Novel compounds and drug conjugates

Abstract

The invention relates to novel PARP1 and PARP2 inhibiting compounds and drug conjugates comprising said compounds and the use of said compounds and drug conjugates in the treatment and prophylaxis of hyperproliferative disorders such as cancer, and to compositions containing said compounds and drug conjugates and processes for their preparation.

Description

[0001] ART-C-P3900PCT

[0002] NOVEL COMPOUNDSAND DRUG CONJUGATES

[0003] FIELD OF THE INVENTION

[0004] The invention relates to novel PARP1 and PARP2 inhibiting compounds and drug conjugates comprising said compounds and the use of said compounds and drug conjugates in the treatment and prophylaxis of hyperproliferative disorders such as cancer, and to compositions containing said compounds and drug conjugates and processes for their preparation.

[0005] BACKGROUND OF THE INVENTION ADP-ribosylation is an NAD+-dependent post-translational protein modification that has a well-characterised role in the DNA damage response. Although there are 17 members of the human poly (ADP-ribose) polymerase (PARP) protein family, PARP1 is responsible for 80-90% of nuclear poly ADP-ribosylation (PARylation), with the remainder attributed mostly to PARP2 (Luscher (2022) FEBS J, 289, 7399). PARP1 and PARP2 act as DNA damage sensors: upon induction of single-strand DNA breaks (SSBs) and double strand DNA breaks (DSBs), they are recruited to chromatin where they catalyse the ADP ribosylation of several proteins. In particular, PARP1 attaches long, branched, negatively charged poly(ADP-ribose) (PAR) chains to itself and to chromatin proteins in the vicinity of the break. The resulting network of chromatin-bound PARylated protein constitutes a scaffold for the recruitment of several proteins with PAR-binding motifs, which act as effectors and regulators of many essential cellular functions (Huang, Mol. Cell, 2022, 82, 2315). Given its key functional role, PARylation is a dynamic and highly controlled chromatin modification that is negatively regulated by dePARylating enzymes, such as poly(ADP-ribose) glycohydrolase (PARG) and ADP-ribosylhydrolase 3 (ARH3) (Pascal (2015) DNA Repair 32, 10). Additionally, autoPARylation, whereby PARP1 PARylates itself, has been linked to the dissociation of PARPs from chromatin in a self-regulatory negative loop (Prokhorova (2021) Nat. Commun. 12, 4055). By inducing PARylation, PARP1 regulates several key cellular processes, such as DNA damage signalling, chromatin remodelling, stability of replication forks, sensing of unligated Okazaki fragments during replication, and more generally, gene transcription, inflammation and metabolism. The pivotal role of PARP1 in the DNA damage response (DDR) is exerted by binding to single strand DNA at sites of SSBs and DSBs and recruiting effectors of DDR (Kanev (2024) Cell Rep. 43(5), 114234).

[0006] The functional link between PARP1 and BRCA1 / 2 has broad implications for cancer biology and therapy (Lord (2016) Nat. Rev. Cancer 16, 110). Germline mutations in the BRCA1 or BRCA2 tumour suppressors are associated with predisposition to several types of solidART-C-P3900PCT

[0007] cancers, and somatic mutations are frequently found in breast, ovarian, and prostate cancer, as well as several other solid tumours (Couch (2014) Science 343, 1466). This is due to the key roles of BRCA1 and BRCA2 in the error-free DSBR mechanism, HR, loss of which leads to genomic instability. However, HR-deficient (HRD) tumours with BRCA1 or BRCA2 mutations, or mutations in other HR genes are often more sensitive to chemotherapeutic DNA damaging agents, and are also more vulnerable to perturbation of alternative DDR pathways, such as the loss of PARP1 function (Murai (2019) Annu. Rev. Cancer Biol. 3, 131). The observation of this synthetic lethality led to the clinical development of a range of pharmacological inhibitors of PARP1, resulting in the FDA approval of olaparib in 2014. A further three PARP inhibitors (PARPi), rucaparib, niraparib and talazoparib, were approved over the following decade, with several others also entering clinical trials (Morganti (2024) JAMA Oncol. 10(5), 658). Indeed, inhibition of PARP1 induces accumulation of toxic DNA DSBs, which cannot be repaired in HRD cells, ultimately resulting in cell death (Murai (2019) Annu. Rev. Cancer Biol. 3, 131).

[0008] All clinically approved PARPi bind in the NAD+ binding site of the catalytic subunit, mimicking the interactions of NAD+ with the binding site (Rudolph (2022) PNAS 119(11), e2121979119; Lord (2017) Science 355(6330), 1152). Initially, it was assumed that the efficacy of PARPi was driven by their ability to out-compete NAD+ binding, thus inhibiting autoPARylation and preventing dissociation of PARP1 from DNA. However, as further structurally diverse PARPi were developed, it became apparent that biochemical inhibitory potency does not always correlate well with synthetic lethality in HRD cells (Shen (2013) Clin. Cancer Res. 19, 5003). This may in part be due to the difficulty of assaying inhibitor potency against PARPs in enzyme turnover assays, due to the high affinity of many PARPi in comparison to the concentration of enzyme required to generate a signal, leading to an underestimation of PARPi potency (Rudolph (2022) PNAS 119(11), e2121979119). However, the observation of dominant negative effects with PARPi e.g. that sensitisation to the alkylating agent MMS is weaker in PARP1 knock-out cells than those treated with PARPi, led to the concept that some PARPi ‘trap’ PARP1 molecules on DNA leading to higher levels of toxic DNA damage and thus, better efficacy (Heacock (2010) DNA Repair 9, 929; Horton (2005) JBC 280, 15773; Helleday (2011) Mol. Oncol. 5, 387; Kedar (2012) Mol. Cancer Res. 10, 360). Two potential mechanisms of increased association of PARP1 to damaged chromatin have been proposed. In the first model, PARP1 is indirectly trapped on DNA as inhibition of PARylation, prevents the conversion of PARP1 into its PARylated form, which has lower affinity for DNA and thus prevents dissociation (Hopkins (2015) Cancer Res. 13, 1465; Rudolph (2018) eLife 7:e37818; Chen (2019) Int. J. Cancer 145, 714). In the second model, allosteric coupling between the catalytic domain and DNA binding domains of PARP1 increases the residence time ofART-C-P3900PCT

[0009] of PARPi. Conversely, this also suggests that highly potent PARPi have the potential to be an effective cancer treatment both within and beyond HRD populations if a therapeutic index can be achieved. A PARPi capable of inducing enhanced retention of PARP1 on chromatin (via a pro-retention mechanism) would cause sufficient accumulation of DNA damage to kill both DDR-proficient and deficient cells, a paradigm previously observed for other chemotherapies, such as topoisomerase inhibitors (TOPOli). However, in order to achieve a sufficient therapeutic index to be a viable cancer treatment, such a PARPi would need to be developed as a tumour-targeted modality, as the broad cytotoxicity of such a compound precludes its systemic delivery as a small molecule drug (Kanev (2024) Cell Rep. 43(5), 114234; LaFargue (2019) Lancet Oncol. 20(1), e15).

[0010] There is therefore a need to provide more effective PARP1 and PARP2 inhibitors for the treatment of hyperproliferative disorders such as cancer.

[0011] SUMMARY OF THE INVENTION

[0012] According to a first aspect of the invention, there is provided a compound of formula (I):

[0013]

[0014] or a tautomeric or a stereochemically isomeric form, a pharmaceutically acceptable salt or a solvate thereof, wherein:

[0015] R1represents hydrogen or methyl;

[0016] R2represents Ci-e alkyl or C3-8 cycloalkyl, wherein said Ci-e alkyl or C3-8 cycloalkyl group is optionally substituted by one or more (e.g. 1 to 11) halogen atoms, Ci-e alkyl or haloCi-6 alkyl; and

[0017] R3represents hydrogen or fluorine.

[0018] According to a second aspect of the invention, there is provided a drug conjugate wherein said drug conjugate comprises:ART-C-P3900PCT

[0019] (a) one or more payloads each comprising a compound of formula (I) as defined herein, which is covalently bound to:

[0020] (b) a linker, which is covalently bound to:

[0021] (c) a tumour cell binding moiety.

[0022] BRIEF DESCRIPTION OF THE FIGURES

[0023] Figure 1: Plots showing changes in fluorescence polarisation over time in the DNA strand exchange assay for PARP1 and PARP2 in response to treatment with Example A1 (Figs. 1A & 1B), Example A2 (Figs. 1C & 1D), olaparib (Figs. 1E & 1F), talazoparib (Figs. 1G & 1H), saruparib (Figs. 1I & 1J), and Pip-6 (Figs. 1K & 1L).

[0024] Figures 2A to 2F: Dose-response plots showing chromatin retention of PARP1 and PARP2 by immunofluorescence in response to differing concentrations of Example A1 (Fig.

[0025] 2A), Example A2 (Fig. 2B), olaparib (Fig. 2C), talazoparib (Fig. 2D), saruparib (Fig. 2E), and Pip-6 (Fig. 2F).

[0026] Figures 2G to 2L: Dose-response plots showing inhibition of PARylation by immunofluorescence in response to differing concentrations of Example A1 (Fig. 2G), Example A2 (Fig. 2H), olaparib (Fig. 2I), talazoparib (Fig. 2J), saruparib (Fig. 2K), and Pip-6 (Fig. 2L).

[0027] Figure 3: Dose-response plots showing cell viability by CTG in response to differing concentrations of Examples C4, 07 and C15 overlaid with Dato-Dxd as positive control in NCI-N87 (Figs. 3A, 3B & 3C), DLD-1 BRCA2 KO (Figs. 3D, 3E & 3F), MCF7 (Figs. 3G, 3H & 3I), PEO1 clone 10 (Figs. 3J & 3K), and SUM149PT (Figs. 3L, 3M & 3N) cells.

[0028] Figure 4: Tumour growth inhibition and body weight plots from a DLD-1 BRCA2- / -mouse xenograft model showing the effect of treatment with differing doses of Examples C1 and C3 (Figs. 4A & 4B), Example C4 (Figs. 4C & 4D), Example 07 (Figs. 4E & 4F), Example C15 (Figs. 4G & 4H) and Example C16 (Figs. 4I & 4J).

[0029] Figure 5: Tumour growth inhibition and body weight plots from an NCI-N87 mouse xenograft model showing the effect of treatment with 1 mg / kg of Example C1 (Figs. 5A & 5B) and differing concentrations of Example C4 (Figs. 5C & 5D), Example 07 (Figs. 5E & 5F), Example C15 (Figs. 5G & 5H) and Example C16 (Figs. 5I & 5J).

[0030] Figure 6: Tumour growth inhibition and body weight plots from a SUM149PT mouse xenograft model showing the effect of treatment with differing concentrations of Example 04, Example C7 and Reference Example 08 (Figs. 6A & 6B).

[0031] Figure 7: Plot showing release of payload over time in the presence and absence of recombinant β-glucuronidase for Examples B1, B3, B4, B5 and B6.

[0032] Figure 8: Native mass spectra showing mass ions and DAR distribution of Sacituzumab (Fig. 8A), LALA-Sacituzumab (Fig. 8B), Example 04 (Fig. 8C), ReferenceART-C-P3900PCT

[0033] Example C5 (Fig. 8D), and Examples C7 (Fig. 8E), C11 (Fig. 8F), C15 (Fig. 8G) and C16 (Fig. 8H).

[0034] Figure 9: Dose-response plots showing differential sensitivity of hTERT RPE-1 WT, PARP1 KO, and PARP1 / 2 dual KO cells in response to treatment with Examples A1 (Fig. 9A) andA2 (Fig. 9B), by CTG.

[0035] DETAILED DESCRIPTION OF THE INVENTION

[0036] PARP1 / PARP2 Inhibiting Compounds

[0037] According to a first aspect of the invention, there is provided a compound of formula (I):

[0038]

[0039] or a tautomeric or a stereochemically isomeric form, a pharmaceutically acceptable salt or a solvate thereof, wherein:

[0040] R1represents hydrogen or methyl;

[0041] R2represents Ci-e alkyl or C3-8 cycloalkyl, wherein said Ci-e alkyl or C3-8 cycloalkyl group is optionally substituted by one or more (e.g. 1 to 11) halogen atoms, Ci-e alkyl or haloCi-6 alkyl; and

[0042] R3represents hydrogen or fluorine.

[0043] Definitions

[0044] The term ‘halo’ or ‘halogen’ as used herein refers to fluorine, chlorine, bromine or iodine.

[0045] The term ‘Ci-e alkyl’ as used herein as a group or part of a group refers to a linear or branched saturated hydrocarbon group containing from 1 to 6 carbon atoms. Examples of such groups include methyl, ethyl, n-propyl, / so-propyl, butyl, / so-butyl, tert-butyl, pentyl, hexyl and the like.ART-C-P3900PCT

[0046] The term ‘Cs-scycloalkyl’ as used herein refers to a saturated monocyclic hydrocarbon ring of 3 to 8 carbon atoms. Examples of such groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl and the like.

[0047] The term ‘optionally substituted’ as used herein refers to a group which may be substituted or unsubstituted by a substituent as herein defined.

[0048] Embodiments

[0049] In one embodiment, R1represents hydrogen.

[0050] In one embodiment, R2represents Ci-e alkyl optionally substituted by one or more (e.g. 1 to 7) halogen atoms (e.g. -CF2-CF3 or -C(F)(F)(Me)).

[0051] In a further embodiment, R2represents Ci-e alkyl optionally substituted by one or more (e.g.

[0052] 1 to 7) halogen atoms (e.g. -CF2-CHF2 or -CF2-CF3). In a further embodiment, R2represents C1.6 alkyl optionally substituted by one or more (e.g. 1 to 7) halogen atoms (e.g. -CF2-CF3).

[0053] In a yet further embodiment, R2represents methyl or ethyl optionally substituted by one or more (e.g. 2 to 5) halogen atoms (e.g. -CF2-CHF2, -CF2-CF3 or -C(F)(F)(Me)). In a yet further embodiment, R2represents methyl or ethyl optionally substituted by one or more (e.g. 2 to 5) halogen atoms (e.g. -CF2-CF3 or -C(F)(F)(Me)).

[0054] In a still yet further embodiment, R2represents ethyl optionally substituted by one or more (e.g. 4 to 5) halogen atoms (e.g. -CF2-CHF2 or -CF2-CF3). In a still yet further embodiment, R2represents ethyl optionally substituted by one or more (e.g. 5) halogen atoms (e.g. -CF2-CF3).

[0055] In one embodiment, R3represents hydrogen. In an alternative embodiment, R3represents fluorine.

[0056] In one embodiment, the invention provides a compound of formula (I) which is the free base of a compound of Examples A1 to A3 or a pharmaceutically acceptable salt or solvate thereof.

[0057] In a further embodiment, the invention provides a compound of formula (I) which is the free base of a compound of Examples A1 or A2 or a pharmaceutically acceptable salt or solvate thereof.ART-C-P3900PCT

[0058] A reference to a compound of the formula (I) and sub-groups thereof also includes ionic forms, salts, solvates, isomers (including geometric and stereochemical isomers), tautomers, N-oxides, esters, prodrugs, isotopes and protected forms thereof, for example, as discussed below; preferably, the salts or tautomers or isomers or N-oxides or solvates thereof; and more preferably, the salts or tautomers or N-oxides or solvates thereof, even more preferably the salts or tautomers or solvates thereof. Hereinafter, compounds and their ionic forms, salts, solvates, isomers (including geometric and stereochemical isomers), tautomers, N-oxides, esters, prodrugs, isotopes and protected forms thereof as defined in any aspect of the invention (except intermediate compounds in chemical processes) are referred to as "compounds of the invention".

[0059] Salts

[0060] Certain compounds of the formula (I) can exist in the form of salts, for example acid addition salts or, in certain cases salts of organic and inorganic bases such as carboxylate, sulfonate and phosphate salts. All such salts are within the scope of this invention, and references to compounds of the formula (I) include the salt forms of the compounds.

[0061] The salts of the present invention can be synthesized from the parent compound that contains a basic or acidic moiety by conventional chemical methods such as methods described in Pharmaceutical Salts: Properties, Selection, and Use, P. Heinrich Stahl (Editor), Camille G. Wermuth (Editor), ISBN: 3-90639-026-8, Hardcover, 388 pages, August 2002. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are used.

[0062] Acid addition salts (mono- or d / -salts) may be formed with a wide variety of acids, both inorganic and organic. Examples of acid addition salts include mono- or d / -salts formed with an acid selected from the group consisting of acetic, 2,2-dichloroacetic, adipic, alginic, ascorbic (e.g. L-ascorbic), L-aspartic, benzenesulfonic, benzoic, 4-acetamidobenzoic, butanoic, (+) camphoric, camphor-sulfonic, (+)-(1S)-camphor-10-sulfonic, capric, caproic, caprylic, cinnamic, citric, cyclamic, dodecylsulfuric, ethane-1,2-disulfonic, ethanesulfonic, 2-hydroxyethanesulfonic, formic, fumaric, galactaric, gentisic, glucoheptonic, D-gluconic, glucuronic (e.g. D-glucuronic), glutamic (e.g. L-glutamic), a-oxoglutaric, glycolic, hippuric, hydrohalic acids (e.g. hydrobromic, hydrochloric, hydriodic), isethionic, lactic (e.g. (+)-L-lactic, (±)-DL-lactic), lactobionic, maleic, malic, (-)-L-malic, malonic, (±)-DL-mandelic,ART-C-P3900PCT

[0063] methanesulfonic, naphthalene-2-sulfonic, naphthalene-1,5-disulfonic, 1-hydroxy-2-naphthoic, nicotinic, nitric, oleic, orotic, oxalic, palmitic, pamoic, phosphoric, propionic, pyruvic, L-pyroglutamic, salicylic, 4-amino-salicylic, sebacic, stearic, succinic, sulfuric, tannic, (+)-L-tartaric, thiocyanic, p-toluenesulfonic, undecylenic and valeric acids, as well as acylated amino acids and cation exchange resins.

[0064] One particular group of salts consists of salts formed from acetic, hydrochloric, hydriodic, phosphoric, nitric, sulfuric, citric, lactic, succinic, maleic, malic, isethionic, fumaric, benzenesulfonic, toluenesulfonic, methanesulfonic (mesylate), ethanesulfonic, naphthalenesulfonic, valeric, acetic, propanoic, butanoic, malonic, glucuronic and lactobionic acids. One particular salt is the hydrochloride salt.

[0065] Where the compounds of the formula (I) contain an amine function, these may form quaternary ammonium salts, for example by reaction with an alkylating agent according to methods well known to the skilled person. Such quaternary ammonium compounds are within the scope of formula (I).

[0066] The compounds of the invention may exist as mono- or d / -salts depending upon the pKaof the acid from which the salt is formed.

[0067] It will be appreciated that for use in medicine the salts of the compounds of formula (I) should be pharmaceutically acceptable. Suitable pharmaceutically acceptable salts will be apparent to those skilled in the art. Pharmaceutically acceptable salts include those described by Berge, Bighley and Monkhouse, J. Pharm. Sci. 1977, 66, pp. 1-19. Such pharmaceutically acceptable salts include acid addition salts formed with inorganic acids e.g. hydrochloric, hydrobromic, sulfuric, nitric or phosphoric acid and organic acids e.g. succinic, maleic, acetic, fumaric, citric, tartaric, benzoic, p-toluenesulfonic, methanesulfonic or naphthalenesulfonic acid. Other salts e.g. oxalates or formates may be used, for example in the isolation of compounds of formula (I) and are included within the scope of this invention. However, salts that are not pharmaceutically acceptable may also be prepared as intermediate forms which may then be converted into pharmaceutically acceptable salts. Such non-pharmaceutically acceptable salts forms, which may be useful, for example, in the purification or separation of the compounds of the invention, also form part of the invention.

[0068] Certain of the compounds of formula (I) may form acid addition salts with one or more equivalents of the acid. The present invention includes within its scope all possible stoichiometric and non-stoichiometric forms.ART-C-P3900PCT

[0069] Solvates

[0070] Those skilled in the art of organic chemistry will appreciate that many organic compounds can form complexes with solvents in which they are reacted or from which they are precipitated or crystallized. These complexes are known as “solvates”. For example, a complex with water is known as a “hydrate”. Pharmaceutically acceptable solvates of the compound of the invention are within the scope of the invention. In one embodiment, the pharmaceutically acceptable solvates of the compounds of the invention include the hydrate thereof.

[0071] In one embodiment, said crystalline form of the compounds of formula (I) is a cocrystal or coformer. Such a cocrystal or coformer may be prepared using water-soluble molecules such as saccharin, caffeine, nicotinamide or carboxylic acids. Coformers may be prepared as described in Emami S et al (2018) BioImpacts 8(4), 305-320, the techniques of which are herein incorporated by reference.

[0072] It will be understood that the invention includes pharmaceutically acceptable derivatives of compounds of formula (I) and that these are included within the scope of the invention.

[0073] As used herein "pharmaceutically acceptable derivative" includes any pharmaceutically acceptable ester or salt of such ester of a compound of formula (I) which, upon administration to the recipient is capable of providing (directly or indirectly) a compound of formula (I) or an active metabolite or residue thereof.

[0074] N-Oxides

[0075] Compounds of the formula (I) containing an amine function may also form N-oxides. A reference herein to a compound of the formula (I) that contains an amine function also includes the N-oxide.

[0076] Where a compound contains several amine functions, one or more than one nitrogen atom may be oxidised to form an N-oxide. Particular examples of N-oxides are the N-oxides of a tertiary amine or a nitrogen atom of a nitrogen-containing heterocycle.

[0077] N-Oxides can be formed by treatment of the corresponding amine with an oxidizing agent such as hydrogen peroxide or a per-acid (e.g. a peroxycarboxylic acid), see for example Advanced Organic Chemistry, by Jerry March, 4th Edition, Wiley Interscience. More particularly, N-oxides can be made by the procedure of L. W Deady (Syn. Commun. 1977,ART-C-P3900PCT

[0078] 7, 509-514) in which the amine compound is reacted with m-chloroperoxybenzoic acid (mCPBA), for example, in an inert solvent such as dichloromethane.

[0079] It will be appreciated by those skilled in the art that certain protected derivatives of compounds of formula (I), which may be made prior to a final deprotection stage, may not possess pharmacological activity as such, but may, in certain instances, be administered orally or parenterally and thereafter metabolised in the body to form compounds of the invention which are pharmacologically active. Such derivatives may therefore be described as “prodrugs”. All such prodrugs of compounds of the invention are included within the scope of the invention. Examples of pro-drug functionality suitable for the compounds of the present invention are described in Drugs of Today, 19, 9, 1983, 499-538 and in Topics in Chemistry, Chapter 31, pp. 306-316 and in “Design of Prodrugs" by H. Bundgaard, Elsevier, 1985, Chapter 1 (the disclosures in which documents are incorporated herein by reference). It will further be appreciated by those skilled in the art, that certain moieties, known to those skilled in the art as “pro-moieties”, for example as described by H. Bundgaard in “Design of Prodrugs" (the disclosure in which document is incorporated herein by reference) may be placed on appropriate functionalities when such functionalities are present within compounds of the invention.

[0080] Also included within the scope of the compound and various salts of the invention are polymorphs thereof.

[0081] Enantiomers

[0082] Where chiral centres are present in compounds of formula (I), the present invention includes within its scope all possible enantiomers and diastereoisomers, including mixtures thereof. The different isomeric forms may be separated or resolved one from the other by conventional methods, or any given isomer may be obtained by conventional synthetic methods or by stereospecific or asymmetric syntheses. The invention also extends to any tautomeric forms or mixtures thereof.

[0083] The subject invention also includes all pharmaceutically acceptable isotopically-labelled compounds which are identical to those recited in formula (I) but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number most commonly found in nature.ART-C-P3900PCT

[0084] Examples of isotopes suitable for inclusion in the compounds of the invention comprise isotopes of hydrogen, such as2H (D) and3H (T), carbon, such as11C,13C and14C, chlorine, such as36CI, fluorine, such as18F, iodine, such as123l,125l and1311, nitrogen, such as13N and15N, oxygen, such as15O,17O and18O, phosphorus, such as32P, and sulfur, such as35S.

[0085] Certain isotopically-labelled compounds of formula (I), for example, those incorporating a radioactive isotope, are useful in drug and / or substrate tissue distribution studies. The compounds of formula (I) can also have valuable diagnostic properties in that they can be used for detecting or identifying the formation of a complex between a labelled compound and other molecules, peptides, proteins, enzymes or receptors. The detecting or identifying methods can use compounds that are labelled with labelling agents such as radioisotopes, enzymes, fluorescent substances, luminous substances (for example, luminol, luminol derivatives, luciferin, aequorin and luciferase) etc. The radioactive isotopes tritium, i.e.3H (T), and carbon-14, i.e.14C, are particularly useful for this purpose in view of their ease of incorporation and ready means of detection.

[0086] Substitution with heavier isotopes such as deuterium, i.e.2H (D), may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements, and hence may be preferred in some circumstances.

[0087] Substitution with positron emitting isotopes, such as11C,18F,15O and13N, can be useful in Positron Emission Topography (PET) studies for examining target occupancy.

[0088] Isotopically-labelled compounds of formula (I) can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the accompanying Examples and Preparations using appropriate isotopically-labelled reagents in place of the non-labelled reagent previously employed.

[0089] Since the compounds of formula (I) are intended for use in pharmaceutical compositions it will readily be understood that they are each preferably provided in substantially pure form, for example at least 60% pure, more suitably at least 75% pure and preferably at least 85%, especially at least 98% pure (% are given on a weight for weight basis). Impure preparations of the compounds may be used for preparing the more pure forms used in the pharmaceutical compositions.ART-C-P3900PCT

[0090] Processes

[0091] According to a further aspect of the present invention there is provided a process for the preparation of compounds of formula (I) and derivatives thereof. The following schemes are examples of synthetic schemes that may be used to synthesise the compounds of the invention. In the following schemes reactive groups can be protected with protecting groups and de-protected according to well established techniques.

[0092] According to a further aspect of the invention there is provided a process for preparing a compound of formula (I) as herein defined which comprises:

[0093] (a) reacting a compound of formula (II):

[0094] R3

[0095]

[0096] (II)

[0097] wherein R1, R2and R3are as defined herein, with a suitable reagent, such as hydrazine hydrate; or

[0098] (b) interconversion of a compound of formula (I) or protected derivative thereof to a further compound of formula (I) or protected derivative thereof; or

[0099] (c) deprotection of a protected derivative of a compound of formula (I); or

[0100] (d) optional formation of a pharmaceutically acceptable salt of a compound of formula (I).

[0101] Process (a) typically comprises reacting a compound of formula (II) with a suitable reagent, such as hydrazine hydrate, in the presence of a suitable solvent (such as ethanol). An example of such a process is described in Example A1.

[0102] Compounds of formula (II) may be prepared in accordance with the procedures described herein. For example, compounds of formula (II) may be prepared in accordance with the experimental procedure described in Example A1.ART-C-P3900PCT

[0103] A wide range of well known functional group interconversions for process (b) are known by a person skilled in the art for converting a precursor compound to a compound of formula (I) and are described in Advanced Organic Chemistry by Jerry March, 4thEdition, John Wiley & Sons, 1992. For example possible metal catalysed functionalisations such as using organotin reagents (the Stille reaction), Grignard reagents and reactions with nitrogen nucleophiles are described in ‘Palladium Reagents and Catalysts’ [Jiro Tsuji, Wiley, ISBN 0-470-85032-9] and Handbook of OrganoPalladium Chemistry for Organic Synthesis [Volume 1, Edited by Ei-ichi Negishi, Wiley, ISBN 0-471-31506-0], It will be appreciated that interconversions form the majority of the experimental procedures described herein in detail for preparing compounds of formula (I).

[0104] If appropriate, the reactions described herein are followed or preceded by one or more reactions known to the skilled of the art and are performed in an appropriate order to achieve the requisite substitutions on each of the variables defined herein to afford other compounds of formula (I). Non-limiting examples of such reactions whose conditions can be found in the literature include:

[0105] protection of reactive functions,

[0106] deprotection of reactive functions,

[0107] halogenation,

[0108] dehalogenation,

[0109] dealkylation,

[0110] alkylation of amine, aniline, alcohol and phenol,

[0111] Mitsunobu reaction on hydroxyl groups,

[0112] cycloaddition reactions on appropriate groups,

[0113] reduction of nitro, esters, cyano, aldehydes,

[0114] transition metal-catalyzed coupling reactions,

[0115] acylation,

[0116] sulfonylation / introduction of sulfonyl groups,

[0117] saponification / hydrolysis of esters groups,

[0118] amidification or transesterification of ester groups,

[0119] esterification or amidification of carboxylic groups,

[0120] halogen exchange,

[0121] nucleophilic substitution with amine, thiol or alcohol,

[0122] reductive amination,

[0123] oxime formation on carbonyl and hydroxylamine groups,

[0124] S-oxidation,ART-C-P3900PCT

[0125] N-oxidation,

[0126] salification.

[0127] It is recognised that the sequence of reactions involving aryl coupling and reduction may be varied. It is also recognised that a wide range of palladium based catalysts are suitable for conducting aryl coupling reactions.

[0128] It may also be recognised that isomer separation may occur at any suitable stage in the synthetic sequence. It should be stressed that such chiral separation forms a key aspect of the invention and that such separation may be conducted in accordance with the methodology described herein or may be conducted in accordance with known methodology. It is also recognised that it may be beneficial to temporarily form a protected derivative of an intermediate in the synthesis, for example, a Boc-protected amine, or S EM -protected amide, in order to facilitate chromatographic separation, chiral resolution or to give improved solubility or yields in particular steps.

[0129] In many of the reactions described above, it may be necessary to protect one or more groups to prevent reaction from taking place at an undesirable location on the molecule. Examples of protecting groups, and methods of protecting and de-protecting functional groups, can be found in Protective Groups in Organic Synthesis (T. Green and P. Wuts; 4th Edition; John Wiley and Sons, 2007).

[0130] A hydroxy group may be protected, for example, as an ether (-OR) or an ester (-OC(=O)R), for example, as: a tert-butyl ether; a tetrahydropyranyl (THP) ether; a benzyl, benzhydryl (diphenylmethyl), or trityl (triphenylmethyl) ether; a trimethylsilyl or tert-butyldimethylsilyl ether; or an acetyl ester (-OC(=O)CH3).

[0131] An amine group may be protected, for example, as an amide (-NRCO-R) or a carbamate (-NRCO-OR), for example, as: a methyl amide (-NHCO-CH3); a benzyl carbamate (-NHCO-OCH2C6H5, -NH-Cbz or NH-Z); as a tert-butyl carbamate (-NHCOOC(CH3)3, NH-Boc); a 2-biphenyl-2-propyl carbamate (-NHCO-OC CHs Ce^CeHs, NH-Boc), as a 9-fluorenylmethyl carbamate (-NH-Fmoc), as a 6-nitroveratryl carbamate (-NH-Nvoc), as a 2-trimethylsilylethyl carbamate (-NH-Teoc), as a 2,2,2-trichloroethyl carbamate (-NH-Troc), as an allyl carbamate (-NH-Alloc), or as a 2(-phenylsulfonyl)ethyl carbamate (-NH-Psec).ART-C-P3900PCT

[0132] Other protecting groups for amines, such as cyclic amines and heterocyclic N-H groups, include toluenesulfonyl (tosyl) and methanesulfonyl (mesyl) groups, benzyl groups such as a para-methoxybenzyl (PMB) group and tetrahydropyranyl (THP) groups.

[0133] A carboxylic acid group may be protected as an ester for example, as: an C1.7 alkyl ester (e.g. a methyl ester; a tert-butyl ester); a Ci.yhaloalkyl ester (e.g. a C1.7 trihaloalkyl ester); a triCi-7 alkylsilyl-Ci-7 alkyl ester; or a C5-20 aryl-C alkyl ester (e.g. a benzyl ester; a nitrobenzyl ester; para-methoxybenzyl ester.

[0134] It will be understood by those skilled in the art that certain compounds of the invention can be converted into other compounds of the invention according to standard chemical methods.

[0135] Pharmaceutically acceptable salts may be prepared conventionally by reaction with the appropriate acid or acid derivative.

[0136] According to a further aspect of the invention, there is provided a compound of formula (I) or a tautomeric or a stereochemically isomeric form, a pharmaceutically acceptable salt or a solvate thereof, prepared by a process as defined herein.

[0137]

[0138] According to a second aspect of the invention, there is provided a drug conjugate wherein said drug conjugate comprises:

[0139] (a) one or more payloads each comprising a compound of formula (I) as defined herein, which is covalently bound to:

[0140] (b) a linker, which is covalently bound to:

[0141] (c) a tumour cell binding moiety.

[0142] It will be appreciated that said payload, said linker and said tumour cell binding moiety are radicals which are linked or conjugated to each other via a covalent bond.

[0143] Drug conjugates (such as antibody drug conjugates (ADCs) or peptide drug conjugates) are a rapidly expanding therapeutic modality designed to specifically target cancer cells by combining the tumour selectivity of a tumour cell binding moiety such as a peptide (i.e. a bicyclic peptide) or an antibody (i.e. a monoclonal antibody (mAb)) or an antibody fragment with the cell-killing ability of highly cytotoxic compounds.ART-C-P3900PCT

[0144] ADCs are typically composed of a mAb targeting a tumour-specific antigen, covalently bound to one or more cytotoxic compounds known as payloads via a linker. Mechanistically, the mAb targets and binds a cancer-specific cell surface antigen with high tumour specificity. Upon binding, the ADC is then internalised inside the cancer cell and trafficked to the lysosome, where it is either catabolised, or the linker is enzymatically cleaved to release the cytotoxic payload (Tsuchikama (2024) Nat. Rev. Clin. Oncol. 21, 203). Additional tumour specificity can be achieved by incorporating a substrate for a tumour-selective enzymatic process within the linker, such as a β-glucuronidase trigger (Jeffrey (2006) Bioconjugate Chem. 17(3), 831). Once liberated from the ADC, the payload can exert its cytotoxic effect, killing the cancer cell. Upon cell death, the payload molecules are released into the surrounding tumour tissue and, if sufficiently permeable, can further exert their effect on neighbouring cancer cells, a phenomenon known as the bystander effect (Staudacher (2017) Br. J. Cancer 117, 1736). The resulting tumour-specificity of ADCs compared to systemic delivery of cytotoxic small molecules has unlocked new clinical opportunities and has accelerated interest in ADCs as a valuable targeted therapeutic modality. Indeed, in the last few years several ADCs have been developed and clinically approved for the treatment of haematological and solid cancers (Liu (2024) Mol. Cancer 23, 62).

[0145] The past few years have seen rapid progress in the ADC field, with efforts predominantly focussed on developing new antibodies and linkers to optimise the tumour-specific delivery of well-characterised cytotoxic payloads. In fact, the vast majority of all clinically approved ADCs to-date utilise payloads that fall into one of only three categories, namely: tubulin poisons; topoisomerase inhibitors; or DNA-damaging agents. As a result, payload-specific mechanisms of resistance have emerged (Moganzo (2016) Mol. Cancer Ther. 15(12), 2825). Furthermore, there is limited evidence in the literature of payload optimisation using medicinal chemistry design principles to address the specific requirements of ADCs beyond cytotoxicity. There is therefore an opportunity to develop novel payloads with improved profiles by specifically optimising parameters such as LogD, permeability, efflux, and metabolic stability, among others, with utility as an ADC payload in-mind.

[0146] Clinically approved ADCs have achieved significant improvements in progression free survival, and occasionally overall survival, but long lasting curative treatments remain elusive for many patients. The lack of variety and broad clinical activity of existing ADC payloads represents a significant limitation of the currently available treatment options for patients (Fu (2022) Signal Transduct. Target Ther. 7, 93). Therefore, the development of new ADCs utilising novel payloads with differentiated mechanisms of action, such as pro-retention PARPIi forART-C-P3900PCT

[0147] example, could significantly improve the scope and patient benefit of ADC-based cancer therapies.

[0148] The current invention relates to the discovery of novel PARP inhibitors of formula (I) as defined herein which are differentiated from current clinical PARP inhibitors in terms of mechanism of action (as described herein), potency and utility in both targeted HR-deficient cancers, and in broader cancer populations beyond BRCA deficiency. It is believed that the compounds of formula (I) have properties which make them particularly useful as novel payloads for drug conjugates (such as ADCs or peptide drug conjugates, e.g. bicyclic peptide drug conjugates) and other similar modalities. Surprisingly, we have found that high cell potency in the presence and absence of BRCA deficiency is obtained by PARPi which both prevent DNA strand exchange on PARP1 and potently bind to PARP2. In addition to their high potency, which is a fundamental requirementof a drug conjugate payload, the compounds of formula (I) also have moderate permeability with limited efflux and low metabolic stability, both of which are potentially beneficial characteristics of a drug conjugate payload (Beaumont (2022) Xenobiotica 52, 8, 770). Furthermore, in contrast to current clinical PARP inhibitors, the compounds of formula (I) incorporate a phenol as a handle for ready attachment to a drug conjugate linker moiety.

[0149] Payloads

[0150] It will be appreciated that the one or more payloads of the drug conjugate of the second aspect of the invention comprises one or more compounds of formula (I) or a tautomeric or a stereochemical ly isomeric form, a pharmaceutically acceptable salt or a solvate thereof. All aspects and embodiments described hereinbefore under the heading “PARP1 / PARP2 Inhibiting Compounds” apply equally here for the payloads.

[0151] In one embodiment, the one or more payloads of said drug conjugate comprise one or more compounds of formula (I) which are the free base of a compound of Examples A1 to A3 or a pharmaceutically acceptable salt or solvate thereof.

[0152] In one embodiment, the drug conjugate comprises between 1 and 10, such as 4 or 8 payloads comprising a compound of formula (I).

[0153] It will be appreciated that the one or more payloads within said drug conjugate may comprise the same or a different structure of a compound of formula (I). In one embodiment, said one or more payloads each comprise the same compound of formula (I).ART-C-P3900PCT

[0154] Linkers

[0155] It will be appreciated that the linker of said drug conjugate will typically comprise any suitable linking moiety configured for linking the payload compound of formula (I) to the tumour cell binding moiety, ideally, as mentioned above, via the hydroxy substituent of the phenol group of said payload compound of formula (I).

[0156] The linker is a bifunctional or multifunctional moiety which can be used to attach one or more payload compounds of formula (I) to the tumour cell binding moiety to form the drug conjugates of the present invention. The connecting group attaching the linker to the payload compound will typically consist of a self-immolative group such as an appropriately substituted carbamate or hemiaminal which incorporates the phenolic oxygen of the payload. Attachment of the linker to the tumour cell binding moiety will typically comprise a covalent bond between a cysteine thiol, or an amine, for example an / V-terminus or amino acid side chain such as lysine, and a suitable functional group on the linker, such as a maleimide or haloacetamide. The linker may be further functionalised with one or more groups that modulate properties including solubility; lipophilicity; and aggregation.

[0157] The linkers are preferably stable under extracellular conditions, such that the drug conjugate remains intact prior to transport or other mechanism of delivery into a target cancer cell. Upon internalisation into the cancer cell, the linker may be cleaved by an enzyme, such as a lysosomal enzyme; hydrolysis; catabolism; or other enzymatic process to release one or more payload compounds of formula (I). In one embodiment, the linker contains a moiety that is a substrate for a lysosomal enzyme, such as cathepsin, or β-glucuronidase. The mechanism of linker cleavage may be facilitated by incorporation of a self-immolative spacer unit, such as a diaminocarbamoyl moiety, which may accelerate the release of payload following the initial metabolic activation of the linker.

[0158] Thus, in one embodiment the linker comprises an -L1-L2-L3- moiety, wherein:

[0159] Li represents a tumour cell binding moiety attachment moiety;

[0160] L2 represents a self-immolative trigger moiety; and

[0161] L3 represents a payload attachment moiety.

[0162] It will be appreciated that references herein to “tumour cell binding moiety attachment moiety” include any moiety capable of attaching to a tumour cell binding moiety, such as a peptide, antibody or antibody fragment. In particular, the moiety will typically comprise a moiety which is capable of forming a functional group via a covalent bond with a thiol or amine group within a cysteine residue of said peptide, antibody or antibody fragment.ART-C-P3900PCT

[0163] Suitable examples of tumour cell binding moiety attachment moieties include a maleimide, dibromopyridazinedione or haloacetamide group, such as a maleimide or dibromopyridazinedione group.

[0164] It will also be appreciated that references herein to “self-immolative trigger moiety” include any moiety which is capable of enabling “traceless” cleavage when used in conjunction with a suitable enzymatic, tumour-specific release mechanism.

[0165] In one embodiment, the self-immolative trigger moiety is a moiety that is a substrate for a lysosomal enzyme, such as cathepsin, or β-glucuronidase. In a further embodiment, the self-immolative trigger moiety comprises a glucuronide containing moiety. The advantage of using such a glucuronide moiety is that upon in vivo administration, it will be exposed to β-glucuronidase enzymes which will lead to the destruction of the glucuronide moiety, thereby causing self-immolation and consequent release of the attached, adjacent component, i.e. the payload of formula (I).

[0166] It will also be appreciated that references herein to “payload attachment moiety” include any moiety which is capable of forming a covalent bond with a substituent within the payload of the compound of formula (I). For example, if the substituent is an amine group, then the payload attachment moiety may typically comprise a carbamate group. Alternatively, if the substituent is a hydroxy group, then the payload attachment moiety may typically comprise a diaminocarbamoyl moiety, or a methylene alkoxy carbamate (MAC). It will be appreciated that depending upon the nature of the payload attachment moiety, it may be present as a portion of the self-immolative trigger moiety.

[0167] Optionally, the linker comprises an additional moiety between the tumour cell binding moiety attachment moiety of Li and the self-immolative trigger moiety of L2. Thus, in a further embodiment the linker comprises an -Li-Lia-L2-I_3- moiety, wherein:

[0168] Li represents a tumour cell binding moiety attachment moiety;

[0169] Lia represents a spacer moiety;

[0170] L2 represents a self-immolative trigger moiety; and

[0171] L3 represents a payload attachment moiety.

[0172] In this embodiment, Li, L2 and L3 are as defined hereinbefore and Liarepresents any suitable spacer moiety, such as a moiety comprising one or more PEG groups which may be linear or branched. The advantage of such PEG groups provides modulation of lipophilicity and masking of the hydrophobicity of the payload of compound of formula (I).ART-C-P3900PCT

[0173] Examples of suitable linkers include those within the compounds of Examples C1 to C16.

[0174] Thus, in one embodiment, the linker of the drug conjugate is selected from any of linkers (i) to (vii):

[0175]

[0176] ART-C-P3900PCT

[0177]

[0178] (vii);

[0179] wherein “(a)” represents the attachment point to the payload compound of formula (I) as defined herein, and “(c)” represents the tumour cell binding moiety, and wherein each “S” represents a sulfur atom within a cysteine residue of said tumour cell binding moiety.ART-C-P3900PCT

[0180] In a further embodiment, the linker of the drug conjugate is selected from any of linkers (i) to (iii):

[0181]

[0182] wherein “(a)” represents the attachment point to the payload compound of formula (I) as defined herein, and “(c)” represents the tumour cell binding moiety, and wherein each “S” represents a sulfur atom within a cysteine residue of said tumour cell binding moiety.ART-C-P3900PCT

[0183] In a further alternative embodiment, the linker of the drug conjugate is selected from any of linkers (iv) to (vii):

[0184]

[0185] ART-C-P3900PCT

[0186] wherein “(a)” represents the attachment point to the payload compound of formula (I) as defined herein, and “(c)” represents the tumour cell binding moiety, and wherein each “S” represents a sulfur atom within a cysteine residue of said tumour cell binding moiety.

[0187] In a yet further alternative embodiment, the linker of the drug conjugate is linker (iv):

[0188]

[0189] (iv);

[0190] wherein “(a)” represents the attachment point to the payload compound of formula (I) as defined herein, and “(c)” represents the tumour cell binding moiety, and wherein each “S” represents a sulfur atom within a cysteine residue of said tumour cell binding moiety.

[0191] In an alternative embodiment, the linker of the drug conjugate is selected from any of linkers (i)a, (i)b, (ii)a, (ii)b, (iii)a, (iii)b, (v)a, (v)b, (vii)aor (vii)b:

[0192]

[0193] ART-C-P3900PCT

[0194] HO OH OH OH

[0195]

[0196] ART-C-P3900PCT

[0197] 5

[0198]

[0199] ART-C-P3900PCT

[0200]

[0201] wherein “(a)” represents the attachment point to the payload compound of formula (I) as defined herein, and “(c)” represents the tumour cell binding moiety, and wherein each “S” represents a sulfur atom within a cysteine residue of said tumour cell binding moiety.

[0202] In a further embodiment, the linker of the drug conjugate is selected from any of linkers (i)a, (i)b, (ii)a, (ii)b, (iii)a, or (iii)b:

[0203]

[0204] ART-C-P3900PCT

[0205] HO OH OH OH

[0206]

[0207] ART-C-P3900PCT

[0208] (iii)a; or

[0209]

[0210] wherein “(a)” represents the attachment point to the payload compound of formula (I) as defined herein, and “(c)” represents the tumour cell binding moiety, and wherein each “S” represents a sulfur atom within a cysteine residue of said tumour cell binding moiety.

[0211] In a further alternative embodiment, the linker of the drug conjugate is selected from any of linkers (v)a, (v)b, (vii)aor (vii)b:

[0212]

[0213] ART-C-P3900PCT

[0214]

[0215] wherein “(a)” represents the attachment point to the payload compound of formula (I) as defined herein, and “(c)” represents the tumour cell binding moiety, and wherein each “S” represents a sulfur atom within a cysteine residue of said tumour cell binding moiety.

[0216] It will be apparent that linkers (i)a, (i)b, (ii)a, (ii)b, (iii)a, (iii)b, (v)a, (v)b, (vii)aor (vii)bcorrespond to hydrolysed equivalents of linkers (i) to (vii). Without being bound by theory, it is believed that these hydrolysed equivalents contribute to a more stable drug conjugate, due to the reduced capacity for premature deconjugation via retro-Michael addition of the cysteine from the tumour cell binding moiety to which the linker is attached.

[0217] Tumour Cell Binding MoietiesART-C-P3900PCT

[0218] It will be appreciated that the tumour cell binding moiety may be any suitable moiety capable of binding to a tumour cell, in particular a given epitope upon such a tumour cell. As described hereinbefore, suitable examples include peptides (i.e. bicyclic peptides), antibodies (i.e. monoclonal antibodies (mAbs)) or antibody fragments.

[0219] References herein to the terms “antibody” or “antibodies” refer to molecules or active fragments of molecules that bind to known antigens, particularly to immunoglobulin molecules and to immunologically active portions of immunoglobulin molecules, i.e. molecules that contain a binding site that immunospecifically binds an antigen. The immunoglobulin according to the invention can be of any class (IgG, IgM, IgD, IgE, IgA and IgY) or subclass (e.g. lgG1, lgG2, lgG3, lgG-4, lgA1 and lgA2) or subclasses (isotypes) of immunoglobulin molecule (e.g. IgG in lgG1, lgG2, lgG3, and lgG4, or IgA in lgA1 and lgA2).

[0220] Within the scope of the present invention the terms “antibody” or “antibodies” include monoclonal, polyclonal, chimeric, single chain, bispecific, human and humanized antibodies as well as active fragments thereof. Examples of active fragments of molecules that bind to known antigens include Fab, F(ab')2, scFvand Fv fragments, including the products of an Fab immunoglobulin expression library and epitope-binding fragments of any of the antibodies and fragments mentioned above.

[0221] As used herein, the term “monoclonal antibody” refers to an antibody that is mass produced in the laboratory from a single clone and that recognizes only one antigen. Monoclonal antibodies are typically made by fusing a normally short-lived, antibody-producing B cell to a fast-growing cell, such as a cancer cell (sometimes referred to as an “immortal” cell). The resulting hybrid cell, or hybridoma, multiplies rapidly, creating a clone that produces large quantities of the antibody. For the purpose of the present invention, “monoclonal antibody” is also to be understood to comprise antibodies that are produced by a mother clone which has not yet reached full monoclonality.

[0222] As used herein, the term “chimeric antibody” refers to a monoclonal antibody comprising a variable region, i.e., binding region, from mouse and at least a portion of a constant region derived from a different source or species, usually prepared by recombinant DNA techniques. Chimeric antibodies comprising a mouse variable region and a human constant region are exemplary embodiments. Such mouse / human chimeric antibodies are the product of expressed immunoglobulin genes comprising DNA segments encoding mouse immunoglobulin variable regions and DNA segments encoding human immunoglobulin constant regions. Other forms of “chimeric antibodies” encompassed by the invention areART-C-P3900PCT

[0223] those in which the class or subclass has been modified or changed from that of the original antibody. Such “chimeric” antibodies are also referred to as “class-switched antibodies”. Methods for producing chimeric antibodies involve conventional recombinant DNA and gene transfection techniques now well known in the art. See, e.g. Morrison, S. L., etal., Proc. Natl. Acad Sci. USA 81 (1984) 6851-6855; U. S. Pat. No. 5,202,238 and U. S. Pat. No. 5,204,244.

[0224] As used herein the term “humanized antibody” or “humanized version of an antibody” refers to antibodies in which the framework or “complementarity determining regions” (CDR) have been modified to comprise the CDR of an immunoglobulin of different specificity as compared to that of the parent immunoglobulin. In some exemplary embodiments, the CDRs of the VH and VL are grafted into the framework region of human antibody to prepare the “humanized antibody.” See e.g. Riechmann, L., et al., Nature 332 (1988) 323-327; and Neuberger, M. S., et al., Nature 314 (1985) 268-270. The heavy and light chain variable framework regions can be derived from the same or different human antibody sequences. The human antibody sequences can be the sequences of naturally occurring human antibodies. Human heavy and light chain variable framework regions are listed e.g. in Lefranc, M.-P., Current Protocols in Immunology (2000) — Appendix 1P A.1P.1-A.1P.37 and are accessible via IMGT, the international ImMunoGeneTics information System® (http: / / imgt.cines.fr) or via http: / / vbase.mrc-cpe.cam.ac.uk, for example. Optionally the framework region can be modified by further mutations. Exemplary CDRs correspond to those representing sequences recognizing the antigens noted above for chimeric antibodies. In some embodiments, such humanized version is chimerized with a human constant region. The term “humanized antibody” as used herein also comprises such antibodies which are modified in the constant region to generate the properties according to the invention, especially in regard to C1q binding and / or FcR binding, e.g. by “class switching” i.e. change or mutation of Fc parts (e.g. from lgG1 to lgG4 and / or lgG1 / lgG4 mutation).

[0225] As used herein the term “human antibody” is intended to include antibodies having variable and constant regions derived from human germ line immunoglobulin sequences. Human antibodies are well-known in the state of the art (van Dijk, M. A., and van de Winkel, J. G., Curr. Opin. Chem. Biol. 5 (2001) 368-374). Human antibodies can also be produced in transgenic animals (e.g. mice) that are capable, upon immunization, of producing a full repertoire or a selection of human antibodies in the absence of endogenous immunoglobulin production. Transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice results in the production of human antibodies upon antigen challenge (see, e.g. Jakobovits, A., etal., Proc. Natl. Acad. Sci. USA 90 (1993) 2551-2555; Jakobovits, A., et al., Nature 362 (1993) 255-258; Brueggemann, M. D., et al., Year Immunol. 1 (1993) 33-40).ART-C-P3900PCT

[0226] Human antibodies can also be produced in phage display libraries (Hoogenboom, H. R., and Winter, G., J. Mol. Biol. 227 (1992) 381-388; Marks, J. D., etal., J. Mol. Biol. 222 (1991) 581-597). The techniques of Cole, A., et al. and Boerner, P., et al. are also available for the preparation of human monoclonal antibodies (Cole, A., et al., Monoclonal Antibodies and Cancer Therapy, Liss, A. R. (1985) p. 77; and Boerner, P., et al., J. Immunol. 147 (1991) 86-95). As already mentioned, according to the invention the term “human antibody” as used herein also comprises such antibodies which are modified in the constant region to generate the properties according to the invention, for example in regard to C1q binding and / or FcR binding, e.g. by “class switching” i.e. change or mutation of Fc parts (e.g. from IgG 1 to lgG4 and / or lgG1 / lgG4 mutation).

[0227] As used herein “single chain antibody” refers to single chain Fv molecules (scFv), wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site (Bird et al., 1988, Science 242:423-426, Huston et al., 1988, Proc. Natl. Acad. Sci. U. S. A. 85:5879-5883 or a bispecific single chain Fv (WO 03 / 11161).

[0228] As used herein the term “bispecific antibodies” refers to antibodies that bind to two (or more) different antigens.

[0229] As used herein the term “antibody fragments” refers to a portion of a full-length antibody, for example possibly a variable domain thereof, or at least an antigen binding site thereof. Examples of antibody fragments include diabodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. scFv antibodies are, e.g. described in Huston, J. S., Methods in Enzymol. 203 (1991) 46-88. Antibody fragments can be derived from an antibody of the invention by a number of art-known techniques. For example, purified monoclonal antibodies can be cleaved with an enzyme, such as pepsin, and subjected to HPLC gel filtration. The appropriate fraction containing Fab fragments can then be collected and concentrated by membrane filtration and the like. For further description of general techniques for the isolation of active fragments of antibodies, see for example, Khaw, B. A. et al. J. Nucl. Med. 23:1011-1019 (1982); Rousseaux etal. Methods Enzymology, 121:663-69, Academic Press, 1986.

[0230] As used herein the term “specific” and “specifically” are used interchangeably to indicate that other biomolecules do not significantly bind to the antibody that is specifically binding to the biomolecule of interest. In some embodiments, the level of binding to a biomolecule otherART-C-P3900PCT

[0231] than a peptide comprising an epitope within a peptide results in a negligible (e.g. not determinable) binding affinity by means of ELISA or an affinity determination.

[0232] By “negligible binding” a binding is meant, which is at least about 85%, particularly at least about 90%, more particularly at least about 95%, even more particularly at least about 98%, but especially at least about 99% and up to 100% less than the binding to a peptide comprising an epitope within a peptide.

[0233] As used herein the term “epitope” refers to a site on a target molecule (e.g. an antigen, such as a protein) to which an antigen-binding molecule (e.g. an antibody or antibody fragment) binds. Epitopes can be formed both from contiguous or adjacent noncontiguous residues (e.g. amino acid residues) of the target molecule. Epitopes formed from contiguous residues (e.g. amino acid residues) typically are also called linear epitopes. An epitope typically includes at least 5 and up to about 12 residues, mostly between 6 and 10 residues (e.g. amino acid residues).

[0234] As used herein, the term “CDR” refers to the hypervariable region of an antibody. The term “hypervariable region”, “HVR”, or “HV”, when used herein refers to the regions of an antibody variable domain which are hypervariable in sequence and / or form structurally defined loops. Generally, antibodies comprise six hypervariable regions; three in the VH (H1, H2, H3), and three in the VL (L1, L2, L3). A number of hypervariable region delineations are in use and are encompassed herein. The Kabat Complementarity Determining Regions are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The letters “HC” and “LC” preceding the term “CDR” refer, respectively, to a CDR of a heavy chain and a light chain.

[0235] In one embodiment, the antibody is a polyclonal antibody. In one embodiment, the antibody is a humanized antibody, a human antibody, a murine antibody or a chimeric antibody.

[0236] In one embodiment, the antigen binding fragment thereof is an antigen-binding fragment (Fab) or a single-chain variable fragment (scFv). In a further embodiment, said fragment is selected from the group consisting of Fab, Fab', F(ab)2, F(ab')2, and scFv.

[0237] It will be appreciated that the antibody or antigen binding fragments of the present invention will be configured to bind to a therapeutic target which is a tumour (i.e. cancer) cell.ART-C-P3900PCT

[0238] In one embodiment, the antibody or antigen binding fragments are configured to bind to a tumour (i.e. cancer) cell. In a further embodiment, the antibody or antigen binding fragments specifically bind to a tumour-associated antigen whose cell surface expression on a tumour cell is different to its expression on a healthy cell.

[0239] In a preferred embodiment, the antibody or antigen binding fragment binds to a target on a tumour (i.e. cancer) cell. Preferred targets include: BAFF-R, CA-125, carbonic anhydrase IX, CD2, CD7, CD19, CD20, CD22, CD23, CD25, CD30, CD33, CD37, CD38, CD40, CD47, CD52, CD56, CD70, CD71, CD73, CD74, CD79b, CD80, CD86, CD123, CD135, CD137, CD269, CD276, CD307, CD319, CEACAM, c-met, CXCR4, DR5, EGFR, EpCAM, ErbB2 / HER2, ErbB3 / HER3, ErbB4 / HER3, FOLR1, FLT-3, IGF-1R, InsR, KIT, LeY, LGR-5, mesothelin, MIP-1A, MIP-1B, MUC1, MUC2, MUC3, MUC4, MUC5ac, Nectin-4, P-cadherin, PD-1, PD1L, PDGFRa, PSCA, PSMA, ROR1, TF, TRAIL receptor (R1 and R2), Trop-2 and VEGFR.

[0240] In a yet preferred embodiment, the antibody or antigen binding fragment binds to a target on a tumour (i.e. cancer) cell selected from: CD19, CD22, CD30, CD33, CD79b, ErbB2 / HER2, FOLR1, Nectin-4, TF and Trop-2. Such targets are those being targeted by existing approved antibody drug conjugates (ADCs).

[0241] In a still yet preferred embodiment, the antibody or antigen binding fragment binds to a target on a tumour (i.e. cancer) cell which is Trop-2.

[0242] In one embodiment, the antibody or antigen binding fragment is selected from one currently used within an existing approved antibody drug conjugate (ADC), such as brentuximab (a chimeric anti-CD30 monoclonal antibody), enfortumab (a human anti-Nectin-4 monoclonal antibody), gemtuzumab (a humanized anti-CD33 monoclonal antibody), inotuzumab (a humanized anti-CD22 monoclonal antibody), loncastuximab (a humanized anti-CD19 monoclonal antibody), mirvetuximab (a chimeric anti-folate receptor alpha (FOLR1) monoclonal antibody), moxetumomab (a mouse anti-CD22 monoclonal antibody), polatuzumab (a humanized anti-CD79b monoclonal antibody), sacituzumab (a humanized anti-Trop-2 monoclonal antibody), LALA-sacituzumab (a humanized anti-Trop-2 monoclonal antibody), tisotumab (a human anti-tissue factor (TF) monoclonal antibody) and trastuzumab (a humanized anti-ErbB2 / HER2 monoclonal antibody). LALA-Sacituzumab is Sacituzumab with a double L to A mutation at positions L234 and L235 on the heavy chain (full sequence listed in Example C15 below).ART-C-P3900PCT

[0243] In one embodiment, the antibody or antigen binding fragment is selected from sacituzumab or LALA-sacituzumab. Data is presented herein which provides evidence that these antibodies demonstrated excellent results when formulated within the drug conjugates of the invention.

[0244] In a further embodiment, the antibody or antigen binding fragment is selected from sacituzumab. Data is presented herein which provides evidence that this antibody demonstrated excellent results when formulated within the drug conjugates of the invention.

[0245] In a further alternative embodiment, the antibody or antigen binding fragment is selected from LALA-sacituzumab. Data is presented herein which provides evidence that this antibody demonstrated excellent results when formulated within the drug conjugates of the invention.

[0246] In particular, it will be appreciated that the LALA-sacituzumab antibody described herein constitutes a novel antibody and therefore forms an additional aspect of the invention.

[0247] Thus, according to a further aspect of the invention, there is provided an antibody which comprises a heavy chain as set forth in SEQ ID NO: 5 and a light chain as set forth in SEQ ID NO: 4.

[0248] In one embodiment, the invention provides a drug conjugate which is a compound of any one of Examples C1 to 04, 06 to 07 and 09 to 016.

[0249] In a further embodiment, the invention provides a drug conjugate which is a compound of any one of Examples 01, 02 or 03.

[0250] In a further embodiment, the invention provides a drug conjugate which is a compound of any one of Examples 04, 07, 015 or 016.

[0251] In a yet further embodiment, the invention provides a drug conjugate which is a compound of Example 015.

[0252] In a yet further embodiment, the invention provides a drug conjugate which is other than a compound of Example 015.

[0253] It will be appreciated that the drug conjugates may be prepared by the skilled person using standard procedures.ART-C-P3900PCT

[0254] In particular, the process will comprise attaching a linker (as defined herein) to the payload (as defined herein, i.e. a compound of formula (I)) followed by attachment of the tumour cell binding moiety (as defined herein, i.e. a peptide, antibody or antibody fragment). Thus, according to a further aspect of the invention, there is provided a process for preparing a drug conjugate as defined herein which comprises the steps of:

[0255] (a) attaching a linker as defined herein to the compound of formula (I) as defined herein, followed by

[0256] (b) attachment of the tumour cell binding moiety as defined herein.

[0257] In particular, the process comprises preparing a drug conjugate of any one of Examples C1 to C16. More particularly, the process comprises preparing a drug conjugate of Example C15.

[0258] More particularly, the drug conjugates of the invention may be prepared using the procedures described herein for Examples C1 to C16. Yet more particularly, the drug conjugates of the invention may be prepared using the procedures described herein for Example C15.

[0259] Thus, according to a further aspect of the invention, there is provided a drug conjugate prepared by a process as defined herein. More particularly, there is provided a drug conjugate of any one of Examples C1 to C16 prepared by a process as defined herein. Yet more particularly, there is provided a drug conjugate of Example C15 prepared by a process as defined herein.

[0260] It will be appreciated that certain intermediates used in the preparation of the drug conjugates of the invention may constitute novel compounds and form part of the invention.

[0261] Thus, according to a further aspect of the invention there is provided an intermediate selected from a compound of B1 to B10. According to a further aspect of the invention there is provided an intermediate selected from a compound of B1 to B3. In one embodiment, there is provided an intermediate selected from B4 or B6.

[0262] Pharmaceutical Compositions

[0263] While it is possible for the compounds of formula (I) and drug conjugates of the invention to be administered alone, it is preferable to present them as a pharmaceutical composition (e.g. formulation). In one embodiment this is a sterile pharmaceutical composition.ART-C-P3900PCT

[0264] Thus, the present invention further provides pharmaceutical compositions, as defined above, and methods of making a pharmaceutical composition comprising (e.g. admixing) at least one compound of formula (I) (and sub-groups thereof as defined herein) or at least one drug conjugate, together with one or more pharmaceutically acceptable excipients and optionally other therapeutic or prophylactic agents, as described herein.

[0265] Thus, according to a further aspect of the invention, there is provided a pharmaceutical composition comprising the compound of formula (I) as defined herein or the drug conjugate as defined herein in combination with one or more pharmaceutically acceptable excipients.

[0266] The pharmaceutically acceptable excipient(s) can be selected from, for example, carriers (e.g. a solid, liquid or semi-solid carrier), adjuvants, diluents, fillers or bulking agents, granulating agents, coating agents, release-controlling agents, binding agents, disintegrants, lubricating agents, preservatives, antioxidants, buffering agents, suspending agents, thickening agents, flavouring agents, sweeteners, taste masking agents, stabilisers or any other excipients conventionally used in pharmaceutical compositions. Examples of excipients for various types of pharmaceutical compositions are set out in more detail below.

[0267] The term “pharmaceutically acceptable” as used herein pertains to compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of a subject (e.g. human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. Each carrier, excipient, etc. must also be “acceptable” in the sense of being compatible with the other ingredients of the formulation.

[0268] Pharmaceutical compositions containing compounds of formula (I) and drug conjugates can be formulated in accordance with known techniques, see for example, Remington’s Pharmaceutical Sciences, Mack Publishing Company, Easton, PA, USA.

[0269] The pharmaceutical compositions can be in any form suitable for oral, parenteral, topical, intranasal, intrabronchial, sublingual, ophthalmic, otic, rectal, intra-vaginal, or transdermal administration. Where the compositions are intended for parenteral administration, they can be formulated for intravenous, intramuscular, intraperitoneal, subcutaneous administration or for direct delivery into a target organ or tissue by injection, infusion or other means of delivery. The delivery can be by bolus injection, short term infusion or longer term infusion and can be via passive delivery or through the utilisation of a suitable infusion pump or syringe driver.ART-C-P3900PCT

[0270] Pharmaceutical formulations adapted for parenteral administration include aqueous and nonaqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats, co-solvents, surface active agents, organic solvent mixtures, cyclodextrin complexation agents, emulsifying agents (for forming and stabilizing emulsion formulations), liposome components for forming liposomes, gellable polymers for forming polymeric gels, lyophilisation protectants and combinations of agents for, inter alia, stabilising the active ingredient in a soluble form and rendering the formulation isotonic with the blood of the intended recipient. Pharmaceutical formulations for parenteral administration may also take the form of aqueous and nonaqueous sterile suspensions which may include suspending agents and thickening agents (R. G. Strickly, Solubilizing Excipients in oral and injectable formulations, Pharmaceutical Research, Vol 21(2) 2004, p 201-230).

[0271] The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules, vials and prefilled syringes, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. In one embodiment, the formulation is provided as an active pharmaceutical ingredient in a bottle for subsequent reconstitution using an appropriate diluent.

[0272] The pharmaceutical formulation can be prepared by lyophilising a compound of formula (I), or sub-groups thereof, or the drug conjugate. Lyophilisation refers to the procedure of freeze-drying a composition. Freeze-drying and lyophilisation are therefore used herein as synonyms.

[0273] Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.

[0274] Pharmaceutical compositions of the present invention for parenteral injection can also comprise pharmaceutically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use.

[0275] Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as sunflower oil, safflower oil, corn oil or olive oil), and injectable organic esters such as ethylART-C-P3900PCT

[0276] oleate. Proper fluidity can be maintained, for example, by the use of thickening or coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0277] The compositions of the present invention may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include agents to adjust tonicity such as sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

[0278] In one particular embodiment of the invention, the pharmaceutical composition is in a form suitable for i.v. administration, for example by injection or infusion. For intravenous administration, the solution can be dosed as is, or can be injected into an infusion bag (containing a pharmaceutically acceptable excipient, such as 0.9% saline or 5% dextrose), before administration.

[0279] In another particular embodiment, the pharmaceutical composition is in a form suitable for sub-cutaneous (s.c.) administration.

[0280] Pharmaceutical dosage forms suitable for oral administration include tablets (coated or uncoated), capsules (hard or soft shell), caplets, pills, lozenges, syrups, solutions, powders, granules, elixirs and suspensions, sublingual tablets, wafers or patches such as buccal patches.

[0281] Thus, tablet compositions can contain a unit dosage of active compound together with an inert diluent or carrier such as a sugar or sugar alcohol, e.g. lactose, sucrose, sorbitol or mannitol; and / or a non-sugar derived diluent such as sodium carbonate, calcium phosphate, calcium carbonate, or a cellulose or derivative thereof such as microcrystalline cellulose (MCC), methyl cellulose, ethyl cellulose, hydroxypropyl methyl cellulose, and starches such as corn starch. Tablets may also contain such standard ingredients as binding and granulating agents such as polyvinylpyrrolidone, disintegrants (e.g. swellable crosslinked polymers such as crosslinked carboxymethylcellulose), lubricating agents (e.g. stearates), preservatives (e.g. parabens), antioxidants (e.g. BHT), buffering agents (for exampleART-C-P3900PCT

[0282] phosphate or citrate buffers), and effervescent agents such as citrate / bicarbonate mixtures. Such excipients are well known and do not need to be discussed in detail here.

[0283] Tablets may be designed to release the drug either upon contact with stomach fluids (immediate release tablets) or to release in a controlled manner (controlled release tablets) over a prolonged period of time or with a specific region of the Gl tract.

[0284] Capsule formulations may be of the hard gelatin or soft gelatin variety and can contain the active component in solid, semi-solid, or liquid form. Gelatin capsules can be formed from animal gelatin or synthetic or plant derived equivalents thereof.

[0285] The solid dosage forms (e.g. tablets, capsules etc.) can be coated or un-coated. Coatings may act either as a protective film (e.g. a polymer, wax or varnish) or as a mechanism for controlling drug release or for aesthetic or identification purposes. The coating (e.g. a Eudragit ™ type polymer) can be designed to release the active component at a desired location within the gastro-intestinal tract. Thus, the coating can be selected so as to degrade under certain pH conditions within the gastrointestinal tract, thereby selectively release the compound in the stomach or in the ileum, duodenum, jejenum or colon.

[0286] Instead of, or in addition to, a coating, the drug can be presented in a solid matrix comprising a release controlling agent, for example a release delaying agent which may be adapted to release the compound in a controlled manner in the gastrointestinal tract. Alternatively the drug can be presented in a polymer coating e.g. a polymethacrylate polymer coating, which may be adapted to selectively release the compound under conditions of varying acidity or alkalinity in the gastrointestinal tract. Alternatively, the matrix material or release retarding coating can take the form of an erodible polymer (e.g. a maleic anhydride polymer) which is substantially continuously eroded as the dosage form passes through the gastrointestinal tract. In another alternative, the coating can be designed to disintegrate under microbial action in the gut. As a further alternative, the active compound can be formulated in a delivery system that provides osmotic control of the release of the compound. Osmotic release and other delayed release or sustained release formulations (for example formulations based on ion exchange resins) may be prepared in accordance with methods well known to those skilled in the art.

[0287] The compound of formula (I) or drug conjugate may be formulated with a carrier and administered in the form of nanoparticles, the increased surface area of the nanoparticles assisting their absorption. In addition, nanoparticles offer the possibility of direct penetration into the cell. Nanoparticle drug delivery systems are described in “Nanoparticle TechnologyART-C-P3900PCT

[0288] for Drug Delivery”, edited by Ram B Gupta and Uday B. Kompella, Informa Healthcare, ISBN 9781574448573, published 13thMarch 2006. Nanoparticles for drug delivery are also described in J. Control. Release, 2003, 91 (1-2), 167-172, and in Sinha etal., Mol. Cancer Ther. August 1, (2006) 5, 1909.

[0289] The pharmaceutical compositions typically comprise from approximately 1% (w / w) to approximately 95% (w / w) active ingredient and from 99% (w / w) to 5% (w / w) of a pharmaceutically acceptable excipient or combination of excipients. Particularly, the compositions comprise from approximately 20% (w / w) to approximately 90%,% (w / w) active ingredient and from 80% (w / w) to 10% of a pharmaceutically acceptable excipient or combination of excipients. The pharmaceutical compositions comprise from approximately 1% to approximately 95%, particularly from approximately 20% to approximately 90%, active ingredient. Pharmaceutical compositions according to the invention may be, for example, in unit dose form, such as in the form of ampoules, vials, suppositories, pre-filled syringes, dragees, tablets or capsules.

[0290] The pharmaceutically acceptable excipient(s) can be selected according to the desired physical form of the formulation and can, for example, be selected from diluents (e.g. solid diluents such as fillers or bulking agents; and liquid diluents such as solvents and cosolvents), disintegrants, buffering agents, lubricants, flow aids, release controlling (e.g. release retarding or delaying polymers or waxes) agents, binders, granulating agents, pigments, plasticizers, antioxidants, preservatives, flavouring agents, taste masking agents, tonicity adjusting agents and coating agents.

[0291] The skilled person will have the expertise to select the appropriate amounts of ingredients for use in the formulations. For example, tablets and capsules typically contain 0-20% disintegrants, 0-5% lubricants, 0-5% flow aids and / or 0-99% (w / w) fillers / or bulking agents (depending on drug dose). They may also contain 0-10% (w / w) polymer binders, 0-5% (w / w) antioxidants, 0-5% (w / w) pigments. Slow release tablets would in addition contain 0-99% (w / w) release-controlling (e.g. delaying) polymers (depending on dose). The film coats of the tablet or capsule typically contain 0-10% (w / w) polymers, 0-3% (w / w) pigments, and / or 0-2% (w / w) plasticizers.

[0292] Parenteral formulations typically contain 0-20% (w / w) buffers, 0-50% (w / w) cosolvents, and / or 0-99% (w / w) Water for Injection (WFI) (depending on dose and if freeze dried).

[0293] Formulations for intramuscular depots may also contain 0-99% (w / w) oils.ART-C-P3900PCT

[0294] Pharmaceutical compositions for oral administration can be obtained by combining the active ingredient with solid carriers, if desired granulating a resulting mixture, and processing the mixture, if desired or necessary, after the addition of appropriate excipients, into tablets, dragee cores or capsules. It is also possible for them to be incorporated into a polymer or waxy matrix that allow the active ingredients to diffuse or be released in measured amounts.

[0295] The compounds of the invention can also be formulated as solid dispersions. Solid dispersions are homogeneous extremely fine disperse phases of two or more solids. Solid solutions (molecularly disperse systems), one type of solid dispersion, are well known for use in pharmaceutical technology (see (Chiou and Riegelman, J. Pharm. Sci., 60, 1281 -1300 (1971)) and are useful in increasing dissolution rates and increasing the bioavailability of poorly water-soluble drugs.

[0296] This invention also provides solid dosage forms comprising the solid solution described above. Solid dosage forms include tablets, capsules, chewable tablets and dispersible or effervescent tablets. Known excipients can be blended with the solid solution to provide the desired dosage form. For example, a capsule can contain the solid solution blended with (a) a disintegrant and a lubricant, or (b) a disintegrant, a lubricant and a surfactant. In addition, a capsule can contain a bulking agent, such as lactose or microcrystalline cellulose. A tablet can contain the solid solution blended with at least one disintegrant, a lubricant, a surfactant, a bulking agent and a glidant. A chewable tablet can contain the solid solution blended with a bulking agent, a lubricant, and if desired an additional sweetening agent (such as an artificial sweetener), and suitable flavours. Solid solutions may also be formed by spraying solutions of drug and a suitable polymer onto the surface of inert carriers such as sugar beads (‘nonpareils’). These beads can subsequently be filled into capsules or compressed into tablets.

[0297] The pharmaceutical formulations may be presented to a patient in “patient packs” containing an entire course of treatment in a single package, usually a blister pack. Patient packs have an advantage over traditional prescriptions, where a pharmacist divides a patient’s supply of a pharmaceutical from a bulk supply, in that the patient always has access to the package insert contained in the patient pack, normally missing in patient prescriptions. The inclusion of a package insert has been shown to improve patient compliance with the physician’s instructions.

[0298] Compositions for topical use and nasal delivery include ointments, creams, sprays, patches, gels, liquid drops and inserts (for example intraocular inserts). Such compositions can be formulated in accordance with known methods.ART-C-P3900PCT

[0299] Examples of formulations for rectal or intra-vaginal administration include pessaries and suppositories which may be, for example, formed from a shaped moldable or waxy material containing the active compound. Solutions of the active compound may also be used for rectal administration.

[0300] Compositions for administration by inhalation may take the form of inhalable powder compositions or liquid or powder sprays, and can be administrated in standard form using powder inhaler devices or aerosol dispensing devices. Such devices are well known. For administration by inhalation, the powdered formulations typically comprise the active compound together with an inert solid powdered diluent such as lactose.

[0301] The compounds of the formula (I) or drug conjugates will generally be presented in unit dosage form and, as such, will typically contain sufficient compound to provide a desired level of biological activity. For example, a formulation may contain from 1 nanogram to 2 grams of active ingredient, e.g. from 1 nanogram to 2 milligrams of active ingredient. Within these ranges, particular sub-ranges of compound are 0.1 milligrams to 2 grams of active ingredient (more usually from 10 milligrams to 1 gram, e.g. 50 milligrams to 500 milligrams), or 1 microgram to 20 milligrams (for example 1 microgram to 10 milligrams, e.g. 0.1 milligrams to 2 milligrams of active ingredient).

[0302] For oral compositions, a unit dosage form may contain from 1 milligram to 2 grams, more typically 10 milligrams to 1 gram, for example 50 milligrams to 1 gram, e.g. 100 miligrams to 1 gram, of active compound.

[0303] The active compound will be administered to a patient in need thereof (for example a human or animal patient) in an amount sufficient to achieve the desired therapeutic effect.

[0304] Anti-Cancer Treatment

[0305] The compounds of the invention, subgroups and examples thereof, are inhibitors of PARP1 and / or PARP2, and which may be useful in preventing or treating disease states or conditions described herein. In addition, the compounds of the invention, and subgroups thereof, will be useful in preventing or treating diseases or condition mediated by PARP1 and / or PARP2. References to the preventing or prophylaxis or treatment of a disease state or condition such as cancer include within their scope alleviating or reducing the incidence of cancer.ART-C-P3900PCT

[0306] Thus, for example, it is envisaged that the compounds of the invention will be useful in alleviating or reducing the incidence of cancer.

[0307] The compounds are generally administered to a subject in need of such administration, for example a human or animal patient, particularly a human.

[0308] The compounds of the present invention may be useful for the treatment of the adult population. The compounds of the present invention may be useful for the treatment of the pediatric population.

[0309] According to a further aspect of the invention there is a provided a compound of formula (I) as defined herein or a drug conjugate as defined herein, for use in the treatment of cancer.

[0310] According to a further aspect of the invention there is a provided a compound of formula (I) as defined herein or a drug conjugate as defined herein, for use in the treatment of tumours which overexpress PARP1 and / or PARP2.

[0311] A further aspect provides the use of a compound of formula (I) as defined herein or a drug conjugate as defined herein for the manufacture of a medicament for the treatment of a disease or condition as described herein, in particular cancer.

[0312] A further aspect provides a method of treatment of a disease or condition as described herein, in particular cancer, which comprises administering to a patient in need thereof a compound of formula (I) as defined herein or a drug conjugate as defined herein.

[0313] Thus, according to a further aspect of the invention there is provided a method of treating cancer in a patient, the method comprising administering to the patient a therapeutically effective amount of a compound of formula (I) as defined herein or the drug conjugate as defined herein.

[0314] According to a further aspect of the invention there is provided a method of treating cancer in a patient, the method comprising administering to the patient a therapeutically effective amount of a pharmaceutical composition as defined herein.

[0315] Examples of cancers (and their benign counterparts) which may be treated (or inhibited) include, but are not limited to tumours of epithelial origin (adenomas and carcinomas of various types including adenocarcinomas, squamous carcinomas, transitional cellART-C-P3900PCT

[0316] carcinomas and other carcinomas) such as carcinomas of the bladder and urinary tract, breast, gastrointestinal tract (including the esophagus, stomach (gastric), small intestine, colon, rectum and anus), liver (hepatocellular carcinoma), gall bladder and biliary system, exocrine pancreas, kidney, lung (for example adenocarcinomas, small cell lung carcinomas, non-small cell lung carcinomas, bronchioalveolar carcinomas and mesotheliomas), head and neck (for example cancers of the tongue, buccal cavity, larynx, pharynx, nasopharynx, tonsil, salivary glands, nasal cavity and paranasal sinuses), ovary, fallopian tubes, peritoneum, vagina, vulva, penis, cervix, myometrium, endometrium, thyroid (for example thyroid follicular carcinoma), adrenal, prostate, skin and adnexae (for example melanoma, basal cell carcinoma, squamous cell carcinoma, keratoacanthoma, dysplastic naevus); haematological malignancies (i.e. leukemias, lymphomas) and premalignant haematological disorders and disorders of borderline malignancy including haematological malignancies and related conditions of lymphoid lineage (for example acute lymphocytic leukemia [ALL], chronic lymphocytic leukemia [CLL], B-cell lymphomas such as diffuse large B-cell lymphoma [DLBCL], follicular lymphoma, Burkitt’s lymphoma, mantle cell lymphoma, MALT lymphoma, T-cell lymphomas and leukaemias, natural killer [NK] cell lymphomas, Hodgkin’s lymphomas, hairy cell leukaemia, monoclonal gammopathy of uncertain significance, plasmacytoma, multiple myeloma, and post-transplant lymphoproliferative disorders), and haematological malignancies and related conditions of myeloid lineage (for example acute myelogenous leukemia [AML], chronic myelogenous leukemia [CML], chronic myelomonocytic leukemia [CMML], hypereosinophilic syndrome, myeloproliferative disorders such as polycythaemia vera, essential thrombocythaemia and primary myelofibrosis, myeloproliferative syndrome, myelodysplastic syndrome, and promyelocytic leukemia); tumours of mesenchymal origin, for example sarcomas of soft tissue, bone or cartilage such as osteosarcomas, fibrosarcomas, chondrosarcomas, rhabdomyosarcomas, leiomyosarcomas, liposarcomas, angiosarcomas, Kaposi’s sarcoma, Ewing’s sarcoma, synovial sarcomas, epithelioid sarcomas, gastrointestinal stromal tumours, benign and malignant histiocytomas, and dermatofibrosarcoma protuberans; tumours of the central or peripheral nervous system (for example astrocytomas, gliomas and glioblastomas, meningiomas, ependymomas, pineal tumours and schwannomas); endocrine tumours (for example pituitary tumours, adrenal tumours, islet cell tumours, parathyroid tumours, carcinoid tumours and medullary carcinoma of the thyroid); ocular and adnexal tumours (for example retinoblastoma); germ cell and trophoblastic tumours (for example teratomas, seminomas, dysgerminomas, hydatidiform moles and choriocarcinomas); and paediatric and embryonal tumours (for example medulloblastoma, neuroblastoma, Wilms tumour, and primitive neuroectodermal tumours); or syndromes, congenital or otherwise, which leave the patient susceptible to malignancy (for example Xeroderma Pigmentosum).ART-C-P3900PCT

[0317] Many diseases are characterized by persistent and unregulated angiogenesis. Chronic proliferative diseases are often accompanied by profound angiogenesis, which can contribute to or maintain an inflammatory and / or proliferative state, or which leads to tissue destruction through the invasive proliferation of blood vessels. Tumour growth and metastasis have been found to be angiogenesis-dependent. Compounds of the invention may therefore be useful in preventing and disrupting initiation of tumour angiogenesis. In particular, the compounds of the invention may be useful in the treatment of metastasis and metastatic cancers.

[0318] Metastasis or metastatic disease is the spread of a disease from one organ or part to another non-adjacent organ or part. The cancers which can be treated by the compounds of the invention include primary tumours (i.e. cancer cells at the originating site), local invasion (cancer cells which penetrate and infiltrate surrounding normal tissues in the local area), and metastatic (or secondary) tumours ie. tumours that have formed from malignant cells which have circulated through the bloodstream (haematogenous spread) or via lymphatics or across body cavities (trans-coelomic) to other sites and tissues in the body.

[0319] Particular cancers include hepatocellular carcinoma, melanoma, oesophageal, renal, colon, colorectal, lung e.g. mesothelioma or lung adenocarcinoma, breast, bladder, gastrointestinal, ovarian and prostate cancers.

[0320] The compounds may also be useful in the treatment of tumour growth, pathogenesis, resistance to chemo- and radio-therapy by sensitising cells to chemotherapy and as an anti-metastatic agent.

[0321] The potency of the compounds of the invention as inhibitors of PARP1 and / or PARP2 can be measured using the biological and biophysical assays set forth in the examples herein and the level of affinity exhibited by a given compound can be defined in terms of the IC50 value. Particular compounds of the present invention are compounds having an IC50 value of less than 1 M, more particularly less than 0.1 pM.

[0322] The compounds will typically be administered in amounts that are therapeutically or prophylactically useful and which generally are non-toxic. However, in certain situations (for example in the case of life threatening diseases), the benefits of administering a compound of formula (I) or the drug conjugate may outweigh the disadvantages of any toxic effects orART-C-P3900PCT

[0323] side effects, in which case it may be considered desirable to administer compounds in amounts that are associated with a degree of toxicity.

[0324] The compounds may be administered over a prolonged term to maintain beneficial therapeutic effects or may be administered for a short period only. Alternatively, they may be administered in a continuous manner or in a manner that provides intermittent dosing (e.g. a pulsatile manner).

[0325] A typical daily dose of the compound of formula (I) or the drug conjugate can be in the range from 100 picograms to 100 milligrams per kilogram of body weight, more typically 5 nanograms to 25 milligrams per kilogram of bodyweight, and more usually 10 nanograms to 15 milligrams per kilogram (e.g. 10 nanograms to 10 milligrams, and more typically 1 microgram per kilogram to 20 milligrams per kilogram, for example 1 microgram to 10 milligrams per kilogram) per kilogram of bodyweight although higher or lower doses may be administered where required. The compound of the formula (I) or the drug conjugate can be administered on a daily basis or on a repeat basis every 2, or 3, or 4, or 5, or 6, or 7, or 10 or 14, or 21, or 28 days for example.

[0326] The compounds of the invention may be administered orally in a range of doses, for example 1 to 1500 mg, 2 to 800 mg, or 5 to 500 mg, e.g. 2 to 200 mg or 10 to 1000 mg, particular examples of doses including 10, 20, 50 and 80 mg. The compound may be administered once or more than once each day. The compound can be administered continuously (i.e. taken every day without a break for the duration of the treatment regimen). Alternatively, the compound can be administered intermittently (i.e. taken continuously for a given period such as a week, then discontinued for a period such as a week and then taken continuously for another period such as a week and so on throughout the duration of the treatment regimen). Examples of treatment regimens involving intermittent administration include regimens wherein administration is in cycles of one week on, one week off; or two weeks on, one week off; or three weeks on, one week off; or two weeks on, two weeks off; or four weeks on two weeks off; or one week on three weeks off - for one or more cycles, e.g. 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more cycles.

[0327] In one particular dosing schedule, a patient will be given an infusion of a compound of the formula (I) or the drug conjugate for periods of one hour daily for up to ten days in particular up to five days for one week, and the treatment repeated at a desired interval such as two to four weeks, in particular every three weeks.ART-C-P3900PCT

[0328] More particularly, a patient may be given an infusion of a compound of the formula (I) or the drug conjugate for periods of one hour daily for 5 days and the treatment repeated every three weeks.

[0329] In another particular dosing schedule, a patient is given an infusion over 30 minutes to 1 hour followed by maintenance infusions of variable duration, for example 1 to 5 hours, e.g. 3 hours.

[0330] In a further particular dosing schedule, a patient is given a continuous infusion for a period of 12 hours to 5 days, an in particular a continuous infusion of 24 hours to 72 hours.

[0331] In another particular dosing schedule, a patient is given the compound orally once a week.

[0332] In another particular dosing schedule, a patient is given the compound orally once-daily for between 7 and 28 days such as 7, 14 or 28 days.

[0333] In another particular dosing schedule, a patient is given the compound orally once-daily for 1 day, 2 days, 3 days, 5 days or 1 week followed by the required amount of days off to complete a one or two week cycle.

[0334] In another particular dosing schedule, a patient is given the compound orally once-daily for 2 weeks followed by 2 weeks off.

[0335] In another particular dosing schedule, a patient is given the compound orally once-daily for 2 weeks followed by 1 week off.

[0336] In another particular dosing schedule, a patient is given the compound orally once-daily for 1 week followed by 1 week off.

[0337] Ultimately, however, the quantity of compound administered and the type of composition used will be commensurate with the nature of the disease or physiological condition being treated and will be at the discretion of the physician.

[0338] It will be appreciated that PARP1 and / or PARP 2 inhibitors can be used as a single agent or in combination with other anticancer agents. Combination experiments can be performed, for example, as described in Chou TC, Talalay P. Quantitative analysis of dose-effectART-C-P3900PCT

[0339] relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regulat 1984;22: 27-55.

[0340] The compounds as defined herein can be administered as the sole therapeutic agent or they can be administered in combination therapy with one of more other compounds (or therapies) for treatment of a particular disease state, for example a neoplastic disease such as a cancer as hereinbefore defined. For the treatment of the above conditions, the compounds of the invention may be advantageously employed in combination with one or more other medicinal agents, more particularly, with other anti-cancer agents or adjuvants (supporting agents in the therapy) in cancer therapy. Examples of other therapeutic agents or treatments that may be administered together (whether concurrently or at different time intervals) with the compounds of the formula (I) or the drug conjugate include but are not limited to:

[0341] • Topoisomerase I inhibitors;

[0342] • Antimetabolites;

[0343] • Tubulin targeting agents;

[0344] • DNA binder and topoisomerase II inhibitors;

[0345] • Alkylating agents;

[0346] • Monoclonal antibodies;

[0347] • Anti-hormones;

[0348] • Signal Transduction inhibitors;

[0349] • Proteasome inhibitors;

[0350] • DNA methyl transferase inhibitors;

[0351] • Cytokines and retinoids;

[0352] • Chromatin targeted therapies;

[0353] • Radiotherapy; and

[0354] • Other therapeutic or prophylactic agents.

[0355] Particular examples of anti-cancer agents or adjuvants (or salts thereof), include but are not limited to any of the agents selected from groups (i)-(xlvi), and optionally group (xlvii), below: (i) Platinum compounds, for example cisplatin (optionally combined with amifostine), carboplatin or oxaliplatin;

[0356] (ii) Taxane compounds, for example paclitaxel, paclitaxel protein bound particles (Abraxane™), docetaxel, cabazitaxel or larotaxel;

[0357] (iii) Topoisomerase I inhibitors, for example camptothecin compounds, for example camptothecin, irinotecan(CPT11), SN-38, ortopotecan;ART-C-P3900PCT

[0358] (iv) Topoisomerase II inhibitors, for example anti-tumour epipodophyllotoxins or podophyllotoxin derivatives for example etoposide, or teniposide;

[0359] (v) Vinca alkaloids, for example vinblastine, vincristine, liposomal vincristine (Onco-TCS), vinorelbine, vindesine, vinflunine or vinvesir;

[0360] (vi) Nucleoside derivatives, for example 5-fluorouracil (5-Fll, optionally in combination with leucovorin), gemcitabine, capecitabine, tegafur, UFT, S1, cladribine, cytarabine (Ara-C, cytosine arabinoside), fludarabine, clofarabine, or nelarabine;

[0361] (vii) Antimetabolites, for example clofarabine, aminopterin, or methotrexate, azacitidine, cytarabine, floxuridine, pentostatin, thioguanine, thiopurine, 6-mercaptopurine, or hydroxyurea (hydroxycarbamide);

[0362] (viii) Alkylating agents, such as nitrogen mustards or nitrosourea, for example cyclophosphamide, chlorambucil, carmustine (BCNll), bendamustine, thiotepa, melphalan, treosulfan, lomustine (CCNll), altretamine, busulfan, dacarbazine, estramustine, fotemustine, ifosfamide (optionally in combination with mesna), pipobroman, procarbazine, streptozocin, temozolomide, uracil, mechlorethamine, methylcyclohexylchloroethylnitrosurea, or nimustine (ACNll);

[0363] (ix) Anthracyclines, anthracenediones and related drugs, for example daunorubicin, doxorubicin (optionally in combination with dexrazoxane), liposomal formulations of doxorubicin (eg. Caelyx™, Myocet™, Doxil™), idarubicin, mitoxantrone, epirubicin, amsacrine, or valrubicin;

[0364] (x) Epothilones, for example ixabepilone, patupilone, BMS-310705, KOS-862 and ZK-EPO, epothilone A, epothilone B, desoxyepothilone B (also known as epothilone D or KOS- 862), aza-epothilone B (also known as BMS-247550), aulimalide, isolaulimalide, or luetherobin;

[0365] (xi) DNA methyl transferase inhibitors, for example temozolomide, azacytidine or decitabine, or SGI-110;

[0366] (xii) Antifolates, for example methotrexate, pemetrexed disodium, or raltitrexed;

[0367] (xiii) Cytotoxic antibiotics, for example antinomycin D, bleomycin, mitomycin C, dactinomycin, carminomycin, daunomycin, levamisole, plicamycin, or mithramycin; (xiv) Tubulin-binding agents, for example combrestatin, colchicines or nocodazole;

[0368] (xv) Signal Transduction inhibitors such as Kinase inhibitors (e.g. EGFR (epithelial growth factor receptor) inhibitors, VEGFR (vascular endothelial growth factor receptor) inhibitors, PDGFR (platelet-derived growth factor receptor) inhibitors, MTKI (multi target kinase inhibitors), Raf inhibitors, mTOR inhibitors for example imatinib mesylate, erlotinib, gefitinib, dasatinib, lapatinib, dovotinib, axitinib, nilotinib, vandetanib, vatalinib, pazopanib, sorafenib, sunitinib, temsirolimus, everolimus (RAD 001), vemurafenib (PLX4032 / RG7204), dabrafenib, encorafenib or an IKB kinase inhibitor such as SAR-ART-C-P3900PCT

[0369] 113945, bardoxolone, BMS-066, BMS-345541, IMD-0354, IMD-2560, or lMD-1041, or MEK inhibitors such as Selumetinib (AZD6244) and Trametinib (GSK121120212); (xvi) Aurora kinase inhibitors for example AT9283, barasertib (AZD1152), TAK-901, MK0457 (VX680), cenisertib (R-763), danusertib (PHA-739358), alisertib (M LN-8237), or MP- 470;

[0370] (xvii) CDK inhibitors for example AT7519, roscovitine, seliciclib, alvocidib (flavopiridol), dinaciclib (SCH-727965), 7-hydroxy-staurosporine (UCN-01), JNJ-7706621, BMS- 387032 (a.k.a. SNS-032), PHA533533, PD332991, ZK-304709, orAZD-5438;

[0371] (xviii) PKA / B inhibitors and PKB (akt) pathway inhibitors for example AKT inhibitors such as KRX-0401 (perifosine / NSC 639966), ipatasertib (GDC-0068; RG-7440), afuresertib (GSK-2110183; 2110183), MK-2206, MK-8156, AT13148, AZD-5363, triciribine phosphate (VQD-002; triciribine phosphate monohydrate (API-2; TCN-P; TCN-PM; VD- 0002), RX-0201, NL-71-101, SR-13668, PX-316, AT13148, AZ-5363, Semaphore, SF1126, or Enzastaurin HCI (LY317615) or MTOR inhibitors such as rapamycin analogues such as RAD 001 (everolimus), CCI 779 (temsirolemus), AP23573 and ridaforolimus, sirolimus (originally known as rapamycin), AP23841 and AP23573, calmodulin inhibitors e.g. CBP-501 (forkhead translocation inhibitors), enzastaurin HCI (LY317615) or PI3K Inhibitors such as dactolisib (BEZ235), buparlisib (BKM-120; NVP- BKM-120), BYL719, copanlisib (BAY-80-6946), ZSTK-474, CUDC-907, apitolisib (GDC- 0980; RG-7422), pictilisib (pictrelisib, GDC-0941, RG-7321), GDC-0032, GDC-0068, GSK-2636771, idelalisib (formerly CAL-101, GS 1101, GS-1101), MLN1117 (INK1117), MLN0128 (INK128), IPI-145 (INK1197), LY-3023414, ipatasertib, afuresertib, MK-2206, MK-8156, LY-3023414, LY294002, SF1126 or PI-103, or sonolisib (PX-866);

[0372] (xix) Hsp90 inhibitors for example AT13387, herbimycin, geldanamycin (GA), 17-allylamino- 17-desmethoxygeldanamycin (17-AAG) e.g. NSC-330507, Kos-953 and CNF-1010, 17- dimethylaminoethylamino-17-demethoxygeldanamycin hydrochloride (17-DMAG) e.g. NSC-707545 and Kos-1022, NVP-AUY922 (VER-52296), NVP-BEP800, CNF-2024 (BIIB-021 an oral purine), ganetespib (STA-9090), SNX-5422 (SC-102112) or IPI-504; (xx) Monoclonal Antibodies (unconjugated or conjugated to radioisotopes, toxins or other agents), antibody derivatives and related agents, such as anti-CD, anti-VEGFR, anti- HER2, anti-CTLA4, anti-PD-1 or anti-EGFR antibodies, for example rituximab (CD20), ofatumumab (CD20), ibritumomab tiuxetan (CD20), GA101 (CD20), tositumomab (CD20), epratuzumab (CD22), lintuzumab (CD33), gemtuzumab ozogamicin (CD33), alemtuzumab (CD52), galiximab (CD80), trastuzumab (HER2 antibody), pertuzumab (HER2), trastuzumab-DM1 (HER2), ertumaxomab (HER2 and CD3), cetuximab (EGFR), panitumumab (EGFR), necitumumab (EGFR), nimotuzumab (EGFR), bevacizumab (VEGF), catumaxumab (EpCAM and CD3), abagovomab (CA125),ART-C-P3900PCT

[0373] farletuzumab (folate receptor), elotuzumab (CS1), denosumab (RANK ligand), figitumumab (IGF1R), CP751.871 (IGF1R), mapatumumab (TRAIL receptor), metMAB (met), mitumomab (GD3 ganglioside), naptumomab estafenatox (5T4), siltuximab (IL6), or immunomodulating agents such as CTLA-4 blocking antibodies and / or antibodies against PD-1 and PD-L1 and / or PD-L2 for example ipilimumab (CTLA4), MK-3475 (pembrolizumab, formerly lambrolizumab, anti-PD-1), nivolumab (anti-PD-1), BMS- 936559 (anti- PD-L1), MPDL320A, AMP-514 or MEDI4736 (anti-PD-L1), or tremelimumab (formerly ticilimumab, CP-675,206, anti-CTLA-4);

[0374] (xxi) Estrogen receptor antagonists or selective estrogen receptor modulators (SERMs) or inhibitors of estrogen synthesis, for example tamoxifen, fulvestrant, toremifene, droloxifene, faslodex, or raloxifene;

[0375] (xxii) Aromatase inhibitors and related drugs, such as exemestane, anastrozole, letrazole, testolactone aminoglutethimide, mitotane or vorozole;

[0376] (xxiii) Antiandrogens (i.e. androgen receptor antagonists) and related agents for example bicalutamide, nilutamide, flutamide, cyproterone, or ketoconazole;

[0377] (xxiv) Hormones and analogues thereof such as medroxyprogesterone, diethylstilbestrol (a.k.a. diethylstilboestrol) or octreotide;

[0378] (xxv) Steroids for example dromostanolone propionate, megestrol acetate, nandrolone (decanoate, phenpropionate), fluoxymestrone or gossypol,

[0379] (xxvi) Steroidal cytochrome P45017alpha-hydroxylase-17,20-lyase inhibitor (CYP17), e.g.

[0380] abiraterone;

[0381] (xxvii) Gonadotropin releasing hormone agonists or antagonists (GnRAs) for example abarelix, goserelin acetate, histrelin acetate, leuprolide acetate, triptorelin, buserelin, or deslorelin;

[0382] (xxviii) Glucocorticoids, for example prednisone, prednisolone, dexamethasone;

[0383] (xxix) Differentiating agents, such as retinoids, rexinoids, vitamin D or retinoic acid and retinoic acid metabolism blocking agents (RAMBA) for example accutane, alitretinoin, bexarotene, or tretinoin;

[0384] (xxx) Farnesyltransferase inhibitors for example tipifarnib;

[0385] (xxxi) Chromatin targeted therapies such as histone deacetylase (HDAC) inhibitors for example panobinostat, resminostat, abexinostat, vorinostat, romidepsin, belinostat, entinostat, quisinostat, pracinostat, tefinostat, mocetinostat, givinostat, CU DC-907, CUDC-101, ACY-1215, MGCD-290, EVP-0334, RG-2833, 4SC-202, romidepsin, AR-42 (Ohio State University), CG-200745, valproic acid, CKD-581, sodium butyrate, suberoylanilide hydroxamide acid (SAHA), depsipeptide (FR 901228), dacinostat (NVP- LAQ824), R306465 / JNJ-16241199, JNJ-26481585, trichostatin A, chlamydocin, A-173, JNJ-MGCD-0103, PXD-101, or apicidin;ART-C-P3900PCT

[0386] (xxxii) Proteasome Inhibitors for example bortezomib, carfilzomib, delanzomib (CEP- 18770), ixazomib (MLN -9708), oprozomib (ONX-0912) or marizomib;

[0387] (xxxiii) Photodynamic drugs for example porfimer sodium or temoporfin;

[0388] (xxxiv) Marine organism-derived anticancer agents such as trabectidin;

[0389] (xxxv) Radiolabelled drugs for radioimmunotherapy for example with a beta particle-emitting isotope (e.g. Iodine -131, Yittrium-90) or an alpha particle-emitting isotope (e.g.

[0390] Bismuth-213 or Actinium-225) for example ibritumomab or Iodine tositumomab;

[0391] (xxxvi) Telomerase inhibitors for example telomestatin;

[0392] (xxxvii) Matrix metalloproteinase inhibitors for example batimastat, marimastat, prinostat or metastat;

[0393] (xxxviii) Recombinant interferons (such as interferon-y and interferon a) and interleukins (e.g. interleukin 2), for example aldesleukin, denileukin diftitox, interferon alfa 2a, interferon alfa 2b, or peginterferon alfa 2b;

[0394] (xxxix) Selective immunoresponse modulators for example thalidomide, or lenalidomide; (xl) Therapeutic Vaccines such as sipuleucel-T (Provenge) or OncoVex;

[0395] (xli) Cytokine-activating agents include Picibanil, Romurtide, Sizofiran, Virulizin, or Thymosin;

[0396] (xlii) Arsenic trioxide;

[0397] (xliii) Inhibitors of G-protein coupled receptors (GPCR) for example atrasentan;

[0398] (xliv) Enzymes such as L-asparaginase, pegaspargase, rasburicase, or pegademase;

[0399] (xlv) DNA repair inhibitors such as PARP inhibitors for example, olaparib, velaparib, iniparib, rucaparib (AG-014699 or PF-01367338), talazoparib or AG-014699;

[0400] (xlvi)DNA damage response inhibitors such as ATM inhibitors AZD0156 MS3541, ATR inhibitors AZD6738, M4344, M6620wee1 inhibitor AZD1775;

[0401] (xlvii) Agonists of Death receptor (e.g. TNF-related apoptosis inducing ligand (TRAIL) receptor), such as mapatumumab (formerly HGS-ETR1), conatumumab (formerly AMG 655), PRO95780, lexatumumab, dulanermin, CS-1008, apomab or recombinant TRAIL ligands such as recombinant Human TRAIL / Apo2 Ligand;

[0402] (xlviii) Prophylactic agents (adjuncts); i.e. agents that reduce or alleviate some of the side effects associated with chemotherapy agents, for example

[0403] - anti-emetic agents,

[0404] - agents that prevent or decrease the duration of chemotherapy-associated neutropenia and prevent complications that arise from reduced levels of platelets, red blood cells or white blood cells, for example interleukin-11 (e.g. oprelvekin), erythropoietin (EPO) and analogues thereof (e.g. darbepoetin alfa), colonystimulating factor analogs such as granulocyte macrophage-colony stimulating factorART-C-P3900PCT

[0405] (GM-CSF) (e.g. sargramostim), and granulocyte-colony stimulating factor (G-CSF) and analogues thereof (e.g. filgrastim, pegfilgrastim),

[0406] - agents that inhibit bone resorption such as denosumab or bisphosphonates e.g. zoledronate, zoledronic acid, pamidronate and ibandronate,

[0407] - agents that suppress inflammatory responses such as dexamethasone, prednisone, and prednisolone,

[0408] - agents used to reduce blood levels of growth hormone and IGF-I (and other hormones) in patients with acromegaly or other rare hormone-producing tumours, such as synthetic forms of the hormone somatostatin e.g. octreotide acetate, - antidote to drugs that decrease levels of folic acid such as leucovorin, or folinic acid, - agents for pain e.g. opiates such as morphine, diamorphine and fentanyl,

[0409] - non-steroidal anti-inflammatory drugs (NSAID) such as COX-2 inhibitors for example celecoxib, etoricoxib and lumiracoxib,

[0410] - agents for mucositis e.g. palifermin,

[0411] - agents for the treatment of side-effects including anorexia, cachexia, oedema or thromoembolic episodes, such as megestrol acetate.

[0412] In one embodiment the anticancer agent is selected from recombinant interferons (such as interferon-y and interferon a) and interleukins (e.g. interleukin 2), for example aldesleukin, denileukin diftitox, interferon alfa 2a, interferon alfa 2b, or peginterferon alfa 2b; interferon-a2 (500 p / ml) in particular interferon-P; and signal transduction inhibitors such as kinase inhibitors (e.g. EGFR (epithelial growth factor receptor) inhibitors, VEGFR (vascular endothelial growth factor receptor) inhibitors, PDGFR (platelet-derived growth factor receptor) inhibitors, MTKI (multi target kinase inhibitors), Raf inhibitors, mTOR inhibitors for example imatinib mesylate, erlotinib, gefitinib, dasatinib, lapatinib, dovotinib, axitinib, nilotinib, vandetanib, vatalinib, pazopanib, sorafenib, sunitinib, temsirolimus, everolimus (RAD 001), vemurafenib (PLX4032 / RG7204), dabrafenib, encorafenib or an IKB kinase inhibitor such as SAR-113945, bardoxolone, BMS-066, BMS-345541, IMD-0354, IMD-2560, or IMD-1041, or MEK inhibitors such as Selumetinib (AZD6244) and Trametinib (GSK121120212), in particular Raf inhibitors (e.g. vemurafenib) or MEK inhibitors (e.g. trametinib).

[0413] Each of the compounds present in the combinations of the invention may be given in individually varying dose schedules and via different routes. As such, the posology of each of the two or more agents may differ: each may be administered at the same time or at different times. A person skilled in the art would know through his or her common general knowledge the dosing regimes and combination therapies to use. For example, theART-C-P3900PCT

[0414] compound of the invention may be using in combination with one or more other agents which are administered according to their existing combination regimen. Examples of standard combination regimens are provided below.

[0415] The taxane compound is advantageously administered in a dosage of 50 to 400 mg per square meter (mg / m2) of body surface area, for example 75 to 250 mg / m2, particularly for paclitaxel in a dosage of about 175 to 250 mg / m2and for docetaxel in about 75 to 150 mg / m2per course of treatment.

[0416] The camptothecin compound is advantageously administered in a dosage of 0.1 to

[0417] 400 mg per square meter (mg / m2) of body surface area, for example 1 to 300 mg / m2, particularly for irinotecan in a dosage of about 100 to 350 mg / m2and for topotecan in about 1 to 2 mg / m2per course of treatment.

[0418] The anti-tumour podophyllotoxin derivative is advantageously administered in a dosage of 30 to 300 mg per square meter (mg / m2) of body surface area, for example 50 to 250mg / m2, particularly for etoposide in a dosage of about 35 to 100 mg / m2and for teniposide in about 50 to 250 mg / m2per course of treatment.

[0419] The anti-tumour vinca alkaloid is advantageously administered in a dosage of 2 to

[0420] 30 mg per square meter (mg / m2) of body surface area, particularly for vinblastine in a dosage of about 3 to 12 mg / m2, for vincristine in a dosage of about 1 to 2 mg / m2, and for vinorelbine in dosage of about 10 to 30 mg / m2per course of treatment.

[0421] The anti-tumour nucleoside derivative is advantageously administered in a dosage of 200 to 2500 mg per square meter (mg / m2) of body surface area, for example 700 to

[0422] 1500 mg / m2, particularly for 5-Fll in a dosage of 200 to 500mg / m2, for gemcitabine in a dosage of about 800 to 1200 mg / m2and for capecitabine in about 1000 to

[0423] 2500 mg / m2per course of treatment.

[0424] The alkylating agents such as nitrogen mustard or nitrosourea is advantageously administered in a dosage of 100 to 500 mg per square meter (mg / m2) of body surface area, for example 120 to 200 mg / m2, particularly for cyclophosphamide in a dosage of about 100 to 500 mg / m2, for chlorambucil in a dosage of about 0.1 to 0.2 mg / kg, for carmustine in a dosage of about 150 to 200 mg / m2, and for lomustine in a dosage of about 100 to 150 mg / m2per course of treatment.ART-C-P3900PCT

[0425] The anti-tumour anthracycline derivative is advantageously administered in a dosage of 10 to 75 mg per square meter (mg / m2) of body surface area, for example 15 to

[0426] 60 mg / m2, particularly for doxorubicin in a dosage of about 40 to 75 mg / m2, for daunorubicin in a dosage of about 25 to 45mg / m2, and for idarubicin in a dosage of about 10 to 15 mg / m2per course of treatment.

[0427] The antiestrogen agent is advantageously administered in a dosage of about 1 to 100 mg daily depending on the particular agent and the condition being treated. Tamoxifen is advantageously administered orally in a dosage of 5 to 50 mg, particularly 10 to 20 mg twice a day, continuing the therapy for sufficient time to achieve and maintain a therapeutic effect. Toremifene is advantageously administered orally in a dosage of about 60mg once a day, continuing the therapy for sufficient time to achieve and maintain a therapeutic effect.

[0428] Anastrozole is advantageously administered orally in a dosage of about 1mg once a day. Droloxifene is advantageously administered orally in a dosage of about 20-1 OOmg once a day. Raloxifene is advantageously administered orally in a dosage of about 60mg once a day. Exemestane is advantageously administered orally in a dosage of about 25mg once a day.

[0429] Antibodies are advantageously administered in a dosage of about 1 to 5 mg per square meter (mg / m2) of body surface area, or as known in the art, if different. Trastuzumab is advantageously administered in a dosage of 1 to 5 mg per square meter (mg / m2) of body surface area, particularly 2 to 4mg / m2per course of treatment.

[0430] Where the compound of the formula (I) or the drug conjugate is administered in combination therapy with one, two, three, four or more other therapeutic agents (particularly one or two, more particularly one), the compounds can be administered simultaneously or sequentially. In the latter case, the two or more compounds will be administered within a period and in an amount and manner that is sufficient to ensure that an advantageous or synergistic effect is achieved. When administered sequentially, they can be administered at closely spaced intervals (for example over a period of 5-10 minutes) or at longer intervals (for example 1, 2, 3, 4 or more hours apart, or even longer periods apart where required), the precise dosage regimen being commensurate with the properties of the therapeutic agent(s). These dosages may be administered for example once, twice or more per course of treatment, which may be repeated for example every 7, 14, 21 or 28 days.

[0431] In one embodiment is provided a compound of formula (I) or the drug conjugate for the manufacture of a medicament for use in therapy wherein said compound is used inART-C-P3900PCT

[0432] combination with one, two, three, or four other therapeutic agents. In another embodiment is provided a medicament for treating cancer which comprises a compound of formula (I) or the drug conjugate wherein said medicament is used in combination with one, two, three, or four other therapeutic agents. The invention further provides use of a compound of formula (I) or the drug conjugate for the manufacture of a medicament for enhancing or potentiating the response rate in a patient suffering from a cancer where the patient is being treated with one, two, three, or four other therapeutic agents.

[0433] It will be appreciated that the particular method and order of administration and the respective dosage amounts and regimes for each component of the combination will depend on the particular other medicinal agent and compound of the present invention being administered, their route of administration, the particular tumour being treated and the particular host being treated. The optimum method and order of administration and the dosage amounts and regime can be readily determined by those skilled in the art using conventional methods and in view of the information set out herein.

[0434] The weight ratio of the compound according to the present invention and the one or more other anticancer agent(s) when given as a combination may be determined by the person skilled in the art. Said ratio and the exact dosage and frequency of administration depends on the particular compound according to the invention and the other anticancer agent(s) used, the particular condition being treated, the severity of the condition being treated, the age, weight, gender, diet, time of administration and general physical condition of the particular patient, the mode of administration as well as other medication the individual may be taking, as is well known to those skilled in the art. Furthermore, it is evident that the effective daily amount may be lowered or increased depending on the response of the treated subject and / or depending on the evaluation of the physician prescribing the compounds of the instant invention. A particular weight ratio for the present compound of formula (I) or the drug conjugate and another anticancer agent may range from 1 / 10 to 10 / 1, more in particular from 1 / 5 to 5 / 1, even more in particular from 1 / 3 to 3 / 1.

[0435] The compounds of the invention may also be administered in conjunction with non-chemotherapeutic treatments such as radiotherapy, photodynamic therapy, gene therapy; surgery and controlled diets.

[0436] The compounds of the present invention also have therapeutic applications in sensitising tumour cells for radiotherapy and chemotherapy. Hence the compounds of the present invention can be used as "radiosensitizer" and / or “chemosensitizer” or can be given inART-C-P3900PCT

[0437] combination with another "radiosensitizer" and / or “chemosensitizer”. In one embodiment the compound of the invention is for use as chemosensitiser.

[0438] The term "radiosensitizer" is defined as a molecule administered to patients in therapeutically effective amounts to increase the sensitivity of the cells to ionizing radiation and / or to promote the treatment of diseases which are treatable with ionizing radiation.

[0439] The term “chemosensitizer” is defined as a molecule administered to patients in therapeutically effective amounts to increase the sensitivity of cells to chemotherapy and / or promote the treatment of diseases which are treatable with chemotherapeutics.

[0440] In one embodiment the compound of the invention is administered with a "radiosensitizer" and / or “chemosensitizer”. In one embodiment the compound of the invention is administered with an "immune sensitizer".

[0441] The term “immune sensitizer” is defined as a molecule administered to patients in therapeutically effective amounts to increase the sensitivity of cells to a PARP1 and / or PARP2 inhibitor.

[0442] Many cancer treatment protocols currently employ radiosensitizers in conjunction with radiation of x-rays. Examples of x-ray activated radiosensitizers include, but are not limited to, the following: metronidazole, misonidazole, desmethylmisonidazole, pimonidazole, etanidazole, nimorazole, mitomycin C, RSU 1069, SR 4233, EO9, RB 6145, nicotinamide, 5-bromodeoxyuridine (BLIdR), 5- iododeoxyuridine (ILIdR), bromodeoxycytidine, fluorodeoxyuridine (FudR), hydroxyurea, cisplatin, and therapeutically effective analogs and derivatives of the same.

[0443] Photodynamic therapy (PDT) of cancers employs visible light as the radiation activator of the sensitizing agent. Examples of photodynamic radiosensitizers include the following, but are not limited to: hematoporphyrin derivatives, Photofrin, benzoporphyrin derivatives, tin etioporphyrin, pheoborbide-a, bacteriochlorophyll-a, naphthalocyanines, phthalocyanines, zinc phthalocyanine, and therapeutically effective analogs and derivatives of the same.

[0444] Radiosensitizers may be administered in conjunction with a therapeutically effective amount of one or more other compounds, including but not limited to: compounds of the invention; compounds which promote the incorporation of radiosensitizers to the target cells; compounds which control the flow of therapeutics, nutrients, and / or oxygen to the targetART-C-P3900PCT

[0445] cells; chemotherapeutic agents which act on the tumour with or without additional radiation; or other therapeutically effective compounds for treating cancer or other diseases.

[0446] Chemosensitizers may be administered in conjunction with a therapeutically effective amount of one or more other compounds, including but not limited to: compounds of the invention; compounds which promote the incorporation of chemosensitizers to the target cells; compounds which control the flow of therapeutics, nutrients, and / or oxygen to the target cells; chemotherapeutic agents which act on the tumour or other therapeutically effective compounds for treating cancer or other disease. Calcium antagonists, for example verapamil, are found useful in combination with antineoplastic agents to establish chemosensitivity in tumor cells resistant to accepted chemotherapeutic agents and to potentiate the efficacy of such compounds in drug-sensitive malignancies.

[0447] Examples of immune sensitizers include the following, but are not limited to: immunomodulating agents, for example monoclonal antibodies such as immune checkpoint antibodies [e.g. CTLA-4 blocking antibodies and / or antibodies against PD-1 and PD-L1 and / or PD-L2 for example ipilimumab (CTLA4), MK-3475 (pembrolizumab, formerly lambrolizumab, anti-PD-1), nivolumab (anti-PD-1), BMS-936559 (anti- PD-L1), MPDL320A, AMP-514 or MEDI4736 (anti-PD-L1), or tremelimumab (formerly ticilimumab, CP-675,206, anti-CTLA-4)]; or Signal Transduction inhibitors; or cytokines (such as recombinant interferons); or oncolytic viruses; or immune adjuvants (e.g. BCG).

[0448] Immune sensitizers may be administered in conjunction with a therapeutically effective amount of one or more other compounds, including but not limited to: compounds of the invention; compounds which promote the incorporation of immune sensitizers to the target cells; compounds which control the flow of therapeutics, nutrients, and / or oxygen to the target cells; therapeutic agents which act on the tumour or other therapeutically effective compounds for treating cancer or other disease.

[0449] For use in combination therapy with another chemotherapeutic agent, the compound of the formula (I) or the drug conjugate and one, two, three, four or more other therapeutic agents can be, for example, formulated together in a dosage form containing two, three, four or more therapeutic agents i.e. in a unitary pharmaceutical composition containing all agents. In an alternative embodiment, the individual therapeutic agents may be formulated separately and presented together in the form of a kit, optionally with instructions for their use.ART-C-P3900PCT

[0450] In one embodiment is provided a combination of a compound of formula (I) or the drug conjugate with one or more (e.g. 1 or 2) other therapeutic agents (e.g. anticancer agents as described above). In a further embodiment is provided a combination of a PARP1 and / or PARP2 inhibitor as described herein and a PI3K / AKT pathway inhibitor selected from: apitolisib, buparlisib, Copanlisib, pictilisib, ZSTK-474, CUDC-907, GSK-2636771, LY-3023414, ipatasertib, afuresertib, MK-2206, MK-8156, Idelalisib, BEZ235 (dactolisib), BYL719, GDC- 0980, GDC-0941, GDC-0032 and GDC-0068.

[0451] In another embodiment is provided a compound of formula (I) or the drug conjugate in combination with one or more (e.g. 1 or 2) other therapeutic agents (e.g. anticancer agents) for use in therapy, such as in the prophylaxis or treatment of cancer.

[0452] In one embodiment the pharmaceutical composition comprises a compound of formula (I) or the drug conjugate together with a pharmaceutically acceptable carrier and optionally one or more therapeutic agent(s).

[0453] In another embodiment the invention relates to the use of a combination according to the invention in the manufacture of a pharmaceutical composition for inhibiting the growth of tumour cells.

[0454] In a further embodiment the invention relates to a product containing a compound of formula (I) or the drug conjugate and one or more anticancer agent, as a combined preparation for simultaneous, separate or sequential use in the treatment of patients suffering from cancer.

[0455] EXAMPLES

[0456] The invention will now be illustrated, but not limited, by reference to the specific embodiments described in the following examples.

[0457] Abbreviations

[0458] Ac Acetyl

[0459] ADC Antibody-drug conjugate

[0460] aq. Aqueous

[0461] Bn Benzyl

[0462] Boc terf-Butyloxycarbonyl

[0463] CMPI 2-Chloro-1 -methylpyridinium iodide

[0464] DAR Drug-antibody ratio

[0465] DCC / V, / V'-DicyclohexylcarbodiimideART-C-P3900PCT

[0466] DCM Dichloromethane

[0467] DI PEA / V, / V-Diisopropylethylamine

[0468] DMAP 4-Dimethylaminopyridine

[0469] DMF Dimethylformamide

[0470] DMSO Dimethylsulfoxide

[0471] EEDQ Ethyl 2-ethoxyquinoline-1(2 / 7)-carboxylate

[0472] Et Ethyl

[0473] EtOH Ethanol

[0474] EtOAc Ethyl acetate

[0475] FA Formic acid

[0476] FLR1 Flow Reactor 1

[0477] FLR2 Flow Reactor 2

[0478] Fmoc ((9 / 7-fluoren-9-yl)methoxy)carbonyl

[0479] h Hour(s)

[0480] HATU 1-[Bis(dimethylamino)methylene]-1 / 7-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate

[0481] HMDS Bis(trimethylsilyl)amine

[0482] HOBt Hydroxybenzotriazole

[0483] HPLC High-performance liquid chromatography

[0484] I PA Propan-2-ol

[0485] Me Methyl

[0486] MeCN Acetonitrile

[0487] MeOH Methanol

[0488] min Minutes

[0489] n-Bu n-Butyl

[0490] NIS / V-iodosuccinimide

[0491] NMR Nuclear magnetic resonance

[0492] PBS Phosphate-buffered saline

[0493] PE Petroleum ether

[0494] PFA Perfluoroalkoxy alkane

[0495] Ph Phenyl

[0496] Prep Preparative

[0497] PS80 Polysorbate-80

[0498] rt Room temperature or ambient temperature

[0499] s Seconds

[0500] SFC Supercritical fluid chromatography

[0501] SS Stainless steelART-C-P3900PCT

[0502] sat. Saturated solution

[0503] TBS terf-Butyldiphenylsilyl

[0504] t-Bu terf-Butyl

[0505] TCEP 3,3',3"-Phosphanetriyltripropionic acid

[0506] TEA Triethylamine

[0507] TFA Trifluoroacetic acid

[0508] THF Tetrahydrofuran

[0509] TMEDA / V, / V, / \ / ’, / \ / ’-Tetramethylethane-1,2-diamine

[0510] TMS Trimethylsilyl

[0511] Typical Preparative HPLC method

[0512] For purification of samples by HPLC the following columns were typically used; SunFire C18, Xtimate C18, Phenomenex Gemini, Phenomenex Synergi C18, Phenomenex Luna, Waters Xbridge C18, Boston Prime C18 and Shim-pack C18. Typical mobile phases used were water and MeCN, with either acidic or basic additives, such as formic acid (0.1% v / v) or ammonium hydroxide (0.05% v / v). A typical method started with 95% water:5% MeCN and decreasingly polar ratios of water and MeCN over a period of 5 to 12 min, with a typical flow rate of 25 mL / min.

[0513] Typical SEC-HPLC method

[0514] For analysis of samples by SEC-HPLC, the typical column used was a TSKgel G3000SWXL (7.8 x 300 mm, 5 pm) column at 25 °C. Typical mobile phase was 85% 25 mM aq. sodium phosphate at pH 6.8, containing 0.15 M sodium chloride: 15% I PA, with a flow rate of 0.8 mL / min over 18 min. Samples were analysed by DAD at 280 nm.

[0515] Intermediate 1: tert-Butyldimethylsilyl dimethyl phosphite

[0516] ° a OTBS

[0517] ^-OMe - ►

[0518] H

[0519]

[0520] OMe MeO' ^OMe

[0521] Step a. To a mixture of dimethyl phosphonate (120 g, 1.09 mol) and tert-butyldimethylsilyl chloride (180 g, 1200 mmol) in DCM (800 mL) was added TEA (220 g, 2180 mmol) at 0 °C. The reaction mixture was stirred at 20 °C for 16 h. Upon completion, the reaction mixture was filtered, the filtrate was evaporated and the resulting residue was re-dissolved in a mixture of PE / EtOAc = 20 / 1 (300 mL). The mixture was filtered, the filtrate was evaporated and the crude product was distilled in vacuo (43 - 46 °C, 20 mm Hg / oil pump) to give the title compound (195 g, 79% yield) as a colorless oil.

[0522] 1H NMR (400 MHz, CDCh) 6 ppm 3.49 (s, 3H), 3.46 (s, 3H), 0.94 (s, 9H), 0.19 (s, 6H).ART-C-P3900PCT

[0523] Intermediate 2: Dimethyl (7-hydroxy-3-oxo-1,3-dihydroisobenzofuran-1-yl)phosphonate

[0524]

[0525] Step a. 3-Hydroxybenzoic acid (15.0 g, 108 mmol) was added to thionyl chloride (40 mL) at 25 °C, after which DMF (793 mg, 10.8 mmol) was added dropwise at 25 °C. The reaction mixture was stirred at 80 °C for 1 h. Upon completion, the reaction mixture was evaporated, the residue re-dissolved in toluene (100 mL) and the mixture was evaporated again to give 3-hydroxybenzoyl chloride (17 g, 90% yield) as a yellow gum which was used without further purification.

[0526] Step b. To a mixture of 3-hydroxy benzoyl chloride (17.0 g, 108 mmol) in THF (120 mL) was added diethylamine (23.8 g, 325 mmol) at 0 °C. The reaction mixture was stirred at 20 °C for 16 h. Upon completion, the reaction mixture was evaporated, and the residue was re-dissolved in DCM (100 mL), washed with H2O (200 mL), sat. aq. NaHCCh (100 mL), 20% citric acid (120 mL) and brine (150 mL). The organic layer was dried over Na2SO4 and evaporated to give / V, / V-diethyl-3-hydroxybenzamide (20.0 g, 95% yield) as a brown gum which was used without further purification.

[0527] m / z ES+ [M+H]+194.1;1H NMR (400 MHz, CDCh) 5 ppm 8.25 (br s, 1H), 7.16 (t, J= 8.0 Hz, 1 H), 6.89 (s, 1 H), 6.83 - 6.73 (m, 2H), 3.56 - 3.49 (m, 2H), 3.31 - 3.25 (m, 2H), 1.28 - 1.24 (m, 3H), 1.12 - 1.08 (m, 3H).

[0528] Step c. To a mixture of / V, / V-diethyl-3-hydroxybenzamide (14.0 g, 72.4 mmol), K2CO3 (20.1 g, 145 mmol) and KI (1.20 g, 7.24 mmol) in acetone (300 mL) was added BnBr (18.5 g, 108 mmol) at 0 °C. The reaction mixture was stirred at 20 °C for 16 h, followed by 50 °C for 2 h. Upon completion, the reaction mixture was evaporated, and the residue was re-dissolved in EtOAc (50 mL), washed with 10% NaOH (50 mL) and brine (50 mL). The organic layer was dried over Na2SO4, evaporated and purified by column chromatography (PE / EtOAc = 4 / 1) to give 3-(benzyloxy)- / V, / \ / -diethylbenzamide (11.0 g, 53% yield) as a light yellow oil.

[0529] m / z ES+ [M+H]+284.1;1H NMR (400 MHz, CDCh) 6 ppm 7.47 - 7.28 (m, 6H), 7.03 - 6.92 (m, 3H), 5.09 (s, 2H), 3.61 - 3.45 (m, 2H), 3.35 - 3.22 (m, 2H), 1.30 - 1.21 (m, 3H), 1.18 - 1.04 (m, 3H).ART-C-P3900PCT

[0530] Step d. This step was conducted under continuous flow conditions: Solution 1 (3-(benzyloxy)- / V, / V-diethylbenzamide (25.0 g, 88.2 mmol) and TMEDA (12.3 g, 105 mmol) in THF (500 mL); 42.4 mL / min) and Solution 2 (n-BuLi (1.6 M in THF, 165 mL); 3.36 mL / min) were added to Flow Reactor 1 {FLR1, PFA, Coils reactor, 1 / 16”, 2 mL, -40 °C, retention time = 2.5 s}. Then Solution 3 (DMF (19.3 g, 264 mmol, 20.3 mL) in THF (50 mL); 5.52 mL / min) was added to Flow Reactor 2 {FLR2, PFA, Coils reactor, 1 / 8”, 4 mL, -40 °C, retention time = 3.7 s}. Upon completion, the reaction mixture was quenched with sat. aq. NH4CI (200 mL) at 0 °C. The aqueous mixture was extracted with EtOAc (600 mL) and the organic layer was dried over Na2SC>4, evaporated and purified by column chromatography (PE / EtOAc = 3 / 1) to give 3-(benzyloxy)- / V, / V-diethyl-2-formylbenzamide (12.0 g, 43% yield) as a yellow gum.

[0531] m / z ES+ [M+H]+312.1;1H NMR (400 MHz, CDCh) 5 ppm 10.57 (s, 1H), 7.53 (dd, J= 8.4, 7.6 Hz, 1H), 7.50 - 7.31 (m, 5H), 7.07 (d, J= 8.0 Hz, 1H), 6.87 (d, J = 7.6 Hz, 1H), 5.22 (s, 2H), 3.60 (q, J = 7.2 Hz, 2H), 3.08 (q, J = 7.2 Hz, 2H), 1.33 (t, J = 7.2 Hz, 3H), 1.02 (t, J = 7.2 Hz, 3H).

[0532] Step e. A mixture of 3-(benzyloxy)- / V, / V-diethyl-2-formylbenzamide (9.00 g, 28.9 mmol) and Intermediate 1 (18.0 g, 80.3 mmol) in THF (80 mL) was stirred at 70 °C for 16 h. Upon completion, the reaction mixture was evaporated to give dimethyl ((2-(benzyloxy)-6-(diethylcarbamoyl)phenyl)((terf-butyldimethylsilyl)oxy)methyl)phosphonate (15.0 g, 95% yield) as a yellow solid which was used without further purification.

[0533] m / z ES+ [M+H]+536.6;1H NMR (400 MHz, CDCh) 6 ppm 7.57 (d, J= 7.6 Hz, 2H), 7.39 - 7.33 (m, 2H), 7.33 - 7.28 (m, 1 H), 7.26 - 7.21 (m, 1 H), 6.95 (d, J = 8.4 Hz, 1 H), 6.78 (d, J = 7.6 Hz, 1H), 5.32 (d, J= 15.6 Hz, 1H), 5.21 - 5.11 (m, 2H), 3.87 (qd, J= 13.6, 7.2 Hz, 1H), 3.68 (d, J = 10.4 Hz, 3H), 3.57 (d, J= 10.4 Hz, 3H), 3.31 (qd, J= 14.8, 7.2 Hz, 1H), 3.20 (qd, J= 13.6, 7.2 Hz, 1H), 3.05 (qd, J= 14.4, 7.2 Hz, 1H), 1.25 (t, J= 7.2 Hz, 3H), 1.06 (t, J= 7.2 Hz, 3H), 0.84 (s, 9H), 0.17 (s, 3H), -0.04 (s, 3H).

[0534] Step f. Methanesulfonic acid (5.38 g, 56.0 mmol) was added dropwise to a solution of dimethyl ((2-(benzyloxy)-6-(diethylcarbamoyl)phenyl)((terf-butyldimethylsilyl)oxy)methyl)phosphonate (15.0 g, 28.0 mmol) in MeOH (100 mL). The reaction mixture was stirred at 20 °C for 7 h. Upon completion, the reaction mixture was evaporated, the residue re-dissolved in H2O (500 mL) and the pH of the mixture was adjusted to pH 8 using sat. aq. NaHCC. The aqueous mixture was extracted with EtOAc (2 x 250 mL). The combined organic layers were dried over Na2SO4 and evaporated to give dimethyl (7-(benzyloxy)-3-oxo-1,3-dihydroisobenzofuran-1-yl)phosphonate (9.30 g, 95% yield) as a light yellow solid.

[0535] m / z ES+ [M+H]+349.0;1H NMR (400 MHz, CDCh) 6 ppm 7.58 - 7.48 (m, 4H), 7.44 - 7.33 (m, 3H), 7.22 (d, J= 7.6 Hz, 1H), 5.79 (d, J= 9.6 Hz, 1H), 5.23 (s, 2H), 3.74 (d, J= 10.4 Hz, 3H), 3.70 (d, J= 11.2 Hz, 3H).ART-C-P3900PCT

[0536] Step g. This step was conducted under continuous flow conditions: Solution 1 (dimethyl (7-(benzyloxy)-3-oxo-1,3-dihydroisobenzofuran-1-yl)phosphonate (5.00 g, 14.3 mmol) in MeOH (100 mL); 0.3 mL / min) was pumped through Flow Reactor 1 packed with granular 5% Pd / ALOs (3.00 g) {FLR1, SS, fixed bed, 6.35 (1 / 4”) mm, 20 mL, 25 °C, retention time 3.3 min}. The H2 back pressure regulator was adjusted to 15 psi, with an H2 flow rate of 20 mL / min.

[0537] Solution 1 was recirculated twice. Upon completion, the reaction mixture was evaporated and the residue was purified by column chromatography (PE / EtOAc = 1 / 1) to give the title compound (3.50 g, 94% yield) as a white solid.

[0538] m / z ES+ [M+H]+258.9;1H NMR (400 MHz, CDCb) 5 ppm 9.09 (s, 1H), 7.59 - 7.47 (m, 2H), 7.29 (dd, J= 7.6, 1.2 Hz, 1H), 5.74 (d, J= 8.4 Hz, 1H), 4.05 (d, J= 10.8 Hz, 3H), 3.59 (d, J = 10.8 Hz, 3H).

[0539] Intermediate 3a: 4-Fluoro-3-(6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzaldehyde

[0540]

[0541] Step a. To a solution of 5-methylpyrazin-2-amine (CAS: 5521-58-4; 70 g, 641 mmol) and 2-bromo-1,1 -dimethoxyethane (CAS: 7252-83-7; 249 g, 1.48 mol) in EtOH (1.2 L) and H2O (70 mL) was added 40% aq. HBr (155 g, 769.73 mmol). The reaction mixture was stirred at 85 °C for 3 h 30 min. Upon completion, the reaction mixture was evaporated, and the residue was re-dissolved in MeOH (1 L). The mixture was poured into EtOAc (4.5 L) and stirred vigorously. The precipitated solid was filtered and washed with EtOAc (1 L) and PE (1 L). The solid was suspended in H2O (1.5 L) and the pH of the mixture was adjusted to pH 9 with 28% NH4OH. The aqueous mixture was extracted with DCM (12 x 500 mL). The combined organic layers were dried over Na2SO4and evaporated to give 6-methylimidazo[1,2-a]pyrazine (69 g, 80% yield) as a yellow solid.

[0542] 1H NMR (400 MHz, DMSO-cfe) 6 ppm 8.96 (s, 1H), 8.41 (s, 1H), 8.03 (s, 1H), 7.76 (s, 1H), 2.41 (s, 3H).

[0543] Step b. This step was conducted under continuous flow conditions: Solution 1 (6-methylimidazo[1,2-a]pyrazine (69 g, 518 mmol) in MeOH (1380 mL); 2.5 mL / min) was pumpedART-C-P3900PCT

[0544] through Flow Reactor 1 packed with granular 8% Pd(OH)2 / AhO3 (72.7 g) {FLR1, SS, fixed bed, 6.35 (1 / 4”) mm, 50 mL, 65 °C, retention time 20 min}. The H2 back pressure regulator was adjusted to 1.5 MPa, with an H2 flow rate of 90 mL / min. Upon completion, the reaction mixture was evaporated to give 6-methyl-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine (71 g, 99% yield) as a yellow oil.

[0545] m / z ES+ [M+H]+138.1;1H NMR (400 MHz, DMSO-cfe) 5 ppm 6.96 (d, J = 0.8 Hz, 1H), 6.80 (d, J= 0.8 Hz, 1H), 3.91 - 3.87 (m, 1H), 3.91 - 3.84 (m, 1H), 3.82 - 3.73 (m, 1H), 3.42 (t, J= 11.2 Hz, 1H), 2.94 - 3.06 (m, 1H), 1.12 (d, J= 6.8 Hz, 3H).

[0546] Step c. To a solution of 6-methyl-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine (30 g, 218 mmol) and NaHCOs (45.9 g, 546 mmol) in THF (500 mL) and H2O (100 mL) was added BOC2O (119 g, 546 mmol). The reaction mixture was stirred at 25 °C for 48 h. Upon completion, the reaction mixture was filtered, the filtrate was poured into sat. brine (300 mL) and extracted with EtOAc (3 x 200 mL). The combined organic layers were dried over Na2SO4 and evaporated. The crude product was triturated (PE / EtOAc = 8 / 1 (180 mL)) at 25 °C for 2 h to give tert-butyl 6-methyl-5,6-dihydroimidazo[1,2-a]pyrazine-7(8 / - / )-carboxylate (39 g, 75% yield) as a yellow solid.

[0547] m / z ES+ [M+H]+238.2;1H NMR (400 MHz, CDCh) 6 ppm 7.09 (d, J= 0.8 Hz, 1H), 6.90 (d, J = 0.8 Hz, 1H), 4.70 (d, J = 16.8 Hz, 1H), 4.62 (d, J = 4.0 Hz, 1H), 4.21 (d, J = 17.2 Hz, 1H), 4.08 - 3.99 (m, 1H), 3.98 - 3.89 (m, 1H), 1.43 (s, 9H), 1.03 (d, J= 6.8 Hz, 3H).

[0548] Step d. To a solution of terf-butyl 6-methyl-5,6-dihydroimidazo[1,2-a]pyrazine-7(8 / 7)-carboxylate (37 g, 155 mmol) in DMF (370 mL) and AcOH (74 mL) was added NIS (42.1 g, 187 mmol). The reaction mixture was stirred at 80 °C for 2 h. Upon completion, the reaction mixture was poured into sat. aq. K2CO3 (1.5 L) and extracted with EtOAc (3 x 300 mL). The combined organic layers were washed with sat. brine (2 x 300 mL), dried over Na2SO4and evaporated. The crude product was triturated (PE / EtOAc = 2 / 1 (300 mL)) at 25 °C for 2 h to give terf-butyl 3-iodo-6-methyl-5,6-dihydroimidazo[1,2-a]pyrazine-7(8 / 7)-carboxylate (39 g, 68% yield) as a light yellow solid.

[0549] m / z ES+ [M+H]+363.9;1H NMR (400 MHz, DMSO-cfe ) 6 ppm 7.14 (s, 1H), 5.04 (d, J = 17.6 Hz, 1H), 4.87 (br s, 1H), 4.31 (d, J = 17.2 Hz, 1H), 3.96 - 3.84 (m, 1H), 3.79 - 3.67 (m, 1H), 1.49 (s, 9H), 1.19 (d, J= 7.2 Hz, 3H).

[0550] Step e. A mixture of ferf-butyl 3-iodo-6-methyl-5,6-dihydroimidazo[1,2-a]pyrazine-7(8 / 7)-carboxylate (8.00 g, 22.0 mmol), 1,1,1,2,2-pentafluoro-2-iodoethane (CAS: 354-64-3; 43.3 g, 176 mmol) and Cu (8.40 g, 132 mmol) in DMF (80 mL) was stirred at 130 °C for 16 h. Upon completion, the reaction mixture was filtered, the filtrate was diluted with H2O (400 mL), 28% NH4OH (50 mL) was added, and the aqueous mixture was extracted with EtOAc (2 x 400 mL). The combined organic layers were dried over Na2SO4, evaporated and purified by columnART-C-P3900PCT

[0551] chromatography (PE / EtOAc = 15 / 1) to give tert-butyl 6-methyl-3-(perfluoroethyl)-5,6-dihydroimidazo[1,2-a]pyrazine-7(8 / - / )-carboxylate (2.50 g, 31% yield) as a light yellow solid. m / z ES+ [M+H]+356.1;1H NMR (400 MHz, CDCh) 6 ppm 7.43 (s, 1H), 5.05 (d, J = 17.6 Hz, 1H), 4.95 - 4.77 (m, 1H), 4.37 (d, J = 17.6 Hz, 1H), 4.20 - 4.10 (m, 1H), 4.07 - 3.94 (m, 1H), 1.51 (s, 9H), 1.20 (d, J= 6.8 Hz, 3H).

[0552] Step f. To a mixture of terf-butyl 6-methyl-3-(perfluoroethyl)-5,6-dihydroimidazo[1,2-a]pyrazine-7(8 / - / )-carboxylate (2.30 g, 6.47 mmol) in MeOH (3 mL) was added 2 M HCI in 1,4-dioxane (15 mL). The reaction mixture was stirred at 40 °C for 2 h. Upon completion, the reaction mixture was evaporated to give 6-methyl-3-(perfluoroethyl)-5, 6,7,8-tetrahydroimidazo[1,2-a]pyrazine hydrochloride (1.95 g, 95% yield) as a light yellow solid, m / z ES+ [M+H]+256.1;1H NMR (400 MHz, DMSO-cfe) 6 ppm 10.88 - 10.17 (m, 2H), 7.69 (s, 1H), 4.60 - 4.53 (m, 1H), 4.52 - 4.48 (m, 1H), 4.45 (d, J = 4.4 Hz, 1H), 4.19 - 4.09 (m, 1H), 3.96 (d, J = 4.8 Hz, 1 H), 1.49 (d, J = 6.4 Hz, 3H).

[0553] Step g. To a mixture of 2-fluoro-5-formylbenzoic acid (CAS: 550363-85-4; 400 mg, 2.38 mmol), 6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine hydrochloride (832 mg, 2.86 mmol) and DIPEA (1.54 g, 11.9 mmol) in DMF (10 mL) was added CMPI (790 mg, 3.09 mmol). The reaction mixture was stirred at 25 °C for 30 min. Upon completion, the reaction mixture was quenched with H2O (0.5 mL), evaporated and purified by reverse phase flash chromatography (H2O (0.1% FA) / MeCN) to give the title compound (0.9 g, 93% yield) as a yellow solid.

[0554] m / z ES+ [M+H]+406.0;1H NMR (400 MHz, CDCh) 6 ppm 10.00 (s, 1H), 8.09 - 8.02 (m, 1H), 8.01 (d, J = 5.2 Hz, 1 H), 7.53 - 7.39 (m, 1 H), 7.35 (t, J = 8.4 Hz, 1 H), 5.77 - 5.46 (m, 1 H), 4.78 - 4.62 (m, 1H), 4.58 - 4.16 (m, 2H), 4.15 - 3.98 (m, 1H), 1.46 - 1.25 (m, 3H).

[0555] Intermediate 3b: (S)-3-(6-Methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzaldehyde

[0556]

[0557] Steps a-e. These 5 steps were conducted as described in Intermediate 3a, steps a-e.ART-C-P3900PCT

[0558] The enantiomers were separated by chiral SFC (column: Daicel ChiralPak AD (250x50 mm; lOpm); mobile phase: A: 80% Heptane; B: 20% EtOH (0.1% NH4OH), isocratic elution to afford the second eluting enantiomer as tert-butyl (S)-6-methyl-3-(perfluoroethyl)-5,6-dihydroimidazo[1,2-a]pyrazine-7(8 / - / )-carboxylate (4.4 g, 28% yield) as a white solid. m / z ES+ [M+H]+356.1;1H NMR (400 MHz, CDCh) 5 ppm 7.43 (s, 1H), 5.05 (d, J = 17.6 Hz, 1H), 4.95 - 4.77 (m, 1H), 4.37 (d, J = 17.6 Hz, 1H), 4.20 - 4.10 (m, 1H), 4.07 - 3.94 (m, 1H), 1.51 (s, 9H), 1.20 (d, J= 6.8 Hz, 3H).

[0559] Step f. This step was conducted in a similar manner to Intermediate 3a, step f.

[0560] Step g. To a mixture of 3-formylbenzoic acid (CAS: 619-21-6; 41 mg, 0.27 mmol) and (S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine hydrochloride (80 mg, 0.27 mmol) in DMF (1 mL) was added HATU (125 mg, 0.33 mmol) and DIPEA (106 mg, 0.82 mmol) at 25 °C. The reaction mixture was stirred at 25 °C for 3 h. Upon completion, the reaction mixture was evaporated, and the residue was purified by prep-HPLC to give the title compound (85 mg, 80% yield) as a yellow solid.

[0561] m / z ES+ [M+H]+388.0;1H NMR (400 MHz, CDCh) 6 ppm 10.08 (s, 1H), 8.05 - 8.00 (m, 1H), 7.98 (s, 1H), 7.76 - 7.71 (m, 1H), 7.71 - 7.64 (m, 1H), 7.43 (s, 1H), 5.72 - 4.71 (m, 2H), 4.60 (d, J= 17.2 Hz, 1H), 4.28 - 4.19 (m, 1H), 4.07 (d, J= 12.8 Hz, 1H), 1.38 (d, J= 7.2 Hz, 3H).

[0562] Intermediate 3c: 4-Fluoro-3-(6-methyl-3-(1, 1,2, 2-tetrafluoroethyl)-5, 6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzaldehyde

[0563]

[0564] Step a. This step was conducted as described in Intermediate 3a, step a.

[0565] Step b. To a solution of 6-methylimidazo[1,2-a]pyrazine (10.0 g, 75.1 mmol) in MeCN (200 mL) was added NIS (18.6 g, 82.6 mmol). The reaction mixture was stirred at 20 °C for 16 h. Upon completion, the mixture was evaporated, diluted with H2O (100 mL) and extracted with EtOAc (2 x 100 mL). The combined organic layers were washed with brine (100 mL), dried over Na2SC>4, evaporated and purified by column chromatography (PE / EtOAc = 1 / 1) to give 3-iodo-6-methylimidazo[1,2-a]pyrazine (3.5 g, 18% yield) as a white solid.ART-C-P3900PCT

[0566] m / z ES+ [M+H]+259.9;1H NMR (400 MHz, CDCh) 6 ppm 8.93 (s, 1H), 7.87 (s, 1H), 7.82 (s, 1H), 2.61 (d, J= 0.8 Hz, 3H).

[0567] Step c. A mixture of cuprous thiocyanate (CAS: 1111-67-7; 790 mg, 6.5 mmol), CsF (2.5 g, 16.2 mmol) and (difluoromethyl)trimethylsilane (CAS: 65864-64-4; 2.0 g, 16.2 mmol) in DMF (25 mL) was stirred at 50 °C for 1 h under a N2 atmosphere. (Bromodifluoromethyl)trimethylsilane (CAS: 115262-01-6; 1.06 g, 5.2 mmol) and NaOAc (426 mg, 5.2 mmol) were then added and the reaction mixture was stirred at 30 °C for an additional 1 h under a N2 atmosphere. The reaction mixture was filtered under a N2 atmosphere and the filtrate was added to 3-iodo-6-methylimidazo[1,2-a]pyrazine (1.0 g, 3.9 mmol). The resulting reaction mixture was stirred at 60 °C for 16 h under a N2 atmosphere. Upon completion, the reaction mixture was poured into H2O (300 mL). 25% aq. NH4OH (5 mL) was added and the resulting mixture was extracted with EtOAc (2 x 100 mL). The combined organic layers were washed with brine (100 mL), dried over Na2SO4, evaporated and purified by column chromatography (PE / EtOAc = 4 / 1) to give 6-methyl-3-(1,1,2,2-tetrafluoroethyl)imidazo[1,2-a]pyrazine (450 mg, 29% yield) as a yellow gum.

[0568] m / z ES+ [M+H]+234.1;1H NMR (400 MHz, DMSO-cfe) 6 ppm 9.20 (s, 1H), 8.44 (s, 1H), 8.17 (s, 1H), 7.23 - 6.90 (m, 1H), 2.51 (s, 3H).

[0569] Step d. This step was conducted under continuous flow conditions: Solution 1: 6-methyl-3-(1,1,2,2-tetrafluoroethyl)imidazo[1,2-a]pyrazine (450 mg, 1.93 mmol) in a mixed solvent of THF (22.5 mL) and MeOH (22.5 mL) was pumped through Flow Reactor 1 packed with granular catalyst Pd(OH)2 / Al2O3 (3.00 g, 5.5% purity) {FLR1, SS, Fixed bed, 6.350 (1 / 4”) mm, 5 mL, 45 °C }. The H2 back pressure regulator was adjusted to 1.5 MPa, with an H2 flow rate of 20 mL / min. Upon completion, the reaction mixture was evaporated to give 6-methyl-3-(1,1,2,2-tetrafluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine (430 mg, 93% yield) as a yellow gum.

[0570] m / z ES+ [M+H]+238.0;1H NMR (400 MHz, CDCh) 5 ppm 7.30 (s, 1H), 6.13 - 5.85 (m, 1H), 4.28 - 4.23 (m, 1H), 4.14 (d, J = 3.2 Hz, 1H), 4.12 - 4.09 (m, 1H), 3.61 (t, J = 11.6 Hz, 1H), 3.28 - 3.20 (m, 1 H), 1.30 (d, J = 6.4 Hz, 3H).

[0571] Step e. This step was conducted in a similar manner to Intermediate 3a, step g, using 2-fluoro-5-formylbenzoic acid (CAS: 550363-85-4).

[0572] m / z ES+ [M+H]+388.2;1H NMR (400 MHz, CDCh) 6 ppm 10.00 (s, 1H), 8.16 - 7.84 (m, 2H), 7.48 - 7.30 (m, 2H), 6.20 - 5.84 (m, 1H), 5.79 - 5.45 (m, 1H), 4.66 (br s, 1H), 4.64 - 4.17 (m, 2H), 4.17 - 4.00 (m, 1H), 1.45 - 1.26 (m, 3H).

[0573] Intermediate 4: (2S,3R,4S,5S,6S)-2-(2-(3-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)propanamido)-4-((((S)-2-ART-C-P3900PCT

[0574] ((methylamino)methyl)pyrrolidine-1-carbonyl)oxy)methyl)phenoxy)-6- (methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate 2,2,2-trifluoroacetate

[0575]

[0576] Step a. To a solution of (2S,3 / ?,4S,5S,6S)-2-(4-(hydroxymethyl)-2-nitrophenoxy)-6- (methoxycarbonyl)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (CAS: 148579-94-6; 10 g, 20.6 mmol) in DCM (110 mL) was added 1 / - / -imidazole (2.1 g, 30.9 mmol) and tert-butyldimethylsilyl chloride (4.66 g, 30.90 mmol, 1.5 equiv). The reaction mixture was stirred at rt for 4 h under a N2 atmosphere. Upon completion, the reaction mixture was quenched by sat. aq. NaHCCh (100 mL) and extracted with DCM (3 x 100 mL). The combined organic layers were washed with brine, dried over Na2SO4, evaporated and purified by column chromatography (PE / EtOAc = 1 / 0 to 1 / 1) to give (2S,3 / ?,4S,5S,6S)-2-(4-(((tert-butyldimethylsilyl)oxy)methyl)-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (11.2 g, 91% yield) as a white solid.ART-C-P3900PCT

[0577] m / z ES+ [M+Na]+622.3;1H NMR (400 MHz, DMSO-cfe) 67.79 (d, J= 2.1 Hz, 1H), 7.62 (dd, J = 8.7, 2.1 Hz, 1H), 7.41 (d, J= 8.7 Hz, 1H), 5.71 (d, J= 7.8 Hz, 1H), 5.50 - 5.42 (m, 1H), 5.15 - 5.03 (m, 2H), 4.76 - 4.69 (m, 3H), 3.64 (s, 3H), 2.04 - 1.97 (m, 9H), 0.90 (s, 9H), 0.08 (s, 6H).

[0578] Step b. The following process was conducted in 4 batches.

[0579] To a solution of (2S,3R,4S,5S,6S)-2-(4-(((tert-butyldimethylsilyl)oxy)methyl)-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (1 g, 1.7 mmol) in DMF (10 mL) was added 4,4'-bipyridine (40 mg, 0.26 mmol) and B2(OH)4 (600 mg, 6.7 mmol). The reaction mixture was stirred at rt for 10 min under an Ar atmosphere. Upon completion, the reaction mixtures from the four batches were combined and poured onto ice. The aqueous mixture was filtered and the filter cake was purified by reverse-phase flash chromatography (H2O / MeCN) to give (2S,3R,4S,5S,6S)-2-(2-amino-4-(((terf-butyldimethylsilyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (3.5 g, 92% yield) as a white solid, m / z ES+ [M+H]+570.4;1H NMR (400 MHz, DMSO-cfe) 66.75 (d, J= 8.2 Hz, 1H), 6.58 (d, J = 2.0 Hz, 1H), 6.39 (dd, J= 8.2, 2.0 Hz, 1H), 5.45 - 5.38 (m, 1H), 5.35 (d, J= 7.9 Hz, 1H), 5.08 - 4.95 (m, 2H), 4.61 (d, J = 10.0 Hz, 1H), 4.57 (s, 2H), 4.46 (s, 2H), 3.59 (s, 3H), 2.04 - 1.92 (m, 9H), 0.83 (s, 9H), 0.00 (s, 6H).

[0580] Step c. To a solution of (2S,3R,4S,5S,6S)-2-(2-amino-4-(((tert-butyldimethylsilyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (3.76 g, 6.6 mmol) in DCM (100 mL) was added / V-Fmoc-?-alanine (CAS: 35737-10-1; 2.47 g, 7.9 mmol) and EEDQ (3.26 g, 13.2 mmol). The reaction mixture was stirred at rt for 4 h under a N2 atmosphere. Upon completion, the reaction mixture was evaporated and the residue was purified by column chromatography (PE / EtOAc = 1 / 0 to 3 / 2) to give (2S,3R,4S,5S,6S)-2-(2-(3-((((9 / 7-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)-4-(((terf-butyldimethylsilyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2 / 7-pyran-3,4, 5-triyl triacetate (4.01 g, 70% yield) as a white solid.

[0581] m / z ES+ [M+H]+863.3;1H NMR (300 MHz, DMSO-cfe) 58.64 (s, 1 H), 7.87 - 7.73 (m, 3H), 7.63 (d, J = 7.5 Hz, 2H), 7.39 - 7.19 (m, 5H), 6.98 (s, 2H), 5.56 - 5.38 (m, 2H), 5.17 - 5.07 (m, 1H), 5.04 - 4.95 (m, 1H), 4.65 (d, J = 9.9 Hz, 1H), 4.55 (s, 2H), 4.27 - 4.11 (m, 3H), 3.57 (s, 3H), 3.22 (s, 2H), 2.51 - 2.46 (m, 2H), 2.00 - 1.89 (m, 9H), 0.82 (s, 9H), 0.00 (s, 6H).

[0582] Step d. To a solution of (2S,3R,4S,5S,6S)-2-(2-(3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)-4-(((terf-butyldimethylsilyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2 / 7-pyran-3, 4, 5-triyl triacetate (4 g, 4.6 mmol) in THF (80 mL) at 0 °C under a N2 atmosphere was added TEA 3HF (5.98 g, 37.1 mmol). The reaction mixture was warmed to rt and stirred for 1 h. Upon completion, the reaction mixture was evaporated and the residue was purified by column chromatography (PE / EtOAc = 1 / 0 to 1 / 1) to give (2S,3R,4S,5S,6S)-2-(2-(3-((((9 / 7-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)-4-ART-C-P3900PCT

[0583] (hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (2.78 g, 80% yield) as a white solid.

[0584] m / z ES+ [M+H]+749.2;1H NMR (300 MHz, DMSO-cfe) 68.72 (s, 1 H), 7.93 - 7.79 (m, 3H), 7.69 (d, J = 7.4 Hz, 2H), 7.46 - 7.27 (m, 5H), 7.09 - 6.99 (m, 2H), 5.62 - 5.43 (m, 2H), 5.24 - 5.12 (m, 2H), 5.11 - 5.01 (m, 1H), 4.72 (d, J= 9.9 Hz, 1H), 4.41 (d, J= 5.5 Hz, 2H), 4.34 - 4.16 (m, 3H), 3.63 (s, 3H), 3.32 - 3.22 (m, 2H), 2.59 - 2.52 (m, 2H), 2.03 - 1.97 (m, 9H).

[0585] Step e. To a solution of (2S,3R,4S,5S,6S)-2-(2-(3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)-4-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (2.39 g, 3.2 mmol) in DCM (40 mL) was added TEA (647 mg, 6.4 mmol) and bis(4-nitrophenyl) carbonate (CAS: 5070-13-3; 1.94 g, 6.4 mmol) at 0 °C. The reaction mixture was warmed to rt and stirred for 2 h under a N2 atmosphere. Upon completion, the reaction mixture was evaporated and directly purified by column chromatography (PE / EtOAc = 1 / 0 to 1 / 1) to give (2S,3R,4S,5S,6S)-2-(2-(3-((((9 / 7-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)-4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (1.94 g, 67% yield) as a white solid.

[0586] m / z ES+ [M+H]+914.2;1H NMR (300 MHz, DMSO-cfe) 68.83 (s, 1H), 8.38 - 8.26 (m, 2H), 8.04 - 7.95 (m, 1H), 7.88 (d, J = 7.5 Hz, 2H), 7.69 (d, J = 7.4 Hz, 2H), 7.62 - 7.52 (m, 2H), 7.46 -7.35 (m, 3H), 7.36 - 7.20 (m, 3H), 7.13 (d, J= 8.5 Hz, 1H), 5.64 (d, J= 7.8 Hz, 1H), 5.56 - 5.45 (m, 1H), 5.28 - 5.15 (m, 3H), 5.12 - 5.02 (m, 1H), 4.75 (d, J = 10.0 Hz, 1H), 4.35 - 4.16 (m, 3H), 3.64 (s, 3H), 3.32 - 3.25 (m, 2H), 2.58 - 2.53 (m, 2H), 2.03 - 1.98 (m, 9H).

[0587] Step f. To a solution of (2S,3R,4S,5S,6S)-2-(2-(3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)-4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (300 mg, 0.33 mmol) in DMF (3 mL) was added tert-butyl (S)-methyl(pyrrolidin-2-ylmethyl)carbamate (CAS: 172477-91-7; 84 mg, 0.4 mmol) in DMF (1 mL) and DIPEA (85 mg, 0.66 mmol) dropwise at 0 °C. The reaction mixture was warmed to rt and stirred for 2 h under a N2 atmosphere. Upon completion, the reaction mixture was directly purified by reverse-phase flash chromatography (H2O / MeCN) to give (2S,3R,4S,5S,6S)-2-(2-(3-((((9 / 7-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)-4-((((S)-2-(((tert-butoxycarbonyl)(methyl)amino)methyl)pyrrolidine-1-carbonyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (130 mg, 40% yield) as a white solid.

[0588] m / z ES+ [M+H]+989.4;1H NMR (300 MHz, DMSO-cfe) 58.76 (s, 1H), 7.89 (d, J= 7.5 Hz, 3H), 7.69 (d, J= 7.5 Hz, 2H), 7.46 - 7.26 (m, 5H), 7.18 - 7.02 (m, 2H), 5.60 (d, J= 7.9 Hz, 1H), 5.54 - 5.45 (m, 1H), 5.23 - 5.14 (m, 1H), 5.11 - 4.89 (m, 3H), 4.73 (d, J= 10.0 Hz, 1H), 4.34 - 4.16ART-C-P3900PCT

[0589] (m, 3H), 3.99 - 3.92 (m, 1 H), 3.63 (s, 3H), 3.35 - 3.12 (m, 6H), 2.82 - 2.65 (m, 3H), 2.56 - 2.52 (m, 2H), 2.02 - 1.99 (m, 9H), 1.88 - 1.66 (m, 4H), 1.42 - 1.31 (m, 9H).

[0590] Step g. To a solution of (2S,3R,4S,5S,6S)-2-(2-(3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)-4-((((S)-2-(((terf-butoxycarbonyl)(methyl)amino)methyl)pyrrolidine-1-carbonyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (100 mg, 0.1 mmol) in DCM (1 mL) was added TFA (1 mL) dropwise at 0 °C. The reaction mixture was warmed to 25 °C and stirred for 2 h under a N2 atmosphere. Upon completion, the reaction mixture was evaporated to give the title compound (98 mg, crude) as a light yellow gum which was used without further purification.

[0591] m / z ES+ [M+H]+889.3.

[0592] Intermediate 5: (2S,3 / ?,4S,5S,6S)-2-(2-(2-((((9H-Fluoren-9-yl)methoxy)carbonyl)(methyl)amino)acetamido)-4-((((S)-2-((methylamino)methyl)pyrrolidine-1-carbonyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate 2,2,2-trifluoroacetate

[0593] F i moc

[0594] Nk

[0595] TFA

[0596] AcO'

[0597]

[0598] OAc

[0599] The title compound was prepared in a similar manner to Intermediate 4, using Fmoc- / V-methylglycine (CAS: 77128-70-2) in step c.

[0600] m / z ES+ [M+H]+889.4.

[0601] Intermediate 6: (2S,3 / ?,4S,5S,6S)-2-(4-((((Chloromethyl)(2-(methylsulfonyl)ethyl)carbamoyl)oxy)methyl)-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetateART-C-P3900PCT

[0602]

[0603] Step a. To a solution of (2S,3R,4S,5S,6S)-2-(4-(hydroxymethyl)-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (CAS: 148579-94-6; 5 g, 10.3 mmol) in DCM (60 mL) were added pyridine (1.63 g, 20.6 mmol) and 4-nitrophenyl carbonochloridate (CAS: 7693-46-1, 4.15 g, 20.6 mmol) at 0 °C. The reaction mixture was warmed to rt and stirred for 1 h under an N2 atmosphere. Upon completion, the reaction mixture was quenched with sat. aq. NaHCCh (50 mL) and extracted with DCM (3 x 60 mL). The combined organic layers were washed with brine, dried over Na2SO4, evaporated and purified by column chromatography (PE / EtOAc = 1 / 0 to 1 / 1) to give (2S,3S,4S,5R,6S)-2-(methoxycarbonyl)-6-(2-nitro-4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenoxy)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (6.07 g, 90% yield) as a white solid.

[0604] m / z ES+ [M+NH4]+668.1.1H NMR (300 MHz, DMSO-cfe) 68.38 - 8.27 (m, 2H), 8.05 (d, J = 2.1 Hz, 1H), 7.82 (dd, J= 8.8, 2.2 Hz, 1H), 7.64 - 7.54 (m, 2H), 7.48 (d, J= 8.7 Hz, 1H), 5.79 (d, J = 7.7 Hz, 1H), 5.52 - 5.43 (m, 1H), 5.33 (s, 2H), 5.18 - 5.05 (m, 2H), 4.76 (d, J= 9.8 Hz, 1H), 3.64 (s, 3H), 2.06 - 1.97 (m, 9H).

[0605] Step b. To a solution of (2S,3S,4S,5R,6S)-2-(methoxycarbonyl)-6-(2-nitro-4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenoxy)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (5 g, 7.7 mmol) and 2-(methylsulfonyl)ethan-1 -amine hydrochloride (CAS: 104458-24-4, 1.47 g, 9.2 mmol) in DCM (50 mL) were added DMAP (94.0 mg, 0.77 mmol) and TEA (1.79 g, 17.7 mmol) at 0 °C. The reaction mixture was warmed to rt and stirred for 4 h under an N2 atmosphere. Upon completion, the reaction mixture was evaporated and the residue was purified by column chromatography (DCM / EtOAc = 1 / 0 to 5 / 1) to give (2S,3S,4S,5R,6S)-2-(methoxycarbonyl)-6-(4-((((2-(methylsulfonyl)ethyl)carbamoyl)oxy)methyl)-2-nitrophenoxy)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (4.1 g, 84% yield) as a white solid.

[0606] m / z ES+ [M+Na]+657.05.1H NMR (400 MHz, CD3CN) 57.83 (s, 1H), 7.62 (d, J= 8.8 Hz, 1H), 7.44 (d, J = 8.7 Hz, 1H), 5.95 (s, 1H), 5.48 - 5.38 (m, 2H), 5.31 - 5.21 (m, 2H), 5.10 (s, 2H), 4.43 (d, J = 9.7 Hz, 1H), 3.70 (s, 3H), 3.62 - 3.52 (m, 2H), 3.23 (t, J = 6.6 Hz, 2H), 2.91 (s, 3H), 2.07 - 1.99 (m, 9H).ART-C-P3900PCT

[0607] Step c. To a solution of (2S,3S,4S,5R,6S)-2-(methoxycarbonyl)-6-(4-((((2- (methylsulfonyl)ethyl)carbamoyl)oxy)methyl)-2-nitrophenoxy)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (3.4 g, 5.4 mmol) in DCM (40 mL) was added paraformaldehyde (228 mg, 7.6 mmol) and TMSCI (914 mg, 8.4 mmol) at 0 °C. The reaction mixture was warmed to rt and stirred for 4 h under an Ar atmosphere. Upon completion, the reaction mixture was evaporated to give the title compound (3.5 g crude) as a light-yellow gum that was used without further purification.

[0608] m / z ES+ [M+Na]+701.0 (quenched by MeOH).

[0609] Intermediate 7a: (2S,3 / ?,4S,5S,6S)-2-(4-(Bromomethyl)-2-(((tert- butoxycarbonyl)amino)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate

[0610]

[0611] Step a. The following process was conducted in 6 batches.

[0612] Under an N2 atmosphere, cone. aq. H2SO4 (20 mL) was slowly added to a solution of 4- hydroxybenzaldehyde (CAS: 123-08-0, 5 g, 40.9 mmol) and 2-chloro- / V- (hydroxymethyl)acetamide (CAS: 2832-19-1, 4.81 g, 38.9 mmol) inAcOH (30 mL) atO °C. The reaction mixture was stirred at rt for 16 h. Upon completion, the reaction mixtures from the six batches were combined and slowly poured onto ice. The aqueous mixture was extracted with EtOAc (3 x 300 mL). The combined organic layers were washed with brine (600 mL), dried over Na2SC>4 and evaporated to give 2-chloro- / V-(5-formyl-2-hydroxybenzyl)acetamide (31.5 g, crude) as a red liquid, that was used without further purification.

[0613] Step b. The following process was conducted in 2 batches.ART-C-P3900PCT

[0614] Under an N2 atmosphere, cone. aq. HCI (104 mL) was slowly added to a solution of 2-chloro- / V-(5-formyl-2-hydroxybenzyl)acetamide (13 g, 57.3 mmol) in 1,4-dioxane (104 mL) at 0 °C. The reaction mixture was stirred under reflux for 1 h. Upon completion, the solvent was evaporated and the residue was re-dissolved in 1,4-dioxane (65 mL) and H2O (65 mL). The aqueous mixture was cooled to 0 °C, and TEA (17 g, 168.3 mmol) and BOC2O (13.2 g, 60.6 mmol) were added. The reaction mixture was stirred at rt for 16 h. Upon completion, the reaction mixtures from the two batches were combined and poured into H2O (400 mL) and extracted with EtOAc (3 x400 mL). The combined organic layers were washed with brine (800 mL), dried over Na2SO4, evaporated and purified by column chromatography (PE / EtOAc = 1 / 0 to 1 / 0.7) to give tert-butyl (5-formyl-2-hydroxybenzyl)carbamate (16 g, 56% yield over 2 steps) as a white solid.

[0615] m / z ES+ [M+H]+252.1;1H NMR (300 MHz, DMSO-cfe) 6 10.74 (s, 1H), 9.80 (s, 1H), 7.72 -7.60 (m, 2H), 7.38 - 7.27 (m, 1H), 7.03 - 6.96 (m, 1H), 4.24 - 4.10 (m, 2H), 1.42 (s, 9H). Step c. The following process was conducted in 4 batches.

[0616] A mixture of tert-butyl (5-formyl-2-hydroxybenzyl)carbamate (4 g, 15.9 mmol), (2R,3R,4S,5S,6S)-2-bromo-6-(methoxycarbonyl)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (CAS: 21085-72-3; 5.82 g, 14.7 mmol) and Ag2O (6.81 g, 29.4 mmol) in MeCN (50 mL) was stirred at rt for 48 h. Upon completion, the reaction mixtures from the four batches were combined, filtered, and the filtrate was evaporated and purified by column chromatography (PE / EtOAc = 1 / 0 to 1 / 0.8) to give (2S,3R,4S,5S,6S)-2-(2-(((tert-butoxycarbonyl)amino)methyl)-4-formylphenoxy)-6-(methoxycarbonyl)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (12 g, 33% yield) as a white solid.

[0617] m / z ES+ [M+Na]+590.3.

[0618] Step d. To a solution of (2S,3R,4S,5S,6S)-2-(2-(((tert-butoxycarbonyl)amino)methyl)-4-formylphenoxy)-6-(methoxycarbonyl)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (6 g, 10.6 mmol) in MeCN (30 mL) and IPA (30 mL) at 0 °C was added NaBH4 (441 mg, 11.7 mmol). The reaction mixture was stirred at rt for 1 h. Upon completion, the reaction mixture was filtered, and the filtrate was evaporated and purified by reverse-phase flash chromatography (H2O (0.01% FA) / MeCN) to give (2S,3R,4S,5S,6S)-2-(2-(((tert-butoxycarbonyl)amino)methyl)-4-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (5.7 g, 95% yield) as a white solid.

[0619] m / z ES+ [M+Na]+592.3;1H NMR (300 MHz, DMSO-cfe) 67.24 - 7.10 (m, 3H), 7.03 - 6.95 (m, 1H), 5.60 - 5.42 (m, 2H), 5.21 - 5.02 (m, 3H), 4.77 - 4.68 (m, 1H), 4.46 - 4.37 (m, 2H), 4.07 -3.92 (m, 2H), 3.64 (s, 3H), 2.13 - 1.93 (m, 9H), 1.40 (s, 9H).

[0620] Step e. Under an N2 atmosphere, CBr4 (1.4 g, 4.23 mmol) was added dropwise to a solution of (2S,3R,4S,5S,6S)-2-(2-(((terf-butoxycarbonyl)amino)methyl)-4-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (1 g, 1.69 mmol) and PPhs (1.11ART-C-P3900PCT

[0621] g, 4.23 mmol) in THF (5 mL) and DCM (5 mL) at 0 °C. The reaction mixture was stirred at rt for 16 h. Upon completion, the reaction mixture was evaporated and purified by column chromatography (PE / EtOAc = 1 / 0 to 1 / 1) to give the title compound (830 mg, 75% yield) as a white solid.

[0622] m / z ES+ [M+Na]+654.0, 656.1;1H NMR (300 MHz, DMSO-cfe) 67.42 - 7.16 (m, 3H), 7.07 -6.96 (m, 1 H), 5.73 - 5.59 (m, 1 H), 5.55 - 5.44 (m, 1 H), 5.24 - 5.03 (m, 2H), 4.83 - 4.54 (m, 3H), 4.11 - 3.87 (m, 2H), 3.64 (s, 3H), 2.09 - 1.95 (m, 9H), 1.40 (s, 9H).

[0623] Intermediate 7b: (2S,3 / ?,4S,5S,6S)-2-(2-(2-((((9H-Fluoren-9-yl)methoxy)carbonyl)(methyl)amino)acetamido)-4-(bromomethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate

[0624]

[0625] Step a. To a solution of (2S,3 / ?,4S,5S,6S)-2-(4-(hydroxymethyl)-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (CAS: 148579-94-6; 10 g, 20.6 mmol) in DCM (110 mL) was added 1 / - / -imidazole (2.1 g, 30.9 mmol) and tert-butyldimethylsilyl chloride (4.66 g, 30.90 mmol, 1.5 equiv). The reaction mixture was stirred at rt for 4 h underART-C-P3900PCT

[0626] an N2 atmosphere. Upon completion, the reaction mixture was quenched by sat. aq. NaHCCfe (100 mL) and extracted with DCM (3 x 100 mL). The combined organic layers were washed with brine, dried over Na2SO4, evaporated and purified by column chromatography (PE / EtOAc = 1 / 0 to 1 / 1) to give (2S,3R,4S,5S,6S)-2-(4-(((tert-butyldimethylsilyl)oxy)methyl)-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (11.2 g, 91% yield) as a white solid.

[0627] m / z ES+ [M+Na]+622.3;1H NMR (400 MHz, DMSO-cfe) 67.79 (d, J= 2.1 Hz, 1H), 7.62 (dd, J= 8.7, 2.1 Hz, 1H), 7.41 (d, J= 8.7 Hz, 1H), 5.71 (d, J= 7.8 Hz, 1H), 5.50 - 5.42 (m, 1H), 5.15 - 5.03 (m, 2H), 4.76 - 4.69 (m, 3H), 3.64 (s, 3H), 2.04 - 1.97 (m, 9H), 0.90 (s, 9H), 0.08 (s, 6H).

[0628] Step b. The following process was conducted in 4 batches.

[0629] To a solution of (2S,3R,4S,5S,6S)-2-(4-(((tert-butyldimethylsilyl)oxy)methyl)-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (1 g, 1.7 mmol) in DMF (10 mL) was added 4,4'-bipyridine (40 mg, 0.26 mmol) and B2(OH)4 (600 mg, 6.7 mmol). The reaction mixture was stirred at rt for 10 min under an Ar atmosphere. Upon completion, the reaction mixtures from the four batches were combined and poured onto ice. The aqueous mixture was filtered and the filter cake was purified by reverse-phase flash chromatography (H2O / MeCN) to give (2S,3R,4S,5S,6S)-2-(2-amino-4-(((terf-butyldimethylsilyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (3.5 g, 92% yield) as a white solid, m / z ES+ [M+H]+570.4;1H NMR (400 MHz, DMSO-cfe) 66.75 (d, J= 8.2 Hz, 1H), 6.58 (d, J = 2.0 Hz, 1H), 6.39 (dd, J= 8.2, 2.0 Hz, 1H), 5.45 - 5.38 (m, 1H), 5.35 (d, J= 7.9 Hz, 1H), 5.08 - 4.95 (m, 2H), 4.61 (d, J = 10.0 Hz, 1H), 4.57 (s, 2H), 4.46 (s, 2H), 3.59 (s, 3H), 2.04 - 1.92 (m, 9H), 0.83 (s, 9H), 0.00 (s, 6H).

[0630] Step c. To a solution of (2S,3R,4S,5S,6S)-2-(2-amino-4-(((tert-butyldimethylsilyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (6 g, 10.53 mmol) and / \ / -(((9 / 7-fluoren-9-yl)methoxy)carbonyl)- / \ / -methylglycine (CAS: 77128-70-2, 3.6 g, 11.6 mmol) in DCM (70 mL) was added EEDQ (5.2 g, 21.06 mmol) at rt. The resulting mixture was stirred at rt for 2 h. Upon completion, the mixture was evaporated and purified by column chromatography (PE / EtOAc = 1 / 0 to 1 / 1) to give (2S,3R,4S,5S,6S)-2-(2-(2-((((9 / 7-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)acetamido)-4-(((terf-butyldimethylsilyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2 / 7-pyran-3,4, 5-triyl triacetate (6 g, 66% yield) as a white solid.

[0631] m / z ES+ [M+H]+863.3.1H NMR (300 MHz, CDCI3) 58.28 (s, 1H), 8.15 - 8.00 (m, 1H), 7.85 -7.49 (m, 3H), 7.45 - 7.27 (m, 3H), 7.25 - 7.01 (m, 2H), 6.94 (d, J= 8.4 Hz, 1H), 5.46 - 5.19 (m, 2H), 5.07 (d, J = 7.5 Hz, 1H), 4.68 (s, 2H), 4.43 (s, 2H), 4.35 - 4.18 (m, 2H), 4.17 - 4.03 (m, 2H), 3.85 - 3.59 (m, 3H), 3.13 (s, 3H), 2.15 - 1.95 (m, 8H), 1.26 (s, 3H), 0.95 - 0.80 (m, 9H), 0.09 (s, 6H).ART-C-P3900PCT

[0632] Step d. To a solution of (2S,3?,4S,5S,6S)-2-(2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)acetamido)-4-(((tert-butyldimethylsilyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (6.8 g, 7.88 mmol) in THF (68 mL) at 0 °C under an N2 atmosphere was added TEA 3HF (6.8 mL, 41.7 mmol). The reaction mixture was stirred at rt for 3 h. Upon completion, the mixture was evaporated and purified by column chromatography (PE / EtOAc = 1 / 0 to 1 / 1.5) to give (2S,3R,4S,5S,6S)-2-(2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)acetamido)-4-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (4.9 g, 83% yield) as a white solid.

[0633] m / z ES+ [M+H]+749.2.1H NMR (300 MHz, DMSO-cfe) 68.95 - 8.82 (m, 1H), 7.96 - 7.82 (m, 3H), 7.69 (d, J= 7.4 Hz, 1H), 7.61 (d, J = 7.5 Hz, 1H), 7.50 - 7.27 (m, 3H), 7.25 - 7.14 (m, 1H), 7.07 (d, J = 9.6 Hz, 2H), 5.60 (dd, J = 11.6, 7.9 Hz, 1H), 5.55 - 5.45 (m, 1H), 5.25 - 5.13 (m, 2H), 5.11 - 5.00 (m, 1H), 4.71 (d, J = 10.0 Hz, 1H), 4.48 - 4.39 (m, 2H), 4.33 (s, 1H), 4.26 -4.10 (m, 2H), 4.07 (s, 1H), 3.62 (d, J = 6.5 Hz, 3H), 2.95 (s, 3H), 2.11 - 1.91 (m, 9H).

[0634] Step e. To a solution of (2S,3R,4S,5S,6S)-2-(2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)acetamido)-4-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (2 g, 2.67 mmol) and PPhs (1.75 g, 6.68 mmol) in THF (20 mL) and DCM (20 mL) at 0 °C under an N2 atmosphere was added CBr4 (2.2 g, 6.68 mmol) dropwise. The reaction mixture was warmed to rt and stirred for 16 h. Upon completion, the reaction mixture was evaporated and purified by column chromatography (PE / EtOAc = 1 / 0 to 1 / 1) to give (2S,3R,4S,5S,6S)-2-(2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)acetamido)-4-(bromomethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (2.04 g, 94% yield) as a white solid.

[0635] m / z ES+ [M+H]+811.2, 813.1.1H NMR (300 MHz, DMSO) 59.19 - 8.88 (m, 1H), 8.23 - 7.96 (m, 1H), 7.95 - 7.81 (m, 2H), 7.75 - 7.54 (m, 2H), 7.51 - 7.04 (m, 6H), 5.83 - 5.62 (m, 1H), 5.60 - 5.45 (m, 1H), 5.45 - 5.37 (m, 1H), 5.30 - 5.16 (m, 1H), 5.14 - 5.00 (m, 1H), 4.82 - 4.65 (m, 2H), 4.39 - 4.15 (m, 4H), 4.14 - 4.07 (m, 1H), 3.69 - 3.55 (m, 3H), 2.95 (s, 3H), 2.03 - 1.96 (m, 9H).

[0636] Intermediate 8: 2,5-Dioxopyrrolidin-1-yl 3-(4,5-dibromo-2-methyl-3,6-dioxo-3,6-dihydropyridazin-1(2H)-yl)propanoate

[0637] o

[0638]

[0639] ART-C-P3900PCT

[0640] 0

[0641]

[0642] Step a. A solution of BOC2O (3.64 g, 16.7 mmol) in DCM (15 mL) was added dropwise over 30 min to a solution of methylhydrazine sulfate (1 g, 6.9 mmol) in I PA (15 mL). The reaction mixture was stirred at rt for 16 h. Upon completion, the reaction mixture was evaporated and purified by reverse-phase flash chromatography (H2O (0.01% FA) / MeCN) to give di-te / f-butyl 1-methylhydrazine-1,2-dicarboxylate (1.17 g, 68% yield) as a white solid.

[0643] m / z ES- [M-H]- 245.1;1H NMR (300 MHz, DMSO-cfe) 5 9.17 - 8.55 (m, 1H), 3.01 - 2.85 (m, 3H), 1.50 - 1.27 (m, 18H).

[0644] Step b. To a solution of di-te / f-butyl 1-methylhydrazine-1,2-dicarboxylate (1.1 g, 4.47 mmol) in t-BuOH (11 mL) was added 10% aq. NaOH (0.2 mL). The reaction mixture was stirred at rt for 10 min, after which tert-butyl acrylate (1.72 g, 13.4 mmol) was added and the reaction mixture was stirred at 60 °C for an additional 20 h. Upon completion, the reaction mixture was poured into H2O (50 mL) and extracted with EtOAc (3 x 50 mL). The combined organic layers were washed with brine (100 mL), dried over Na2SO4, evaporated and purified by reversephase flash chromatography (H2O (0.01% FA) / MeCN) to give di-terf-butyl 1-(3-(te / f-butoxy)-3-oxopropyl)-2-methylhydrazine-1,2-dicarboxylate (1.32 g, 79% yield) as a colourless liquid, m / z ES+ [M+H]+375.2;1H NMR (300 MHz, DMSO-cfe) 63.79 - 3.64 (m, 1H), 3.53 - 3.36 (m, 1H), 2.98 - 2.85 (m, 3H), 2.48 - 2.37 (m, 2H), 1.46 - 1.31 (m, 27H).

[0645] Step c. A solution of 3, 4-dibromofuran-2, 5-dione (CAS: 1122-12-9, 710 mg, 2.8 mmol) in AcOH (20 mL) was stirred under reflux for 30 min, after which di-terf-butyl 1-(3-(te / f-butoxy)-3-oxopropyl)-2-methylhydrazine-1,2-dicarboxylate (900 mg, 2.4 mmol) was added and the reaction mixture was stirred under reflux for an additional 4 h. Upon completion, the reaction mixture was evaporated and purified by reverse-phase flash chromatography (H2O (0.01% FA) / MeCN) to give 3-(4,5-dibromo-2-methyl-3,6-dioxo-3,6-dihydropyridazin-1(2 / - / )-yl)propanoic acid (650 mg, 76% yield) as a white solid.

[0646] m / z ES+ [M+H]+354.8, 356.9, 358.9;1H NMR (400 MHz, DMSO-cfe) 5 12.52 (s, 1H), 4.33 -4.19 (m, 2H), 3.56 (s, 3H), 2.71 - 2.56 (m, 2H).

[0647] Step d. To a solution of 3-(4,5-dibromo-2-methyl-3,6-dioxo-3,6-dihydropyridazin-1(2 / - / )-yl)propanoic acid (600 mg, 1.7 mmol) and / V-hydroxysuccinimide (CAS: 6066-82-6; 215 mg, 1.9 mmol) in THF (12 mL) was added DCC (286 mg, 1.9 mmol). The reaction mixture was stirred at rt for 16 h. Upon completion, the reaction mixture was filtered and the filtrate was evaporated and purified by column chromatography (PE / EtOAc = 1 / 0 to 1 / 2.5) to give the title compound (480 mg, 62% yield) as a white solid.ART-C-P3900PCT

[0648] m / z ES+ [M+H]+451.9, 453.9, 455.9;1H NMR (400 MHz, DMSO-cfe) 6 4.45 - 4.34 (m, 2H), 3.58 (s, 3H), 3.22 - 3.15 (m, 2H), 2.77 (s, 4H).

[0649] Example A1: (S)-4-(4-Fluoro-3-(6-methyl-3-(perfluoroethyl)-5, 6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)-5-hydroxyphthalazin-1(2H)-one

[0650]

[0651] Step a. Under an N2 atmosphere, LiHMDS (1 M in THF, 1.55 mL) was added to a solution of Intermediate 2 (200 mg, 0.77 mmol) in THF (40 mL) at -65 °C. The reaction mixture was stirred at -65 °C for 30 min, after which Intermediate 3a (273 mg, 0.67 mmol) in THF (5 mL) was added. The reaction mixture was stirred at -65 °C for an additional 30 min, then warmed to 25 °C and stirred for a further 16 h. Upon completion, the reaction mixture was poured into sat. aq. NH4CI (100 mL) at 0 °C, and the aqueous mixture was extracted with EtOAc (3 x 80 mL). The combined organic layers were dried over Na2SO4, evaporated and purified by reverse phase flash chromatography (H2O (0.1% FA) / MeCN) to give 3-(4-fluoro-3-(6-methyl- 3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzylidene)-4-hydroxyisobenzofuran-1(3 / - / )-one (300 mg, 82% yield) as a yellow solid.

[0652] m / z ES+ [M+H]+538.2.

[0653] Step b. Hydrazine hydrate (0.91 g, 18.1 mmol) was added to a solution of 3-(4-fluoro-3-(6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzylidene)- 4-hydroxyisobenzofuran-1(3 / - / )-one (0.3 g, 0.56 mmol) in EtOH (5 mL). The reaction mixture was stirred at 80 °C for 16 h. Upon completion, sat. aq. NH4CI (0.3 mL) was added and the mixture was evaporated and purified by reverse phase flash chromatography (H2O (0.1% FA) / MeCN) to give 4-(4-fluoro-3-(6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)-5-hydroxyphthalazin-1(2 / - / )-one (280 mg, 90% yield) as a white solid.

[0654] m / z ES+ [M+H]+552.2.ART-C-P3900PCT

[0655] The enantiomers were separated by chiral SFC (column: Daicel ChiralPak AD (250x30 mm; 10 m); mobile phase: A: CO2; B: EtOH (0.1% NH4OH) to afford the first eluting enantiomer as the title compound (129 mg, 46% yield) as a white solid.

[0656] m / z ES+ [M+H]+552.1;1H NMR (400 MHz, DMSO-cfe) 6 ppm 12.43 (s, 1H), 10.90 (br s, 1H), 7.76 - 7.64 (m, 1H), 7.63 - 7.51 (m, 2H), 7.39 - 7.15 (m, 4H), 5.31 - 5.25 (m, 1H), 4.67 - 4.41 (m, 3H), 4.40 - 3.67 (m, 3H), 1.28 - 0.96 (m, 3H).

[0657] Example A2: (S)-5-Hydroxy-4-(3-(6-methyl-3-(perfluoroethyl)-5, 6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)phthalazin-1(2H)-one

[0658] o

[0659]

[0660] Steps a-b. These 2 steps were conducted in a similar manner to Example A1, steps a-b, using Intermediate 3b in step a.

[0661] m / z ES+ [M+H]+534.2;1H NMR (400 MHz, DMSO-cfe) 6 ppm 12.44 (s, 1H), 11.12 - 10.52 (m, 1H), 7.70 (d, J = 7.2 Hz, 1H), 7.62 - 7.47 (m, 2H), 7.42 - 7.35 (m, 1H), 7.35 - 7.24 (m, 3H), 7.20 (d, J = 8.0 Hz, 1H), 5.31 - 4.87 (m, 1H), 4.72 - 4.26 (m, 4H), 4.23 - 4.11 (m, 1H), 4.04 -3.84 (m, 1H), 1.09 (s, 3H).

[0662] Example A3: (S)-4-(4-Fluoro-3-(6-methyl-3-(1, 1,2, 2-tetrafluoroethyl)-5, 6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)-5-hydroxyphthalazin-1(2H)-one

[0663]

[0664] ART-C-P3900PCT

[0665] Step a. To a mixture of Intermediate 3c (140 mg, 0.36 mmol) and Intermediate 2 (93 mg, 0.36 mmol) in THF (20 mL) at 0 °C was added DBU (121 mg, 0.80 mmol). The reaction mixture was stirred at 20 °C for 2 h. Upon completion, the reaction mixture was poured into sat. aq. NH4CI (100 mL) at 0 °C and extracted with EtOAc (3 x 40 mL). The combined organic layers were dried over Na2SO4 and evaporated to give 3-(4-fluoro-3-(6-methyl-3-(1,1,2,2-tetrafluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzylidene)-4-hydroxyisobenzofuran-1(3 / - / )-one (180 mg, 95% yield) as a yellow solid.

[0666] m / z ES+ [M+H]+520.3.

[0667] Step b. This step was conducted in a similar manner to Example A1, step b. The enantiomers were separated by chiral SFC (column: DAICELCHIRALPAKAD (250x30 mm, 10 m); mobile phase: A: 65% CO2; B: 35% EtOH (0.1% NH4OH) to afford the first eluting enantiomer as the title compound (69 mg, 43% yield) as a white solid.

[0668] m / z ES+ [M+H]+534.1;1H NMR (400 MHz, DMSO-cfe) 6 ppm 12.44 (s, 1H), 10.90 (br s, 1H), 7.70 (dd, J = 7.6, 0.8 Hz, 1 H), 7.59 (t, J = 8.0 Hz, 1 H), 7.46 - 7.37 (m, 1 H), 7.33 (d, J = 3.6 Hz, 1H), 7.31 - 7.27 (m, 1H), 7.27 - 7.19 (m, 2H), 7.06 - 6.70 (m, 1H), 5.27 (d, J = 17.6 Hz, 1H), 4.61 - 4.38 (m, 3H), 4.38 - 3.85 (m, 3H), 1.23 - 0.98 (m, 3H).

[0669] Example B1: (2S,3S,4S,5R,6S)-6-(2-(3-(3-(2-(2-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethoxy)propanamido)propanamido)-4-((((S)-2-(((((4-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)-1-oxo-1,2-dihydrophthalazin-5-yl)oxy)carbonyl)(methyl)amino)methyl)pyrrolidine-1-carbonyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid o o

[0670]

[0671] ART-C-P3900PCT

[0672]

[0673] Step a. To a solution of Example A1 (500 mg, 0.91 mmol) and 4-nitrophenyl carbonochloridate (CAS: 7693-46-1, 274 mg, 1.36 mmol) inTHF (15 mL) was added DIPEA (352 mg, 2.72 mmol) dropwise at 0 °C under an Ar atmosphere. The reaction mixture was warmed to rt and stirred for 3 h. Upon completion, the reaction mixture was evaporated and the residue was purified by reverse-phase flash chromatography (H2O (0.01% FA) / MeCN) to give (S)-4-(4-fluoro-3-(6-ART-C-P3900PCT

[0674] methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)-1-oxo-1,2-dihydrophthalazin-5-yl (4-nitrophenyl) carbonate (350 mg, 54% yield) as a white solid. m / z ES+ [M+H]+717.2;1H NMR (300 MHz, DMSO-cfe) 6 12.85 (s, 1H), 8.36 (d, J= 9.1 Hz, 2H), 8.29 (d, J= 7.8 Hz, 1H), 8.13 - 8.04 (m, 1H), 7.99 - 7.88 (m, 1H), 7.67 (d, J= 9.1 Hz, 2H), 7.58 - 7.43 (m, 2H), 7.31 - 7.17 (m, 2H), 5.36 - 5.12 (m, 1H), 4.69 - 4.25 (m, 4H), 4.17 - 3.94 (m, 2H), 1.19 - 0.76 (m, 3H).

[0675] Step b. To a solution of (S)-4-(4-fluoro-3-(6-methyl-3-(perfluoroethyl)-5, 6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)-1-oxo-1,2-dihydrophthalazin-5-yl (4-nitrophenyl) carbonate (30 mg, 0.04 mmol) in DMF (0.6 mL) were added Intermediate 4 (57 mg, 0.06 mmol) in DMF (0.6 mL), HOBt (8.5 mg, 0.05 mmol) in DMF (0.3 mL) and DIPEA (16 mg, 0.11 mmol) in DMF (0.3 mL) at 0 °C. The reaction mixture was warmed to 25 °C and stirred for 6 h under an Ar atmosphere. Upon completion, the reaction mixture was directly purified by reverse-phase flash chromatography (H2O (0.01% TFA) / MeCN) to give (2S,3R,4S,5S,6S)-2-(2-(3-((((9 / 7-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)-4-((((S)-2-(((((4-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)-1 -oxo-1, 2-dihydrophthalazin-5-yl)oxy)carbonyl)(methyl)amino)methyl)pyrrolidine-1-carbonyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (25 mg, 41% yield) as a white solid.

[0676] m / z ES+ [M+H]+1466.50;1H NMR (300 MHz, DMSO-cfe) 6 12.70 (s, 1H), 8.77 (s, 1H), 8.42 (s, 1H), 8.22 - 8.09 (m, 1H), 7.95 - 7.83 (m, 3H), 7.84 - 7.72 (m, 1H), 7.71 - 7.60 (m, 2H), 7.58 -7.49 (m, 1H), 7.46 - 7.35 (m, 3H), 7.35 - 7.18 (m, 4H), 7.16 - 6.85 (m, 3H), 5.67 - 5.56 (m, 1H), 5.55 - 5.44 (m, 1H), 5.29 - 5.13 (m, 2H), 5.12 - 4.88 (m, 3H), 4.78 - 4.67 (m, 1H), 4.62 - 4.17 (m, 6H), 4.15 - 3.97 (m, 2H), 3.68 - 3.54 (m, 3H), 3.34 - 3.19 (m, 7H), 3.21 - 2.65 (m, 6H), 2.20 - 1.83 (m, 8H), 1.89 - 1.60 (m, 4H), 1.08 - 0.74 (m, 3H).

[0677] Step c. To a solution of (2S,3R,4S,5S,6S)-2-(2-(3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)-4-((((S)-2-(((((4-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)-1-oxo-1,2-dihydrophthalazin-5-yl)oxy)carbonyl)(methyl)amino)methyl)pyrrolidine-1-carbonyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (25 mg, 0.02 mmol) in THF (0.22 mL) and MeOH (0.22 mL) was added 1 M aq. LiOH (0.22 mL, 0.22 mmol) dropwise at 0 °C. The reaction mixture was stirred at 0 °C for 2 h under an N2 atmosphere. Upon completion, the reaction mixture was directly purified by reverse-phase flash chromatography (H2O (0.01% FA) / MeCN) to give (2S,3S,4S,5?,6S)-6-(2-(3-aminopropanamido)-4-((((S)-2-(((((4-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)-1 -oxo-1, 2-dihydrophthalazin-5-ART-C-P3900PCT

[0678] yl)oxy)carbonyl)(methyl)amino)methyl)pyrrolidine-1-carbonyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (16 mg, 85% yield) as a white solid. m / z ES+ [M+H]+1104.4;1H NMR (300 MHz, DMSO-cfe) 6 12.75 (s, 1H), 9.63 (s, 1H), 8.37 (s, 1H), 8.29 - 8.09 (m, 2H), 7.87 - 7.73 (m, 1H), 7.63 - 7.48 (m, 2H), 7.41 - 7.03 (m, 5H), 5.35 -5.14 (m, 2H), 5.12 - 4.77 (m, 3H), 4.69 - 4.44 (m, 2H), 4.43 - 4.24 (m, 2H), 4.21 - 3.91 (m, 4H), 3.83 - 3.30 (m, 8H), 3.23 - 2.92 (m, 6H), 2.90 - 2.62 (m, 4H), 1.89 - 1.67 (m, 4H), 1.03 - 0.74 (m, 3H).

[0679] Step d. To a mixture of (2S,3S,4S,5R,6S)-6-(2-(3-aminopropanamido)-4-((((S)-2-(((((4-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)-1 -oxo-1, 2-dihydrophthalazin-5-yl)oxy)carbonyl)(methyl)amino)methyl)pyrrolidine-1-carbonyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (10 mg, 0.01 mmol) and 2,5-dioxopyrrolidin- 1-yl 3-(2-(2-(2,5-dioxo-2,5-dihydro-1 / 7-pyrrol-1-yl)ethoxy)ethoxy)propanoate (CAS: 1433997-01-3; 5 mg, 0.01 mmol) in DMF (1 mL) was added DIPEA (2 mg, 0.02 mmol) in DMF (0.1 mL) dropwise at 0 °C. The reaction mixture was warmed to 25 °C and stirred for 2 h under an Ar atmosphere. Upon completion, the reaction mixture was directly purified by reverse-phase flash chromatography (H2O (0.01% FA) / MeCN) to give the title compound (5 mg, 37% yield) as a white solid.

[0680] m / z ES+ [M+H]+1343.2;1H NMR (300 MHz, DMSO-cfe) 6 12.70 (s, 1H), 9.28 - 9.03 (m, 1H), 8.27 - 7.88 (m, 3H), 7.85 - 7.73 (m, 1H), 7.62 - 7.46 (m, 2H), 7.46 - 6.93 (m, 8H), 5.93 - 5.62 (m, 1H), 5.42 - 5.16 (m, 2H), 5.12 - 4.70 (m, 3H), 4.68 - 3.95 (m, 6H), 3.82 - 3.68 (m, 1H), 3.61 - 3.39 (m, 13H), 3.29 - 3.08 (m, 7H), 3.07 - 2.97 (m, 2H), 2.88 - 2.65 (m, 2H), 2.63 - 2.53 (m, 2H), 2.35 - 2.17 (m, 2H), 1.94 - 1.58 (m, 4H), 1.10 - 0.73 (m, 3H).

[0681] Example B2: (2S,3S,4S,5 / ?,6S)-6-(2-(2-(3-(2,5-Dioxo-2,5-dihydro-1 H-pyrrol-1 -y I )-N-methylpropanamido)acetamido)-4-((((S)-2-(((((4-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)-1-oxo-1,2-dihydrophthalazin-5-yl)oxy)carbonyl)(methyl)amino)methyl)pyrrolidine-1-carbonyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid

[0682]

[0683] ART-C-P3900PCT

[0684]

[0685] Step a. This step was conducted as described in Example B1, step a.

[0686] Steps b-c. These 2 steps were conducted in a similar manner to Example B1, steps b-c, using Intermediate 5 in step b.

[0687] m / z ES+ [M+H]+1104.5;1H NMR (300 MHz, DMSO-cfe) 6 12.87 - 12.63 (m, 1H), 9.84 (s, 1H), 8.30 - 7.98 (m, 2H), 7.94 - 7.66 (m, 1H), 7.61 - 7.47 (m, 2H), 7.46 - 6.88 (m, 6H), 5.33 - 5.13 (m, 2H), 5.12 - 4.88 (m, 2H), 4.88 - 4.67 (m, 1H), 4.63 - 4.25 (m, 3H), 4.22 - 3.97 (m, 4H), 3.88 - 3.51 (m, 6H), 3.50 - 3.30 (m, 5H), 3.26 - 2.91 (m, 6H), 2.86 - 2.63 (m, 2H), 1.98 - 1.60 (m, 4H), 1.20 - 0.74 (m, 3H).

[0688] Step d. To a mixture of (2S,3S,4S,5F?,6S)-6-(4-((((S)-2-(((((4-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)-1-oxo-1,2-dihydrophthalazin-5-yl)oxy)carbonyl)(methyl)amino)methyl)pyrrolidine-1-carbonyl)oxy)methyl)-2-(2-(methylamino)acetamido)phenoxy)-3,4,5-trihydroxytetrahydro-2 / 7-pyran-2-carboxylic acid (80 mg, 0.07 mmol), 2,5-dioxopyrrolidin-1-yl 3-(2,5-dioxo-2,5-dihydro-1 / 7-pyrrol-1-yl)propanoate (CAS: 55750-62-4; 29 mg, 0.11 mmol) and HOBt (12 mg, 0.09 mmol) in DMF (1.5 mL) was added DIPEA (28 mg, 0.22 mmol) in DMF (0.3 mL) dropwise at 0ART-C-P3900PCT

[0689] °C. The reaction mixture was warmed to 25 °C and stirred for 2 h under an Ar atmosphere. Upon completion, the reaction mixture was directly purified by reverse-phase flash chromatography (H2O (0.01% FA) / MeCN) to give the title compound (45 mg, 49% yield) as a white solid.

[0690] m / z ES+ [M+H]+1255.4;1H NMR (400 MHz, DMSO-cfe) 6 12.69 (s, 1H), 9.30 - 9.05 (m, 1H), 8.26 - 8.08 (m, 2H), 7.88 - 7.71 (m, 1H), 7.60 - 7.48 (m, 1H), 7.45 - 7.17 (m, 3H), 7.15 - 6.91 (m, 5H), 5.86 - 5.71 (m, 1H), 5.46 - 5.16 (m, 2H), 5.11 - 4.73 (m, 3H), 4.66 - 3.98 (m, 8H), 3.97 - 3.83 (m, 1H), 3.72 - 3.57 (m, 2H), 3.51 - 3.36 (m, 4H), 3.32 - 3.10 (m, 4H), 3.09 - 2.91 (m, 4H), 2.89 - 2.72 (m, 2H), 2.73 - 2.61 (m, 2H), 2.60 - 2.51 (m, 2H), 2.03 - 1.52 (m, 4H), 1.29 -0.76 (m, 3H).

[0691] Example B3: (2S,3S,4S,5 / ?,6S)-6-(2-(3-(3-(2-(2-(2,5-Dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)ethoxy)ethoxy)propanamido)propanamido)-4-((((((4-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)-1-oxo-1,2-dihydrophthalazin-5-yl)oxy)methyl)(2- (methylsulfonyl)ethyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H- pyran-2-carboxylic acid

[0692]

[0693] ART-C-P3900PCT

[0694]

[0695] Step a. To a solution of Example A1 (48 mg, 0.09 mmol) and DIPEA (33 mg, 0.26 mmol) in DCM (1 mL) was added Intermediate 6 (88 mg, 0.13 mmol) in DCM (1 mL) dropwise at 0 °CART-C-P3900PCT

[0696] under an Ar atmosphere. The reaction mixture was warmed to rt and stirred for 4 h. Upon completion, the reaction mixture was quenched with sat. aq. NaHCCfe (50 iL) and evaporated. The residue was purified by reverse-phase flash chromatography (H2O (0.01% FA) / MeCN) to give (2S,3R,4S,5S,6S)-2-(4-((((((4-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)-1 -oxo-1, 2-dihydrophthalazin-5-yl)oxy)methyl)(2-(methylsulfonyl)ethyl)carbamoyl)oxy)methyl)-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (70 mg, 68% yield) as a white solid.

[0697] m / z ES+ [M+H]+1198.5;1H NMR (300 MHz, DMSO-cfe) 5 12.64 (s, 1H), 8.07 - 7.82 (m, 2H), 7.81 - 7.68 (m, 2H), 7.64 - 7.52 (m, 1H), 7.50 - 7.33 (m, 2H), 7.32 - 7.01 (m, 3H), 5.84 - 5.68 (m, 1H), 5.54 - 5.34 (m, 3H), 5.33 - 5.21 (m, 1H), 5.19 - 5.04 (m, 4H), 4.84 - 4.69 (m, 1H), 4.56 - 4.26 (m, 3H), 4.21 - 3.96 (m, 2H), 3.82 - 3.55 (m, 6H), 3.50 - 3.38 (s, 2H), 3.08 - 2.90 (m, 3H), 2.08 - 1.93 (m, 9H), 1.20 - 0.88 (m, 3H).

[0698] Step b. To a solution of (2S,3R,4S,5S,6S)-2-(4-((((((4-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)-1-oxo-1,2-dihydrophthalazin-5-yl)oxy)methyl)(2-(methylsulfonyl)ethyl)carbamoyl)oxy)methyl)-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (70 mg, 0.06 mmol) in DMF (1.5 mL) were added 4,4'-bipyridine (0.9 mg, 0.01 mmol) and B2(OH)4 (21 mg, 0.23 mmol). The reaction mixture was stirred at rt for 10 min. Upon completion, the reaction mixture was directly purified by reverse-phase flash chromatography (H2O (0.01% FA) / MeCN) to give (2S,3R,4S,5S,6S)-2-(2-amino-4-((((((4-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)-1-oxo-1,2-dihydrophthalazin-5-yl)oxy)methyl)(2-(methylsulfonyl)ethyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (59 mg, 86% yield) as a white solid.

[0699] m / z ES+ [M+H]+1168.4;1H NMR (300 MHz, DMSO-cfe) 5 12.64 (s, 1H), 7.99 - 7.83 (m, 1H), 7.79 - 7.65 (m, 1H), 7.61 - 7.51 (m, 1H), 7.50 - 7.35 (m, 1H), 7.32 - 6.99 (m, 3H), 6.90 - 6.64 (m, 2H), 6.62 - 6.41 (m, 1H), 5.55 - 5.34 (m, 4H), 5.32 - 5.18 (m, 1H), 5.17 - 5.05 (m, 2H), 5.04 - 4.87 (m, 2H), 4.78 - 4.59 (m, 3H), 4.55 - 4.27 (m, 3H), 4.26 - 3.79 (m, 3H), 3.72 - 3.55 (m, 5H), 3.43 - 3.36 (m, 2H), 3.06 - 2.89 (m, 3H), 2.08 - 1.92 (m, 9H), 1.26 - 0.89 (m, 3H).

[0700] Step c. To a solution of (2S,3R,4S,5S,6S)-2-(2-amino-4-((((((4-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)-1-oxo-1,2-dihydrophthalazin-5-yl)oxy)methyl)(2-(methylsulfonyl)ethyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (50 mg, 0.04 mmol) in DCM (2 mL) were added / V-Fmoc- / 3-alanine (CAS: 35737-10-1, 27 mg, 0.09 mmol) and EEDQ (32 mg, 0.13 mmol) at 0 °C. The reaction mixture was warmed to rt and stirred for 4 h under an Ar atmosphere. Upon completion, the reaction mixture was evaporated and the residue wasART-C-P3900PCT

[0701] purified by reverse-phase flash chromatography (H2O (0.01% FA) / MeCN) to give (2S,3R,4S,5S,6S)-2-(2-(3-((((9 / 7-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)-4-((((((4-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)-1 -oxo-1, 2-dihydrophthalazin-5-yl)oxy)methyl)(2-(methylsulfonyl)ethyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (37 mg, 59% yield) as a white solid.

[0702] m / z ES+ [M+H]+1461.6;1H NMR (300 MHz, DMSO-cfe) 6 12.62 (s, 1H), 9.02 - 8.80 (m, 1H), 7.93 - 7.81 (m, 4H), 7.67 - 7.54 (m, 4H), 7.45 - 7.30 (m, 4H), 7.21 - 7.03 (m, 6H), 5.76 - 5.58 (m, 1H), 5.58 - 5.46 (m, 1H), 5.45 - 5.27 (m, 3H), 5.27 - 5.16 (m, 2H), 5.15 - 5.01 (m, 3H), 4.79 - 4.65 (m, 1H), 4.49 - 4.26 (m, 5H), 4.26 - 4.18 (m, 2H), 4.16 - 4.04 (m, 3H), 4.04 - 3.97 (m, 1H), 3.96 - 3.57 (m, 5H), 3.49 - 3.41 (m, 2H), 3.00 - 2.82 (m, 5H), 2.08 - 1.92 (m, 9H), 1.23 -1.14 (m, 3H).

[0703] Step d. To a solution of (2S,3R,4S,5S,6S)-2-(2-(3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)-4-((((((4-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)-1-oxo-1,2-dihydrophthalazin-5-yl)oxy)methyl)(2-(methylsulfonyl)ethyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (32 mg, 0.02 mmol) in THF (0.29 mL) and methanol (0.29 mL) was added 1 M aq. LiOH (0.29 mL, 0.29 mmol) dropwise at 0 °C. The reaction mixture was stirred at 0 °C for 2 h under a N2 atmosphere. Upon completion, the reaction mixture was directly purified by reverse-phase flash chromatography (H2O (0.01% FA) / MeCN) to give (2S,3S,4S,5R,6S)-6-(2-(3-aminopropanamido)-4-((((((4-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)-1 -oxo-1, 2-dihydrophthalazin-5-yl)oxy)methyl)(2-(methylsulfonyl)ethyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2 / 7-pyran-2-carboxylic acid (20 mg, 83% yield) as a white solid.

[0704] m / z ES+ [M+H]+1099.4;1H NMR (300 MHz, DMSO-cfe) 6 12.65 (s, 1H), 9.83 (s, 1H), 8.39 -8.07 (m, 1 H), 7.98 - 7.83 (m, 1 H), 7.80 - 7.64 (m, 1 H), 7.62 - 7.51 (m, 1 H), 7.51 - 7.37 (m, 1 H), 7.34 - 6.93 (m, 5H), 5.55 - 5.19 (m, 4H), 5.15 - 5.01 (m, 2H), 4.89 - 4.70 (m, 1H), 4.58 - 4.24 (m, 4H), 4.23 - 3.91 (m, 4H), 3.75 - 3.32 (m, 11 H), 3.05 - 2.93 (m, 3H), 2.49 - 2.36 (m, 4H), 1.26 - 0.95 (m, 3H).

[0705] Step e. To a solution of (2S,3S,4S,5R,6S)-6-(2-(3-aminopropanamido)-4-((((((4-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)- 1 -oxo-1, 2-dihydrophthalazin-5-yl)oxy)methyl)(2-(methylsulfonyl)ethyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2 / 7-pyran- 2-carboxylic acid (13 mg, 0.012 mmol) and 2,5-dioxopyrrolidin-1-yl 3-(2-(2-(2,5-dioxo-2,5-dihydro-1 / 7-pyrrol-1-yl)ethoxy)ethoxy)propanoate (CAS: 1433997-01-3; 6 mg, 0.02 mmol) in DMF (1 mL) were added DIPEA (5 mg, 0.04 mmol) and HOBt (2 mg, 0.01 mmol) at 0 °C. TheART-C-P3900PCT

[0706] reaction mixture was warmed to 25 °C and stirred for 5 h under an Ar atmosphere. Upon completion, the reaction mixture was directly purified by reverse-phase flash chromatography (H2O (0.01% FA) / MeCN) to give the title compound (6 mg, 38% yield) as a white solid. m / z ES+ [M+H]+1338.2;1H NMR (300 MHz, DMSO-cfe) 6 12.63 (s, 1H), 9.52 - 9.08 (m, 1H), 8.25 - 8.08 (m, 1H), 7.95 - 7.84 (m, 1H), 7.78 - 7.64 (m, 1H), 7.60 - 7.51 (m, 1H), 7.49 - 7.36 (m, 1H), 7.32 - 7.07 (m, 4H), 7.06 - 6.91 (m, 3H), 5.78 - 5.65 (m, 1H), 5.49 - 5.16 (m, 4H), 5.16 - 4.99 (m, 2H), 4.92 - 4.76 (m, 1H), 4.64 - 3.99 (m, 7H), 3.89 - 3.65 (m, 3H), 3.63 - 3.36 (m, 16H), 3.14 - 2.92 (m, 4H), 2.88 - 2.77 (m, 2H), 2.68 - 2.55 (m, 4H), 1.24 - 0.94 (m, 3H).

[0707] Example B4: (2S,3S,4S,5 / ?,6S)-6-(2-((3-(4,5-Dibromo-2-methyl-3,6-dioxo-3,6-dihydropyridazin-1(2H)-yl)propanamido)methyl)-4-(((4-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)-1-oxo-1,2-dihydrophthalazin-5-yl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid

[0708] O

[0709]

[0710] ART-C-P3900PCT

[0711]

[0712] Step a. A mixture of Intermediate 7a (960 mg, 1.52 mmol), Example A1 (700 mg, 1.27 mmol) and K2CO3 (700 mg, 5.07 mmol) in DMF (10 mL) was stirred at rt for 2 h. Upon completion, the reaction mixture was filtered and the filtrate was directly purified by reverse-phase flash chromatography (H2O (0.01% FA) / MeCN) to give (2S,3F?,4S,5S,6S)-2-(2-(((tert-butoxycarbonyl)amino)methyl)-4-(((4-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)-1 -oxo-1, 2-dihydrophthalazin-5-yl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (1.1 g, 78% yield) as a white solid.

[0713] m / z ES+ [M+H]+1103.3.1H NMR (300 MHz, DMSO-cfe) 6 12.63 (s, 1H), 7.90 - 7.80 (m, 1H), 7.77 - 7.67 (m, 1 H), 7.57 (s, 1 H), 7.50 - 7.38 (m, 1 H), 7.33 - 6.85 (m, 7H), 5.63 (d, J = 7.8 Hz, 1H), 5.56 - 5.45 (m, 1H), 5.36 - 5.01 (m, 5H), 4.81 - 4.67 (m, 1H), 4.60 - 4.27 (m, 3H), 4.23 -3.71 (m, 5H), 3.62 (s, 3H), 2.11 - 1.95 (m, 9H), 1.40 - 0.91 (m, 12H).

[0714] Step b. To a solution of (2S,3R,4S,5S,6S)-2-(2-(((tert-butoxycarbonyl)amino)methyl)-4-(((4-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)-1 -oxo-1, 2-dihydrophthalazin-5-yl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (1.1 g, 1.00 mmol) in DCM (5 mL) at 0 °C was added TFA (5 mL) dropwise. The reaction mixture was stirred at rt for 1 h. UponART-C-P3900PCT

[0715] completion, the reaction mixture was evaporated to give (2S,3R,4S,5S,6S)-2-(2-(aminomethyl)-4-(((4-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)-1 -oxo-1, 2-dihydrophthalazin-5-yl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (880 mg, crude) as a white solid that was used without further purification.

[0716] m / z ES+ [M+H]+1003.3.1H NMR (300 MHz, DMSO-cfe) 6 12.64 (s, 1H), 7.91 - 7.81 (m, 1H), 7.78 - 7.67 (m, 1 H), 7.57 (s, 1 H), 7.52 - 7.31 (m, 2H), 7.29 - 6.86 (m, 6H), 5.65 (d, J = 7.8 Hz, 1H), 5.57 - 5.46 (m, 1H), 5.39 - 5.01 (m, 6H), 4.80 - 4.68 (m, 1H), 4.57 - 4.29 (m, 4H), 3.83 -3.67 (m, 4H), 3.60 (s, 3H), 2.14 - 1.90 (m, 9H), 1.27 - 0.71 (m, 3H).

[0717] Step c. To a solution of (2S,3R,4S,5S,6S)-2-(2-(aminomethyl)-4-(((4-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)-1-oxo- 1.2-dihydrophthalazin-5-yl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (880 mg, 0.88 mmol) in THF (3.7 mL) and MeOH (3.7 mL) at 0 °C was added 1 M aq. LiOH (3.7 mL, 3.7 mmol) dropwise. The reaction mixture was stirred at rt for 90 min. Upon completion, the reaction mixture was quenched with FA (0.14 mL) and then directly purified by reverse-phase flash chromatography (H2O (0.01% FA) / MeCN) to give (2S,3S,4S,5R,6S)-6-(2-(aminomethyl)-4-(((4-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)-1-oxo-1,2-dihydrophthalazin-5-yl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2 / 7-pyran-2-carboxylic acid (700 mg, 92% yield) as a white solid.

[0718] m / z ES+ [M+H]+863.3;1H NMR (300 MHz, DMSO-cfe) 6 12.71 - 12.38 (m, 1H), 8.84 - 8.12 (m, 1H), 7.92 - 6.50 (m, 9H), 5.40 - 4.96 (m, 4H), 4.77 - 4.57 (m, 1H), 4.52 - 4.28 (m, 3H), 4.27 -3.92 (m, 3H), 3.89 - 3.66 (m, 2H), 3.37 - 3.03 (m, 9H), 1.25 - 0.92 (m, 3H).

[0719] Step d. To a solution of (2S,3S,4S,5R,6S)-6-(2-(aminomethyl)-4-(((4-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)-1-oxo- 1.2-dihydrophthalazin-5-yl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2 / 7-pyran-2-carboxylic acid (700 mg, 0.81 mmol) in DMF (8 mL) was added Intermediate 8 (735 mg, 1.63 mmol) at 0 °C. The reaction mixture was stirred at rt for 2 h. Upon completion, the reaction mixture was directly purified by reverse-phase flash chromatography (H2O (0.01% FA) / MeCN) to give the title compound (550 mg, 56% yield) as a white solid.

[0720] m / z ES+ [M+H]+1199.0, 1201.0, 1202.0, 1203.0;1H NMR (300 MHz, DMSO-cfe) 5 12.60 (s, 1H), 8.81 - 8.27 (m, 1H), 7.90 - 7.81 (m, 1H), 7.79 - 7.68 (m, 1H), 7.61 - 7.53 (m, 1H), 7.52 -7.43 (m, 1 H), 7.36 - 7.23 (m, 1 H), 7.22 - 6.85 (m, 5H), 5.69 - 5.47 (m, 1 H), 5.40 - 5.03 (m, 4H), 5.00 - 4.77 (m, 1H), 4.61 - 4.13 (m, 7H), 4.15 - 3.67 (m, 4H), 3.62 - 3.47 (m, 3H), 3.45 - 3.33 (m, 5H), 2.60 - 2.52 (m, 2H), 1.23 - 0.95 (m, 3H).ART-C-P3900PCT

[0721] Example B5: (2S,3S,4S,5R,6S)-6-(2-((3-(2,5-Dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)propanamido)methyl)-4-(((4-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5, 6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)-1 -oxo-1,2-dihydrophthalazin-5-yl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid

[0722]

[0723] Steps a-c. These 3 steps were conducted as described in Example B4, steps a-c.

[0724] Step d. To a solution of (2S,3S,4S,5R,6S)-6-(2-(aminomethyl)-4-(((4-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)-1-oxo-1,2-dihydrophthalazin-5-yl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2 / 7-pyran-2-carboxylic acid (15 mg, 0.02 mmol) and 2,5-dioxopyrrolidin-1-yl 3-(2,5-dioxo-2,5-dihydro-1 / 7-pyrrol-1-yl)propanoate (CAS: 55750-62-4; 10 mg, 0.04 mmol) in DMF (0.8 mL) was added DI PEA (9 mg, 0.07 mmol) at 0 °C. The reaction mixture was stirred at rt for 2 h. Upon completion, the reaction mixture was directly purified by reverse-phase flash chromatography (H2O (0.01% FA) / MeCN) to give the title compound (6.5 mg, 37% yield) as a white solid. m / z ES+ [M+H]+1014.2;1H NMR (300 MHz, DMSO-cfe) 6 12.59 (s, 1H), 7.89 - 7.80 (m, 1H), 7.77 - 7.67 (m, 1H), 7.60 - 7.43 (m, 2H), 7.40 - 7.24 (m, 1H), 7.23 - 6.86 (m, 8H), 5.61 - 5.43 (m, 1H), 5.34 - 5.03 (m, 4H), 4.99 - 4.76 (m, 1H), 4.63 - 4.15 (m, 6H), 4.13 - 3.71 (m, 3H), 3.69 - 3.51 (m, 3H), 3.48 - 3.32 (m, 4H), 2.47 - 2.32 (m, 2H), 1.22 - 0.87 (m, 3H).ART-C-P3900PCT

[0725] Example B6: (2S,3S,4S,5 / ?,6S)-6-(2-((3-(4,5-Dibromo-2-methyl-3,6-dioxo-3,6-dihydropyridazin-1(2H)-yl)propanamido)methyl)-4-(((4-(3-((S)-6-methyl-3- (perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)-1-oxo-1,2-dihydrophthalazin-5-yl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid

[0726] o

[0727]

[0728] ART-C-P3900PCT

[0729]

[0730] Step a. A mixture of Intermediate 7a (1.6 g, 2.5 mmol), Example A2 (1.13 g, 2.1 mmol) and K2CO3 (582 mg, 4.2 mmol) in DMF (16 mL) was stirred at rt for 4 h. Upon completion, the reaction mixture was filtered and directly purified by reverse-phase flash chromatography (H2O (0.01% FA) / MeCN) to give (2S,3R,4S,5S,6S)-2-(2-(((ferf-butoxycarbonyl)amino)methyl)-4-(((4-(3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)-1 -oxo-1, 2-dihydrophthalazin-5-yl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (1.7 g, 62% yield) as a white solid. m / z ES+ [M+H]+1085.4;1H NMR (300 MHz, DMSO-cfe) 6 12.62 (s, 1H), 7.85 (d, J = 7.8 Hz, 1H), 7.76 - 7.66 (m, 1H), 7.55 (s, 1H), 7.44 (d, J= 8.2 Hz, 1 H), 7.28 - 7.11 (m, 4H), 7.08 - 6.89 (m, 4H), 5.66 - 5.57 (m, 1 H), 5.57 - 5.46 (m, 1 H), 5.20 - 5.00 (m, 5H), 4.80 - 4.70 (m, 1 H), 4.46 - 4.29 (m, 3H), 4.15 - 3.77 (m, 5H), 3.63 (s, 3H), 2.14 - 1.91 (m, 9H), 1.34 (s, 9H), 1.01 (s, 3H).

[0731] Step b. To a solution of (2S,3R,4S,5S,6S)-2-(2-(((tert-butoxycarbonyl)amino)methyl)-4-(((4-(3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)-1 -oxo-1, 2-dihydrophthalazin-5-yl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (1.7 g, 1.6 mmol) in DCM (15 mL) at 0 °C was added TFA (5 mL). The reaction mixture was stirred at rt for 2 h. Upon completion,ART-C-P3900PCT

[0732] the reaction mixture was evaporated and purified by reverse-phase flash chromatography (H2O (0.01% FA) / MeCN) to give (2S,3F?,4S,5S,6S)-2-(2-(aminomethyl)-4-(((4-(3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)-1-oxo-1,2-dihydrophthalazin-5-yl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (1.3 g, 84% yield) as a white solid.

[0733] m / z ES+ [M+H]+985.4;1H NMR (300 MHz, DMSO-cfe) 6 12.66 (s, 1H), 7.92 - 7.83 (m, 1H), 7.77 - 7.68 (m, 1H), 7.59 - 7.55 (m, 1H), 7.48 - 7.38 (m, 2H), 7.32 - 7.24 (m, 2H), 7.20 - 6.98 (m, 4H), 5.72 - 5.64 (m, 1 H), 5.60 - 5.47 (m, 1 H), 5.32 - 4.96 (m, 5H), 4.80 - 4.72 (m, 1 H), 4.54 - 4.27 (m, 4H), 4.20 - 4.06 (m, 1 H), 3.97 - 3.77 (m, 3H), 3.62 (s, 3H), 2.09 - 1.99 (m, 9H), 1.05 (s, 3H).

[0734] Step c. To a solution of (2S,3R,4S,5S,6S)-2-(2-(aminomethyl)-4-(((4-(3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)-1-oxo-1,2-dihydrophthalazin-5-yl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (1.3 g, 1.32 mmol) in THF (13.2 mL) and MeOH (13.2 mL) was added 1 M aq. LiOH (13 mL, 13.2 mmol) dropwise at 0 °C. The reaction mixture was stirred at rt for 90 min. Upon completion, the reaction mixture was quenched with FA (0.5 mL), partially evaporated to about 20 mL and directly purified by reverse-phase flash chromatography (H2O (0.01% FA) / MeCN) to give (2S,3S,4S,5F?,6S)-6-(2-(aminomethyl)-4-(((4-(3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)-1-oxo-1,2-dihydrophthalazin-5-yl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2 / 7-pyran-2-carboxylic acid (1 g, 90% yield) as a white solid.

[0735] m / z ES+ [M+H]+845.2;1H NMR (300 MHz, DMSO-cfe) 6 12.63 (s, 1H), 7.83 - 7.68 (m, 1H), 7.60 - 7.45 (m, 3H), 7.40 - 7.22 (m, 3H), 7.14 - 6.95 (m, 4H), 5.79 - 5.00 (m, 5H), 4.71 - 4.58 (m, 1H), 4.55 - 4.34 (m, 3H), 4.28 - 4.01 (m, 3H), 3.95 - 3.69 (m, 2H), 3.33 - 3.09 (m, 8H), 1.05 (s, 3H).

[0736] Step d. A mixture of (2S,3S,4S,5R,6S)-6-(2-(aminomethyl)-4-(((4-(3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)-1-oxo-1,2-dihydrophthalazin-5-yl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2 / 7-pyran-2-carboxylic acid (290 mg, 0.34 mmol) and Intermediate 8 (232 mg, 0.52 mmol) in DMF (3 mL) was stirred at rt for 4 h. Upon completion, the reaction mixture was directly purified by reversephase flash chromatography (H2O (0.01% FA) / MeCN) to give the title compound (268 mg, 66% yield) as a white solid.

[0737] m / z ES+ [M+H]+1181.1, 1183.1, 1184.1, 1185.1;1H NMR (400 MHz, DMSO-cfe) 5 12.63 (s, 1 H), 8.60 (s, 1 H), 7.90 - 7.81 (m, 1 H), 7.78 - 7.68 (m, 1 H), 7.60 - 7.52 (m, 1 H), 7.50 - 7.42 (m, 1H), 7.41 - 7.19 (m, 4H), 7.16 - 7.00 (m, 3H), 6.99 - 6.86 (m, 1H), 5.65 - 5.50 (m, 1H), 5.36 -5.22 (m, 1H), 5.18 - 4.98 (m, 3H), 4.95 -4.85 (m, 1H), 4.48 - 4.35 (m, 3H), 4.31 - 4.18 (m, 4H),ART-C-P3900PCT

[0738] 4.18 - 4.01 (m, 2H), 3.91 - 3.71 (m, 2H), 3.56 - 3.41 (m, 5H), 3.33 - 3.18 (m, 2H), 2.60 - 2.52 (m, 2H), 1.00 (s, 3H).

[0739] Example B7: (2S,3S,4S,5 / ?,6S)-6-(2-(2-(3-(4,5-Dibromo-2-methyl-3,6-dioxo-3,6-dihydropyridazin-1(2H)-yl)- / V-methylpropanamido)acetamido)-4-(((4-(4-fluoro-3-((S)-6- methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)- 1-oxo-1,2-dihydrophthalazin-5-yl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H- pyran-2-carboxylic acid

[0740] o

[0741] I

[0742]

[0743] IART-C-P3900PCT

[0744]

[0745] Step a. A mixture of Intermediate 7b (990 mg, 1.22 mmol), Example A1 (674 mg, 1.22 mmol) and K2CO3 (673 mg, 4.88 mmol) in DMF (20 mL) was stirred at rt for 4 h. Upon completion, the reaction mixture was filtered and directly purified by reverse-phase flash chromatography (H2O (0.01% FA) / MeCN) to give (2S,3R,4S,5S,6S)-2-(2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)acetamido)-4-(((4-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)-1-oxo-1,2-dihydrophthalazin-5-yl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (Fmoc product; 133 mg, 8% yield) as a white solid and (2S,3R,4S,5S,6S)-2-(4-(((4-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)-1 -oxo-1, 2-dihydrophthalazin-5-yl)oxy)methyl)-2-(2-(methylamino)acetamido)phenoxy)-6-(methoxycarbonyl)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (Des-Fmoc product; 290 mg, 22% yield) as a white solid.

[0746] Fmoc product: m / z ES+ [M+H]+1282.5.1H NMR (400 MHz, DMSO-d6) 5 12.73 - 12.52 (m, 1H), 9.13 - 8.88 (m, 1H), 7.92 - 7.83 (m, 4H), 7.81 - 7.66 (m, 2H), 7.59 - 7.54 (m, 1H), 7.52 -7.37 (m, 3H), 7.37 - 7.22 (m, 3H), 7.15 -6.88 (m, 5H), 6.25- 5.98 (m, 1H), 5.83- 5.60 (m, 1H), 5.57 - 5.44 (m, 1H), 5.39 - 5.01 (m, 5H), 4.79 - 4.67 (m, 1H), 4.55 - 3.81 (m, 10H), 3.76 - 3.65 (m, 1H), 3.64 - 3.53 (m, 2H), 3.00 - 2.79 (m, 3H), 2.15 - 1.85 (m, 9H), 1.31 - 1.09 (m, 3H).ART-C-P3900PCT

[0747] Des-Fmoc product: m / z ES+ [M+H]+1060.3.

[0748] Step b. To a solution of (2S,3R,4S,5S,6S)-2-(2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)acetamido)-4-(((4-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)-1-oxo-1,2-dihydrophthalazin-5-yl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (Fmoc product; 133 mg, 0.10 mmol) and (2S,3R,4S,5S,6S)-2-(4-(((4-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)-1 -oxo-1, 2-dihydrophthalazin-5-yl)oxy)methyl)-2-(2-(methylamino)acetamido)phenoxy)-6-(methoxycarbonyl)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (Des-Fmoc product; 290 mg, 0.27 mmol) in THF (4.8 mL) and MeOH (4.8 mL) at 0 °C was added 1 M aq. LiOH (4.8 mL, 4.8 mmol) dropwise. The reaction mixture was stirred at rt for 90 min. Upon completion, the reaction mixture was quenched by FA (0.18 mL) and directly purified by reverse-phase flash chromatography (H2O (0.01% FA) / MeCN) to give (2S,3S,4S,5 / ?,6S)-6-(4-(((4-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)-1 -oxo-1, 2-dihydrophthalazin-5-yl)oxy)methyl)-2-(2-(methylamino)acetamido)phenoxy)-3,4,5-trihydroxytetrahydro-2 / 7-pyran-2-carboxylic acid (280 mg, 82% yield) as a white solid.

[0749] m / z ES+ [M+H]+920.2.1H NMR (400 MHz, DMSO-d6) 6 12.62 (s, 1H), 9.91 (s, 1H), 8.28 -8.09 (m, 1 H), 7.89 - 7.80 (m, 1 H), 7.78 - 7.68 (m, 1 H), 7.60 - 7.53 (m, 1 H), 7.52 - 7.40 (m, 1 H), 7.26 - 6.83 (m, 5H), 5.76 - 4.96 (m, 6H), 4.91 - 4.72 (m, 1H), 4.62 - 4.28 (m, 4H), 4.22 - 3.87 (m, 4H), 3.79 - 3.56 (m, 5H), 3.37 - 3.20 (m, 2H), 2.49 - 2.45 (m, 2H), 1.26 - 1.02 (m, 3H). Step c. This step was conducted in a similar manner to Example B4, step d.

[0750] m / z ES+ [M+H]+1255.9, 1257.9, 1258.9, 1259.9;1H NMR (400 MHz, DMSO-cfe) 6 12.54 (s, 1H), 9.52 - 9.11 (m, 1H), 8.26 - 8.02 (m, 1H), 7.83 - 7.74 (m, 1H), 7.72 - 7.59 (m, 1H), 7.57 -7.35 (m, 2H), 7.23 - 6.72 (m, 5H), 5.94 - 5.53 (m, 1H), 5.38 - 4.96 (m, 4H), 4.91 - 4.60 (m, 1H), 4.55 - 4.05 (m, 7H), 4.02 - 3.78 (m, 2H), 3.57 - 3.45 (m, 7H), 3.27 - 3.16 (m, 3H), 3.03 - 2.89 (m, 2H), 2.81 - 2.72 (m, 2H), 2.71 - 2.56 (m, 1H), 1.12 - 0.73 (m, 3H).

[0751] Example B8: (2S,3S,4S,5 / ?,6S)-6-(2-(2-(3-(2,5-Dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)-Af-methylpropanamido)acetamido)-4-(((4-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)-1 -oxo-1,2-dihydrophthalazin-5-yl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acidART-C-P3900PCT

[0752]

[0753] Steps a-b. These 2 steps were conducted as described in Example B7, steps a-b.

[0754] Step c. This step was conducted in a similar manner to Example B2, step d.

[0755] m / z ES+ [M+H]+1071.2.1H NMR (400 MHz, DMSO-d6) 5 12.61 (s, 1H), 9.34 - 9.10 (m, 1H), 8.30 - 8.10 (m, 1H), 7.87 - 7.80 (m, 1H), 7.78 - 7.67 (m, 1H), 7.59 - 7.52 (m, 1H), 7.50 - 7.39 (m, 1H), 7.19 - 6.83 (m, 7H), 5.87 - 5.67 (m, 1H), 5.43 - 5.19 (m, 2H), 5.17 - 5.00 (m, 2H), 4.98 - 4.83 (m, 1H), 4.45 - 4.29 (m, 3H), 4.26 - 4.13 (m, 2H), 4.11 - 3.83 (m, 3H), 3.66 - 3.57 (m, 2H), 3.51 - 3.34 (m, 6H), 3.07 - 2.80 (m, 3H), 2.73 - 2.64 (m, 1H), 2.60 - 2.53 (m, 1H), 1.14 -0.73 (m, 3H).

[0756] Example B9: (2S,3S,4S,5 / ?,6S)-6-(2-(2-(3-(4,5-Dibromo-2-methyl-3,6-dioxo-3,6-dihydropyridazin-1(2H)-yl)-N-methylpropanamido)acetamido)-4-(((4-(3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)-1-oxo-1,2-dihydrophthalazin-5-yl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acidART-C-P3900PCT

[0757]

[0758] The title compound was prepared in a similar manner to Example B7, using Example A2 in step a.

[0759] m / z ES+ [M+H]+1238.1, 1240.0, 1241.0, 1242.0.1H NMR (400 MHz, DMSO-d6) 5 12.63 (s, 1H), 9.36 - 9.12 (m, 1H), 8.24 - 8.11 (m, 1H), 7.89 - 7.82 (m, 1H), 7.78 - 7.68 (m, 1H), 7.60 -7.41 (m, 2H), 7.30 - 7.17 (m, 2H), 7.10 -6.92 (m, 3H), 6.90-6.75 (m, 1H), 5.79- 5.67 (m, 1H), 5.39 - 5.25 (m, 1H), 5.16 - 5.07 (m, 2H), 4.98 - 4.83 (m, 1H), 4.46 - 4.07 (m, 10H), 3.90 - 3.76 (m, 2H), 3.63 - 3.52 (m, 5H), 3.03 (s, 3H), 2.84 (s, 3H), 2.75 - 2.64 (m, 1H), 2.50 - 2.46 (m, 1H), 0.99 (s, 3H).

[0760] Example B10: (2S,3S,4S,5 / ?,6S)-6-(2-(2-(3-(2,5-Dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)- / V- methylpropanamido)acetamido)-4-(((4-(3-((S)-6-methyl-3-(perfluoroethyl)-5, 6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)-1 -oxo-1,2-dihydrophthalazin-5-yl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid

[0761] o

[0762]

[0763] The title compound was prepared in a similar manner to Example B8, using Example A2 in step a.ART-C-P3900PCT

[0764] m / z ES+ [M+H]+1053.4.1H NMR (300 MHz, DMSO-d6) 5 12.61 (s, 1H), 9.45 - 9.02 (m, 1H), 8.27 - 8.10 (m, 1H), 7.90 - 7.82 (m, 1H), 7.78 - 7.67 (m, 1H), 7.59 - 7.50 (m, 1H), 7.50 - 7.42 (m, 1H), 7.29 - 7.17 (m, 2H), 7.12 - 6.89 (m, 5H), 6.88 - 6.78 (m, 1H), 5.86 - 5.62 (m, 1H), 5.42 - 5.25 (m, 1H), 5.22 - 5.00 (m, 3H), 4.99 - 4.84 (m, 1H), 4.56 - 4.00 (m, 7H), 3.98 - 3.74 (m, 2H), 3.70 - 3.55 (m, 2H), 3.50 - 3.31 (m, 5H), 3.02 (s, 2H), 2.89 - 2.78 (m, 1 H), 2.75 - 2.64 (m, 1H), 2.62 - 2.53 (m, 1H), 1.16 - 0.89 (m, 3H).

[0765] Preparation of Example C1: Sacituzumab-PARPi ADC (DAR 8.0)

[0766]

[0767] wherein Ab represents Sacituzumab and wherein “S” represents a sulfur atom within a cysteine residue of said Sacituzumab.

[0768] Sacituzumab has the following sequence:

[0769] Heavy chain (HC) QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEP TYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 3).

[0770] Light chain (LC) DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRF SGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIKRTVAAPSVFIFPPSDEQLKART-C-P3900PCT

[0771] SGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 4).

[0772] Sacituzumab was purchased commercially and reconstituted with PBS pH 7.4 to yield a 6.0 mg / mL solution.

[0773] 8 molar equiv. of 1 mM aq. TCEP relative to the antibody was added at rt, and the antibody was left to reduce at 37 °C for 3 h with gentle shaking.

[0774] DMSO was added to formulate the reduced antibody to 10% v / v DMSO, after which 12 molar equiv. of Example B1 was added dropwise from a 10 mM DMSO stock, and the reduced antibody was allowed to conjugate at 25 °C for 90 min with gentle shaking.

[0775] 24 molar equiv. of 10 mM aq. cysteine was added at rt to quench unreacted Example B1. The resulting ADC was purified through ultrafiltration (50KD ultrafiltration tube) using PBS pH 7.4 to remove small molecules and DMSO co-solvent. The purified ADC was concentrated to 3.0 mg / mL by membrane centrifugation and formulated to 0.02% v / v PS80.

[0776] The title compound was sampled for QC testing, with the below specification:

[0777] Quantity 17 mg

[0778] Average DAR 7.9

[0779] Endotoxin < 1 EU / mL

[0780] Purity (SEC-HPLC) 96.9%

[0781] Concentration 3.0 mg / mL

[0782] Residual Example A1 < 2%

[0783]

[0784] Preparation of Example C2: Sacituzumab-PARPi ADC (DAR 8.0)

[0785] o

[0786]

[0787] OH OH wherein Ab represents Sacituzumab and wherein “S” represents a sulfur atom within a cysteine residue of said Sacituzumab.ART-C-P3900PCT

[0788] Example C2 was prepared in a similar manner to Example C1, using Example B2 as the payload-linker, and was sampled for QC testing, with the below specification:

[0789] Quantity 7 mg

[0790] Average DAR 7.8

[0791] Endotoxin < 1 EU / mL

[0792] Purity (SEC-HPLC) 97.3%

[0793] Concentration 2.8 mg / mL

[0794] Residual Example A1 < 2%

[0795]

[0796] Preparation of Example C3: Sacituzumab-PARPi ADC (DAR 8.0)

[0797]

[0798] wherein Ab represents Sacituzumab and wherein “S” represents a sulfur atom within a cysteine residue of said Sacituzumab.

[0799] Example C3 was prepared in a similar manner to Example C1, using Example B3 as the payload-linker, and was sampled for QC testing, with the below specification:

[0800] Quantity 16 mg

[0801] Average DAR 7.9

[0802] Endotoxin < 1 EU / mL

[0803] Purity (SEC-HPLC) 96.8%

[0804] Concentration 2.9 mg / mL

[0805] Residual Example A1 < 2%

[0806]

[0807] Preparation of Example C4: Sacituzumab-PARPi ADC (DAR 4.0)ART-C-P3900PCT

[0808]

[0809] wherein Ab represents Sacituzumab and wherein each “S” represents a sulfur atom within a cysteine residue of said Sacituzumab.

[0810] Sacituzumab was generated via transient expression in CHO K1 cells at a titer of 1.6 g / L for 7 days and purified by affinity chromatography (MabSelect SuRe, Cytiva), cation exchange chromatography (POROS XS, Thermo Fisher Scientific) and ultrafiltration to give a 18.2 mg / mL solution in PBS (pH 7.2).

[0811] 8 molar equiv. of 1 mM aq. TCEP relative to the antibody was added at rt, and the antibody was left to reduce at 37 °C for 2 h with gentle shaking.

[0812] DMSO was added to formulate the reduced antibody to 10% v / v DMSO, after which 4.9 molar equiv. of Example B4 was added dropwise from a 10 mM DMSO stock, and the reduced antibody was allowed to conjugate at 25 °C for 90 min with gentle shaking.

[0813] The resulting ADC was purified through ultrafiltration (50KD ultrafiltration tube) using Histidine pH 6.0 to remove small molecules and DMSO co-solvent. The purified ADC was concentrated to 3.0 mg / mL by membrane centrifugation.

[0814] The title compound was sampled for QC testing, with the below specification:

[0815] Quantity 84 mg

[0816] Average DAR 4.1

[0817] Endotoxin < 1 EU / mL

[0818] Purity (SEC-HPLC) 98.3%

[0819] Concentration 2.9 mg / mL

[0820] Residual Example A1 < 2%

[0821]

[0822] Preparation of isotype control Reference Example C5: lgG1-kappa-PARPi ADC (DAR 4.0)ART-C-P3900PCT

[0823]

[0824] wherein Ab represents non-targeting antibody, lgG1-kappa and wherein each “S” represents a sulfur atom within a cysteine residue of said lgG1 -kappa.

[0825] Reference Example C5 was prepared in a similar manner to Example C4, using lgG1 -kappa as the antibody, 5.2 molar equiv. of Example B4, and was sampled for QC testing, with the below specification:

[0826] Quantity 21 mg

[0827] Average DAR 4.1

[0828] Endotoxin < 1 EU / mL

[0829] Purity (SEC-HPLC) 98.2

[0830] Concentration 2.9 mg / mL

[0831] Residual Example A1 < 2%

[0832]

[0833] Preparation of Example C6: Sacituzumab-PARPi ADC (DAR 4.0)

[0834]

[0835] wherein Ab represents Sacituzumab and wherein “S” represents a sulfur atom within a cysteine residue of said Sacituzumab.

[0836] Sacituzumab was produced according to the protocol described in Example C4.ART-C-P3900PCT

[0837] 2.6 molar equiv. of 1 mM aq. TCEP relative to the antibody was added at rt, and the antibody was left to reduce at 37 °C for 2 h with gentle shaking.

[0838] DMSO was added to formulate the reduced antibody to 10% v / v DMSO, after which 6 molar equiv. of Example B5 was added dropwise from a 10 mM DMSO stock, and the reduced antibody was allowed to conjugate at 25 °C for 90 min with gentle shaking.

[0839] 12 molar equiv. of 10 mM aq. cysteine was added at rt to quench unreacted Example B5. The resulting ADC was purified through ultrafiltration (50KD ultrafiltration tube) using Histidine pH 6.0 to remove small molecules and DMSO co-solvent. The purified ADC was concentrated to 3.0 mg / mL by membrane centrifugation.

[0840] The title compound was sampled for QC testing, with the below specification:

[0841] Quantity 17 mg

[0842] Average DAR 4.1

[0843] Endotoxin < 1 EU / mL

[0844] Purity (SEC-HPLC) 98.5%

[0845] Concentration 3.0 mg / mL

[0846] Residual Example A1 < 2%

[0847]

[0848] Preparation of Example C7: Sacituzumab-PARPi ADC (DAR 4.0)

[0849]

[0850] wherein Ab represents Sacituzumab and wherein each “S” represents a sulfur atom within a cysteine residue of said Sacituzumab.

[0851] Sacituzumab was produced according to the protocol described in Example C4.

[0852] 8 molar equiv. of 1 mM aq. TCEP relative to the antibody was added at rt, and the antibody was left to reduce at 37 °C for 2 h with gentle shaking.

[0853] DMSO was added to formulate the reduced antibody to 10% v / v DMSO, after which 5.5 molar equiv. of Example B6 was added dropwise from a 10 mM DMSO stock, and the reduced antibody was allowed to conjugate at 25 °C for 90 min with gentle shaking.ART-C-P3900PCT

[0854] The resulting ADC was purified through ultrafiltration (50KD ultrafiltration tube) using Histidine pH 6.0 to remove small molecules and DMSO co-solvent. The purified ADC was concentrated to 3.0 mg / mL by membrane centrifugation.

[0855] The title compound was sampled for QC testing, with the below specification:

[0856] Quantity 23 mg

[0857] Average DAR 4.1

[0858] Endotoxin < 1 EU / mL

[0859] Purity (SEC-HPLC) 98.8%

[0860] Concentration 3.0 mg / mL

[0861] Residual Example A2 < 2%

[0862]

[0863] Preparation of isotype control Reference Example C8: lgG1-kappa-PARPi ADC (DAR 4.0)

[0864]

[0865] wherein Ab represents non-targeting antibody, lgG1-kappa and wherein each “S” represents a sulfur atom within a cysteine residue of said lgG1 -kappa.

[0866] Reference Example C8 was prepared in a similar manner to Example C7, using I gG1 -kappa as the antibody, and was sampled for QC testing, with the below specification:

[0867] Quantity 140 mg

[0868] Average DAR 3.87

[0869] Endotoxin < 0.12 EU / mg

[0870] Purity (SEC-HPLC) 97.59

[0871] Concentration 13.86 mg / mL

[0872] Residual Example A2 < 0.19%

[0873]

[0874] Preparation of Example C9: Sacituzumab-PARPi ADC (DAR 4.0)ART-C-P3900PCT

[0875]

[0876] wherein Ab represents Sacituzumab and wherein each “S” represents a sulfur atom within a cysteine residue of said Sacituzumab.

[0877] Example C9 was prepared in a similar manner to Example C7, using Example B7 (4.25 molar equiv. instead of 5.5 molar equiv) as the payload-linker, and was sampled for QC testing, with the below specification:

[0878] Quantity 12.6 mg

[0879] Average DAR 4.0

[0880] Endotoxin < 1 EU / mL

[0881] Purity (SEC-HPLC) 98.5%

[0882] Concentration 3.0 mg / mL

[0883] Residual Example A1 < 2%

[0884]

[0885] Preparation of Example C10: Sacituzumab-PARPi ADC (DAR 8.0)

[0886]

[0887] wherein Ab represents Sacituzumab and wherein “S” represents a sulfur atom within a cysteine residue of said Sacituzumab.

[0888] Sacituzumab was produced according to the protocol described in Example C4.ART-C-P3900PCT

[0889] 8 molar equiv. of 1 mM aq. TCEP relative to the antibody was added at rt, and the antibody was left to reduce at 37 °C for 2 h with gentle shaking.

[0890] DMSO was added to formulate the reduced antibody to 10% v / v DMSO, after which 12 molar equiv. of Example B8 was added dropwise from a 10 mM DMSO stock, and the reduced antibody was allowed to conjugate at 25 °C for 90 min with gentle shaking.

[0891] 24 molar equiv. of 10 mM aq. cysteine was added at rt to quench unreacted Example B8. The resulting ADC was purified through ultrafiltration (50KD ultrafiltration tube) using Histidine pH 6.0 to remove small molecules and DMSO co-solvent. The purified ADC was concentrated to 3.0 mg / mL by membrane centrifugation.

[0892] The title compound was sampled for QC testing, with the below specification:

[0893] Quantity 14.4 mg

[0894] Average DAR 8.0

[0895] Endotoxin < 1 EU / mL

[0896] Purity (SEC-HPLC) 98.5%

[0897] Concentration 3.0 mg / mL

[0898] Residual Example A1 < 2%

[0899]

[0900] Preparation of Example C11: Sacituzumab-PARPi ADC (DAR 4.0)

[0901]

[0902] wherein Ab represents Sacituzumab and wherein “S” represents a sulfur atom within a cysteine residue of said Sacituzumab.

[0903] Sacituzumab was produced according to the protocol described in Example C4.

[0904] 3.0 molar equiv. of 1 mM aq. TCEP relative to the antibody was added at rt, and the antibody was left to reduce at 37 °C for 2 h with gentle shaking.

[0905] DMSO was added to formulate the reduced antibody to 10% v / v DMSO, after which 6 molar equiv. of Example B8 was added dropwise from a 10 mM DMSO stock, and the reduced antibody was allowed to conjugate at 25 °C for 90 min with gentle shaking.ART-C-P3900PCT

[0906] 12 molar equiv. of 10 mM aq. cysteine was added at rt to quench unreacted Example B8.

[0907] The resulting ADC was purified through ultrafiltration (50KD ultrafiltration tube) using Histidine pH 6.0 to remove small molecules and DMSO co-solvent. The purified ADC was concentrated to 3.0 mg / mL by membrane centrifugation.

[0908] The title compound was sampled for QC testing, with the below specification:

[0909] Quantity 12.5 mg

[0910] Average DAR 4.1

[0911] Endotoxin < 1 EU / mL

[0912] Purity (SEC-HPLC) 98.7%

[0913] Concentration 3.0 mg / mL

[0914] Residual Example A1 < 2%

[0915]

[0916] Preparation of Example C12: Sacituzumab-PARPi ADC (DAR 4.0)

[0917]

[0918] wherein Ab represents Sacituzumab and wherein each “S” represents a sulfur atom within a cysteine residue of said Sacituzumab.

[0919] Example C12 was prepared in a similar manner to Example C7, using Example B9 as the payload-linker, and was sampled for QC testing, with the below specification:

[0920] Quantity 12 mg

[0921] Average DAR 4.2

[0922] Endotoxin < 1 EU / mL

[0923] Purity (SEC-HPLC) 98.4%

[0924] Concentration 2.9 mg / mL

[0925] Residual Example A2 < 2%

[0926]

[0927] Preparation of Example C13: Sacituzumab-PARPi ADC (DAR 8.0)ART-C-P3900PCT

[0928]

[0929] wherein Ab represents Sacituzumab and wherein “S” represents a sulfur atom within a cysteine residue of said Sacituzumab.

[0930] Example C13 was prepared in a similar manner to Example C10, using Example B10 as the payload-linker, and was sampled for QC testing, with the below specification:

[0931] Quantity 12 mg

[0932] Average DAR 7.8

[0933] Endotoxin < 1 EU / mL

[0934] Purity (SEC-HPLC) 98.0%

[0935] Concentration 3.0 mg / mL

[0936] Residual Example A2 < 2%

[0937]

[0938] Preparation of Example C14: Sacituzumab-PARPi ADC (DAR 4.0)

[0939]

[0940] wherein Ab represents Sacituzumab and wherein “S” represents a sulfur atom within a cysteine residue of said Sacituzumab.

[0941] Example C14 was prepared in a similar manner to Example C11, using Example B10 as the payload-linker, and was sampled for QC testing, with the below specification:ART-C-P3900PCT

[0942] Quantity 11 mg

[0943] Average DAR 4.0

[0944] Endotoxin < 1 EU / mL

[0945] Purity (SEC-HPLC) 97.6%

[0946] Concentration 3.0 mg / mL

[0947] Residual Example A2 < 2%

[0948]

[0949] Preparation of Example C15: LALA-Sacituzumab-PARPi ADC (DAR 4.0)

[0950]

[0951] wherein Ab represents Sacituzumab with a double L toA mutation at positions L234 and L235 (Ell numbering according to Kabat (Kabat, E. A. (1991)) on the Heavy Chain to give L234A / L235A-Sacituzumab (LALA-Sacituzumab) and wherein each “S” represents a sulfur atom within a cysteine residue of said LALA-Sacituzumab.

[0952] LALA-Sacituzumab has the following sequence:

[0953] Heavy chain (HC) QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEP TYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGG PSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 5).

[0954] Light chain (LC)ART-C-P3900PCT

[0955] DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRF SGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIKRTVAAPSVFIFPPSDEQLK SGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 4).

[0956] LALA-Sacituzumab was generated via transient expression in CHO K1 cells at a titer of 1.25 g / L for 7 days and purified by affinity chromatography (MabSelect SuRe, Cytiva), cation exchange chromatography (POROS XS, Thermo Fisher Scientific) and ultrafiltration to give a 12.5 mg / mL solution in PBS (pH 7.2).

[0957] LALA-Sacituzumab was reconstituted with PBS pH 7.4 to yield a 6.0 mg / mL solution.

[0958] 8 molar equiv. of 1 mM aq. TCEP relative to the antibody was added at rt, and the antibody was left to reduce at 37 °C for 2 h with gentle shaking.

[0959] DMSO was added to formulate the reduced antibody to 10% v / v DMSO, after which 4.7 molar equiv. of Example B4 was added dropwise from a 10 mM DMSO stock, and the reduced antibody was allowed to conjugate at 25 °C for 90 min with gentle shaking.

[0960] The resulting ADC was purified through ultrafiltration (50KD ultrafiltration tube) using Histidine pH 6.0 to remove small molecules and DMSO co-solvent. The purified ADC was concentrated to 2.8 mg / mL by membrane centrifugation.

[0961] The title compound was sampled for QC testing, with the below specification:

[0962] Quantity 120 mg

[0963] Average DAR 4.0

[0964] Endotoxin < 1 EU / mL

[0965] Purity (SEC-HPLC) 97.9%

[0966] Concentration 2.8 mg / mL

[0967] Residual Example A1 < 2%

[0968]

[0969] Preparation of Example C16: LALA-Sacituzumab-PARPi ADC (DAR 4.0)

[0970]

[0971] ART-C-P3900PCT

[0972] wherein Ab represents LALA-Sacituzumab and wherein each “S” represents a sulfur atom within a cysteine residue of said LALA-Sacituzumab.

[0973] Example C16 was prepared in a similar manner to Example C15, using Example B6 as the payload-linker, and was sampled for QC testing, with the below specification:

[0974] Quantity 23 mg

[0975] Average DAR 4.0

[0976] Endotoxin < 1 EU / mL

[0977] Purity (SEC-HPLC) 97.3%

[0978] Concentration 2.6 mg / mL

[0979] Residual Example A2 < 2%

[0980]

[0981] BIOLOGICAL EXAMPLES

[0982] Unless otherwise stated, all reagents were purchased from commercial vendors and used as received without further purification. Except where indicated, all values quoted for KD, KI, IC50, EC50 and AUG are the mean of > 2 independent replicates.

[0983] Biological Example 1: PARP1 and PARP2 FRET binding assays

[0984] FRET probe

[0985] 3-(2-{(1E,3E,5E)-5-[3,3-dimethyl-5-sulfo-1-(3-sulfopropyl)-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dien-1-yl}-3-[6-({8-[4-(4-{3-[(8-methoxy-4-oxo-3,4-dihydrophthalazin-1-yl)methyl]benzoyl}-2-oxopiperazin-1-yl)anilino]-8-oxooctyl}amino)-6-oxohexyl]-3-methyl-5-sulfo-3H-indol-1-ium-1-yl)propane-1 -sulfonate was synthesised according to the literature procedure described in J. Med. Chem. 2021, 64(19), 14498-14512.

[0986] PARP proteins

[0987] Human PARP1 and PARP2 were expressed in insect cells, purified, aliquoted and stored at -80 °C until required. PARP1 (aa2-1014) was purified by affinity chromatography (NiNTA and Heparin) followed by a size exclusion chromatography step. PARP2 (aa1-583) was purified by affinity chromatography (GST) followed by tag cleavage and a final size exclusion chromatography step.

[0988] FRET labelling of PARP proteins

[0989] Lanthascreen Thiol Reactive Tb chelate (Invitrogen #PV3580) and protein (PARP1 or PARP2 as required) which had been thawed on ice were combined in a 1:1 molar ratio and left at room temperature in the dark for 15 minutes to react. Unreacted Tb chelate was then removed by buffer exchange on a Zeba column previously equilibrated with enzyme storage bufferART-C-P3900PCT

[0990] (PARP1: 50 mM Hepes pH 7.5, 300 mM NaCI, 0.5 mM TCEP, 10% Glycerol; PARP2: 25 mM HEPES pH 8.0, 150 mM NaCI, 0.1 mM TCEP, 1 mM EDTA, 0.025% Igepal), according to the manufacturer’s instructions (Thermo Scientific Zeba 7K MWCO #89882). Protein and Tb chelate concentrations were determined by measuring the absorbance at 280 and 343 nm according to the manufacturer’s instructions (Invitrogen #PV3580). The labelled PARP1 and PARP2 were split into aliquots and kept at -80°C for future use.

[0991] PARP1 dumbbell DNA preparation

[0992] 5’-GCTGAGCTTCTGGTGAAGCTCAGCTCGCGGCAGCTGGTGCTGCCGCGA-3’ (SEQ ID NO: 1), which was based on Langelier et al. (Langelier et al., Nature Communications (2018), 9(884)) was synthesized by Integrated DNA Technologies. The oligo was resuspended in annealing buffer (20 mM Tris pH 7.5, 50 mM NaCI) using the volume recommended by the manufacturer to generate a 100 pM solution. The concentration of the resuspended oligo was determined using the A260 (with a buffer blank subtraction) and extinction coefficient supplied by the manufacturer. Once resuspended, 100 pL DNA aliquots were heated to 65 °C for 15 mins before the heating block was switched off and left to cool to room temperature to allow the DNA to self-anneal. Once cooled the samples were pooled and stored as 20 pL aliquots at -80 °C.

[0993] PARP FRET binding assays

[0994] All assays were carried out in phosphate buffered saline (PBS) supplemented with 0.01% v / v Tween-20 and 0.5 mM tris(2-carboxyethyl)phosphine which were freshly prepared just prior to use.

[0995] Compounds which had been pre dissolved in DMSO to give 10 mM stocks were dispensed into white 384-shallow well microplates (Greiner #784075) using a D300e (Tecan) or Mosquito LV (SPT Labtech). Compounds were dispensed either as an 11 -point dose response with a 3-fold serial dilution or a 22-point dose response with a 2-fold serial dilution. The volume of DMSO was adjusted to ensure that each well contained 1% v / v DMSO in the final assay (100 nL DMSO).

[0996] 2x reaction mixtures of Tb labeled PARP1 (2.4 nM), PARP2 (2.4 nM), PARP1 + DNA (2.4 nM PARP1, 40 nM DNA) or PARP2 + DNA (2.4 nM PARP2, 40 nM DNA) and 2x reaction mixture of probe (200 nM) were prepared in assay buffer. 5 pL of the relevant 2x PARP protein (or PARP + DNA mix) and 5 pL of 2x probe were dispensed into the appropriate wells of the preprepared assay ready plates using a Multidrop Combi (Thermo Fisher Scientific). Immediately after dispensing, the plate was spun in a Megafuge 16R benchtop centrifuge (Thermo FisherART-C-P3900PCT

[0997] Scientific) at 300 RPM for 10 seconds. The plates were covered and allowed to incubate at room temperature in the dark for 2 hours. Plates were then read on a PHERAstar FSX instrument (BMG Labtech) using the TR FRET optic module, acceptor 337 / 665 nm, donor 337 / 620, 50 flashes.

[0998] Data analysis

[0999] All data analysis was carried out using Genedata Screener software. IC50 values were calculated using the SmartFit function. Where compounds displayed biphasic behaviour, IC50S were calculated using the Biphasic fit function and only the most potent of the two IC50 reported.

[1000] To account for competition by the probe, a Cheng-Prusoff correction to determine compound KD was made for all biochemical data by dividing IC50 obtained from Genedata by (1+[probe] / probe KD). Values for probe KD are shown in Table 1 below.

[1001] Table 1: FRET probe Kps

[1002] Assay Probe KD(nM)

[1003] PARP1 2.6

[1004] PARP1 + DNA 1.5

[1005] PARP2 3.9

[1006] PARP2 + DNA 8.5

[1007]

[1008] Results

[1009] The results of the PARP1 and PARP2 FRET binding assays are shown below in Table 2.

[1010] Table 2: Results of Biological Example 1

[1011] Example PARP1 KD PARP1 + PARP2 KD PARP2 +

[1012] (nM) DNA KD (nM) (nM) DNA KD (nM) Example A1 0.059 0.006 0.015 0.039

[1013] Example A2 0.071 0.004 0.014 0.031

[1014] Example A3 - 0.007 - 0.007

[1015] Olaparib 0.47 14 0.096 5.0

[1016] Talazoparib 0.60 0.030 15 9.9

[1017] Saruparib 0.29 0.027 520 3,300

[1018] Pip-6 0.79 0.021 0.019 0.030

[1019]

[1020] ART-C-P3900PCT

[1021] The results of Biological Example 1 confirm that the tested compounds of the invention are highly potent binders of PARP1 and PARP2 in the presence and absence of DNA. The compounds of the invention are more potent binders of PARP1 in the presence of DNA than literature examples such as Pip-6. Surprisingly, the observed increase in potency is specific to PARP1 and there is no corresponding increase in the PARP2 potency relative to Pip-6.

[1022] Biological Example 2: PARP1 and PARP2 DNA strand exchange assays

[1023] DNA strand exchange assays were based on the method described by Langelier et al., Nature Communications (2018), 9(884). DNA oligos were synthesized by Integrated DNA Technologies and prepared as described in Biological Example 1. The identities of the oligos used are shown below in Table 3.

[1024] Table 3: Oligos used in DNA strand exchange assays

[1025] PARP1 5'- probe GCTGAGC / X / TCTGGTGAAGCTCAGCTCGCGGCAGCTGGTGCTGCCGC GA-3' (SEQ ID NO: 2)

[1026] PARP 5’- unlabelled GCTGAGCTTCTGGTGAAGCTCAGCTCGCGGCAGCTGGTGCTGCCGCG DNA A-3’ (SEQ ID NO: 1)

[1027] PARP2 5'- Probe pGCTGAGC / X / TCTGGTGAAGCTCAGCTCGCGGCAGCTGGTGCTGCCG CGA-3' (SEQ ID NO: 2)

[1028] PARP2 5’- unlabelled pGCTGAGCTTCTGGTGAAGCTCAGCTCGCGGCAGCTGGTGCTGCCGC DNA GA-3’ (SEQ ID NO: 1)

[1029]

[1030] wherein X represents a [18F]-3-fluoro-l-a-methyl-tyrosine (FAMT) label.

[1031] All assays were carried out in freshly prepared assay buffer (12 mM HEPES pH 8, 8 mM MgCh, 0.05 mg / mL BSA, 5.7 mM p-mercaptoethanol, 4% glycerol and 60 mM KOI).

[1032] PARP1 strand exchange assay

[1033] Compounds which had been pre dissolved in DMSO to give 10 mM stocks were dispensed into black 384-shallow well microplates (Greiner #784076) using a D300e (Tecan). Compounds were dispensed to give a final assay concentration of 5 pM. The volume of DMSO was adjusted to ensure that each well contained 1% v / v DMSO in the final assay (100 nL DMSO).ART-C-P3900PCT

[1034] 2x reaction mixtures of PARP and probe DNA (20 nM PARP1 DNA probe and 40 nM PARP1) and unlabelled PARP1 DNA (200 nM) were made in assay buffer. 5 pL of the PARP1 and probe DNA mix are dispensed into the appropriate wells of the pre-prepared assay ready plate using an Integra multichannel pipette along with control wells containing only 2x probe DNA. The plate was then covered and left to equilibrate for 30 mins. The plate is then transferred to a PHERAstar FSX instrument equipped with injectors (BMG Labtech). 5 pL of unlabelled 2x DNA (or buffer for the no exchange control) was injected into the appropriate wells and the FP signal monitored until the labelled DNA had fully dissociated using an FI-FP optic module (FP 540-20 590-20 590-20) and the gain set to 35 mP using the labelled DNA. Cycle time and assay duration were dependent on dissociation speed (typically 1-20 s cycle times and run for 4-90 mins).

[1035] PARP2 strand exchange assay

[1036] Compounds which had been pre-dissolved in DMSO to give 10 mM stocks were dispensed into black 384-shallow well microplates (Greiner #784076) using a DC1 and Mosquito (SPT Labtech). Compounds were initially prediluted at 1 in 20 in DMSO using the DC1 (SPT Labtech), then dispensed to give a final assay concentration of 5 pM using the Mosquito (SPT Labtech). Each well contained 1% v / v DMSO in the final assay (100 nL DMSO).

[1037] 2x reaction mixtures of PARP and probe DNA (20 nM PARP2 DNA probe and 40 nM PARP2) and unlabelled PARP2 DNA (200 nM) were prepared in assay buffer. 5 pL of the 2x PARP2 and probe DNA mix are dispensed into the appropriate wells of the pre-prepared assay ready plate using a Tempest (Formulatrix) along with full exchange control wells containing only 2x probe DNA. The plate was then covered and left to equilibrate for 30 mins before 5 pL of the unlabelled PARP2 DNA mix (or buffer for the no exchange control) were dispensed into the appropriate wells using a Tempest (Formulatrix). 10 pL of paraffin oil (Molecular dimensions #MD2-03) was then dispensed into reaction wells using the Integra multichannel pipette. The plate was then spun at 500Xg for 1 minute before being measured on a PHERAstar FSX (BMG Labtech) using an FI-FP optic module (FP 540-20590-20590-20) and the gain set to 35 mP using the labelled DNA. Measurements were taken every 300s for 650 cycles.

[1038] Data

[1039] All data analysis was carried out using Genedata Screener software. The dissociation rate of labelled DNA in the presence and absence of compound was determined using the Slow-Binding (Local Fit) Aggregation Method in the Mechanistic Analysis Extension. The residenceART-C-P3900PCT

[1040] time was calculated as the reciprocal of the dissociation rate. Residence time is reported relative to the DMSO control.

[1041] Results

[1042] The results of the PARP1 and PARP2 DNA strand exchange assays are shown in Figure 1 and summarised below in Table 4. The data shown are the fold-change (FC) in residence time of the labelled DNA substrate on PARP in the presence of compound relative to DMSO control.

[1043] Table 4: Results of Biological Example 2

[1044] Example PARP1 FC PARP2 FC

[1045] Example A1 59 1.9

[1046] Example A2 n.d. 2.3

[1047] Olaparib 0.9 1.1

[1048] Talazoparib 8.5 1.6

[1049] Saruparib <0.9 0.9

[1050] Pip-6 27 1.5

[1051]

[1052] *n.d. = not determined (dissociation rate was slower than the assay length); indicates prolongation of residence time of PARP on DNA, but quantification was not possible.

[1053] The results of Biological Example 2 indicate that the tested compounds of the invention induce a significantly prolonged residence time of PARP1 on DNA, which is consistent with a proretention mechanism. This pro-retention mechanism is not evident with either saruparib or olaparib and the effect of compounds of the invention is stronger than with talazoparib. The rate of PARP1 strand exchange in the presence of saruparib was too fast to be accurately measured under the assay conditions, hence its value being given as <0.9. Interestingly, the magnitude of the pro-retention effect (i.e. the FC) of compound Example A1 is double that of Pip-6. The magnitude of the pro-retention effect of compound Example A2 was too large to measure accurately under these assay conditions. Surprisingly, the compounds of the invention do not induce the same magnitude of prolongation of residence time of PARP2 on DNA, which suggests that the pro-retention behaviour of the compounds is specific to PARP1.

[1054] Biological Example 3: Immunofluorescence assay to measure chromatin retention of PARP1 and PARP2 and inhibition of PARylation

[1055] Cells were seeded at a density of 8,000 cells per well into 96-well plates. Methyl methanesulfonate (MMS) was added at a final concentration of 0.01%, then PARPi were added with D300e (Tecan) at final concentration range between 0.1 nM and 1 pM andART-C-P3900PCT

[1056] incubated for 4 hours in culture conditions (37°C and 5% CO2). Cell media was then removed and a pre-extraction step was performed for 3 min at room temperature with cold cytoskeleton (CSK) buffer supplemented with 0.5% Triton X-100. Cells were then fixed with 4% paraformaldehyde (PFA) at room temperature for 10 minutes and were washed twice with phosphate-buffered saline (PBS) before permeabilization with ice-cold methanol for 10 minutes at 4°C. Cells were then washed twice with PBS and blocked in blocking buffer (PBS + 0.5% Triton X-100 + 0.5% BSA) for 1 hour at room temperature. Immunostaining of PARP1, PARP2 or Poly / Mono-ADP Ribosylation was performed by adding the primary antibody diluted 1:1000 in blocking buffer and incubated overnight at 4°C. Cells were then washed three times with PBS-TX (PBS + 0.1% Triton X-100) then incubated for 1 hour at room temperature with secondary goat anti-rabbit AlexaFluor 488 antibody diluted 1:5000 in blocking buffer containing 4',6-diamidino-2-phenylindole (DAPI) diluted 1:10000, to counterstain nuclei. Cells were then washed three times with PBS-TX and then imaged in PBS using confocal microscopy (Operetta CLS, Revvity) using a long working distance 20X objective, capturing 5 fields of view per well with 5 Z-slices and a Z-step of 1.2 pm. The nuclear fluorescent intensity was measured and analysed using Harmony v5.1 (Revvity).

[1057] In this assay, PARP1 and PARP2 signal intensity in the nuclei assess the level of the two proteins bound to the chromatin and was used to estimate the level of PARP1 and PARP2 chromatin retention induced by the test compounds. The intensity of the nuclear staining for Poly / Mono-ADP Ribosylation was used to measure the level of PARylation and the inhibitory effect of the test compounds. Dose-response curves, EC50 and area under the curve (AUC), were generated with GeneData Software. The units of AUC are expressed as % activity x concentration in pM (%A*pM).

[1058] Results

[1059] The results of the immunofluorescence chromatin retention and PARylation assay are shown in Figure 2 and are summarised below in Table 5.

[1060] Table 5: Results of Biological Example 3

[1061] Example PARP1 PARP2 PARP1 PARP2 PARP1 PARyla Chromatin Chromatin Chromatin Chromatin Selectivity tion retention retention retention retention (AUC) EC50 EC50(nM) EC50(nM) AUC AUC (nM)

[1062] (%A*pM) (%A*pM)

[1063] Example A1 1.4 2.2 340 270 1.3 0.98

[1064]

[1065] ART-C-P3900PCT

[1066] Example A2 0.45 0.70 340 330 1.0 0.42 Olaparib 1,800 2,000 140 80 1.7 31 Talazoparib 3.2 24 260 170 1.5 2.1 Saruparib 1.7 > 10,000 170 0.44 > 380 0.40 Pip-6 0.77 1.8 430 360 1.2 0.56

[1067]

[1068] The results of Biological Example 3 indicate that the tested compounds of the invention are potent inducers of PARP1 and PARP2 retention on chromatin, and inhibit PARylation at concentrations similar to clinically active PARPi. Surprisingly, removal of the fluoro-substituent on the central phenyl ring results in a modest improvement in potency for chromatin retention of PARP1 and PARP2, as well as for PARylation.

[1069] Biological Example 4: Cell viability assays with compounds of the invention in a BRCA2 DLD-1 isogenic model

[1070] Cells were grown at 37 °C in 5% CO2 in the appropriate medium as specified by the manufacturer. Cells in exponential growth phase with < 20 consecutive passages of culture were used to perform viability assays.

[1071] Cell viability evaluation was performed using the CellTiter-Glo Luminescent Cell Viability Assay kit (G7570, Promega). Test compounds in eight 3-fold serial dilutions were dispensed using a Tecan dispenser to 96-well white flat-bottomed assay plates (3610, Corning). The final concentration of DMSO was 0.1% (v / v) in each well. Cells were seeded in 175 pL media per well. The plates were incubated at 37 °C in 5% CO2 for 7 days. On day 7, the plates were removed from the incubator and equilibrated at room temperature for 30 min. 50 pl CellTiter-Glo was added to each assay well. Plates are then shaken for 4 min at 450 rpm and left to stand at room temperature for 25 minutes. Luminescence was recorded with a CLARIOstar plate reader.

[1072] All data analysis was carried out using Genedata Screener software. EC50 values were calculated using the SmartFit function. Quoted values are relative EC50, calculated as the concentration of test compound that induces a 50% growth inhibition between the minimal and maximal observed responses. Where compounds displayed biphasic behaviour, EC50 values were calculated using the Biphasic fit function and only the most potent of the two EC50 values is reported.

[1073] ResultsART-C-P3900PCT

[1074] The results of Biological Example 4 are shown below in Table 6.

[1075] Table 6: Results of Biological Example 4

[1076] Example DLD-1 WT EC50(nM) DLD-1 BRCA2-Z- EC50(nM)

[1077] Example A1 0.18 0.036

[1078] Example A2 0.20 0.026

[1079] Olaparib > 1,000 46

[1080] Talazoparib 27 0.64

[1081] Saruparib > 1,000 1.00

[1082] Pip-6 1.16 0.15

[1083]

[1084] The results of Biological Example 4 show that the tested compounds of the invention are highly cytotoxic to DLD-1 WT and DLD-1 BRCA2- / - cells in vitro. In contrast to olaparib, talazoparib and saruparib, compounds of the invention are highly cytotoxic to cancer cells that are HR proficient, as well as showing the expected sensitivity in BRCA2-deficient cells, suggesting that they may have broader clinical utility against a wider range of cancers beyond BRCA.

[1085] Biological Example 5: Cell viability assays with compounds of the invention in a range of cancer cell lines

[1086] Cells were grown and tested for viability under the conditions described in Biological Example 4 above. Where specified, single clones were isolated from single cell seeding of the parent population.

[1087] Results

[1088] The results of Biological Example 5 are summarised below in Table 7.

[1089] Table 7: Results of Biological Example 5

[1090] Viability EC5o (nM)

[1091] Cell line Tissue HR status Examples Saruparib Talazoparib Pip-6

[1092] A1 A2 A2780 Ovary HRP 2.2 1.5 0.16 0.036 0.016 A549 Lung HRP > 1000 52 0.85 0.27 0.13 A549 BRCA1

[1093] Lung 0.50 0.63 0.063 0.035 0.025 BRCA1 KO null

[1094] HCT116 Colon HRP 4.0 2.3 0.20 0.049 0.032 HT-29 Colon HRP > 1000 58 1.15 0.39 0.70

[1095]

[1096] ART-C-P3900PCT

[1097] BRCA1

[1098] JHOS-2 Ovary > 1000 134 0.74 0.21 0.19 mut

[1099] BRCA1

[1100] JHOS-4 Ovary > 1000 0.57 0.13 0.041 0.070 null

[1101] RAD51C

[1102] KP-363T Colon 0.66 0.58 0.09 0.032 0.008 null

[1103] MCF7 Breast HRP > 1000 13 0.41 0.080 0.079 MDA-MB- BRCA1

[1104] Breast 0.69 0.37 0.07 0.030 0.012 436 null

[1105] MDA-MB- Breast PALB2 mut 10 6.3 0.32 0.073 0.050 468

[1106] NCI-H460 Lung HRP 2.5 2.3 0.24 0.075 0.031 NCI-N87 Stomach HRP > 1000 286 2.10 0.33 0.40 PEO1 BRCA2

[1107] Ovary 0.86 0.77 0.21 0.060 0.020 clone 10 null

[1108] BRCA2

[1109] PEO4 Ovary > 1000 77 0.66 0.31 0.18 revertant

[1110] BRCA1

[1111] SUM149PT Breast 6.31 1.58 0.16 0.040 0.020

[1112] A11q

[1113] BRCA1

[1114] UWB1.289 Ovary 2.9 1.7 0.27 0.043 0.027

[1115] A11q

[1116] BRCA1

[1117] UWB1.289

[1118] Ovary over- 100 1.0 0.18 0.15 + BRCA1

[1119] expressed

[1120]

[1121] The results of Biological Example 5 show that the tested compounds of the invention are highly cytotoxic to a wide variety of cancer cell lines of diverse lineage and HR status, suggesting that they may have clinical utility in HRD populations and against a broader range of cancers beyond BRCA-deficiency.

[1122] Biological Example 6: Cell viability assays with ADCs of the invention

[1123] Cell viability assays were performed as described in Biological Example 4 using serial dilutions of the indicated ADCs. Expression levels of TROP2 and HER2 were assessed using flow cytometry. Cells (0.5 x 106) were harvested and washed with cold phosphate buffered saline (PBS) (PAN-Biotech, P04-36500), followed by a wash with 0.5% bovine serum albumin (BSA) (PAN-Biotech, P06-1391500). Cells were then incubated with rabbit Alexa Fluor 647- conjugated anti-human TROP2 (1:100; Novus Biologicals, NBP2-89492AF647) and mouse FITC-conjugated anti-human HER2 (1:100, BD Biosciences, 340553) antibodies, or rabbitART-C-P3900PCT

[1124] A ADC EC ( / L)ngm5olexa Fluor 647-conjugated IgG (1:100, Novus Biologicals, NBP2-24891AF647) and mouse FITC-conjugated IgGlK (1:100, BD Biosciences, 550616) isotype controls on ice for 30 minutes. After washing with PBS twice, fluorescence was measured using a Flow Cytometer BD LSRFortessa (SORP) and analysed using FlowJo. The mean fluorescence intensity was calculated and normalised to the isotype-matched control-stained cells. The percent of positive cells was also extrapolated.

[1125] Results

[1126] The results of the cell viability assays are shown in Figure 3 and are summarised below in Table 8.

[1127] Table 8: Results of Biological Example 6

[1128] NCI- DLD-1 PEO1

[1129] DLD-1 WT MCF7 SUM149PT N87 BRCA2 KO clone 10

[1130] TROP2 levels

[1131] High Low Low Medium Very high Medium (% positive

[1132] (92.0%) (9.8%) (7.8%) (33.4%) (99.6%) (42.5%) cells)

[1133] HER2 levels

[1134] High Medium / Low Medium / Low Very low Very low Very low (% positive

[1135] (70.6%) (15.8%) (10.4%) (1.8%) (2.5%) (1.0%) cells)

[1136] BRCA2 BRCA1 HR status HRP HRP BRCA2 null HRP

[1137] null A11q Example C1 144 - 2300 52 22 34 Example C2 40 - 1000 51 5.4 23 Example C3 34 - 248 100 4.6 21 Example C4 105 >10,000 3492 376 8.2 1725 Example C7 96 >10,000 1621 35 6.6 475 Example C10 55 - 920 36 - - Example C12 70 >10,000 3980 11 6.4 18 Example C15 125 >10,000 3810 859 - 3440 Dato-Dxd

[1138] (Datopotamab 210 >10,000 2518 257 4.9 718 deruxtecan)

[1139] Enhertu

[1140] (Trastuzumab 25 >10,000 1100 >10000 2900 - deruxtecan)

[1141]

[1142] ART-C-P3900PCT

[1143] The results of Biological Example 6 show that the tested ADCs of the invention are highly cytotoxic to a variety of cancer cells of different lineage in vitro. Additionally, the results confirm that the magnitude of the effect of the ADC is dependent on both the expression levels of the target antigen and the HR status of the cancer cell.

[1144] Biological Example 7: In vivo efficacy of ADCs of the invention in a DLD-1 BRCA2- / -mouse xenograft model

[1145] Balb / c nude, female mice were inoculated subcutaneously at the right flank with DLD1 BRCA2- / - tumour cells (10 x 106) in 0.2 mLof PBS with Matrigel (1:1) for tumour development. The animals were assigned to the groups described in Table 9 below, when average tumour volume reached approximately 150 mm3and dosed intravenously with a single administration of the ADCs. Tumour sizes were measured twice weekly in two dimensions with a caliper, and the volume was expressed in mm3, using the formula: V = 0.5 a x b2where a and b are the long and short diameters of the tumour, respectively. The tumour size was then used for TGI calculation. Body weight was measured twice per week and the study was ended on the day indicated in Table 9.

[1146] TGI was calculated for each group using the formula: TGI (%) = [1 -(Ti-T0) / (Vi-V0)] *100; Ti is the average tumour volume of a treatment group on a given day, TO is the average tumour volume of the treatment group on the day of treatment start, Vi is the average tumour volume of the vehicle control group on the same day with Ti, and V0 is the average tumour volume of the vehicle group on the day of treatment start. Tumour regression was calculated when TGI was over 100% using the formula: Regression (%) = (T0-Ti) / T0 *100.

[1147] Statistical analysis was performed with GraphPad Prism on tumour readings for all groups using two-way AN OVA and Dunnett’s multiple comparison test.

[1148] Results

[1149] The results of Biological Example 7 are shown in Figure 4 and are summarised below in Table 9. The data in Table 9 were generated over multiple iterations of this study plan and the dosing groups are arranged by study number accordingly. The %TGI for each Example is therefore calculated relative to the vehicle group within the same discrete study.

[1150] Table 9: Dosing regimen and results for Biological Example 7

[1151] Study Group n Treatment Dose %TGI (day) p value 1 1 6 Vehicle - - -

[1152]

[1153] ART-C-P3900PCT

[1154] 2 6 Example C1 10 mg / kg 85% (d25) **

[1155] 3 6 Example C3 3 mg / kg 67% regression (d25) **

[1156] 4 6 Example C3 10 mg / kg 71% regression (d25) **

[1157] 1 6 Vehicle - - -

[1158] 2 6 Example C2 3 mg / kg 50% (d17) ns

[1159] 2 3 6 Example C2 10 mg / kg 95% (d17) *

[1160] 4 6 Example C10 3 mg / kg 98% (d17) *

[1161] 5 6 Example C10 10 mg / kg 64% regression (d17) **

[1162] 1 6 Vehicle - - -

[1163] 2 6 Example C4 3 mg / kg 95% (d21) ***

[1164] 3 6 Example C4 6 mg / kg 100% (d21) ***

[1165] 3 4 6 Example C4 10 mg / kg 51% regression (d21) ***

[1166] 5 6 Example C7 3 mg / kg 92% (d21) ***

[1167] 6 6 Example C7 6 mg / kg 93% (d21) ***

[1168] 7 6 Example C7 10 mg / kg 100% (d21) ***

[1169] 1 6 Vehicle - - -

[1170] 4 2 6 Example C9 3 mg / kg 90% (d18) ****

[1171] 3 6 Example C11 3 mg / kg 79% (d18) ***

[1172] 1 6 Vehicle - - -

[1173] 2 6 Example C12 3 mg / kg 76% (d25) ** 5

[1174] 3 6 Example C12 6 mg / kg 90% (d25) ***

[1175] 4 6 Example C13 3 mg / kg 74% (d25) **

[1176]

[1177] ART-C-P3900PCT

[1178] 5 6 Example C14 3 mg / kg 64% (d25) **

[1179] 1 6 Vehicle - - -

[1180] 2 6 Example C15 3 mg / kg 97% (d23) ****

[1181] 6 3 6 Example C15 6 mg / kg 58% regression (d23) ***

[1182] 4 6 Example C15 10 mg / kg 43% regression (d23) ***

[1183] 5 6 Example C16 10 mg / kg 98% (d23) ***

[1184]

[1185] ns = not significant; * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001

[1186] The results of Biological Example 7 show that the tested ADCs of the invention are efficacious in a mouse xenograft model of colon cancer following a single dose and that the efficacy is dose-proportional where multiple doses have been given. The bodyweight data confirm that the ADCs are well-tolerated at the administered doses for the duration of the study.

[1187] Biological Example 8: In vivo efficacy of ADCs of the invention in an NCI-N87 mouse xenograft model

[1188] Balb / c nude, female mice were inoculated subcutaneously at the right flank with NCI-N87 tumour cells (10 x 106) in 0.2 mL of PBS with Matrigel (1:1) for tumour development. The animals were randomized as described in Table 10 below, when the average tumour volume reached approximately 175 mm3and dosed intravenously with a single administration of the ADCs. Tumour sizes were measured twice weekly in two dimensions with a caliper, and the volume was expressed in mm3, using the formula: V = 0.5 a x b2where a and b are the long and short diameters of the tumour, respectively. The tumour size was then used for TGI calculation, as described above for Biological Example 7. Body weight was measured twice per week and the study was ended on the day indicated in Table 10.

[1189] Statistical analysis was performed with GraphPad Prism on tumour readings for all groups using two-way ANOVA and Sidak's or Dunnett’s multiple comparisons tests.

[1190] Results

[1191] The results of Biological Example 8 are shown in Figure 5 and are summarised below in Table 10. The data in Table 10 were generated over multiple iterations of this study plan and the dosing groups are arranged by study number accordingly. The %TGI for each Example is therefore calculated relative to the vehicle group within the same discrete study.ART-C-P3900PCT

[1192] Table 10: Dosing regimen and results for Biological Example 8

[1193] Study Group n Treatment Dose % TGI (day) p value 1 6 Vehicle (PBS) - - - 1

[1194] 2 6 Example 01 1 mg / kg 50% (d18) ** 1 6 Vehicle - - - 2 2 6 Example 02 2 mg / kg 37% (d21) **

[1195] 3 6 Example 03 2 mg / kg 48% (d21) * 1 6 Vehicle - - - 2 6 Example 04 3 mg / kg 100% (d37) * 3 6 Example 04 6 mg / kg 52% regression (d37) ** 3 4 6 Example 04 10 mg / kg 50% regression (d37) **

[1196] 5 6 Example 07 3 mg / kg 65% (d37) ns 6 6 Example 07 6 mg / kg 81% (d37) * 7 6 Example 07 10 mg / kg 97% (d37) * 1 6 Vehicle - - - 2 6 Example 09 6 mg / kg 55% regression (d34) *** 3 6 Example 010 6 mg / kg 42% regression (d34) *** 4

[1197] 4 6 Example 011 6 mg / kg 33% regression (d34) *** 5 6 Example 013 6 mg / kg 83% (d34) *** 6 6 Example 014 6 mg / kg 76% (d34) ** 1 6 Vehicle - - - 5 2 6 Example 012 3 mg / kg 87% (d25) ns 3 6 Example 012 6 mg / kg 99% (d25) * 1 6 Vehicle - - - 2 6 Example 015 3 mg / kg 57% regression (d21) ** 6 3 6 Example 015 6 mg / kg 62% regression (d21) **

[1198] 4 6 Example 015 10 mg / kg 65% regression (d21) ** 5 6 Example 016 10 mg / kg 11% regression (d21) **

[1199]

[1200] ART-C-P3900PCT

[1201] ns = not significant; * = p < 0.05; ** = p < 0.01; *** = p < 0.001; statistical analysis using two-way ANOVA and Sidak's (study 1) or Dunnett’s (studies 2, 3, 4, 5 & 6) multiple comparisons tests.

[1202] The results of Biological Example 8 show that the tested ADCs of the invention are efficacious in a mouse xenograft model of gastric cancer following a single dose and that the efficacy is dose-proportional where multiple doses have been given. The bodyweight data confirm that the ADCs are well-tolerated at the administered doses for the duration of the study.

[1203] Biological Example 9: In vitro metabolic stability assessment of compounds of the invention

[1204] In vitro metabolic stability assessments were carried out in human microsomes and hepatocytes under industry standard conditions as detailed below.

[1205] Microsomal stability assay

[1206] Stability of compounds was assessed in human liver microsomes (Corning) using the following procedure, where T is ‘Test’ and NCF is ‘No co-factor,’ i.e. without NADPH:

[1207] 1. Empty ‘Incubation’ plates T60 and NCF60 were pre-warmed at 37°C for 10 min.

[1208] 2. Microsome working solution (445 μL) was transferred into pre-warmed ‘Incubation’ plates T60 and NCF60, followed by a 10 min incubation at 37°C.

[1209] 3. Microsome working solution (54 μL) was transferred to a Blank60 plate, followed by the addition of 6 μL NADPH cofactor and 180 μL of stop solution into each well.

[1210] 4. Compound working solution (5 pL) was added to the ‘incubation’ plates (T60 and NCF60) containing microsomes.

[1211] 5. For the ‘Incubation’ plate NCF60, 50 μL of PB buffer was added, the plate was incubated at 37°C for 60 min.

[1212] 6. Stop solution (180 μL) and NADPH working solution (6 μL) were added to the TO plate.

[1213] Then, a mixture (54 μL) was removed from the ‘Incubation’ plate T60 and transferred to the TO plate.

[1214] 7. For the ‘Incubation’ plate T60, NADPH working solution (44 μL) was added, followed by a 60 min incubation at 37°C.

[1215] 8. At 5, 15, 30, 45, and 60 min, 60 μL of each sample at each time point was transferred to a well containing 180 μL of stop solution, followed by mixing.

[1216] 9. All sampling plates were shaken for 10 min, then centrifuged at 3220 xg for 20 min at 4°C.

[1217] 10. The supernatant (80 μL) was transferred into 240 μL of pure water and mixed using a plate shaker for 10 min.ART-C-P3900PCT

[1218] 11. Each bioanalysis plate was sealed and shaken for 10 min prior to LC-MS / MS analysis.

[1219] The reagents used for the microsomal stability assay were prepared as detailed in Table 11 below.

[1220] Table 11: Reagents for the microsomal stability assay

[1221] Reagent Stock cone. Final cone. Test compounds 10 mM 1 μM

[1222] Positive controls 10 mM 1 μM

[1223] Human liver microsomes - 0.5 mg / mL NADPH - 1 mM

[1224]

[1225] Testosterone, Diclofenac and Propafenone were used as positive controls. Stock solutions of test compounds and positive controls were diluted to 100 pM with MeCN. Microsomes were diluted to a concentration of 0.56 mg / mL using a 100 mM potassium phosphate buffer (PB buffer). An appropriate amount of NADPH powder was weighed and diluted to a concentration of 10 mM using a 10 mM MgCl₂ solution. The stop solution consisted of cold (4°C) MeCN containing 250 nM tolbutamide and 250 nM labetalol as internal standards (IS).

[1226] Data analysis

[1227] The percentage of remaining test compound after incubation was calculated using the following equation:

[1228] Peak area ratio of analyte to internal standard at each time point

[1229] %Remaining= — — - -:— - - - -:- - - - — - -::— x 100

[1230] Peak area ratio of analyte to internal standard at zero time point

[1231] The equation of first-order kinetics was used to calculate T1 / 2:

[1232] Ct= C0-e-k'‘

[1233]

[1234] CLint (microsomes) is calculated as follows.

[1235] CLint (mic) = 0.693 / T½ / mg microsome protein per mL

[1236] Hepatocyte stability assay

[1237] Stability of compounds was assessed in human hepatocytes (BiolVT) using the following procedure:ART-C-P3900PCT

[1238] 1. Preparation of intermediate solution: the stock solution of test compound was diluted to 1 mM with DMSO and the stock solution of positive control compound was diluted to 3 mM with DMSO.

[1239] 2. Preparation of working solution: the intermediate solution of test compound was diluted to 100 pM with ACN and the intermediate solution of positive control compound was diluted to 300 pM with ACN.

[1240] 3. Preparation of 0.5 x 106 / mL cells suspension: cryopreserved cells were thawed, isolated and suspended in incubation medium. Then they were diluted with prewarmed incubation medium to 0.5 x 106cells / mL.

[1241] 4. 198 μL of pre-warmed cell suspension was added in 96-well plates.

[1242] 5. 2 μL working solution was added to each well of 96-well plates.

[1243] 6. TO Sample was mixed for about 1 min to achieve a homogenous suspension. Then 25 μL of each sample was transferred into wells containing 125 μL of ice-cold stop solution followed by mixing.

[1244] 7. Incubation plates were incubated at 37°C in a 95% humidified incubator at 5% CO2 with constant shaking.

[1245] 8. At 15, 30, 60 and 90 min, samples were mixed, then 25 μL of each sample at each time point was transferred to wells containing 125 μL of ice-cold stop solution followed by further mixing.

[1246] 9. Medium Control (MC) sample plates (labelled as T0-MC and T90-MC) were prepared in the same way as cell incubation except that medium was used instead of cell suspension.

[1247] 10. The plates were shaken immediately on a plate shaker, then all sample plates were centrifuged at 3220 x g for 20 min.

[1248] 11. After centrifugation, 80 μL / well of supernatant in the sample plate was transferred to another set of pre-labelled 96-well plates containing 240 μL of ultra-pure water.

[1249] 12. Analytical plates were sealed and stored at 4°C prior to LC-MS / MS analysis.

[1250] The reagents used for the hepatocyte stability assay were prepared as detailed in Table 12 below.

[1251] Table 12: Reagents for the hepatocyte stability assay

[1252] Reagent Final cone.

[1253] Test compounds 1 μM

[1254] Positive controls 3 μM

[1255] Human hepatocytes 0.5 x106cells / mL

[1256]

[1257] ART-C-P3900PCT

[1258] MeCN 0.90 %

[1259] DMSO 0.10 %

[1260]

[1261] 7-Ethoxycoumarin and 7-hydroxycoumarin were used as positive controls. The thawing medium was Williams’ Medium E containing 5% fetal bovine serum and 30% Percoll solution and other supplements. The incubation medium was Williams’ Medium E (no phenol red) containing 2 mM L-Glutamine and 25 mM HEPES. The stop solution consisted of cold (4°C) MeCN containing 250 nM tolbutamide and 250 nM labetalol as internal standards (IS).

[1262] Data analysis

[1263] The percentage of remaining test compound after incubation was calculated using the following equation:

[1264] Peak area ratio of analyte to internal standard at each time point

[1265] %Remaining=— — - -:— - - - -:- - - - — - -::— x 100

[1266] Peak area ratio of analyte to internal standard at zero time point

[1267] The equation of first-order kinetics was used to calculate T1 / 2:

[1268] Ct= C0-e-k-‘

[1269]

[1270] CLint (hepatocytes) is calculated as follows.

[1271] CLint (hep) = 0.693 / T½ / million cells per mL

[1272] Results

[1273] The results of Biological Example 9 are shown below in Table 13.

[1274] Table 13: Results of Biological Example 9

[1275] Example CLint microsomes CLint hepatocytes (μL / min / 10⁶

[1276] (pL / min / mg) cells)

[1277] Example A1 370 38

[1278] Example A2 220 50

[1279] Talazoparib < 9.6 < 6.4

[1280] Saruparib < 9.6 < 6.4

[1281] Pip-6 16 < 6.4

[1282]

[1283] The results of Biological Example 9 indicate that the tested compounds of the invention display low in vitro metabolic stability, a characteristic which could be beneficial in facilitating their rapid clearance from systemic circulation following release from an ADC and avoiding payloadART-C-P3900PCT

[1284] related toxicity. Interestingly, removal of the fluoro-substituent on the central phenyl ring increases clearance in human hepatocytes.

[1285] Biological Example 10: In vitro permeability assessment of compounds of the invention In vitro permeability assessments were carried out in an MDCK assay under industry standard conditions.

[1286] MDCK assay

[1287] MDCK II cells (obtained from Piet Borst at the Netherlands Cancer Institute) were seeded onto polycarbonate membranes (PC) in 96-well insert systems at 2.33 x 105cells / cm2for 4- to 7 days to achieve confluent cell monolayer formation. HBSS (Hanks Balanced Salt Solution) containing 10.0 mM HEPES at pH 7.50 ± 0.05 was used as transport buffer in this study.

[1288] Test compounds were tested at 2.00 pM bi-directionally in duplicate. Digoxin, Nadolol and Metoprolol were used as positive controls. Digoxin was tested at 10.0 μM bi-directionally in duplicate, while Nadolol and Metoprolol were tested at 2.00 μM in the A to B direction in duplicate. Final DMSO concentration was adjusted to less than 1.0%. The plate was incubated for 2.5 hours in a CO2 incubator at 37.0°C, with 5.0% CO2 at saturated humidity without shaking. Following incubation, all samples were mixed with stop solution and centrifuged at 3220 xg for 20 minutes. Concentrations of test and control compounds in samples were semi-quantitatively determined by LC-MS / MS methodologies, using area ratio of analyte / internal standard.

[1289] Data analysis

[1290] The apparent permeability coefficient Papp(cm / s) was calculated using the equation:

[1291] Papp=(dCr / dt) X Vr / (A X Co)

[1292] Where dCr / dtis the cumulative concentration of compound in the receiver chamber as a function of time; Vris the solution volume in the receiver chamber (0.0750 mL on the apical side, 0.250 mL on the basolateral side); A is the surface area for the transport, i.e. 0.143 cm2for the area of the monolayer; and Co is the initial concentration in the donor chamber.

[1293] The efflux ratio was calculated using the equation:

[1294] Efflux Ratio = Papp(B-A) / Papp(A-B)ART-C-P3900PCT

[1295] Results

[1296] The results of Biological Example 10 are shown below in Table 14.

[1297] Table 14: Results of Biological Example 10

[1298] Example Papp A-B (10-6Papp B-A (10-6Efflux Ratio

[1299] cm / s) cm / s)

[1300] Example A1 6.4 12 1.8

[1301] Example A2 7.9 15 1.9

[1302] Pip-6 3.8 23 6.2

[1303]

[1304] The results of Biological Example 10 indicate that the tested compounds of the invention display high permeability and minimal efflux, a characteristic which may be beneficial in facilitating the bystander effect following release from an ADC. Surprisingly, removal of the fluoro-substituent on the central phenyl ring results in an increase in permeability.

[1305] Biological Example 11: In v / o efficacy of ADCs of the invention in a SUM149PT mouse xenograft model

[1306] Balb / c nude, female mice were inoculated subcutaneously at the right flank with SUM149PT tumour cells (10 x 106) in 0.2 mL of PBS with Matrigel (1:1) for tumour development. The animals were randomized as described in Table 15 below, when the average tumour volume reached approximately 140 mm3and dosed intravenously with a single administration of the ADCs. Tumour sizes were measured twice weekly in two dimensions with a caliper, and the volume was expressed in mm3, using the formula: V = 0.5 a x b2where a and b are the long and short diameters of the tumour, respectively. The tumour size was then used for TGI calculation, as described above for Biological Example 7. Body weight was measured twice per week and the study was ended on the day indicated in Table 15.

[1307] Statistical analysis was performed with GraphPad Prism on tumour readings for all groups using two-way AN OVA and Dunnett’s multiple comparison test.

[1308] Results

[1309] The results of Biological Example 11 are shown in Figure 6 and are summarised below in Table 15.

[1310] Table 15: Dosing regimen and results for Biological Example 11

[1311]

[1312] Group n Treatment Dose % TGI (day) p valueART-C-P3900PCT

[1313] 1 6 Vehicle - - - 2 6 Example C4 10 mg / kg 69% regression (d28) ****

[1314] 3 6 Example C7 5 mg / kg 76% (d28) ****

[1315] 4 6 Example C7 10 mg / kg 91% (d28) ****

[1316] Reference

[1317] 5 6 10 mg / kg 21% (d28) ns

[1318] Example C8

[1319]

[1320] ns = not significant; **** = p < 0.0001

[1321] The results of Biological Example 11 show that the tested ADCs of the invention are efficacious in a mouse xenograft model of breast cancer following a single dose and that the efficacy is dose-proportional where multiple doses have been given. The data also confirm that the efficacy is dependent on the target antigen, as isotype control Reference Example C8 does not induce tumour growth inhibition. The bodyweight data confirm that the ADCs are well-tolerated at the administered doses for the duration of the study.

[1322] Biological Example 12: B-Glucuronidase release assay

[1323] The selective release of payload upon exposure to β-glucuronidase activity was assessed by LC-MS / MS analysis of samples of linker-payloads incubated in the presence and absence of recombinant β-glucuronidase. The general procedure for this assay is as follows:

[1324] 1. Preparation of 100 mM sodium phosphate (PBS) solution: A basic solution was prepared by dissolving 14.2 g / L Na2HPO4 in deionized water. An acidic solution was prepared by dissolving 12.0 g / L NaH2PO4 in deionized water. The basic solution was titrated with the acidic solution to pH 7.4 and stored at 4 °C for up to 30 days. The pH was checked on the day of experiment and was adjusted if outside the range pH 7.4 ± 0.1.

[1325] 2. Preparation of 10 mM / V-acetylcysteine solution: 1.94 mg / V-acetylcysteine was dissolved in 1.18 mL of the above PBS pH 7.4 solution at rt.

[1326] 3. Preparation of working solution: The stock solutions of compounds were prepared in DMSO at a concentration of 5 mM. 10 pL of 5 mM test compound was transferred into a fresh tube, then 10 pL of 10 mM / V-acetylcysteine and 80 pL of PBS pH 7.4 were added to the tube and mixed well. The pH of the working solution was confirmed to be in the range pH 6.5-7.5, and then the working solutions were left to stand at rt for 1 h.ART-C-P3900PCT

[1327] 4. Preparation of β-glucuronidase solution: 232 mg NaOAc was dissolved in 28.2 mL of deionised water (100 mM) at rt and the pH was adjusted to pH 4.7 by the addition of AcOH. 3.98 mg β-glucuronidase was dissolved in 3.98 mL of the 100 mM NaOAc solution to give a concentration of 1 mg / mL.

[1328] 5. Procedure for stability determination: 10 pL of 50 pM working solutions (500 pM working solution diluted with 100 mM NaOAc), 40 pL of 100 mM NaOAc and 50 pL of 1 mg / mL β-glucuronidase solution were added to low adsorption tubes to achieve a final compound concentration of 5 pM, and β-glucuronidase concentration of 0.5 mg / mL. No enzyme control incubations were prepared in an analogous fashion, adding 90 pL of 100 mM NaOAc to the test compound solution. The assay was performed in duplicate. The reactions were incubated at 37 °C at 70 rpm in a water bath and samples were taken at 0, 1, 3, 5 and 7 h. The initiation of the reactions was staggered so that all time points were terminated with 1000 pL cold quench solution (MeCN with 0.1% formic acid containing internal standards (IS, 100 nM Tolbutamide, 500 nM Labetalol and 2 pM Ketoprofen)) at the same time. Samples were vortexed for 1 min, then centrifuged at rt at 4000 rpm for 10 min. Aliquots of 200 pL of the supernatant were used for LC-MS / MS analysis.

[1329] 6. Procedure for sample analysis: The plate was placed into a well plate autosampler.

[1330] The samples were evaluated by LC-MS / MS analysis and metabolites were detected using HRMS.

[1331] Final working stock concentrations, volumes and ratios are shown in Table 16 below.

[1332] Table 16: Reagent concentrations and volumes for the P-glucuronidase release assay Reagent Stock Cone. Final cone.

[1333] Test compound 5 mM 5 pM

[1334] / V-Acetylcysteine 10 mM 10 pM

[1335] P-glucuronidase - 0.5 mg / mL

[1336]

[1337] PBS - -

[1338] Data analysis

[1339] All calculations were carried out using Microsoft Excel. Peak area ratios were determined from extracted ion chromatograms. Percentages of linker-payload remaining and payload release at each time point were calculated using the following equation:

[1340] Peak area ratio of analyte to internal standard at each time point

[1341] %= - - - - - x 100

[1342] Peak area ratio of analyte to internal standard at zero time pointART-C-P3900PCT

[1343] Results

[1344] The results of the β-glucuronidase release assay are shown in Figure 7 and are summarised below in Table 17.

[1345] Table 17: Results of Biological Example 12

[1346] Linker-Payload Payloac Example Condition* Remaining (%) Released [%)

[1347] 0 h 1 h 3 h 5 h 7 h 0 h 1 h 3 h 5 h 7 h + Glue 100 0.1 0.3 3.8 0.2 0.1 22 39 38 40 B1

[1348] - Glue 100 92 85 82 81 0.0 0.3 1.1 1.4 1.5 + Glue 100 0.3 0.1 0.3 0.1 3.8 45 41 44 43 B3

[1349] - Glue 100 100 99 100 100 0.0 0.1 0.2 0.2 0.3 + Glue 100 0.2 0.0 0.0 0.1 0.0 65 98 100 100 B4

[1350] - Glue 100 95 81 93 87 0.0 0.0 0.0 0.0 0.0 + Glue 100 0.3 0.7 0.0 0.5 1.3 100 100 100 100 B5

[1351] - Glue 100 99 94 88 88 0.1 1.9 4.4 6.0 5.5 + Glue 100 0.4 4.3 0.6 1.8 0.0 55 92 100 93 B6

[1352]

[1353] - Glue 100 100 100 100 97 0.3 0.0 0.2 0.0 0.0 indicates whether the incubation was cone ucted in the presence (+) or absence (-) of recombinant β-glucuronidase enzyme.

[1354] The results of Biological Example 12 confirm that the tested compounds of the invention selectively undergo decomposition to release payload in the presence of β-glucuronidase enzyme. The data also show that the tested compounds of the invention are stable in the absence of β-glucuronidase enzyme and that no payload is released, suggesting that the linker-payloads are resistant to hydrolysis under these conditions.

[1355] Biological Example 13: Native mass spec analysis of ADCs of the invention

[1356] The drug-to-antibody ratio (DAR) of ADCs of the invention was quantified by native mass spec analysis on an Agilent 1290 HPLC coupled with an Agilent MS TOF G6230B mass spectrometer. ADC solutions were injected as prepared without dilution or further sample preparation. The instrument settings were as follows:

[1357] Column: AdvanceBio SEC 200A, 2.1 x 50 mm, 1.9|jm Mobile phase: 100 mM NH4Ac in H2O

[1358] Run time: 5 min

[1359] Ion source: DualAJS ESI

[1360] Scan patterns: PositiveART-C-P3900PCT

[1361] Mass deconvolution range: 2000-7000 Da

[1362] Theoretical mass analysis was performed using Agilent MassHunter Sequence Manager 10.0.

[1363] The DAR was calculated using the following equation:

[1364] ZPeak heiqht x n

[1365] ^ Totai peak height

[1366]

[1367] where n is the number of payloads attached to the mAb.

[1368] Results

[1369] The results of Biological Example 13 are shown in Figure 8 and are summarised below in Table 18.

[1370] Table 18: Results of Biological Example 13

[1371] DAR Theoretical Observed

[1372] Example Height % Height Peak Mass (Da)* Mass (Da)

[1373] Sacituzumab N / A 148131 148132 2.33E+04 100 LALA-Sacituzumab N / A 147963 147966 6.42E+03 100

[1374] 3 151245 151255 7.61E+02 4.27 Example C4 4 152283 152296 1.53E+04 85.49

[1375] 5 153481 153499 1.83E+03 10.24 3 148458 148456 9.42E+02 5.31 Reference

[1376] 4 149499 149496 1.48E+04 83.43 Example C5

[1377] 5 150699 150696 2.00E+03 11.26 2 150176 150173 2.10E+01 0.22 3 151199 151205 2.29E+02 2.36 Example C7

[1378] 4 152222 152224 7.83E+03 80.82 5 153404 153406 1.61E+03 16.61 0 148130 148129 7.01E+02 4.51 2 150271 150274 3.74E+03 24.05 Example C11 3 151341 151346 1.17E+02 0.75

[1379] 4 152411 152417 5.91E+03 38.02 5 153481 153484 1.80E+02 1.16

[1380]

[1381] ART-C-P3900PCT

[1382] 6 154552 154561 3.56E+03 22.88 7 155622 155636 9.56E+01 0.62 8 156692 156704 1.25E+03 8.02 3 151086 151086 9.55E+02 11.14 Example C15 4 152127 152127 6.92E+03 80.75

[1383] 5 153167 153327 6.95E+02 8.11 3 151032 151033 1.04E+03 7.84 Example C16 4 152055 152053 1.10E+04 83.07

[1384] 5 153078 153235 1.21E+03 9.09

[1385]

[1386] *The calculated masses of Sacituzumab and LALA-Sacituzumab correspond to the following modifications: pE(Q)*2, -K*2, G0F*2, where pE(Q) represents a cyclisation of the / V-terminal glutamine residue of the HC; -K represents loss of the C-terminal lysine residue of the HC; and GOF is an N-linked glycan at position N297 of the HC. The corresponding theoretical masses of the resulting ADCs have been calculated accordingly.

[1387] Biological Example 14: Cell viability assays with compounds of the invention in RPE-1 WT, PARP1 KO and PARP1 / PARP2 dual KO lines

[1388] Cells were grown and tested for viability under the conditions described in Biological Example 4 above. Where specified, single clones were isolated from single cell seeding of the parent population.

[1389] Results

[1390] The results of Biological Example 14 are shown in Figure 9 and are summarised below in Table 19.

[1391] Table 19: Results of Biological Example 14

[1392] Viability EC50(nM)

[1393] Cell Line

[1394] Saruparib Talazoparib Example A1 Example A2 hTERT RPE-1 WT > 1000 910 0.53 0.27 hTERT RPE-1

[1395] > 1000 500 3.8 0.65 PARP1 KO clone G7

[1396] hTERT RPE-1

[1397] PARP1_PARP2 KO > 1000 > 1000 > 10 > 10 clone E6

[1398]

[1399] ART-C-P3900PCT

[1400] The results of Biological Example 14 indicate that the cytotoxicity of the tested compounds of the invention is mediated by both PARP1 and PARP2. Loss of PARP1 induces partial resistance to the tested compounds of the invention, loss of both PARP1 and PARP2 together completely ablates cytotoxicity.

Claims

ART-C-P3900PCTCLAIMS1. A compound of formula (I):or a tautomeric or a stereochemically isomeric form, a pharmaceutically acceptable salt or a solvate thereof, wherein:R1represents hydrogen or methyl;R2represents Ci-e alkyl or C3-8 cycloalkyl, wherein said Ci-e alkyl or C3-8 cycloalkyl group is optionally substituted by one or more (e.g. 1 to 11) halogen atoms, Ci-e alkyl or haloCi-6 alkyl; andR3represents hydrogen or fluorine.

2. The compound as defined in claim 1, wherein R1represents hydrogen.

3. The compound as defined in claim 1 or claim 2, wherein R2represents:(a) C1.6 alkyl optionally substituted by one or more halogen atoms; or(b) C1.6 alkyl optionally substituted by 1 to 7 halogen atoms; or(c) methyl or ethyl optionally substituted by 2 to 5 halogen atoms; or(d) ethyl optionally substituted by 5 halogen atoms; or(e) -CF2-CHF2 or -CF2-CF3; or(f) -CF2-CF3.

4. The compound as defined in any one of claims 1 to 3, wherein R3represents hydrogen.

5. The compound as defined in any one of claims 1 to 3, wherein R3represents fluorine.

6. The compound as defined in claim 1, wherein the compound is the free base of a compound of Examples A1 to A3:ART-C-P3900PCT(S)-4-(4-Fluoro-3-(6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)-5-hydroxyphthalazin-1(2 / - / )-one (A1);(S)-5-Hydroxy-4-(3-(6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)phthalazin-1(2 / - / )-one (A2); or(S)-4-(4-Fluoro-3-(6-methyl-3-(1, 1,2,2-tetrafluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)-5-hydroxyphthalazin-1(2 / - / )-one (A3);or a pharmaceutically acceptable salt or solvate thereof.

7. A process for preparing a compound of formula (I) as defined in claim 1 which comprises:(a) reacting a compound of formula (II):R3(II)wherein R1, R2and R3are as defined in claim 1, with a suitable reagent, such as hydrazine hydrate; or(b) interconversion of a compound of formula (I) or protected derivative thereof to a further compound of formula (I) or protected derivative thereof; or(c) deprotection of a protected derivative of a compound of formula (I); or(d) optional formation of a pharmaceutically acceptable salt of a compound of formula (I).

8. A compound of formula (I) or a tautomeric or a stereochemically isomeric form, a pharmaceutically acceptable salt or a solvate thereof, prepared by the process as defined in claim 7.

9. A drug conjugate wherein said drug conjugate comprises:ART-C-P3900PCT(a) one or more payloads each comprising a compound of formula (I) as defined in any one of claims 1 to 6 or 8, which is covalently bound to:(b) a linker, which is covalently bound to:(c) a tumour cell binding moiety.

10. The drug conjugate as defined in claim 9, which comprises between 1 and 10, such as 4 or 8 payloads, each comprising a compound of formula (I).

11. The drug conjugate as defined in claim 9 or claim 10, wherein said one or more payloads each comprise the same compound of formula (I).

12. The drug conjugate as defined in any one of claims 9 to 11, wherein the linker comprises an -L1-L2-L3- moiety, wherein:Li represents a tumour cell binding moiety attachment moiety;L2 represents a self-immolative trigger moiety; andL3 represents a payload attachment moiety.

13. The drug conjugate as defined in any one of claims 9 to 12, wherein the linker comprises an -Li-Lia-L2-Ls- moiety, wherein:Li represents a tumour cell binding moiety attachment moiety;Lia represents a spacer moiety;L2 represents a self-immolative trigger moiety; andL3 represents a payload attachment moiety.

14. The drug conjugate as defined in claim 12 or claim 13, wherein the tumour cell binding moiety attachment moiety comprises a maleimide, dibromopyridazinedione or haloacetamide group, such as a maleimide or dibromopyridazinedione group.

15. The drug conjugate as defined in claim 12 or claim 13, wherein the self-immolative trigger moiety comprises a glucuronide containing moiety.

16. The drug conjugate as defined in claim 12 or claim 13, wherein the payload attachment moiety comprises a carbamate group, such as a diaminocarbamoyl moiety, or a methylene alkoxy carbamate (MAC).

17. The drug conjugate as defined in claim 13, wherein the spacer moiety comprises one or more PEG groups which may be linear or branched.ART-C-P3900PCT18. The drug conjugate as defined in any one of claims 9 to 17, wherein said linker is selected from any of linkers (i) to (vii):ART-C-P3900PCTsuch as linker (iv):ART-C-P3900PCT(iv);wherein “(a)” represents the attachment point to the payload compound of formula (I) as defined in any one of claims 1 to 6 or 8, and “(c)” represents the tumour cell binding moiety, and wherein each “S” represents a sulfur atom within a cysteine residue of said tumour cell binding moiety.

19. The drug conjugate as defined in any one of claims 9 to 17, wherein said linker is selected from any of linkers (i)a, (i)b, (ii)a, (ii)b, (iii)a, (iii)b, (v)a, (v)b, (vii)aor (vii)b:OH OHART-C-P3900PCTHO OH OH OHART-C-P3900PCT5ART-C-P3900PCT(vii)b;wherein “(a)” represents the attachment point to the payload compound of formula (I) as defined in any one of claims 1 to 6 or 8, and “(c)” represents the tumour cell binding moiety, and wherein each “S” represents a sulfur atom within a cysteine residue of said tumour cell binding moiety.

20. The drug conjugate as defined in any one of claims 9 to 19, wherein said tumour cell binding moiety is selected from a peptide, an antibody or an antigen binding fragment.

21. The drug conjugate as defined in claim 20, wherein said antibody or antigen binding fragment binds to a target on a tumour cell selected from: CD19, CD22, CD30, CD33, CD79b, ErbB2 / HER2, FOLR1, Nectin-4, TF and Trop-2.

22. The drug conjugate as defined in claim 20 or claim 21, wherein said antibody or antigen binding fragment binds to a target on a tumour cell which is Trop-2.

23. The drug conjugate as defined in any one of claims 20 to 22, wherein said tumour cell binding moiety is an antibody.

24. The drug conjugate as defined in claim 23, wherein said antibody is a monoclonal antibody.ART-C-P3900PCT25. The drug conjugate as defined in claim 24, wherein said monoclonal antibody is selected from: brentuximab, enfortumab, gemtuzumab, inotuzumab, loncastuximab, mirvetuximab, moxetumomab, polatuzumab, sacituzumab, tisotumab and trastuzumab.

26. The drug conjugate as defined in claim 24 or claim 25, wherein said monoclonal antibody is sacituzumab or LALA-sacituzumab.

27. The drug conjugate as defined in any one of claims 9 to 26, which is a compound of any one of Examples C1 to C4, C6 to C7 and C9 to C16:Example C1: Sacituzumab-PARPi ADC (DAR 8.0)wherein Ab represents Sacituzumab and wherein “S” represents a sulfur atom within a cysteine residue of said Sacituzumab;Example C2: Sacituzumab-PARPi ADC (DAR 8.0)wherein Ab represents Sacituzumab and wherein “S” represents a sulfur atom within a cysteine residue of said Sacituzumab;ART-C-P3900PCTExample C3: Sacituzumab-PARPi ADC (DAR 8.0)wherein Ab represents Sacituzumab and wherein “S” represents a sulfur atom within a cysteine residue of said Sacituzumab;Example C4: Sacituzumab-PARPi ADC (DAR 4.0)wherein Ab represents Sacituzumab and wherein each “S” represents a sulfur atom within a cysteine residue of said Sacituzumab;Example C6: Sacituzumab-PARPi ADC (DAR 4.0)wherein Ab represents Sacituzumab and wherein “S” represents a sulfur atom within a cysteine residue of said Sacituzumab;ART-C-P3900PCTExample C7: Sacituzumab-PARPi ADC (DAR 4.0)wherein Ab represents Sacituzumab and wherein each “S” represents a sulfur atom within a cysteine residue of said Sacituzumab;Example C9: Sacituzumab-PARPi ADC (DAR 4.0)wherein Ab represents Sacituzumab and wherein each “S” represents a sulfur atom within a cysteine residue of said Sacituzumab;Example C10: Sacituzumab-PARPi ADC (DAR 8.0)ART-C-P3900PCTwherein Ab represents Sacituzumab and wherein “S” represents a sulfur atom within a cysteine residue of said Sacituzumab;Example C11: Sacituzumab-PARPi ADC (DAR 4.0)wherein Ab represents Sacituzumab and wherein “S” represents a sulfur atom within a cysteine residue of said Sacituzumab;Example C12: Sacituzumab-PARPi ADC (DAR 4.0)wherein Ab represents Sacituzumab and wherein each “S” represents a sulfur atom within a cysteine residue of said Sacituzumab;Example C13: Sacituzumab-PARPi ADC (DAR 8.0)ART-C-P3900PCTwherein Ab represents Sacituzumab and wherein “S” represents a sulfur atom within a cysteine residue of said Sacituzumab;Example C14: Sacituzumab-PARPi ADC (DAR 4.0)wherein Ab represents Sacituzumab and wherein “S” represents a sulfur atom within a cysteine residue of said Sacituzumab;Example C15: LALA-Sacituzumab-PARPi ADC (DAR 4.0)wherein Ab represents Sacituzumab with a double L to A mutation at positions L234 and L235 (Ell numbering according to Kabat (Kabat, E. A. (1991)) on the HC to give L234A / L235A-ART-C-P3900PCTSacituzumab (LALA-Sacituzumab) and wherein each “S” represents a sulfur atom within a cysteine residue of said LALA-Sacituzumab; andExample C16: LALA-Sacituzumab-PARPi ADC (DAR 4.0)wherein Ab represents LALA-Sacituzumab (as described in Example C15) and wherein each “S” represents a sulfur atom within a cysteine residue of said LALA-Sacituzumab;such as C4, C7, C15 or C16; in particular C15.

28. An antibody which comprises a heavy chain as set forth in SEQ ID NO: 5 and a light chain as set forth in SEQ ID NO:

429. A process for preparing a drug conjugate as defined in any one of claims 9 to 27 which comprises the steps of:(a) attaching a linker as defined in any one of claims 12 to 19 to the compound of formula (I) as defined in any one of claims 1 to 6 or 8, followed by(b) attachment of the tumour cell binding moiety as defined in any one of claims 20 to 26.

30. A drug conjugate prepared by the process as defined in claim 29.

31. An intermediate selected from a compound of Example B1 to B10:Example B1: (2S,3S,4S,5R,6S)-6-(2-(3-(3-(2-(2-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1- yl)ethoxy)ethoxy)propanamido)propanamido)-4-((((S)-2-(((((4-(4-fluoro-3-((S)-6-methyl-3- (perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)-1-oxo-1,2- dihydrophthalazin-5-yl)oxy)carbonyl)(methyl)amino)methyl)pyrrolidine-1- carbonyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2 / 7-pyran-2-carboxylic acid;Example B2: (2S,3S,4S,5R,6S)-6-(2-(2-(3-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)- / V- methylpropanamido)acetamido)-4-((((S)-2-(((((4-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-ART-C-P3900PCT5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)-1-oxo-1,2-dihydrophthalazin-5-yl)oxy)carbonyl)(methyl)amino)methyl)pyrrolidine-1-carbonyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2 / 7-pyran-2-carboxylic acid;Example B3: (2S,3S,4S,5R,6S)-6-(2-(3-(3-(2-(2-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethoxy)propanamido)propanamido)-4-((((((4-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)-1-oxo-1,2-dihydrophthalazin-5-yl)oxy)methyl)(2-(methylsulfonyl)ethyl)carbamoyl)oxy)methyl)phenoxy)-3, 4, 5 -tri hy d roxy tetra hy d ro-2 H- py ra n-2-ca rboxy I i c aci d;Example B4: (2S,3S,4S,5R,6S)-6-(2-((3-(4,5-Dibromo-2-methyl-3,6-dioxo-3,6-dihydropyridazin-1(2 / - / )-yl)propanamido)methyl)-4-(((4-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)-1-oxo-1,2-dihydrophthalazin-5-yl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2 / 7-pyran-2-carboxylic acid;Example B5: (2S,3S,4S,5R,6S)-6-(2-((3-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)methyl)-4-(((4-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)-1 -oxo-1, 2-dihydrophthalazin-5-yl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2 / 7-pyran-2-carboxylic acid;Example B6: (2S,3S,4S,5R,6S)-6-(2-((3-(4,5-Dibromo-2-methyl-3,6-dioxo-3,6-dihydropyridazin-1(2 / - / )-yl)propanamido)methyl)-4-(((4-(3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)-1-oxo-1,2-dihydrophthalazin-5-yl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2 / 7-pyran-2-carboxylic acid;Example B7: (2S,3S,4S,5R,6S)-6-(2-(2-(3-(4,5-Dibromo-2-methyl-3,6-dioxo-3,6-dihydropyridazin-1(2 / - / )-yl)- / V-methylpropanamido)acetamido)-4-(((4-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)-1-oxo-1,2-dihydrophthalazin-5-yl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2 / 7-pyran-2-carboxylic acid;Example B8: (2S,3S,4S,5R,6S)-6-(2-(2-(3-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)- / V-methylpropanamido)acetamido)-4-(((4-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)-1 -oxo-1, 2-dihydrophthalazin-5-yl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2 / 7-pyran-2-carboxylic acid;Example B9: (2S,3S,4S,5R,6S)-6-(2-(2-(3-(4,5-Dibromo-2-methyl-3,6-dioxo-3,6-dihydropyridazin-1(2 / - / )-yl)-N-methylpropanamido)acetamido)-4-(((4-(3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)-1-oxo-1,2-dihydrophthalazin-5-yl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2 / 7-pyran-2-carboxylic acid; orExample B10: (2S,3S,4S,5R,6S)-6-(2-(2-(3-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)- / V-methylpropanamido)acetamido)-4-(((4-(3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-ART-C-P3900PCTtetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)-1 -oxo-1, 2-dihydrophthalazin-5-yl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2 / 7-pyran-2-carboxylic acid;such as B4 or B6.

32. A pharmaceutical composition comprising the compound of formula (I) as defined in any one of claims 1 to 6 or 8 or the drug conjugate as defined in any one of claims 9 to 27 or 30 in combination with one or more pharmaceutically acceptable excipients.

33. A pharmaceutical composition comprising a compound of formula (I) as defined in any of claims 1 to 6 or 8 or the drug conjugate as defined in any one of claims 9 to 27 or 30, in combination with one or more therapeutic agents.

34. A compound of formula (I) as defined in any of claims 1 to 6 or 8, or the drug conjugate as defined in any one of claims 9 to 27 or 30, or the pharmaceutical composition as defined in claim 32 or claim 33 for use in therapy.

35. A compound of formula (I) as defined in any of claims 1 to 6 or 8, or the drug conjugate as defined in any one of claims 9 to 27 or 30, or the pharmaceutical composition as defined in claim 32 or claim 33 for use in the prophylaxis or treatment of cancer.

36. A method of treating cancer in a patient, the method comprising administering to the patient a therapeutically effective amount of a compound of formula (I) as defined in any one of claims 1 to 6 or 8, or the drug conjugate as defined in any one of claims 9 to 27 or 30, or the pharmaceutical composition as defined in claim 32 or claim 33.

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