy-1-methyl-4H -benzo[f] [1,2,4] triazolo [4,3-a] [1 ,4] diazepin-4-yl) -N- (4- (4- (dimethylamino) piperidin-1-yl) phenyl) acetamide):

The pro-drug of the present invention may comprise the corresponding quaternary ammonium derivatives of RT48 ((S) -2- (6- (4-chlorophenyl) -8-methoxy-1-methyl-4H -benzo[f] [1,2,4] triazolo [4,3-a] [1 ,4] diazepin-4-yl)-1-(4-(dimethylamino) piperidin-1-yl) ethan-1-one) or RT53 ((S) -2- (6- (4-chlorophenyl) -8-methoxy-1-methyl-4H -benzo[f] [1,2,4] triazolo [4,3-a] [1,4] diazepin-4-yl) -N- (4- (4- (dimethylamino) piperidin-1-yl) phenyl) acetamide).

The present invention also provides a method of preparing a ligand drug conjugate according to the present invention, wherein the method comprises: i) functionalising a BET inhibitor with a tertiary amine to form a modified BET inhibitor according to the present invention; ii) bonding the modified BET inhibitor formed in step i) to a cleavable bridge; and iii) bonding the cleavable bridge bound to the modified BET inhibitor formed in step ii) to a targeting ligand. Step i) may comprise a condensation reaction between a carboxylic acid precursor of a BET inhibitor and a compound comprising both a primary or secondary amine and a terminal tertiary amine. The primary or secondary amine may react with the carboxylic acid group to form an amide link such that the modified BET inhibitor formed comprises a terminal tertiary amine.

Step ii) may comprise the alkylation of the modified BET inhibitor formed in step i) to form a compound comprising a quaternary amine.

Step iii) may comprise a condensation reaction between the cleavable bridge bound to the modified BET inhibitor formed in step ii) and the targeting ligand, wherein the cleavable bridge bound to the modified BET inhibitor comprises a primary amine and the targeting ligand comprises a carboxylic acid group.

The ligand drug conjugate of the present invention may be used as a medicament. Optionally, the ligand drug conjugate may be used to treat cancers including prostate cancer, brain cancer, breast cancer, colon cancer, pancreatic cancer, liver cancer, lung cancer, ovarian cancer, blood cancer such as multiple myeloma, Burkitt’s lymphoma, AML, MLL or ALL, or bone marrow cancer.

The ligand drug conjugate of the present invention may be used in a method of medical treatment. Optionally, the ligand drug conjugate may be used in a method of treating cancers including prostate cancer, brain cancer, breast cancer, colon cancer, pancreatic cancer, liver cancer, lung cancer, ovarian cancer, blood cancer such as multiple myeloma, Burkitt’s lymphoma, AML, MLL or ALL, or bone marrow cancer.

Examples

Example 1: Conjugation site validation with I-BET762 - fluorescein derivatives

I-BET762 was chemically functionalised with bulky substituents to confirm that the conjugation site could be derivatised while retaining putative ligand-receptor interactions. Fluorescein was used as a model cargo and tethered to I-BET762 through diamine spacers with different lengths. The diamine spacers were prepared via single protection with a t-butyloxycarbonyl (BOC) protecting group as shown below:

The spacers used included a simple propyl chain (13), a 5-atom PEG unit (14) and an 8-atom PEG unit (15). These moieties were considered for their distinct properties in modulating the activity of the construct. In general, longer linkers are preferred for bulky substituents while PEGs in different sizes can contribute to the overall hydrophilicity of the conjugate (Srinivasarao, M., Galliford, C. V. & Low, P. S. Principles in the design of ligand-targeted cancer therapeutics and imaging agents. Nature reviews. Drug discovery 14, 203-219, 2015).

Three different fluorescent-labelled l-BET762-based ligands were then prepared by protecting one of the primary amines of the diamine spacers with a BOC group and coupling the free amine with the carboxylic acid in I-BET762 precursor 12 to produce the intermediate compounds 16, 17 and 18 as shown below. These intermediate compounds were then deprotected and the free amine conjugated with a fluorescein motif bearing a N- hydroxysuccinimide ester (NHS-ester) to produce fluorescein conjugates 19, 20 and 21 as also shown below.

To act as a negative control in biological studies, a probe without the I-BET762 structure (22) was also synthesised as shown below:

Once the fluorescent probes were synthesised, purified and characterised, they were evaluated for their affinity towards BET-bromodomain proteins. These included establishing compound affinity towards BRD2(1), BRD3(1), BRD4(1), and BRD4(2) BET modules using AlphaScreen technology as the detection method. (AlphaScreen specific binding assays were conducted at Cerep, Celle I’Evescault, France.) The retrieved data suggested that overall the compounds were able to bind efficiently to the target proteins despite the presence of a bulky substituent as fluorescein. A higher consistency was found in the binding data for BRD4(1) and BRD4(2) proteins, although against both of these proteins there was a clear decrease in the binding potency. This was evident for all compounds in BRD4(2) with a 6- to 10-fold decrease in comparison with I-BET762 (IC50 = 0.3 mM), and for the PEGylated probes 21 (IC50 = 2.77 μM, 7-fold decrease) and 20 (IC50 = 5.22 μM, 12-fold decrease) in BRD4(1) (Figure 1). Surprisingly, compound 19 comprising a simple propyl chain as spacer was found binding with high affinity to BRD4(1) (IC50 = 0.85 μM). Yet when analysing the affinity of compounds across the different proteins no correlation between spacer chemistry and binding potency was observed. While the latter compound was 16 times more potent (IC50 = 0.24 μM) than I- BET762 (IC50 = 3.9 μM) against BRD2(1), the same compound was unable to induce competitive peptide displacement in BRD3(1) at concentrations as high as 100 μM (Figure 1). If not assay-dependent, these data might suggest different binding requirements to the active site of each domain.

Fluorescein derivatives were prepared since fluorescence-based techniques are important research tools for probing intermolecular interactions. However, the fluorescent-labelled probes were only effective when the cellular membrane was disrupted with Triton X-100 detergent prior to incubation with the fluorescein derivatives (not in live cells). Parallel Membrane Permeability Assay (PAM PA) confirmed that the diffusion of the fluorescein derivatives across cellular membranes is strongly impaired whereas the parent molecule I-BET762 readily diffuses across cellular membranes.

Confocal microscopy analysis on the internalization rate of 21 (e-h) and 22 (a-d) with and without membrane disruption with Triton X-100 detergent prior to incubation showed that all cells treated with compound 21 (Figure 2h) showed a pronounced fluorescent signal, whereas in samples treated with 22 no staining was observed (Figure 2d). In addition, the fluorescent signal of 21 was much stronger in the nuclei where the BET-bromodomain proteins are localized suggesting an efficient targeting in cells. Importantly, cells treated with the non- targeted fluorescent probe 22 were not stained even when the membrane had been disrupted, which emphasises the role of the BET-based effector. The same trend was observed when incubating cells with the fluorescent conjugate 19 comprising only a 3-atom spacer.

Example 2: Modification of I-BET762

As shown in Example 1, it is possible to derivatise the amide group in I-BET762 and retain the desired functionality. Therefore, this position was used to introduce terminal tertiary amines, which in turn enable the formation of pro-drugs and the accommodation of a cleavable bridge.

The free carboxylic acid on I-BET762 precursor 12 allows various synthetic modifications, including the addition of a piperidine ring bearing a terminal tertiary amine to form with (compound 27, hereafter RT48) or without (compound 26, hereafter RT53) a phenyl ring between the piperidine ring and the amide bond formed with the I-BET762 scaffold as shown below:

An analogue bearing a terminal quaternary amine (compound 28, hereafter RT56) instead of the terminal tertiary amine was also synthesised and used as a control with the terminal positively charged group being used to impair membrane permeability and thus most cellular effects as also shown:

A series of biophysical assays were then performed, including differential scanning fluorimetry (DSF), AlphaScreen, and Isothermal Titration Calorimetry (ITC).

DSF: Thermal melting experiments were carried out using an Mx3005p Real Time PCR machine (Stratagene). Proteins were buffered in 10 mM HEPES pH 7.5, 500 mM NaCI and assayed in a 96 well plate at a final concentration of 2 mM in 20 μL volume. Compounds were added at a final concentration of 10 μM. SYPRO Orange (Molecular Probes) was added as a fluorescence probe at a dilution of 1 in 1000. Excitation and emission filters for the SYPRO- Orange dye were set to 465 nm and 590 nm, respectively. The temperature was raised with a step of 3 °C per minute from 25 °C to 96 °C and fluorescence readings were taken at each interval. The temperature dependence of the fluorescence during the protein denaturation process was approximated by the equation:

where AuG is the difference in unfolding free energy between the folded and unfolded state, R is the gas constant and yF and yU are the fluorescence intensity of the probe in the presence of completely folded and unfolded protein respectively. The baselines of the denatured and native state were approximated by a linear fit. The observed temperature shifts, ΔT_m ^obs, were recorded as the difference between the transition midpoints of sample and reference wells containing protein without ligand in the same plate and determined by non-linear least squares fit.

DSF was used to assess binding efficacy and selectivity across different bromodomain families. Figure 3 shows average temperature shifts ( ΔT_m ^obs) in degrees Celsius upon binding of compounds at a final concentration of 10 mM. With the exception of BRDT(1), all compounds led to enhanced melting temperatures, in some cases even when compared to the parent I- BET762 molecule, which suggests a specific and strong binding interaction to a panel of BET proteins. Outside the BET family (BAZ2BA, CREBP, PB1(5) and PCAF) no melting temperature shifts were identified, confirming that these ligands are tailored for the BET family of bromodomain proteins. Target binding affinity was further studied using AlphaScreen technology. As performed in Example 1 , AlphaScreen was run against the BRD2(1), BRD3(1), BRD4(1) and BRD4(2) (see Figure 4). The data obtained suggested that the RT compounds possess binding affinities to BRD domains as good as or even better than I-BET762. In particular, RT53 showed an increase of 4-fold against BRD2(1) (ICso = 19.3 nM) and BRD4(1) (ICso = 18.2 nM) in comparison with the parent molecule I-BET762 (ICso BRD2(1) = 84.9 nM; ICso BRD4(1) = 68.1 nM) (Figure 4A,C). Also, against BRD3(1) an ICso increase of 3-fold was observed for RT53 (IC50 = 67.1 nM) in comparison with I-BET762 (ICso = 214.8 nM, Figure 4B), whereas no significant changes were observed against BRD4(2) (RT53 ICso = 25.9 nM ; I-BET762 ICso = 21.3 nM, Figure 4D). Furthermore, the presence of a positive charge in RT56 did not impair binding. In fact, RT56 also showed a potent binding against BRD4(1) (ICso = 6.6 nM, Figure 4C) and BRD4(2) (ICso = 2.4 nM, Figure 4D).

ITC: Experiments were carried out on an ITC200 titration microcalorimeter from MicroCal™, LLC (GE Healthcare) equipped with a washing module, with a cell volume of 0.2003 ml_ and a 40 μL microsyringe. Experiments were carried out at 15 °C while stirring at 1000 rpm, in ITC buffer (20 mM HEPES pH 7.5 (at 25 °C), 200 mM NaCI). The microsyringe was loaded with a solution of protein sample (340 - 400 mM, in ITC buffer) and was carefully inserted into the calorimetric cell which was filled with an amount of the protein (0.2 ml_, 27-33 mM in ITC buffer). Following baseline equilibration an additional delay of 60 seconds was applied. All titrations were conducted using an initial control injection of 0.3 μL followed by 38 identical injections of 1 mI_ with a duration of 2 seconds (per injection) and a spacing of 120 seconds between injections. The titration experiments were designed in such a fashion, as to ensure complete saturation of the proteins before the final injection. The heat of dilution for the proteins were independent of their concentration and corresponded to the heat observed from the last injection, following saturation of compound binding, thus facilitating the estimation of the baseline of each titration from the last injection. The collected data were corrected for peptide heats of dilution (measured on separate experiments by titrating the proteins into ITC buffer) and deconvoluted using the MicroCal™ Origin software supplied with the instrument to yield enthalpies of binding (AH) and binding constants (KB) in the same fashion to that previously described in detail by Wiseman and co-workers (Wiseman, T., Williston, S., Brandts, J. F. & Lin, L. N. Rapid Measurement of Binding Constants and Heats of Binding Using a New Titration Calorimeter Analytical Biochemistry 179, 131-137, 1989). Thermodynamic parameters were calculated using the basic equation of thermodynamics (AG = AH - T AS = - RTInK_B, where AG, AH and AS are the changes in free energy, enthalpy and entropy of binding respectively). In all cases a single binding site model was employed as supplied with the MicroCal™ Origin software package. Dissociation constants and thermodynamic parameters are listed in Table 1 below.

The binding affinity of compounds was further characterized in solution through ITC. Conversely to AlphaScreen, ITC allows measuring the affinity of binding partners in their native state. The KD values and thermodynamic parameters obtained confirmed that derivatization of BET inhibitors to contain terminal tertiary amine (RT53) or quaternary ammonium (RT56) motifs does not impair binding and can, in fact, lead to more potent BET inhibition. In addition, it was observed that RT53 and RT56 bind to isolated BRD4(1) and BRD4(2) in a similar range of KD values (14 and 21 nM for BRD4(1) and 21 and 18 nM for BRD4(2), respectively, Figure 5, Table 4).

Table 1 : Thermodynamic characterisation and KD values from ITC

Interestingly the interaction of both inhibitors with the second domain of BRD2 and BRD4 is characterized by favourable positive entropy change ( TAS ~ +2 to +3 kcal/mol), while in BRD4(1) a negative TAS is observed (~ -0.4 to ~ -1 kcal/mol), suggesting that the essential mechanisms of molecular recognition and engagement may be distinctive (Table 1). Together this data shows that the synthetic design to convert I-BET762 into a tertiary amine-containing BET inhibitor was successful in that RT53 shows a potent inhibition of BET proteins even when compared to the parent molecule.

Example 3: Biological Evaluation of the I-BET762 derivatives formed in Example 2

The three compounds prepared in Example 2 were profiled against several cancer cell lines to find the most responsive models. A semi-automated small-molecule sensitivity profiling of 26 cancer cell lines was established and their response evaluated after 96 h incubation with compounds in comparison with the parent molecule I-BET762. Cellular response was determined in cell lines deriving from a wide range of tissues including prostate, lung, brain, pancreas, breast, ovary, colon, as well as haematological and bone marrow tumours (see Figure 6).

In the solid tumours tested, potent inhibitory effects were observed mostly in prostate cancer cell lines, particularly in those dependent on AR signalling (LNCaP, VCaP and 22RV1 , IC50 = 0.2 - 2.6 mM). In contrast, AR negative cells (DU 145 and PC3) were overall more resistant to treatment. Surprisingly though, PC3 cells were responsive only to RT53 (IC50 = 3.1 μM), whilst neither RT48 nor I-BET762 affected cellular viability up to the maximum dose tested (10 μM). In AR-positive cell lines the response to RT53 and RT48 treatment was comparable to that of the parent molecule, I-BET762, although RT53 was clearly the most effective amongst the three (RT53 IC50 = 0.2 - 0.7 μM; RT48 IC50 = 0.3 - 2.6 μM; I-BET762 IC50 = 0.2 - 1.5 μM). Conversely, RT56 was ineffective across all cell lines (IC50 > 10 μM), likely due to the quaternary ammonium salt motif impairing compound internalization. This was further confirmed by PAMPA that showed that RT56 does not diffuse across cellular membranes. In regard to membrane permeability, it was also notable that the modest diffusion observed for RT53 (logPe = -5.92) in comparison with I-BET762 (logPe = -4.32), despite RT53 showing equivalent and in some cases even more cancer cell killing activity than the parent molecule. Nevertheless, drug sensitive phenotypes were also partially observed in cell lines from breast (MDA-MB-453, IC50 = 1.3 - 5.0 μM), ovary (CAOV3, IC50 = 0.5 -1.9 μM) and brain tissues (U87-MG, IC50 = 1.72 - 7.93 μM), as well as in all blood cancer cells tested, namely those from acute T cell leukemia (Jurkat, IC50 = 0.3- 1.2 μM), acute myeloblastic leukemia (Kasumi- 1, IC50 = 0.1 - 0.3 μM), and Burkitt’s lymphoma (DAUDI, IC50 = 0.4 - 1.3 μM). Moreover, cancer cell lines derived from lung, pancreas and colon tissues were entirely unresponsive to all compounds (IC50 > 10 μM).

Example 4: Preparation of a liqand-druq conjugate comprising an I-BET762 derivative Solid-phase peptide synthesis was used to synthesize a VC linker using a 2-chlorotrityl resin followed by the sequential addition and deprotection of fluorenylmethoxycarbonyl (FMOC)- citruline-OH and FMOC-valine-OH. The VC dipeptide was then functionalised with an aminophenyl group for derivatisation with RT53, affording the linker-drug conjugate 38 (hereafter VC-PABQ-RT53) as shown below:

It was evaluated whether RT53 could be released from VC-PABQ-RT53 and whether this linker would be stable, and what form of BET inhibitor would be released when a specific triggering event occurred. In a buffered medium containing cathepsin B (pH 5.5, 37 °C) complete liberation of RT53 was observed in 18 h (0% of VC-PABQ-RT53). Figure 7 presents a series of traces detected with HPLC and identified with LC-MS (with the star indicating the VC peak) and clearly shows the proportion of VC and RT53 increasing and the proportion of VC-PABQ-RT53 decreasing over time. Figure 8 shows the rate of RT53 release, estimated from peak area (mAU*s) divided by the sum of free drug and linker-drug conjugates, and confirms that the proportion of VC and RT53 increased and the proportion of VC-PABQ-RT53 decreased over time, but only when in the presence of cathepsin B. This demonstrated the effective controlled release of RT53. HPLC: measurements were performed on a Phenomenex Luna C18 10 μM, 250 x 21.2 mm column at a flow rate of 10 mL/min using linear gradients of 80 % A/ 20 % B to 60 % A/ 40% B over 40 min. (A = Milipore water + 0.1% TFA, B = ACN).

LC-MS: combined HPLC measurements performed as outlined above with mass spectrometry analysis recorded on a Bruker Daltonics microTOF ESI-TOF mass spectrometer. Calculated and exact m/z values are indicated in Daltons.

The linker-drug conjugate VC-PABQ-RT53 was then attached to the PSMA targeting ligand DU PA (42):

Deprotection of VC-PABQ-RT53 with trifluoroacetic acid provided the functional amine for hexafluorophosphate benzotriazole tetramethyl uranium (HBTU)-mediated coupling with DU PA 42 to result in compound 43, which upon tertbutyl deprotection with trifluoroacetic acid for 1.5 h at room temperature afforded the final conjugate 44 (hereafter DUPA-VC-PABQ- RT53), as shown below:

Example 5: Verification of the therapeutic activity of the ligand-drug conjugate comprising an

I-BET762 derivative

Mice bearing subcutaneous LNCaP tumours were randomised into groups (n=5 per group) and then received vehicle or drugs at 12 mg/kg for 7 consecutive days. Surprisingly, treatment with DUPA-VC-PABQ-RT53 conjugate led to a significant reduction in tumour volume (93%, p=0.0001, see Figure 9A) and tumour weight (92%, p=0.0001, see Figure 9B) in comparison with vehicle, whereas the single drugs RT53 and I-BET762 had a much less pronounced effect (approximately 40 and 50% reduction of tumour volume and weight, respectively (see Figure 9). The targeted conjugate DUPA-VC-PAQB-RT53 also showed a stronger tumour growth inhibition in comparison with the linker-drug-conjugate VC-PAQB-RT53 (68%), which suggests a successful DUPA-mediated accumulation of drugs in the PSMA-positive LNCaP tumours. Importantly, all drugs were well tolerated in the mice models at the dose tested and up to the 15 days post drug exposure as indicated by the maintenance of stable body weight (Figure 10).

Claims

Claims:

1. A ligand drug conjugate comprising: a) a targeting ligand; b) a cleavable bridge; and c) a bromodomain and extraterminal (BET) inhibitor.

2. A ligand drug conjugate according to claim 1 wherein a) the targeting ligand comprises a cancer targeting ligand.

3. A ligand drug conjugate according to claim 2 wherein the cancer targeting ligand comprises a prostate cancer targeting ligand, a brain cancer targeting ligand, a breast cancer targeting ligand, a colon cancer targeting ligand, a pancreatic cancer targeting ligand, a liver cancer targeting ligand, a lung cancer targeting ligand, an ovarian cancer targeting ligand, a blood cancer targeting ligand such as a multiple myeloma targeting ligand, a Burkitt’s lymphoma targeting ligand, an acute myeiobiastic leukemia (AML) targeting ligand, a mixed- lineage leukemia (MLL) targeting ligand or an acute lymphoblastic leukemia (ALL) targeting ligand, or a bone marrow cancer targeting ligand.

4. A ligand drug conjugate according to claim 3, wherein the cancer targeting ligand comprises a prostate cancer targeting ligand, optionally a prostate-specific membrane antigen (PSMA) targeting ligand, a transferrin-receptor-expressing prostate cancer targeting ligand, a folate-receptor-expressing prostate cancer targeting ligand, an oncogenic glucose-regulated proteins (GRPs) in prostate cancer (PC-3) targeting ligand, or an LHRH-receptor targeting ligand.

5. A ligand drug conjugate according to claim 4, wherein the PSMA targeting ligand comprises small molecules (including peptides and aptamers) or monoclonal antibodies, optionally 2-[3-(1,3-dicarboxypropyl)-ureido]pentanedioic acid (DUPA), glycosylphosphatidyl- inositol (GPI), Lys-NHCONH-Glu, WQPDTAHHWATL, CYT-356 or J591.

6. A ligand drug conjugate according to claim 4, wherein the transferrin-receptor- targeting ligand comprises transferrin, HAIYPRH (T7) peptide or monoclonal antibodies, optionally 8D3 and RI7-217.

7. A ligand drug conjugate according to claim 4, wherein the folate-receptor- expressing prostate cancer targeting ligand comprises folic acid.

8. A ligand drug conjugate according to claim 4, wherein the oncogenic GRPs in PC-3 targeting ligand comprises an siRNA.

9. A ligand drug conjugate according to claim 3, wherein the cancer targeting ligand comprises a brain cancer targeting ligand, optionally an epidermal growth factor receptor (EGFR) glioblastoma targeting ligand.

10. A ligand drug conjugate according to claim 3, wherein the cancer targeting ligand comprises a breast cancer targeting ligand, optionally an LHRH-receptor targeting ligand, a N-acetyl-galactosamine and b-D galactose targeting ligand, an HER-2-expressing breast cancer targeting ligand or an MCF-7 breast cancer targeting ligand.

11. A ligand drug conjugate according to claim 4 or claim 10, wherein the LHRH-receptor targeting ligand comprises LHRH or (D-Lys⁶)- LHRH.

12. A ligand drug conjugate according to claim 10, wherein the N-acetyl-galactosamine and b-D galactose targeting ligand comprises soybean agglutinin.

13. A ligand drug conjugate according to claim 10, wherein the HER-2-expressing breast cancer targeting ligand comprises an HER-2 antibody.

14. A ligand drug conjugate according to claim 10, wherein the MCF-7 breast cancer targeting ligand comprises folic acid.

15. A ligand drug conjugate according to claim 3, wherein the cancer targeting ligand comprises a colon cancer targeting ligand, optionally an EGFR colorectal cancer targeting ligand or a mucinl -glycoprotein-expressing colon cancer (C26) targeting ligand.

16. A ligand drug conjugate according to claim 9 or claim 15, wherein the EGFR targeting ligand comprises EGFR or an EGFRvlll antibody.

17. A ligand drug conjugate according to claim 15, wherein the C26 targeting ligand comprises aptamers, optionally a 5TR1 aptamer.

18. A ligand drug conjugate according to claim 3, wherein the cancer targeting ligand comprises a lung cancer targeting ligand, optionally a mucin 1 -transmembrane protein targeting ligands or an H69AR (human small cell lung carcinoma) targeting ligand.

19. A ligand drug conjugate according to claim 18, wherein the mucin 1 -transmembrane protein targeting ligand comprises aptamers.

20. A ligand drug conjugate according to claim 18, wherein the H69AR targeting ligand comprises peptides, optionally e.g. JB434.

21. A ligand drug conjugate according to claim 3, wherein the cancer targeting ligand comprises an ovarian cancer targeting ligand, optionally a CD44+ targeting ligand.

22. A ligand drug conjugate according to claim 21 , wherein the CD44+ targeting ligand comprises hyaluronic acid.

23. A ligand drug conjugate according to claim 3, wherein the cancer targeting ligand comprises a blood cancer targeting ligand, optionally a folate-expressing leukemia targeting ligand.

24. A ligand drug conjugate according to claim 21 , wherein the folate-expressing leukemia targeting ligand comprises folic acid.

25. A ligand drug conjugate according to any preceding claim wherein b) the cleavable bridge comprises a linker, optionally a cathepsin B sensitive linker, a disulphide linker or a glucosidase sensitive linker.

26. A ligand drug conjugate according to claim 25 wherein the linker comprises a dipeptide, optionally valine-citruline (VC), phenylalanine-arginine, phenylalanine-lysine or valine-alanine, or b-galactoside.

27. A ligand drug conjugate according to any preceding claim wherein b) the cleavable bridge comprises an aminobenzyl quaternary ammonium salt (PABQ) and a dipeptide VC linker.

28. A ligand drug conjugate according to any preceding claim wherein the ligand drug conjugate further comprises a spacer.

29. A ligand drug conjugate according to claim 28 wherein the spacer comprises an alkyl chain such as a propyl chain or a pentyl chain, or a polyethylene glycol chain such as a 5- atom PEG unit or an 8-atom PEG unit.

30. A ligand drug conjugate according to any preceding claim wherein the BET inhibitor comprises I-BET762, (+)-JQ1, MS417, OXT015, (2), RVX-208, (3), OXFBD02, OXFBD03, I-BET151 , (4), PFI-1 , I-BET726, MS436, XD14, or a modified version thereof:

31. A ligand drug conjugate according to claim 28, wherein the BET inhibitor comprises I-BET762 or a modified version thereof.

32. A modified BET inhibitor, wherein the modified BET inhibitor comprises a terminal tertiary amine.

33. A modified BET inhibitor according to claim 32, wherein the terminal tertiary amine comprises a piperidine ring bound to the terminal tertiary amine or a piperazine ring bound to the terminal tertiary amine.

34. A modified BET inhibitor according to claim 32 or 33, wherein the modified BET inhibitor comprises RT48 ((S) -2- (6- (4-chlorophenyl) -8-methoxy-1-methyl-4H -benzo[f] RT53 ((S) -2- (6- (4-chlorophenyl) -8-methoxy-1-methyl-4H -benzo[f] [1,2,4] triazolo [4,3-a] [1,4] diazepin-4-yl) -N- (4- (4- (dimethylamino) piperidin-1-yl) phenyl) acetamide):

35. A pro-drug of a BET inhibitor, wherein the pro-drug comprises a BET inhibitor and a quaternary amine.

36. A pro-drug of a BET inhibitor according to claim 35, wherein the quaternary amine comprises a quaternary alkylamine or a quaternary aniline.

37. A method of preparing a ligand drug conjugate according to any of claims 1 to 31 , wherein the method comprises: i) functionalising a BET inhibitor with a tertiary amine to form a modified BET inhibitor according to any of claims 32 to 34; ii) bonding the modified BET inhibitor formed in step i) to a cleavable bridge; and iii) bonding the cleavable bridge bound to the modified BET inhibitor formed in step ii) to a targeting ligand.

38. A method of preparing a ligand drug conjugate according to claim 34 wherein step i) comprises a condensation reaction between a carboxylic acid precursor of a BET inhibitor and a compound comprising a primary or secondary amine and a terminal tertiary amine.

39. A method of preparing a ligand drug conjugate according to claim 37 wherein step ii) comprises the alkylation of the modified BET inhibitor formed in step i) to form a compound comprising a quaternary amine.

40. A method of preparing a ligand drug conjugate according to claim 34 wherein step iii) comprises a condensation reaction between the cleavable bridge bound to the modified BET- inhibitor formed in step iii) and the targeting ligand, wherein the cleavable bridge bound to the modified BET-inhibitor comprises a primary amine and the targeting ligand comprises a carboxylic acid group.

41. A ligand drug conjugate according to any of claims 1 to 31 for use as a medicament.

42. A ligand drug conjugate according to claim 41 for use in treating cancer.

43. A ligand drug conjugate according to claim 41 or 42 for use in treating prostate cancer, brain cancer, breast cancer, colon cancer, pancreatic cancer, liver cancer, lung cancer, ovarian cancer, blood cancer such as multiple myeloma, Burkitt’s lymphoma, AML, MLL or ALL, or bone marrow cancer.

44. A ligand drug conjugate according to claim 41, 42 or 43, for use in treating prostate cancer.